Academic Appointments

Administrative Appointments

  • Director, Graduate Program in Cancer Biology (1990 - 2002)
  • Director, Division of Radiation and Cancer Biology (1984 - 2004)

Honors & Awards

  • Bruce Cain Memorial Award, American Association for Cancer Research (1999)
  • Gold Medal, Americal Society for Therapeutic Radiology and Oncology (1999)
  • Failla Memorial Award, Radiation Research Society (2000)
  • Weiss Medal, Association for Radiation Research (2001)
  • Henry S. Kaplan Distinguished Scientist Award, International Association for Radiation Research (2007)

Professional Education

  • B.Sc, Birmingham University, Physics (1963)
  • M.Sc, London University, Radiation biology and physics (1965)
  • Ph.D, Oxford University, Cancer Biology (1968)

Research & Scholarship

Current Research and Scholarly Interests

We seek to understand the mechanisms responsible for the resistance of solid tumors to cancer therapies and to develop strategies to overcome these resistances. The main project is

We are investigating the role of bone marrow derived cells in restoring the tumor vasculature after radiotherapy (which destroys local angiogenesis). This process is known as vasculogenesis. In particular we seek to improve tumor cure rates by radiotherapy by inhibiting the repair of the tumor vasculature in GBM by circulating cells following radiation to the tumors by selective inhibition of the chemokine pathway(s) responsive for the mobilization and capture in the tumor of the circulating proangiogenic cells.

Clinical Trials

  • Whole Brain Radiation Therapy With Standard Temozolomide Chemo-Radiotherapy and Plerixafor in Treating Patients With Glioblastoma Recruiting

    This phase II trial studies how well whole brain radiation therapy works with standard temozolomide chemo-radiotherapy and plerixafor in treating patients with glioblastoma (brain tumor). Radiation therapy uses high energy x-rays to kill tumor cells and shrink tumors. Drugs used in chemotherapy, such as temozolomide, work in different ways to stop the growth of tumor cells, either by killing the cells, by stopping them from dividing, or by stopping them from spreading. Plerixafor is a drug that may prevent recurrence of glioblastoma after radiation treatment. Giving whole brain radiation therapy with standard temozolomide chemo-radiotherapy and plerixafor may work better in treating patients with glioblastoma.

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2020-21 Courses

Graduate and Fellowship Programs


All Publications

  • Radiation Damage to Tumor Vasculature Initiates a Program that Promotes Tumor Recurrences. International journal of radiation oncology, biology, physics Brown, J. M. 2020


    This review, mostly of preclinical data, summarizes the evidence that radiation at doses relevant to radiotherapy initiates a pathway that promotes the reconstitution of the tumor vasculature leading to tumor recurrence. The pathway is not specific to tumors; it promotes repair of damaged and ischemic normal tissues by attracting proangiogenic cells from the bone marrow. For radiation of tumors the pathway comprises: 1) Radiation causes loss of endothelial cells and reduced tumor blood perfusion leading to increased tumor hypoxia and increased levels of hypoxia inducible factor-1 (HIF-1). Alternatively increased HIF-1 levels may arise by reactive oxygen species (ROS) production caused by tumor reoxygenation, 2) Increased HIF-1 levels lead to increased levels in the tumor of the chemokine stromal cell-derived factor-1 (SDF-1, CXCL12), which captures monocytes/macrophages expressing the CXCR4 receptor of CXCL12, 3) The increased levels of tumor associated macrophages (TAMs) become highly proangiogenic (M2 polarized) and restore the tumor vasculature thereby promoting tumor recurrence. The relevance of this pathway for radiotherapy is that it can be blocked in a number of different ways including by inhibitors of monocytes/macrophages, of HIF-1, of CXCL12, of CXCR4, and of CSF-1R, the latter of which is responsible for the M2 polarization of the TAMs. All of these inhibitors produce a robust enhancement of the radiation response of a wide variety of preclinical tumor models. Further, the same inhibitors actually provide protection against radiation damage of several normal tissues. Some of these pathway inhibitors are available clinically and a first-in-human trial of the CXCR4 inhibitor, plerixafor, with radiotherapy of glioblastoma has yielded promising results including an impressive increase in local tumor control. Further clinical trials are warranted.

    View details for DOI 10.1016/j.ijrobp.2020.05.028

    View details for PubMedID 32473180

  • Macrophage exclusion after radiation therapy (MERT): A new and effective way to increase the therapeutic ratio of radiotherapy. Radiotherapy and oncology : journal of the European Society for Therapeutic Radiology and Oncology Brown, J. M., Thomas, R., Nagpal, S., Recht, L. 2019; 144: 159–64


    Here we review a variety of preclinical studies and a first-in-human clinical trial of newly diagnosed glioblastoma (GBM) patients that have investigated the significance of the influx of tumor associated macrophages (TAMs) into tumors after irradiation. We summarize the effects on the response of the tumors and normal tissues to radiation of various agents that either reduce the influx of TAMs into tumors after radiation or change their M1/M2 polarization. The studies show that following irradiation there is an accumulation of bone marrow derived TAMs in the irradiated tumors. These TAMs stimulate the resumption of blood flow in the irradiated tumors thereby promoting recurrence of the tumors. A key mechanism for this accumulation of TAMs is driven by the SDF-1/CXCR4 chemokine pathway though other pathways could also be involved for some tumors. Blocking this pathway to prevent the TAM accumulation in the tumors both enhances tumor response to radiation and protects irradiated tissues. A clinical trial in which the CXCR4 antagonist plerixafor was added to standard therapy of glioblastoma validated the preclinical findings by demonstrating i) reduced blood flow in the irradiated site, and ii) significantly improved tumor local control compared to GBM patients not treated with plerixafor. We conclude that macrophage exclusion after radiation therapy (MERT) is an effective way both to enhance the tumor response to radiation and to protect the irradiated normal tissues. Further clinical trials are warranted.

    View details for DOI 10.1016/j.radonc.2019.11.020

    View details for PubMedID 31812931

  • Dynamin 2, cell trafficking, and the triple-negative paradox. Oncotarget Chernikova, S. B., Game, J. C., Brown, J. M. 2019; 10 (24): 2336–37

    View details for PubMedID 31040924

  • Targeting the Post-Irradiation Tumor Microenvironment in Glioblastoma via Inhibition of CXCL12. Cancers Giordano, F. A., Link, B., Glas, M., Herrlinger, U., Wenz, F., Umansky, V., Brown, J. M., Herskind, C. 2019; 11 (3)


    Radiotherapy is a mainstay in glioblastoma therapy as it not only directly targets tumor cells but also depletes the tumor microvasculature. The resulting intra-tumoral hypoxia initiates a chain of events that ultimately leads to re-vascularization, immunosuppression and, ultimately, tumor-regrowth. The key component of this cascade is overexpression of the CXC-motive chemokine ligand 12 (CXCL12), formerly known as stromal-cell derived factor 1 (SDF-1). We here review the role of CXCL12 in recruitment of pro-vasculogenic and immunosuppressive cells and give an overview on future and current drugs that target this axis.

    View details for PubMedID 30813533

  • Macrophage Exclusion after Radiation Therapy (MERT): A First in Human Phase I/II Trial using a CXCR4 Inhibitor in Glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research Thomas, R. P., Nagpal, S. n., Iv, M. n., Soltys, S. G., Bertrand, S. n., Pelpola, J. S., Ball, R. L., Yang, J. n., Sundaram, V. n., Lavezo, J. L., Born, D. E., Vogel, H. n., Brown, J. M., Recht, L. n. 2019


    Preclinical studies have demonstrated that post-irradiation tumor revascularization is dependent on a stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4)-driven process in which myeloid cells are recruited from bone marrow. Blocking this axis results in survival improvement in preclinical models of solid tumors, including glioblastoma (GBM). We conducted a phase I/II study to determine the safety and efficacy of Macrophage Exclusion after Radiation Therapy (MERT) using the reversible CXCR4 inhibitor plerixafor in newly diagnosed glioblastoma patients.We enrolled 9 patients to the phase I study and an additional 20 patients to phase II using a modified toxicity probability interval (mTPI) design. Plerixafor was continuously infused intravenously via PICC line for four consecutive weeks beginning at day 35 of conventional treatment with concurrent chemo-radiation. Blood serum samples were obtained for pharmacokinetic analysis. Additional studies included relative cerebral blood volume (rCBV) analysis using MRI and histopathology analysis of recurrent tumors.Plerixafor was well tolerated with no drug-attributable grade 3 toxicities observed. At the maximum dose of 400 µg/kg/day, biomarker analysis found suprathreshold plerixafor serum levels and an increase in plasma SDF-1 levels. Median overall survival was 21.3 months (95% Confidence Interval (CI) 15.9, NA) with a progression-free survival of 14.5 months (95% CI 11.8, NA). MRI and histopathology support the mechanism of action to inhibit post-irradiation tumor revascularization.Infusion of the CXCR4 inhibitor plerixafor was well tolerated as an adjunct to standard chemo-irradiation in newly diagnosed GBM patients and improves local control of tumor recurrences.

    View details for DOI 10.1158/1078-0432.CCR-19-1421

    View details for PubMedID 31537527

  • Dynamin impacts homology-directed repair and breast cancer response to chemotherapy. The Journal of clinical investigation Chernikova, S. B., Nguyen, R. B., Truong, J. T., Mello, S. S., Stafford, J. H., Hay, M. P., Olson, A. n., Solow-Cordero, D. E., Wood, D. J., Henry, S. n., von Eyben, R. n., Deng, L. n., Gephart, M. H., Aroumougame, A. n., Wiese, C. n., Game, J. C., Győrffy, B. n., Brown, J. M. 2018


    After the initial responsiveness of triple-negative breast cancers (TNBCs) to chemotherapy, they often recur as chemotherapy-resistant tumors, and this has been associated with upregulated homology-directed repair (HDR). Thus, inhibitors of HDR could be a useful adjunct to chemotherapy treatment of these cancers. We performed a high-throughput chemical screen for inhibitors of HDR from which we obtained a number of hits that disrupted microtubule dynamics. We postulated that high levels of the target molecules of our screen in tumors would correlate with poor chemotherapy response. We found that inhibition or knockdown of dynamin 2 (DNM2), known for its role in endocytic cell trafficking and microtubule dynamics, impaired HDR and improved response to chemotherapy of cells and of tumors in mice. In a retrospective analysis, levels of DNM2 at the time of treatment strongly predicted chemotherapy outcome for estrogen receptor-negative and especially for TNBC patients. We propose that DNM2-associated DNA repair enzyme trafficking is important for HDR efficiency and is a powerful predictor of sensitivity to breast cancer chemotherapy and an important target for therapy.

    View details for PubMedID 30371505

  • The Promise of Targeting Macrophages in Cancer Therapy CLINICAL CANCER RESEARCH Brown, J., Recht, L., Strober, S. 2017; 23 (13): 3241–50


    Cancer therapy has developed around the concept of killing, or stopping the growth of, the cancer cells. Molecularly targeted therapy is the modern expression of this paradigm. Increasingly, however, the realization that the cancer has co-opted the normal cells of the stroma for its own survival has led to the concept that the tumor microenvironment (TME) could be targeted for effective therapy. In this review, we outline the importance of tumor-associated macrophages (TAM), a major component of the TME, in the response of tumors to cancer therapy. We discuss the normal role of macrophages in wound healing, the major phenotypes of TAMs, and their role in blunting the efficacy of cancer treatment by radiation and anticancer drugs, both by promoting tumor angiogenesis and by suppressing antitumor immunity. Finally, we review the many preclinical studies that have shown that the response of tumors to irradiation and anticancer drugs can be improved, sometimes markedly so, by depleting TAMs from tumors or by suppressing their polarization from an M1 to an M2 phenotype. The data clearly support the validity of clinical testing of combining targeting TAMs with conventional therapy. Clin Cancer Res; 23(13); 3241-50. ©2017 AACR.

    View details for PubMedID 28341752

    View details for PubMedCentralID PMC5529121

  • SDF-1 Blockade Enhances Anti-VEGF Therapy of Glioblastoma and Can Be Monitored by MRI NEOPLASIA Deng, L., Stafford, J. H., Liu, S., Chernikova, S. B., Merchant, M., Recht, L., Brown, J. M. 2017; 19 (1): 1-7


    Despite the approval of antiangiogenic therapy for glioblastoma multiforme (GBM) patients, survival benefits are still limited. One of the resistance mechanisms for antiangiogenic therapy is the induction of hypoxia and subsequent recruitment of macrophages by stromal-derived factor (SDF)-1α (CXCL-12). In this study, we tested whether olaptesed pegol (OLA-PEG, NOX-A12), a novel SDF-1α inhibitor, could reverse the recruitment of macrophages and potentiate the antitumor effect of anti-vascular endothelial growth factor (VEGF) therapy. We also tested whether magnetic resonance imaging (MRI) with ferumoxytol as a contrast agent could provide early information on macrophage blockade. Orthotopic human G12 glioblastomas in nude mice and rat C6 glioblastomas were employed as the animal models. These were treated with bevacizumab or B-20, both anti-VEGF antibodies. Rats were MR imaged with ferumoxytol for macrophage detection. Tumor hypoxia and SDF-1α expression were elevated by VEGF blockade. Adding OLA-PEG to bevacizumab or B-20 significantly prolonged the survival of rodents bearing intracranial GBM compared with anti-VEGF therapy alone. Intratumoral CD68+ tumor associated macrophages (TAMs) were increased by VEGF blockade, but the combination of OLA-PEG + VEGF blockade markedly lowered TAM levels compared with VEGF blockade alone. MRI with ferumoxytol as a contrast agent noninvasively demonstrated macrophage reduction in OLA-PEG + anti-VEGF-treated rats compared with VEGF blockade alone. In conclusion, inhibition of SDF-1 with OLA-PEG inhibited the recruitment of TAMs by VEGF blockage and potentiated its antitumor efficacy in GBM. Noninvasive MRI with ferumoxytol as a contrast agent provides early information on the effect of OLA-PEG in reducing TAMs.

    View details for DOI 10.1016/j.neo.2016.11.010

    View details for Web of Science ID 000393058200001

    View details for PubMedID 27940247

    View details for PubMedCentralID PMC5149063

  • Colony stimulating factor 1 receptor inhibition delays recurrence of glioblastoma after radiation by altering myeloid cell recruitment and polarization NEURO-ONCOLOGY Stafford, J. H., Hirai, T., Deng, L., Chernikova, S. B., Urata, K., West, B. L., Brown, J. M. 2016; 18 (6): 797-806


    Glioblastoma (GBM) may initially respond to treatment with ionizing radiation (IR), but the prognosis remains extremely poor because the tumors invariably recur. Using animal models, we previously showed that inhibiting stromal cell-derived factor 1 signaling can prevent or delay GBM recurrence by blocking IR-induced recruitment of myeloid cells, specifically monocytes that give rise to tumor-associated macrophages. The present study was aimed at determining if inhibiting colony stimulating factor 1 (CSF-1) signaling could be used as an alternative strategy to target pro-tumorigenic myeloid cells recruited to irradiated GBM.To inhibit CSF-1 signaling in myeloid cells, we used PLX3397, a small molecule that potently inhibits the tyrosine kinase activity of the CSF-1 receptor (CSF-1R). Combined IR and PLX3397 therapy was compared with IR alone using 2 different human GBM intracranial xenograft models.GBM xenografts treated with IR upregulated CSF-1R ligand expression and increased the number of CD11b+ myeloid-derived cells in the tumors. Treatment with PLX3397 both depleted CD11b+ cells and potentiated the response of the intracranial tumors to IR. Median survival was significantly longer for mice receiving combined therapy versus IR alone. Analysis of myeloid cell differentiation markers indicated that CSF-1R inhibition prevented IR-recruited monocyte cells from differentiating into immunosuppressive, pro-angiogenic tumor-associated macrophages.CSF-1R inhibition may be a promising strategy to improve GBM response to radiotherapy.

    View details for DOI 10.1093/neuonc/nov272

    View details for Web of Science ID 000376151200012

    View details for PubMedID 26538619

  • The Tumor Radiobiology of SRS and SBRT: Are More Than the 5 Rs Involved? INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Brown, J. M., Carlson, D. J., Brenner, D. J. 2014; 88 (2): 254-262


    Stereotactic radiosurgery (SRS) and stereotactic body radiation therapy (SBRT), also known as stereotactic ablative radiation therapy (SABR), are rapidly becoming accepted practice for the radiation therapy of certain tumors. Typically, SRS and SBRT involve the delivery of 1 or a few large-dose fractions of 8 to 30 Gy per fraction: a major paradigm shift from radiation therapy practice over the past 90 years, when, with relatively large amounts of normal tissues receiving high doses, the goal was to maximize tumor response for an acceptable level of normal tissue injury. The development of SRS and SBRT have come about because of technologic advances in image guidance and treatment delivery techniques that enable the delivery of large doses to tumors with reduced margins and high gradients outside the target, thereby minimizing doses to surrounding normal tissues. Because the results obtained with SRS and SBRT have been impressive, they have raised the question whether classic radiobiological modeling, and the linear-quadratic (LQ) model, are appropriate for large doses per fraction. In addition to objections to the LQ model, the possibility of additional biological effects resulting from endothelial cell damage, enhanced tumor immunity, or both have been raised to account for the success of SRS and SBRT. In this review, we conclude that the available preclinical and clinical data do not support a need to change the LQ model or to invoke phenomena over and above the classic 5 Rs of radiobiology and radiation therapy, with the likely exception that for some tumors high doses of irradiation may produce enhanced antitumor immunity. Thus, we suggest that for most tumors, the standard radiobiology concepts of the 5 Rs are sufficient to explain the clinical data, and the excellent results obtained from clinical studies are the result of the much larger biologically effective doses that are delivered with SRS and SBRT.

    View details for DOI 10.1016/j.ijrobp.2013.07.022

    View details for Web of Science ID 000330213700002

    View details for PubMedID 24411596

    View details for PubMedCentralID PMC3893711

  • Blockade of SDF-1 after irradiation inhibits tumor recurrences of autochthonous brain tumors in rats NEURO-ONCOLOGY Liu, S., Alomran, R., Chernikova, S. B., Lartey, F., Stafford, J., Jang, T., Merchant, M., Zboralski, D., Zoellner, S., Kruschinski, A., Klussmann, S., Recht, L., Brown, J. M. 2014; 16 (1): 21-28


    Background Tumor irradiation blocks local angiogenesis, forcing any recurrent tumor to form new vessels from circulating cells. We have previously demonstrated that the post-irradiation recurrence of human glioblastomas in the brains of nude mice can be delayed or prevented by inhibiting circulating blood vessel-forming cells by blocking the interaction of CXCR4 with its ligand stromal cell-derived factor (SDF)-1 (CXCL12). In the present study we test this strategy by directly neutralizing SDF-1 in a clinically relevant model using autochthonous brain tumors in immune competent hosts. Methods We used NOX-A12, an l-enantiomeric RNA oligonucleotide that binds and inhibits SDF-1 with high affinity. We tested the effect of this inhibitor on the response to irradiation of brain tumors in rat induced by n-ethyl-N-nitrosourea. Results Rats treated in utero with N-ethyl-N-nitrosourea began to die of brain tumors from approximately 120 days of age. We delivered a single dose of whole brain irradiation (20 Gy) on day 115 of age, began treatment with NOX-A12 immediately following irradiation, and continued with either 5 or 20 mg/kg for 4 or 8 weeks, doses and times equivalent to well-tolerated human exposures. We found a marked prolongation of rat life span that was dependent on both drug dose and duration of treatment. In addition we treated tumors only when they were visible by MRI and demonstrated complete regression of the tumors that was not achieved by irradiation alone or with the addition of temozolomide. Conclusions Inhibition of SDF-1 following tumor irradiation is a powerful way of improving tumor response of glioblastoma multiforme.

    View details for DOI 10.1093/neuonc/not149

    View details for PubMedID 24335554

  • Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ahn, G., Tseng, D., Liao, C., Dorie, M. J., Czechowicz, A., Brown, J. M. 2010; 107 (18): 8363-8368


    Despite recent advances in radiotherapy, loco-regional failures are still the leading cause of death in many cancer patients. We have previously reported that bone marrow-derived CD11b(+) myeloid cells are recruited to tumors grown in irradiated tissues, thereby restoring the vasculature and tumor growth. In this study, we examined whether neutralizing CD11b monoclonal antibodies could inhibit the recruitment of myeloid cells into irradiated tumors and inhibit their regrowth. We observed a significant enhancement of antitumor response to radiation in squamous cell carcinoma xenografts in mice when CD11b antibodies are administered systemically. Histological examination of tumors revealed that CD11b antibodies reduced infiltration of myeloid cells expressing S100A8 and matrix metalloproteinase-9. CD11b antibodies further inhibited bone marrow-derived cell adhesion and transmigration to C166 endothelial cell monolayers and chemotactic stimuli, respectively, to levels comparable to those from CD11b knockout or CD18 hypomorphic mice. Given the clinical availability of humanized CD18 antibodies, we tested two murine tumor models in CD18 hypomorphic or CD11b knockout mice and found that tumors were more sensitive to irradiation when grown in CD18 hypomorphic mice but not in CD11b knockout mice. When CD18 hypomorphism was partially rescued by reconstitution with the wild-type bone marrow, the resistance of the tumors to irradiation was restored. Our study thus supports the rationale of using clinically available Mac-1 (CD11b/CD18) antibodies as an adjuvant therapy to radiotherapy.

    View details for DOI 10.1073/pnas.0911378107

    View details for Web of Science ID 000277310400058

    View details for PubMedID 20404138

    View details for PubMedCentralID PMC2889597

  • Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice JOURNAL OF CLINICAL INVESTIGATION Kioi, M., Vogel, H., Schultz, G., Hoffman, R. M., Harsh, G. R., Brown, J. M. 2010; 120 (3): 694-705


    Despite the high doses of radiation delivered in the treatment of patients with glioblastoma multiforme (GBM), the tumors invariably recur within the irradiation field, resulting in a low cure rate. Understanding the mechanism of such recurrence is therefore important. Here we have shown in an intracranial GBM xenograft model that irradiation induces recruitment of bone marrow-derived cells (BMDCs) into the tumors, restoring the radiation-damaged vasculature by vasculogenesis and thereby allowing the growth of surviving tumor cells. BMDC influx was initiated by induction of HIF-1 in the irradiated tumors, and blocking this influx prevented tumor recurrence. Previous studies have indicated that BMDCs are recruited to tumors in part through the interaction between the HIF-1-dependent stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4. Pharmacologic inhibition of HIF-1 or of the SDF-1/CXCR4 interaction prevented the influx of BMDCs, primarily CD11b+ myelomonocytes, and the postirradiation development of functional tumor vasculature, resulting in abrogation of tumor regrowth. Similar results were found using neutralizing antibodies against CXCR4. Our data therefore suggest a novel approach for the treatment of GBM: in addition to radiotherapy, the vasculogenesis pathway needs to be blocked, and this can be accomplished using the clinically approved drug AMD3100, a small molecule inhibitor of SDF-1/CXCR4 interactions.

    View details for DOI 10.1172/JCI40283

    View details for PubMedID 20179352

  • Association of reactive oxygen species levels and radioresistance in cancer stem cells NATURE Diehn, M., Cho, R. W., Lobo, N. A., Kalisky, T., Dorie, M. J., Kulp, A. N., Qian, D., Lam, J. S., Ailles, L. E., Wong, M., Joshua, B., Kaplan, M. J., Wapnir, I., Dirbas, F. M., Somlo, G., Garberoglio, C., Paz, B., Shen, J., Lau, S. K., Quake, S. R., Brown, J. M., Weissman, I. L., Clarke, M. F. 2009; 458 (7239): 780-U123


    The metabolism of oxygen, although central to life, produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny, and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably, subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing, CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that, similar to normal tissue stem cells, subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny, which may contribute to tumour radioresistance.

    View details for DOI 10.1038/nature07733

    View details for PubMedID 19194462

  • Matrix metalloproteinase-9 is required for tumor vasculogenesis but not for angiogenesis: Role of bone marrow-derived myelomonocytic cells CANCER CELL Ahn, G., Brown, J. M. 2008; 13 (3): 193-205


    Tumor vasculature is derived from sprouting of local vessels (angiogenesis) and bone marrow (BM)-derived circulating cells (vasculogenesis). By using a model system of transplanting tumors into an irradiated normal tissue to prevent angiogenesis, we found that tumors were unable to grow in matrix metalloproteinase-9 (MMP-9) knockout mice, but tumor growth could be restored by transplantation of wild-type BM. Endothelial progenitor cells did not contribute significantly to this process. Rather, CD11b-positive myelomonocytic cells from the transplanted BM were responsible for tumor growth and the development of immature blood vessels in MMP-9 knockout mice receiving wild-type BM. Our results suggest that MMP-9 could be an important target for adjunct therapy to enhance the response of tumors to radiotherapy.

    View details for DOI 10.1016/j.ccr.2007.11.032

    View details for Web of Science ID 000253932300005

    View details for PubMedID 18328424

    View details for PubMedCentralID PMC2967441

  • In Reply to Song etal, and In Reply to Brown and Carlson. International journal of radiation oncology, biology, physics Grimm, J., Mahadevan, A., Brown, J. M., Carlson, D. J., Brenner, D. J., Lo, S. S., Song, C. W., Cho, L. C. 2021; 110 (1): 253–54

    View details for DOI 10.1016/j.ijrobp.2021.02.021

    View details for PubMedID 33864825

  • Blocking the CXCL12/CXCR4 pathway both radiosensitizes brain metastases in mice and protects against radiation-induced cognitive dysfunction following whole brain irradiation in rats. Brown, M., Shamloo, M., Chernikova, S., Recht, L. AMER ASSOC CANCER RESEARCH. 2021
  • In Regard to Nam et al. International journal of radiation oncology, biology, physics Brown, J. M. 2021; 109 (4): 1124

    View details for DOI 10.1016/j.ijrobp.2020.11.028

    View details for PubMedID 33610292

  • Cell Killing and Chromosome Aberrations by Ionizing Radiations: Brother, Can You Paradigm? International journal of radiation oncology, biology, physics Bedford, J. S., Brown, J. M. 2021; 109 (1): 73–75

    View details for DOI 10.1016/j.ijrobp.2020.08.041

    View details for PubMedID 33308706

  • In Regard to Song et al. International journal of radiation oncology, biology, physics Brown, J. M., Carlson, D. J. 2020

    View details for DOI 10.1016/j.ijrobp.2020.06.075

    View details for PubMedID 33243481

  • Beware of clinical trials of DNA repair inhibitors. International journal of radiation oncology, biology, physics Brown, J. M. 2018

    View details for PubMedID 30529372

  • Report from the SWOG Radiation Oncology Committee: Research Objectives Workshop 2017 CLINICAL CANCER RESEARCH Okunieff, P., Casey-Sawicki, K., Lockney, N. A., Hoppe, B. S., Enderling, H., Pinnix, C., Welsh, J., Krishnan, S., Yothers, G., Browns, M., Knox, S., Bristow, R., Spellman, P., Mitin, T., Nabavizadeh, N., Jaboin, J., Manning, H., Feng, F., Galbraith, S., Solanki, A. A., Harkenrider, M. M., Tuli, R., Decker, R. H., Finkelstein, S. E., Hsu, C. C., Ha, C. S., Jagsi, R., Shumway, D., Daly, M., Wang, T. C., Fitzgerald, T. J., Laurie, F., Marshall, D. T., Raben, D., Constine, L., Thomas, C. R., Kachnic, L. A. 2018; 24 (15): 3500–3509


    The Radiation Therapy Committee of SWOG periodically evaluates its strategic plan in an effort to maintain a current and relevant scientific focus, and to provide a standard platform for future development of protocol concepts. Participants in the 2017 Strategic Planning Workshop included leaders in cancer basic sciences, molecular theragnostics, pharmaceutical and technology industries, clinical trial design, oncology practice, and statistical analysis. The committee discussed high-priority research areas, such as optimization of combined modality therapy, radiation oncology-specific drug design, identification of molecular profiles predictive of radiation-induced local or distant tumor responses, and methods for normal tissue-specific mitigation of radiation toxicity. The following concepts emerged as dominant questions ready for national testing: (i) what is the role of radiotherapy in the treatment of oligometastatic, oligorecurrent, and oligoprogressive disease? (ii) How can combined modality therapy be used to enhance systemic and local response? (iii) Can we validate and optimize liquid biopsy and other biomarkers (such as novel imaging) to supplement current response criteria to guide therapy and clinical trial design endpoints? (iv) How can we overcome deficiencies of randomized survival endpoint trials in an era of increasing molecular stratification factors? And (v) how can we mitigate treatment-related side effects and maximize quality of life in cancer survivors? The committee concluded that many aspects of these questions are ready for clinical evaluation and example protocol concepts are provided that could improve rates of cancer cure and quality of survival. Clin Cancer Res; 24(15); 3500-9. ©2018 AACR.

    View details for PubMedID 29661779

  • The Biology of SBRT: LQ or Something New? INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Brown, J. 2018; 101 (4): 964

    View details for PubMedID 29976509

  • 18F-EF5 Pet-Based Imageable Hypoxia Predicts for Local Control in Tumors Treated With Conformal Radiotherapy Qian, Y., Liu, Y., Von Eyben, R., Carter, J. N., Pollom, E. L., Harris, J. P., Prionas, N. D., Binkley, M. S., Simmons, A., Diehn, M., Chin, F. T., Shultz, D. B., Brown, J., Maxim, P. G., Koong, A. C., Graves, E. E., Loo, B. W. ELSEVIER SCIENCE INC. 2018: E17–E18
  • Combining precision radiotherapy with molecular targeting and immunomodulatory agents: a guideline by the American Society for Radiation Oncology LANCET ONCOLOGY Bristow, R. G., Alexander, B., Baumann, M., Bratman, S. V., Brown, J., Camphausen, K., Choyke, P., Citrin, D., Contessa, J. N., Dicker, A., Kirsch, D. G., Krause, M., Quynh-Thu Le, Milosevic, M., Morris, Z. S., Sarkaria, J. N., Sondel, P. M., Tran, P. T., Wilson, G. D., Willers, H., Wong, R. S., Harari, P. M. 2018; 19 (5): E240–E251


    The practice of radiation oncology is primarily based on precise technical delivery of highly conformal, image-guided external beam radiotherapy or brachytherapy. However, systematic research efforts are being made to facilitate individualised radiation dose prescriptions on the basis of gene-expressssion profiles that reflect the radiosensitivity of tumour and normal tissue. This advance in precision radiotherapy should complement those benefits made in precision cancer medicine that use molecularly targeted agents and immunotherapies. The personalisation of cancer therapy, predicated largely on genomic interrogation, is facilitating the selection of therapies that are directed against driver mutations, aberrant cell signalling, tumour microenvironments, and genetic susceptibilities. With the increasing technical power of radiotherapy to safely increase local tumour control for many solid tumours, it is an opportune time to rigorously explore the potential benefits of combining radiotherapy with molecular targeted agents and immunotherapies to increase cancer survival outcomes. This theme provides the basis and foundation for this American Society for Radiation Oncology guideline on combining radiotherapy with molecular targeting and immunotherapy agents.

    View details for PubMedID 29726389

  • 18F-EF5 PET-based Imageable Hypoxia Predicts Local Recurrence in Tumors Treated With Highly Conformal Radiation Therapy. International journal of radiation oncology, biology, physics Qian, Y. n., Von Eyben, R. n., Liu, Y. n., Chin, F. T., Miao, Z. n., Apte, S. n., Carter, J. N., Binkley, M. S., Pollom, E. L., Harris, J. P., Prionas, N. D., Kissel, M. n., Simmons, A. n., Diehn, M. n., Shultz, D. B., Brown, J. M., Maxim, P. G., Koong, A. C., Graves, E. E., Loo, B. W. 2018


    Tumor hypoxia contributes to radiation resistance. A noninvasive assessment of tumor hypoxia would be valuable for prognostication and possibly selection for hypoxia-targeted therapies. 18F-pentafluorinated etanidazole (18F-EF5) is a nitroimidazole derivative that has demonstrated promise as a positron emission tomography (PET) hypoxia imaging agent in preclinical and clinical studies. However, correlation of imageable hypoxia by 18F-EF5 PET with clinical outcomes after radiation therapy remains limited.Our study prospectively enrolled 28 patients undergoing radiation therapy for localized lung or other tumors to receive pretreatment 18F-EF5 PET imaging. Depending on the level of 18F-EF5 tumor uptake, patients underwent functional manipulation of tumor oxygenation with either carbogen breathing or oral dichloroacetate followed by repeated 18F-EF5 PET. The hypoxic subvolume of tumor was defined as the proportion of tumor voxels exhibiting higher 18F-EF5 uptake than the 95th percentile of 18F-EF5 uptake in the blood pool. Tumors with a hypoxic subvolume ≥ 10% on baseline 18F-EF5 PET imaging were classified as hypoxic by imaging. A Cox model was used to assess the correlation between imageable hypoxia and clinical outcomes after treatment.At baseline, imageable hypoxia was demonstrated in 43% of all patients (12 of 28), including 6 of 16 patients with early-stage non-small cell lung cancer treated with stereotactic ablative radiation therapy and 6 of 12 patients with other cancers. Carbogen breathing was significantly associated with decreased imageable hypoxia, while dichloroacetate did not result in a significant change under our protocol conditions. Tumors with imageable hypoxia had a higher incidence of local recurrence at 12 months (30%) than those without (0%) (P < .01).Noninvasive hypoxia imaging by 18F-EF5 PET identified imageable hypoxia in about 40% of tumors in our study population. Local tumor recurrence after highly conformal radiation therapy was higher in tumors with imageable hypoxia.

    View details for PubMedID 29859786

  • Preclinical Data on Efficacy of 10 Drug-Radiation Combinations: Evaluations, Concerns, and Recommendations TRANSLATIONAL ONCOLOGY Stone, H. B., Bernhard, E. J., Coleman, C., Deye, J., Capala, J., Mitchell, J. B., Brown, J. 2016; 9 (1): 46–56


    Clinical testing of new therapeutic interventions requires comprehensive, high-quality preclinical data. Concerns regarding quality of preclinical data have been raised in recent reports. This report examines the data on the interaction of 10 drugs with radiation and provides recommendations for improving the quality, reproducibility, and utility of future studies. The drugs were AZD6244, bortezomib, 17-DMAG, erlotinib, gefitinib, lapatinib, oxaliplatin/Lipoxal, sunitinib (Pfizer, Corporate headquarters, New York, NY), thalidomide, and vorinostat.In vitro and in vivo data were tabulated from 125 published papers, including methods, radiation and drug doses, schedules of administration, assays, measures of interaction, presentation and interpretation of data, dosimetry, and conclusions.In many instances, the studies contained inadequate or unclear information that would hamper efforts to replicate or intercompare the studies, and that weakened the evidence for designing and conducting clinical trials. The published reports on these drugs showed mixed results on enhancement of radiation response, except for sunitinib, which was ineffective.There is a need for improved experimental design, execution, and reporting of preclinical testing of agents that are candidates for clinical use in combination with radiation. A checklist is provided for authors and reviewers to ensure that preclinical studies of drug-radiation combinations meet standards of design, execution, and interpretation, and report necessary information to ensure high quality and reproducibility of studies. Improved design, execution, common measures of enhancement, and consistent interpretation of preclinical studies of drug-radiation interactions will provide rational guidance for prioritizing drugs for clinical radiotherapy trials and for the design of such trials.

    View details for PubMedID 26947881

  • Fractionated Radiation for Newly Diagnosed Supratentorial Glioblastoma Multiforme: In Regard to Brachman et al. International journal of radiation oncology, biology, physics Brown, M. n., Bernhard, E. n., Mitchel, J. n., Stone, H. n. 2016; 94 (1): 210–11

    View details for PubMedID 26700716

  • Improving the Predictive Value of Preclinical Studies in Support of Radiotherapy Clinical Trials. Clinical cancer research : an official journal of the American Association for Cancer Research Coleman, C. N., Higgins, G. S., Brown, J. M., Baumann, M. n., Kirsch, D. G., Willers, H. n., Prasanna, P. G., Dewhirst, M. W., Bernhard, E. J., Ahmed, M. M. 2016; 22 (13): 3138–47


    There is an urgent need to improve reproducibility and translatability of preclinical data to fully exploit opportunities for molecular therapeutics involving radiation and radiochemotherapy. For in vitro research, the clonogenic assay remains the current state-of-the-art of preclinical assays, whereas newer moderate and high-throughput assays offer the potential for rapid initial screening. Studies of radiation response modification by molecularly targeted agents can be improved using more physiologic 3D culture models. Elucidating effects on the cancer stem cells (CSC, and CSC-like) and developing biomarkers for defining targets and measuring responses are also important. In vivo studies are necessary to confirm in vitro findings, further define mechanism of action, and address immunomodulation and treatment-induced modification of the microenvironment. Newer in vivo models include genetically engineered and patient-derived xenograft mouse models and spontaneously occurring cancers in domesticated animals. Selection of appropriate endpoints is important for in vivo studies; for example, regrowth delay measures bulk tumor killing, whereas local tumor control assesses effects on CSCs. The reliability of individual assays requires standardization of procedures and cross-laboratory validation. Radiation modifiers must be tested as part of clinical standard of care, which includes radiochemotherapy for most tumors. Radiation models are compatible with but also differ from those used for drug screening. Furthermore, the mechanism of a drug as a chemotherapeutic agent may be different from its interaction with radiation and/or radiochemotherapy. This provides an opportunity to expand the use of molecular-targeted agents. Clin Cancer Res; 22(13); 3138-47. ©2016 AACR.

    View details for PubMedID 27154913

  • Innovation in Radiation Oncology Reply INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Brown, J., Adler, J. R. 2015; 93 (4): 935–36

    View details for PubMedID 26530765

  • Is Equipment Development Stifling Innovation in Radiation Oncology? INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Brown, J., Adler, J. R. 2015; 92 (4): 713–14

    View details for PubMedID 26104924

  • Gastrointestinal Toxicities With Combined Antiangiogenic and Stereotactic Body Radiation Therapy. International journal of radiation oncology, biology, physics Pollom, E. L., Deng, L., Pai, R. K., Brown, J. M., Giaccia, A., Loo, B. W., Shultz, D. B., Le, Q. T., Koong, A. C., Chang, D. T. 2015; 92 (3): 568-576


    Combining the latest targeted biologic agents with the most advanced radiation technologies has been an exciting development in the treatment of cancer patients. Stereotactic body radiation therapy (SBRT) is an ablative radiation approach that has become established for the treatment of a variety of malignancies, and it has been increasingly used in combination with biologic agents, including those targeting angiogenesis-specific pathways. Multiple reports have emerged describing unanticipated toxicities arising from the combination of SBRT and angiogenesis-targeting agents, particularly of late luminal gastrointestinal toxicities. In this review, we summarize the literature describing these toxicities, explore the biological mechanism of action of toxicity with the combined use of antiangiogenic therapies, and discuss areas of future research, so that this combination of treatment modalities can continue to be used in broader clinical contexts.

    View details for DOI 10.1016/j.ijrobp.2015.02.016

    View details for PubMedID 26068491

  • High-dose and fractionation effects in stereotactic radiation therapy: Analysis of tumor control data from 2965 patients RADIOTHERAPY AND ONCOLOGY Shuryak, I., Carlson, D. J., Brown, J. M., Brenner, D. J. 2015; 115 (3): 327-334


    Two aspects of stereotactic radiotherapy (SRT) require clarification: First, are tumoricidal mechanisms at high-doses/fraction the same as at lower doses? Second, is single high-dose SRT treatment advantageous for tumor control (TCP) vs. multi-fraction SRT?We analyzed published TCP data for lung tumors or brain metastases from 2965 SRT patients, covering a wide range of doses and fraction numbers. We used: (a) a linear-quadratic model (including heterogeneity), which assumes the same mechanisms at all doses, and (b) alternative models with terms describing distinct tumoricidal mechanisms at high doses.Both for lung and brain data, the LQ model provided a significantly better fit over the entire range of treatment doses than did any of the models requiring extra terms at high doses. Analyzing the data as a function of fractionation (1 fraction vs. >1 fraction), there was no significant effect on TCP in the lung data, whereas for brain data multi-fraction SRT was associated with higher TCP than single-fraction treatment.Our analysis suggests that distinct tumoricidal mechanisms do not determine tumor control at high doses/fraction. In addition, there is evidence suggesting that multi-fraction SRT is superior to single-dose SRT.

    View details for DOI 10.1016/j.radonc.2015.05.013

    View details for Web of Science ID 000358804600007

    View details for PubMedID 26058991

  • Cell Death Identification in Anticancer Therapy-Letter. Cancer research Brown, J. M., Wouters, B. G., Kirsch, D. G. 2015; 75 (17): 3681

    View details for PubMedID 26286478

    View details for PubMedCentralID PMC4558372

  • Inhibition of CXCR7 extends survival following irradiation of brain tumours in mice and rats BRITISH JOURNAL OF CANCER Walters, M. J., Ebsworth, K., Berahovich, R. D., Penfold, M. E., Liu, S., Al Omran, R., Kioi, M., Chernikova, S. B., Tseng, D., Mulkearns-Hubert, E. E., Sinyuk, M., Ransohoff, R. M., Lathia, J. D., Karamchandani, J., Kohrt, H. E., Zhang, P., Powers, J. P., Jaen, J. C., Schall, T. J., Merchant, M., Recht, L., Brown, J. M. 2014; 110 (5): 1179-1188


    In experimental models of glioblastoma multiforme (GBM), irradiation (IR) induces local expression of the chemokine CXCL12/SDF-1, which promotes tumour recurrence. The role of CXCR7, the high-affinity receptor for CXCL12, in the tumour's response to IR has not been addressed.We tested CXCR7 inhibitors for their effects on tumour growth and/or animal survival post IR in three rodent GBM models. We used immunohistochemistry to determine where CXCR7 protein is expressed in the tumours and in human GBM samples. We used neurosphere formation assays with human GBM xenografts to determine whether CXCR7 is required for cancer stem cell (CSC) activity in vitro. RESULTS :CXCR7 was detected on tumour cells and/or tumour-associated vasculature in the rodent models and in human GBM. In human GBM, CXCR7 expression increased with glioma grade and was spatially associated with CXCL12 and CXCL11/I-TAC. In the rodent GBM models, pharmacological inhibition of CXCR7 post IR caused tumour regression, blocked tumour recurrence, and/or substantially prolonged survival. CXCR7 expression levels on human GBM xenograft cells correlated with neurosphere-forming activity, and a CXCR7 inhibitor blocked sphere formation by sorted CSCs.These results indicate that CXCR7 inhibitors could block GBM tumour recurrence after IR, perhaps by interfering with CSCs.

    View details for DOI 10.1038/bjc.2013.830

    View details for Web of Science ID 000332836000011

    View details for PubMedID 24423923

    View details for PubMedCentralID PMC3950859

  • Vasculogenesis: a crucial player in the resistance of solid tumours to radiotherapy BRITISH JOURNAL OF RADIOLOGY Brown, J. M. 2014; 87 (1035)


    Tumours have two main ways to develop a vasculature: by angiogenesis, the sprouting of endothelial cells from nearby blood vessels, and vasculogenesis, the formation of blood vessels from circulating cells. Because tumour irradiation abrogates local angiogenesis, the tumour must rely on the vasculogenesis pathway for regrowth after irradiation. Tumour irradiation produces a marked influx of CD11b(+) myeloid cells (macrophages) into the tumours, and these are crucial to the formation of blood vessels in the tumours after irradiation and for the recurrence of the tumours. This process is driven by increased tumour hypoxia, which increases levels of HIF-1 (hypoxia-inducible factor 1), which in turn upregulates SDF-1 (stromal cell-derived factor 1 or CXCL12), the main driver of the vasculogenesis pathway. Inhibition of HIF-1 or of its downstream target SDF-1 prevents the radiation-induced influx of the CD11b(+) myeloid cells and delays or prevents the tumours from recurring following irradiation. Others and we have shown that with a variety of tumours in both mice and rats, the inhibition of the SDF-1/CXCR4 pathway delays or prevents the recurrence of implanted or autochthonous tumours following irradiation or following treatment with vascular disrupting agents or some chemotherapeutic drugs such as paclitaxel. In addition to the recruited macrophages, endothelial progenitor cells (EPCs) are also recruited to the irradiated tumours, a process also driven by SDF-1. Together, the recruited proangiogenic macrophages and the EPCs reform the tumour vasculature and allow the tumour to regrow following irradiation. This is a new paradigm with major implications for the treatment of solid tumours by radiotherapy.

    View details for DOI 10.1259/bjr.20130686

    View details for Web of Science ID 000334291600011

    View details for PubMedID 24338942

    View details for PubMedCentralID PMC4064599

  • Dose escalation, not “new biology,” can account for the efficacy of stereotactic body radiation therapy with non-small cell lung cancer. In reply to Rao et al. International journal of radiation oncology, biology, physics Brown, J. M., Carlson, D. J., Brenner, D. J. 2014; 89 (3): 693–94

    View details for PubMedID 24929172

  • Lessons Learned from Radiation Oncology Clinical Trials CLINICAL CANCER RESEARCH Liu, F., Okunieff, P., Bernhard, E. J., Stone, H. B., Yoo, S., Coleman, C. N., Vikram, B., Brown, M., Buatti, J., Guha, C. 2013; 19 (22): 6089-6100


    A workshop entitled "Lessons Learned from Radiation Oncology Trials" was held on December 7-8, 2011, in Bethesda, MD, to present and discuss some of the recently conducted radiation oncology clinical trials with a focus on those that failed to refute the null hypothesis. The objectives of this workshop were to summarize and examine the questions that these trials provoked, to assess the quality and limitations of the preclinical data that supported the hypotheses underlying these trials, and to consider possible solutions to these challenges for the design of future clinical trials. Several themes emerged from the discussions: (i) opportunities to learn from null-hypothesis trials through tissue and imaging studies; (ii) value of preclinical data supporting the design of combinatorial therapies; (iii) significance of validated biomarkers; (iv) necessity of quality assurance in radiotherapy delivery; (v) conduct of sufficiently powered studies to address the central hypotheses; and (vi) importance of publishing results of the trials regardless of the outcome. The fact that well-designed hypothesis-driven clinical trials produce null or negative results is expected given the limitations of trial design and complexities of cancer biology. It is important to understand the reasons underlying such null results, however, to effectively merge the technologic innovations with the rapidly evolving biology for maximal patient benefit through the design of future clinical trials.

    View details for DOI 10.1158/1078-0432.CCR-13-1116

    View details for Web of Science ID 000327320900006

    View details for PubMedID 24043463

    View details for PubMedCentralID PMC3965328

  • Inhibiting Vasculogenesis After Radiation: A New Paradigm to Improve Local Control by Radiotherapy SEMINARS IN RADIATION ONCOLOGY Brown, J. M. 2013; 23 (4): 281-287


    Tumors are supported by blood vessels, and it has long been debated whether their response to irradiation is affected by radiation damage to the vasculature. We have shown in preclinical models that, indeed, radiation is damaging to the tumor vasculature and strongly inhibits tumor angiogenesis. However, the vasculature can recover by colonization from circulating cells, primarily proangiogenenic CD11b+ monocytes or macrophages from the bone marrow. This secondary pathway of blood vessel formation, known as vasculogenesis, thus acts to restore the tumor vasculature and allows the tumor to recur following radiation. The stimulus for the influx of these CD11b+ cells into tumors following irradiation is the increased levels of hypoxia-inducible factor-1 in the tumor due to induced tumor hypoxia secondary to blood vessel loss. This increases tumor levels of the chemokine stromal cell-derived factor-1, which has chemokine receptors CXCR4 and CXCR7 on monocytes and endothelial cells thereby capturing these cells in the tumors. The increase in CD11b+ monocytes in tumors following irradiation can be prevented using antibodies or small molecules that inhibit hypoxia-inducible factor-1 or the interaction of stromal cell-derived factor-1 with its receptors. We show that the effect of inhibiting these chemokine-chemokine receptor interactions is a marked increase in the radiation response of transplanted or chemically induced tumors in mice and rats. This strategy of inhibiting vasculogenesis following tumor irradiation is a new paradigm in radiotherapy and suggests that higher levels of local control of tumors in several sites would be achievable with this strategy.

    View details for DOI 10.1016/j.semradonc.2013.05.002

    View details for Web of Science ID 000324352600007

    View details for PubMedID 24012342

    View details for PubMedCentralID PMC3768004

  • Dose Escalation, Not "New Biology," Can Account for the Efficacy of Stereotactic Body Radiation Therapy With Non-Small Cell Lung Cancer INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Brown, J. M., Brenner, D. J., Carlson, D. J. 2013; 85 (5): 1159-1160

    View details for DOI 10.1016/j.ijrobp.2012.11.003

    View details for Web of Science ID 000316790500015

    View details for PubMedID 23517805

    View details for PubMedCentralID PMC3608927

  • The irradiated tumor microenvironment: role of tumor-associated macrophages in vascular recovery. Frontiers in physiology Russell, J. S., Brown, J. M. 2013; 4: 157


    Radiotherapy is an important modality used in the treatment of more than 50% of cancer patients in the US. However, despite sophisticated techniques for radiation delivery as well as the combination of radiation with chemotherapy, tumors can recur. Thus, any method of improving the local control of the primary tumor by radiotherapy would produce a major improvement in the curability of cancer patients. One of the challenges in the field is to understand how the tumor vasculature can regrow after radiation in order to support tumor recurrence, as it is unlikely that any of the endothelial cells within the tumor could survive the doses given in a typical radiotherapy regimen. There is now considerable evidence from both preclinical and clinical studies that the tumor vasculature can be restored following radiotherapy from an influx of circulating cells consisting primarily of bone marrow derived monocytes and macrophages. The radiation-induced influx of bone marrow derived cells (BMDCs) into tumors can be prevented through the blockade of various cytokine pathways and such strategies can inhibit tumor recurrence. However, the post-radiation interactions between surviving tumor cells, recruited immune cells, and the remaining stroma remain poorly defined. While prior studies have described the monocyte/macrophage inflammatory response within normal tissues and in the tumor microenvironment, less is known about this response with respect to a tumor after radiation therapy. The goal of this review is to summarize existing research studies to provide an understanding of how the myelomonocytic lineage may influence vascular recovery within the irradiated tumor microenvironment.

    View details for PubMedID 23882218

    View details for PubMedCentralID PMC3713331

  • The irradiated tumor microenvironment: role of tumor-associated macrophages in vascular recovery FRONTIERS IN PHYSIOLOGY Russell, J. S., Brown, J. M. 2013; 4


    Radiotherapy is an important modality used in the treatment of more than 50% of cancer patients in the US. However, despite sophisticated techniques for radiation delivery as well as the combination of radiation with chemotherapy, tumors can recur. Thus, any method of improving the local control of the primary tumor by radiotherapy would produce a major improvement in the curability of cancer patients. One of the challenges in the field is to understand how the tumor vasculature can regrow after radiation in order to support tumor recurrence, as it is unlikely that any of the endothelial cells within the tumor could survive the doses given in a typical radiotherapy regimen. There is now considerable evidence from both preclinical and clinical studies that the tumor vasculature can be restored following radiotherapy from an influx of circulating cells consisting primarily of bone marrow derived monocytes and macrophages. The radiation-induced influx of bone marrow derived cells (BMDCs) into tumors can be prevented through the blockade of various cytokine pathways and such strategies can inhibit tumor recurrence. However, the post-radiation interactions between surviving tumor cells, recruited immune cells, and the remaining stroma remain poorly defined. While prior studies have described the monocyte/macrophage inflammatory response within normal tissues and in the tumor microenvironment, less is known about this response with respect to a tumor after radiation therapy. The goal of this review is to summarize existing research studies to provide an understanding of how the myelomonocytic lineage may influence vascular recovery within the irradiated tumor microenvironment.

    View details for DOI 10.3389/fphys.2013.00157

    View details for Web of Science ID 000346774000155

    View details for PubMedCentralID PMC3713331

  • R-loops and genomic instability in Bre1 (RNF20/40)-deficient cells CELL CYCLE Chernikova, S. B., Brown, J. M. 2012; 11 (16): 2980-2984


    We have proposed that maintenance of genomic stability may constitute the basis for the tumor-suppressing activity of the Bre1 (RNF20/RNF40) complex. Revisiting the evidence we presented in our recent publication, we discuss the mechanism by which maintenance of genomic stability by the Bre1 complex is achieved through coordination of events during transcription. Among many functions of Bre1, we focus on the two that, when defective, could lead to the formation of R-loops, the RNA:DNA hybrid structures regarded as a major source of genomic instability. Specifically, we discuss the role of Bre1-mediated H2B ubiquitination in the 3'-end processing of replication-associated histone mRNA and in heterochromatic gene silencing and show how disturbance of these two functions may result in the specific pattern of chromosomal abnormalities we observe in the Bre1-depleted cells.

    View details for DOI 10.4161/cc.21090

    View details for Web of Science ID 000308004000014

    View details for PubMedID 22825248

    View details for PubMedCentralID PMC3442907

  • Deficiency in Mammalian Histone H2B Ubiquitin Ligase Bre1 (Rnf20/Rnf40) Leads to Replication Stress and Chromosomal Instability CANCER RESEARCH Chernikova, S. B., Razorenova, O. V., Higgins, J. P., Sishc, B. J., Nicolau, M., Dorth, J. A., Chernikova, D. A., Kwok, S., Brooks, J. D., Bailey, S. M., Game, J. C., Brown, J. M. 2012; 72 (8): 2111-2119


    Mammalian Bre1 complexes (BRE1A/B (RNF20/40) in humans and Bre1a/b (Rnf20/40) in mice) function similarly to their yeast homolog Bre1 as ubiquitin ligases in monoubiquitination of histone H2B. This ubiquitination facilitates methylation of histone H3 at K4 and K79, and accounts for the roles of Bre1 and its homologs in transcriptional regulation. Recent studies by others suggested that Bre1 acts as a tumor suppressor, augmenting expression of select tumor suppressor genes and suppressing select oncogenes. In this study, we present an additional mechanism of tumor suppression by Bre1 through maintenance of genomic stability. We track the evolution of genomic instability in Bre1-deficient cells from replication-associated double-strand breaks (DSB) to specific genomic rearrangements that explain a rapid increase in DNA content and trigger breakage-fusion-bridge cycles. We show that aberrant RNA-DNA structures (R-loops) constitute a significant source of DSBs in Bre1-deficient cells. Combined with a previously reported defect in homologous recombination, generation of R-loops is a likely initiator of replication stress and genomic instability in Bre1-deficient cells. We propose that genomic instability triggered by Bre1 deficiency may be an important early step that precedes acquisition of an invasive phenotype, as we find decreased levels of BRE1A/B and dimethylated H3K79 in testicular seminoma and in the premalignant lesion in situ carcinoma.

    View details for DOI 10.1158/0008-5472.CAN-11-2209

    View details for Web of Science ID 000302905700022

    View details for PubMedID 22354749

    View details for PubMedCentralID PMC3328627

  • Imaging Tumor Sensitivity to a Bioreductive Prodrug: Two for the Price of One! CLINICAL CANCER RESEARCH Brown, J. M. 2012; 18 (6): 1487-1489


    Hypoxia is an important characteristic of many solid tumors and has a major negative effect on treatment response. A way to combat this effect is with drugs called "bioreductive prodrugs" or "hypoxic cytotoxins," which are metabolized under hypoxia to toxic species. However, the patients with hypoxic tumors need to be identified.

    View details for DOI 10.1158/1078-0432.CCR-11-3267

    View details for Web of Science ID 000301672400001

    View details for PubMedID 22317761

  • Inhibiting homologous recombination for cancer therapy CANCER BIOLOGY & THERAPY Chernikova, S. B., Game, J. C., Brown, J. M. 2012; 13 (2): 61-68


    We review the rationale for seeking inhibitors of homologous recombination (HR) repair for use in cancer therapy. Cells use HR as one way to repair DNA double-strand breaks that arise directly from treatments such as radiotherapy, or indirectly during replication when forks encounter other damage. HR occurs during the S and G 2 phases of the cell cycle and is therefore more significant in dividing cancer cells than in non-dividing cells of healthy tissue, giving a potential therapeutic advantage to inhibiting the process. Also, some tumors consist of cells that are defective in other DNA repair pathways, and such cells may be sensitive to HR repair inhibitors because of synthetic lethality, in which blocking two alternative pathways that a cell can use to reach a needed end-point has a much bigger impact than blocking either pathway alone. We review strategies for identifying HR inhibitors and discuss current progress.

    View details for DOI 10.4161/cbt.13.2.18872

    View details for Web of Science ID 000300357700001

    View details for PubMedID 22336907

    View details for PubMedCentralID PMC3336066

  • Overcoming Hypoxia-Induced Apoptotic Resistance through Combinatorial Inhibition of GSK-3 beta and CDK1 CANCER RESEARCH Mayes, P. A., Dolloff, N. G., Daniel, C. J., Liu, J. J., Hart, L. S., Kuribayashi, K., Allen, J. E., Jee, D. I., Dorsey, J. F., Liu, Y. Y., Dicker, D. T., Brown, J. M., Furth, E. E., Klein, P. S., Sears, R. C., El-Deiry, W. S. 2011; 71 (15): 5265-U264


    Tumor hypoxia is an inherent impediment to cancer treatment that is both clinically significant and problematic. In this study, we conducted a cell-based screen to identify small molecules that could reverse the apoptotic resistance of hypoxic cancer cells. Among the compounds, we identified were a structurally related group that sensitized hypoxic cancer cells to apoptosis by inhibiting the kinases GSK-3β and cyclin-dependent kinase (CDK) 1. Combinatorial inhibition of these proteins in hypoxic cancer cells and tumors increased levels of c-Myc and decreased expression of c-IAP2 and the central hypoxia response regulator hypoxia-inducible factor (HIF) 1α. In mice, these compounds augmented the hypoxic tumor cell death induced by cytotoxic chemotherapy, blocking angiogenesis and tumor growth. Taken together, our findings suggest that combinatorial inhibition of GSK-3β and CDK1 augment the apoptotic sensitivity of hypoxic tumors, and they offer preclinical validation of a novel and readily translatable strategy to improve cancer therapy.

    View details for Web of Science ID 000293267600023

    View details for PubMedID 21646472

    View details for PubMedCentralID PMC3667402

  • Targeting SDF-1/CXCR4 to inhibit tumour vasculature for treatment of glioblastomas BRITISH JOURNAL OF CANCER Tseng, D., Vasquez-Medrano, D. A., Brown, J. M. 2011; 104 (12): 1805-1809


    Local recurrence of glioblastomas is a major cause of patient mortality after definitive treatment. This review discusses the roles of the chemokine stromal cell-derived factor-1 and its receptor CXC chemokine receptor 4 (CXCR4) in affecting the sensitivity of glioblastomas to irradiation. Blocking these molecules prevents or delays tumour recurrence after irradiation by inhibiting the recruitment of CD11b+ monocytes/macrophages that participate in revascularising the tumour. We review the literature pertaining to the mechanism by which revascularisation occurs following tumour irradiation using experimental models. Areas of interest and debate in the literature include the process by which endothelial cells die after irradiation and the identity/origin of the cells that reconstitute the tumour blood vessels after injury. Understanding the processes that mediate tumour revascularisation will guide the improvement of clinical strategies for preventing recurrence of glioblastoma after irradiation.

    View details for DOI 10.1038/bjc.2011.169

    View details for Web of Science ID 000291384700001

    View details for PubMedID 21587260

    View details for PubMedCentralID PMC3111201



    Tumor hypoxia has been observed in many human cancers and is associated with treatment failure in radiation therapy. The purpose of this study is to quantify the effect of different radiation fractionation schemes on tumor cell killing, assuming a realistic distribution of tumor oxygenation.A probability density function for the partial pressure of oxygen in a tumor cell population is quantified as a function of radial distance from the capillary wall. Corresponding hypoxia reduction factors for cell killing are determined. The surviving fraction of a tumor consisting of maximally resistant cells, cells at intermediate levels of hypoxia, and normoxic cells is calculated as a function of dose per fraction for an equivalent tumor biological effective dose under normoxic conditions.Increasing hypoxia as a function of distance from blood vessels results in a decrease in tumor cell killing for a typical radiotherapy fractionation scheme by a factor of 10(5) over a distance of 130 μm. For head-and-neck cancer and prostate cancer, the fraction of tumor clonogens killed over a full treatment course decreases by up to a factor of ∼10(3) as the dose per fraction is increased from 2 to 24 Gy and from 2 to 18 Gy, respectively.Hypofractionation of a radiotherapy regimen can result in a significant decrease in tumor cell killing compared to standard fractionation as a result of tumor hypoxia. There is a potential for large errors when calculating alternate fractionations using formalisms that do not account for tumor hypoxia.

    View details for DOI 10.1016/j.ijrobp.2010.10.007

    View details for Web of Science ID 000288471500031

    View details for PubMedID 21183291

    View details for PubMedCentralID PMC3053128

  • Deficiency in Bre1 Impairs Homologous Recombination Repair and Cell Cycle Checkpoint Response to Radiation Damage in Mammalian Cells RADIATION RESEARCH Chernikova, S. B., Dorth, J. A., Razorenova, O. V., Game, J. C., Brown, J. M. 2010; 174 (5): 558-565


    The pathway involving Bre1-dependent monoubiquitination of histone H2B lysine 123, which leads to Dot1-dependent methylation of histone H3 lysine 79 (H3K79me2), has been implicated in survival after exposure to ionizing radiation in Saccharomyces cerevisiae. We found that depletion of mammalian homologs of Bre1 compromises the response to ionizing radiation, leading to increased radiosensitivity and a G(2)/M checkpoint defect. The deficiency in Bre1a/b function was also associated with increased sensitivity to crosslinking drugs and defective formation of Rad51 foci in mouse cells, suggesting a defect in homologous recombinational repair analogous to that seen in Saccharomyces. In budding yeast, H3K79me2 is important for the recruitment of the checkpoint signaling protein Rad9 to sites of double-strand breaks (DSBs). However, in mammalian cells, 53BP1 (the Rad9 ortholog) in addition to H3K79me2 recognizes a different residue, H4K20me2, and some studies argue that it is H4K20me2 and not H3K79me2 that is the preferred target for 53BP1. We show here that depletion of Bre1b specifically reduced dimethylation of H3K79 without affecting dimethylation of H4K20. Thus our data suggest that the observed defects in the radiation response of Bre1a/b-deficient cells are associated with reduced H3K79me2 and not with H4K20me2.

    View details for DOI 10.1667/RR2184.1

    View details for Web of Science ID 000283829800003

    View details for PubMedID 20738173

    View details for PubMedCentralID PMC2988074

  • Pharmacokinetic/Pharmacodynamic Modeling Identifies SN30000 and SN29751 as Tirapazamine Analogues with Improved Tissue Penetration and Hypoxic Cell Killing in Tumors CLINICAL CANCER RESEARCH Hicks, K. O., Siim, B. G., Jaiswal, J. K., Pruijn, F. B., Fraser, A. M., Patel, R., Hogg, A., Liyanage, H. D., Dorie, M. J., Brown, J. M., Denny, W. A., Hay, M. P., Wilson, W. R. 2010; 16 (20): 4946-4957


    Tirapazamine (TPZ) has attractive features for targeting hypoxic cells in tumors but has limited clinical activity, in part because of poor extravascular penetration. Here, we identify improved TPZ analogues by using a spatially resolved pharmacokinetic/pharmacodynamic (SR-PKPD) model that considers tissue penetration explicitly during lead optimization.The SR-PKPD model was used to guide the progression of 281 TPZ analogues through a hierarchical screen. For compounds exceeding hypoxic selectivity thresholds in single-cell cultures, SR-PKPD model parameters (kinetics of bioreductive metabolism, clonogenic cell killing potency, diffusion coefficients in multicellular layers, and plasma pharmacokinetics at well tolerated doses in mice) were measured to prioritize testing in xenograft models in combination with radiation.SR-PKPD-guided lead optimization identified SN29751 and SN30000 as the most promising hypoxic cytotoxins from two different structural subseries. Both were reduced to the corresponding 1-oxide selectively under hypoxia by HT29 cells, with an oxygen dependence quantitatively similar to that of TPZ. SN30000, in particular, showed higher hypoxic potency and selectivity than TPZ in tumor cell cultures and faster diffusion through HT29 and SiHa multicellular layers. Both compounds also provided superior plasma PK in mice and rats at equivalent toxicity. In agreement with SR-PKPD predictions, both were more active than TPZ with single dose or fractionated radiation against multiple human tumor xenografts.SN30000 and SN29751 are improved TPZ analogues with potential for targeting tumor hypoxia in humans. Novel SR-PKPD modeling approaches can be used for lead optimization during anticancer drug development.

    View details for DOI 10.1158/1078-0432.CCR-10-1439

    View details for Web of Science ID 000282877700007

    View details for PubMedID 20732963

    View details for PubMedCentralID PMC3390971



    To evaluate the effect of tumor hypoxia on the expected level of cell killing by regimens of stereotactic ablative radiotherapy (SABR) and to determine the extent to which the negative effect of hypoxia could be prevented using a clinically available hypoxic cell radiosensitizer.We have calculated the expected level of tumor cell killing from regimens of SABR, both with and without the assumption that 20% of the tumor cells are hypoxic, using the standard linear quadratic model and the universal survival curve modification. We compare the results obtained with our own clinical data for lung tumors of different sizes and with published data from other studies. We also have calculated the expected effect on cell survival of adding the hypoxic cell sensitizer etanidazole at clinically achievable drug concentrations. Modeling tumor cell killing with any of the currently used regimens of SABR produces results that are inconsistent with the majority of clinical findings if tumor hypoxia is not considered. However, with the assumption of tumor hypoxia, the expected level of cell killing is consistent with clinical data. For only some of the smallest tumors are the clinical data consistent with no tumor hypoxia, but there could be other reasons for the sensitivity of these tumors. The addition of etanidazole at clinically achievable tumor concentrations produces a large increase in the expected level of tumor cell killing from the large radiation doses used in SABR.The presence of tumor hypoxia is a major negative factor in limiting the curability of tumors by SABR at radiation doses that are tolerable to surrounding normal tissues. However, this negative effect of hypoxia could be overcome by the addition of clinically tolerable doses of the hypoxic cell radiosensitizer etanidazole.

    View details for DOI 10.1016/j.ijrobp.2010.04.070

    View details for Web of Science ID 000282147000002

    View details for PubMedID 20832663

    View details for PubMedCentralID PMC2939040

  • Elusive Identities and Overlapping Phenotypes of Proangiogenic Myeloid Cells in Tumors AMERICAN JOURNAL OF PATHOLOGY Coffelt, S. B., Lewis, C. E., Naldini, L., Brown, J. M., Ferrara, N., De Palma, M. 2010; 176 (4): 1564-1576


    It is now established that bone marrow-derived myeloid cells regulate tumor angiogenesis. This was originally inferred from studies of human tumor biopsies in which a positive correlation was seen between the number of tumor-infiltrating myeloid cells, such as macrophages and neutrophils, and tumor microvessel density. However, unequivocal evidence was only provided once mouse models were used to examine the effects on tumor angiogenesis by genetically or pharmacologically targeting myeloid cells. Since then, identifying the exact myeloid cell types involved in this process has proved challenging because of myeloid cell heterogeneity and the expression of overlapping phenotypic markers in tumors. As a result, investigators often simply refer to them now as "bone marrow-derived myeloid cells." Here we review the findings of various attempts to phenotype the myeloid cells involved and discuss the therapeutic implications of correctly identifying-and thus being able to target-this proangiogenic force in tumors.

    View details for DOI 10.2353/ajpath.2010.090786

    View details for Web of Science ID 000276471500001

    View details for PubMedID 20167863

    View details for PubMedCentralID PMC2843445

  • Henry S. Kaplan Distinguished Scientist Award Lecture 2007. The remarkable yin and yang of tumour hypoxia. International journal of radiation biology Brown, M. n. 2010; 86 (11): 907–17


    To honour perhaps the most influential man in the radiation sciences, Henry S. Kaplan, by reviewing the field of tumour hypoxia, one of his many interests.It was postulated over 50 years ago by Thomlinson and Gray that human solid tumours would contain hypoxic cells and that they would exert a negative influence on the outcome of radiotherapy, a prediction that has been amply validated. In addition to the 'chronic' hypoxia that they proposed we know that tumours also have 'acute' hypoxia produced by the unstable nature of tumour blood flow. The most hypoxic cells in the tumour are the key mediators of the response to large radiation doses (such as are given in stereotactic body radiotherapy), whereas cells at intermediate levels of hypoxia are the most important for standard fractionated radiotherapy. But tumour hypoxia can be exploited to preferentially activate anti-cancer drugs such as tirapazamine in tumours. In addition, hypoxia, through the transcription factor hypoxia inducible factor 1(HIF-1), drives both local angiogenesis and vasculogenesis from circulating cells. We have shown that local tumour irradiation can inhibit angiogenesis, making growth of tumours from surviving cancer cells dependent on vasculogenesis, and that inhibiting key steps in vasculogenesis can markedly sensitize tumours to irradiation.

    View details for PubMedID 20569190

  • Role of endothelial progenitors and other bone marrow-derived cells in the development of the tumor vasculature ANGIOGENESIS Ahn, G., Brown, J. M. 2009; 12 (2): 159-164


    Increasing evidence suggests the importance of bone marrow-derived cells for blood vessel formation (neovascularization) in tumors, which can occur in two mechanisms: angiogenesis and vasculogenesis. Angiogenesis results from proliferation and sprouting of existing blood vessels close to the tumor, while vasculogenesis is believed to arise from recruitment of circulating cells, largely derived from the bone marrow, and de novo clonal formation of blood vessels from these cells. Although bone marrow-derived cells are crucial for neovascularization, current evidence suggests a promotional role of these cells on the existing blood vessels rather than de novo neovascularization in tumors. This is believed to be due to the highly proangiogenic features of these cells. The bone marrow-derived cells are heterogeneous, consisting of many different cell types including endothelial progenitor cells, myeloid cells, lymphocytes, and mesenchymal cells. These cells are highly orchestrated under the influence of the specific tumor microenvironment, which varies depending on the tumor type, thereby tightly regulating neovascularization in the tumors. In this review, we highlight some of the recent findings on each of these cell types by outlining some of the essential proangiogenic cytokines that these cells secrete to promote tumor angiogenesis and vasculogenesis.

    View details for DOI 10.1007/s10456-009-9135-7

    View details for Web of Science ID 000267095000006

    View details for PubMedID 19221886

    View details for PubMedCentralID PMC2863022

  • Roles of DNA repair and reductase activity in the cytotoxicity of the hypoxia-activated dinitrobenzamide mustard PR-104A MOLECULAR CANCER THERAPEUTICS Gu, Y., Patterson, A. V., Atwell, G. J., Chernikova, S. B., Brown, J. M., Thompson, L. H., Wilson, W. R. 2009; 8 (6): 1714-1723


    PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. Its major metabolite, PR-104A, is metabolized to the corresponding hydroxylamine (PR-104H) and amine (PR-104M), resulting in activation of the nitrogen mustard moiety. We characterize DNA damage responsible for cytotoxicity of PR-104A by comparing sensitivity of repair-defective hamster Chinese hamster ovary cell lines with their repair-competent counterparts. PR-104H showed a repair profile similar to the reference DNA cross-linking agents chlorambucil and mitomycin C, with marked hypersensitivity of XPF(-/-), ERCC1(-/-), and Rad51D(-/-) cells but not of XPD(-/-) or DNA-PK(CS)(-/-) cells. This pattern confirmed the expected dependence on the ERCC1-XPF endonuclease, implicated in unhooking DNA interstrand cross-links at blocked replication forks, and homologous recombination repair (HRR) in restarting collapsed forks. However, even under anoxia, the hypersensitivity of XPF(-/-), ERCC1(-/-), and Rad51D(-/-) cells to PR-104A itself was lower than for chlorambucil. To test whether this reflects inefficient PR-104A reduction, a soluble form of human NADPH:cytochrome P450 oxidoreductase was stably expressed in Rad51D(-/-) cells and their HRR-restored counterpart. This expression increased hypoxic metabolism of PR-104A to PR-104H and PR-104M as well as hypoxia-selective cytotoxicity of PR-104A and its dependence on HRR. We conclude that PR-104A cytotoxicity is primarily due to DNA interstrand cross-linking by its reduced metabolites, although under conditions of inefficient PR-104A reduction (low reductase expression or aerobic cells), a second mechanism contributes to cell killing. This study shows that hypoxia, reductase activity, and DNA interstrand cross-link repair proficiency are key variables that interact to determine PR-104A sensitivity.

    View details for DOI 10.1158/1535-7163.MCT-08-1209

    View details for Web of Science ID 000267043100032

    View details for PubMedID 19509245

  • DNA Cross-Links in Human Tumor Cells Exposed to the Prodrug PR-104A: Relationships to Hypoxia, Bioreductive Metabolism, and Cytotoxicity CANCER RESEARCH Singleton, R. S., Guise, C. P., Ferry, D. M., Pullen, S. M., Dorie, M. J., Brown, J. M., Patterson, A. V., Wilson, W. R. 2009; 69 (9): 3884-3891


    PR-104, currently in clinical trial, is converted systemically to the dinitrobenzamide nitrogen mustard prodrug PR-104A, which is reduced selectively in hypoxic cells to cytotoxic hydroxylamine (PR-104H) and amine (PR-104M) metabolites. Here, we evaluate the roles of this reductive metabolism, and DNA interstrand cross-links (ICL), in the hypoxic and aerobic cytotoxicity of PR-104. Using a panel of 9 human tumor cell lines, cytotoxicity was determined by clonogenic assay after a 2-hour aerobic or hypoxic exposure to PR-104A. PR-104H and PR-104M were determined by high performance liquid chromatography/mass spectrometry, and ICL with the alkaline comet assay. Under hypoxia, the relationship between ICL and cell killing was similar between cell lines. Under aerobic conditions, there was a similar relationship between ICL and cytotoxicity, except in lines with very low rates of aerobic reduction of PR-104A (A2780, C33A, H1299), which showed an ICL-independent mechanism of PR-104A cytotoxicity. Despite this, in xenografts from the same lines, the frequency of PR-104-induced ICL correlated with clonogenic cell killing (r(2) = 0.747) with greatest activity in the fast aerobic metabolizers. In addition, changing levels of hypoxia in SiHa tumors modified both ICL frequency and tumor growth delay in parallel. We conclude that both aerobic and hypoxic nitroreduction of PR-104A contribute to the monotherapy antitumor activity of PR-104 in human tumor xenografts, and that ICL are responsible for its antitumor activity and represent a broadly applicable biomarker for tumor cell killing by this novel prodrug.

    View details for DOI 10.1158/0008-5472.CAN-08-4023

    View details for Web of Science ID 000265761900022

    View details for PubMedID 19366798

  • Influence of bone marrow-derived hematopoietic cells on the tumor response to radiotherapy Experimental models and clinical perspectives CELL CYCLE Ahn, G., Brown, J. M. 2009; 8 (7): 970-976


    In this review, we highlight some of recent studies underscoring the importance of the tumor microenvironment, especially the role of bone marrow-derived myeloid cells, in restoring tumor growth after irradiation. Myeloid cells are hematopoietic cells that give rise to monocytes and macrophages in the peripheral blood and tissues. These cells have been shown to be proangiogenic in tumors promoting tumor growth. We also discuss our previously unpublished results on the effect of irradiation on the tumor vasculature including pericyte and basement membrane coverage to the endothelium of tumor blood vessels. We summarize the clinical significance of these studies including the use of MMP-9 inhibitors, administering white blood cell boosters, or planning safety margin of tumor volumes, in order to improve overall clinical benefits in cancer patients treated with radiotherapy.

    View details for Web of Science ID 000265593900007

    View details for PubMedID 19270527

    View details for PubMedCentralID PMC2862685

  • Radiosensitization of Yeast Cells by Inhibition of Histone H4 Acetylation RADIATION RESEARCH Song, S., McCann, K. E., Brown, J. M. 2008; 170 (5): 618-627


    Deletion of genes for proteins involved in histone H4 acetylation produces sensitivity to DNA-damaging agents in both Saccharomyces cerevisiae and mammalian cells. In the present studies, we show that treating wild-type yeast cells with histone acetyl transferase (HAT) inhibitors, which are chemicals that cause a global decrease in histone H4 acetylation, sensitizes the cells to ionizing radiation. Using HAT inhibitors, we have placed histone H4 acetylation into the RAD51-mediated homologous recombination repair pathway. We further show that yeast cells with functionally defective HAT proteins have normal phospho-H2A (gamma-H2A) induction after irradiation but a reduced rate of loss of gamma-H2A. This argues that HAT-defective cells are able to detect DNA double-strand breaks normally but have a defect in the repair of these lesions. We also show that cells treated with HAT inhibitors have intact G1 and G2 checkpoints after exposure to ionizing radiation, suggesting that G1 and G2 checkpoint activation is independent of histone H4 acetylation.

    View details for Web of Science ID 000260591300008

    View details for PubMedID 18959465

  • A role for Lsm1p in response to ultraviolet-radiation damage in Saccharomyces cerevisiae RADIATION RESEARCH Spicakova, T., McCann, K., Brown, J. M. 2008; 170 (4): 411-421


    A genome-wide screen in Saccharomyces cerevisiae identified LSM1 as a new gene affecting sensitivity to ultraviolet (UV) radiation. Lsmlp is a member of a cytoplasmic complex composed of Lsmlp-7p that interacts with the yeast mRNA degradation machinery. To investigate the potential role of Lsmlp in response to UV radiation, we constructed double mutant strains in which LSM1 was deleted in combination with a representative gene from each of three known yeast DNA repair pathways. Our results show that lsm1delta increases the UV-radiation sensitivity of the rad1delta and rad51delta mutants, but not the radl8delta mutant, placing LSM1 within the post-replication repair/damage tolerance pathway (PRR). When combined with other deletions affecting PRR, lsm1delta increases the UV-radiation sensitivity of the rev3delta, rad30delta and pol30-K164R mutants but not rad5delta. Furthermore, the UV-radiation sensitivity phenotype of lsmldelta is partially rescued by mutations in genes involved in 3' to 5' mRNA degradation, and mutations predicted to function in RNA interactions confer the most UV-radiation sensitivity. Together, these results suggest that Lsmlp may confer protection against UV-radiation damage by protecting the 3' ends of mRNAs from exosome-dependent 3' to 5' degradation as part of a novel RAD5-mediated, PCNA-K164 ubiquitylation-independent subpathway of PRR.

    View details for Web of Science ID 000259686300001

    View details for PubMedID 19024647

  • Optimized Clostridium-directed enzyme prodrug therapy improves the antitumor activity of the novel DNA cross-linking agent PR-104 CANCER RESEARCH Liu, S., Ahn, G., Kioi, M., Dorie, M., Patterson, A. V., Brown, J. M. 2008; 68 (19): 7995-8003


    We have previously shown that spores of the nonpathogenic clostridial strain C. sporogenes genetically engineered to express the E. coli-derived cytosine deaminase gene are effective in converting systemically injected nontoxic 5-fluorocytosine into the toxic anticancer drug 5-fluorouracil, thereby producing tumor-specific antitumor activity. To improve the expression of E. coli-derived genes with this system, we first replaced the original fdP promoter in the vector with one of two powerful endogenous clostridial promoters: that of the thiolase gene (thlP) and that for the clostridial transcription factor abrB310 (abrBP). These substitutions improved protein expression levels of the prodrug-activating genes by 2- to 3-fold in comparison with fdP-driven expression. However, despite these strong promoters, we found much higher expression of the nitroreductase (NTR) protein in the E. coli host compared with the clostridial host, which we hypothesized could be the result of different codon use between the two organisms. To test this, we constructed new expression vectors with an artificially synthesized NTR gene using optimized clostridial codons (sNTR). Results from both enzymatic assays and Western blots of cell extracts from clostridial transformants harboring plasmid constructs of thlP-sNTR and abrBP-sNTR showed that the expression and activity of the NTR gene product was increased by approximately 20-fold compared with the original construct. In vivo studies with i.v. administered sNTR-expressing C. sporogenes spores in SiHa tumor-bearing mice showed significantly improved antitumor efficacy when combined with either 5-aziridinyl-2,4-dinitrobenzamide (CB1954) or the novel dinitrobenzamide mustard prodrug, PR-104.

    View details for DOI 10.1158/0008-5472.CAN-08-1698

    View details for Web of Science ID 000260029900037

    View details for PubMedID 18829557

    View details for PubMedCentralID PMC2596930

  • In vivo H-1 magnetic resonance spectroscopy of lactate in patients with Stage IV head and neck squamous cell carcinoma 49th Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology (ASTRO) Le, Q., Koong, A., Lieskovsky, Y. Y., Narasimhan, B., Graves, E., Pinto, H., Brown, J. M., Spielman, D. ELSEVIER SCIENCE INC. 2008: 1151–57


    To investigate in vivo(1)H magnetic resonance spectroscopy imaging of lactate for assessing tumor hypoxia in head and neck cancers and to determine its utility in predicting the response and outcomes.Volume-localized lactate-edited (1)H magnetic resonance spectroscopy at 1.5 T was performed in vivo on involved neck nodes and control subcutaneous tissues in 36 patients with Stage IV head and neck cancer. The signal intensities (SIs) of lactate, choline, and creatine and the choline/creatine ratio were measured. The tumor partial pressure of oxygen (pO(2)) was obtained in the same lymph node before MRS. Patients were treated with either two cycles of induction chemotherapy (tirapazamine, cisplatin, 5-fluorouracil) followed by simultaneous chemoradiotherapy or the same regimen without tirapazamine. The lactate SI and the choline/creatine ratio correlated with the tumor pO(2), nodal response, and locoregional control.The lactate SI was greater for the involved nodes (median, 0.25) than for the subcutaneous tissue (median, 0.04; p = 0.07). No significant correlation was found between the lactate SI and tumor pO(2) (mean, 0.46 +/- 0.10 for hypoxic nodes [pO(2) < or =10 mm Hg, n = 15] vs. 0.36 +/- 0.07 for nonhypoxic nodes [pO(2) >10 mm Hg, n = 21], p = 0.44). A significant correlation was found between the choline/creatine ratios and tumor pO(2) (mean, 2.74 +/- 0.34 for hypoxic nodes vs. 1.78 +/- 0.31 for nonhypoxic nodes, p = 0.02). No correlation was found between the lactate SI and the complete nodal response (p = 0.52) or locoregional control rates.The lactate SI did not correlate with tumor pO(2), treatment response, or locoregional control. Additional research is needed to refine this technique.

    View details for DOI 10.1016/j.ijrobp.2007.11.030

    View details for Web of Science ID 000257299200025

    View details for PubMedID 18258377

    View details for PubMedCentralID PMC2601688

  • High-dose single-fraction radiotherapy: Exploiting a new biology? INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Brown, J. M., Koong, A. C. 2008; 71 (2): 324-325

    View details for DOI 10.1016/j.ijrobp.2008.02.003

    View details for Web of Science ID 000255971100002

    View details for PubMedID 18474308

  • Homologous recombination is the principal pathway for the repair of DNA damage induced by tirapazamine in mammalian cells CANCER RESEARCH Evans, J. W., Chernikova, S. B., Kachnic, L. A., Banath, J. P., Sordet, O., Delahoussaye, Y. M., Treszezamsky, A., Chon, B. H., Feng, Z., Gu, Y., Wilson, W. R., Pommier, Y., Olive, P. L., Powell, S. N., Brown, J. M. 2008; 68 (1): 257-265


    Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-dioxide) is a promising hypoxia-selective cytotoxin that has shown significant activity in advanced clinical trials in combination with radiotherapy and cisplatin. The current study aimed to advance our understanding of tirapazamine-induced lesions and the pathways involved in their repair. We show that homologous recombination plays a critical role in repair of tirapazamine-induced damage because cells defective in homologous recombination proteins XRCC2, XRCC3, Rad51D, BRCA1, or BRCA2 are particularly sensitive to tirapazamine. Consistent with the involvement of homologous recombination repair, we observed extensive sister chromatid exchanges after treatment with tirapazamine. We also show that the nonhomologous end-joining pathway, which predominantly deals with frank double-strand breaks (DSB), is not involved in the repair of tirapazamine-induced DSBs. In addition, we show that tirapazamine preferentially kills mutants both with defects in XPF/ERCC1 (but not in other nucleotide excision repair factors) and with defects in base excision repair. Tirapazamine also induces DNA-protein cross-links, which include stable DNA-topoisomerase I cleavable complexes. We further show that gamma H2AX, an indicator of DNA DSBs, is induced preferentially in cells in the S phase of the cell cycle. These observations lead us to an overall model of tirapazamine damage in which DNA single-strand breaks, base damage, and DNA-protein cross-links (including topoisomerase I and II cleavable complexes) produce stalling and collapse of replication forks, the resolution of which results in DSB intermediates, requiring homologous recombination and XPF/ERCC1 for their repair.

    View details for DOI 10.1158/0008-5472.CAN-06-4497

    View details for Web of Science ID 000252072100033

    View details for PubMedID 18172318

  • What causes the radiation gastrointestinal syndrome?: overview. International journal of radiation oncology, biology, physics Brown, M. n. 2008; 70 (3): 799–800

    View details for PubMedID 18262092

    View details for PubMedCentralID PMC2350186

  • Targeting tumors with hypoxia-activated cytotoxins FRONTIERS IN BIOSCIENCE-LANDMARK Ahn, G., Brown, M. 2007; 12: 3483-3501


    This review focuses on the recent development of hypoxia-activated cytotoxins. Such drugs are prodrugs activated to cytotoxic products in the hypoxic environment of solid tumors (so-called "bioreductive prodrugs"), but can also be activated by radiation (radiation-activated prodrugs). These compounds grew out of research on hypoxic radiosensitizers, which are compounds that can overcome the radiation resistance of hypoxic cells, and we will discuss this area also. The advantages and limitations of each class of the hypoxia-activated cytotoxins are discussed. In addition we will discuss a novel method of targeting drugs to tumors based on anaerobic bacteria, the so-called "clostridia-directed enzyme prodrug therapy" or CDEPT, which also exploits the hypoxic environment of solid tumors.

    View details for DOI 10.2741/2329

    View details for Web of Science ID 000246157900059

    View details for PubMedID 17485316

  • Tumor hypoxia in cancer therapy OXYGEN BIOLOGY AND HYPOXIA Brown, J. M. 2007; 435: 297-?


    Human solid tumors are invariably less well-oxygenated than the normal tissues from which they arose. This so-called tumor hypoxia leads to resistance to radiotherapy and anticancer chemotherapy as well as predisposing for increased tumor metastases. In this chapter, we examine the resistance of tumors to radiotherapy produced by hypoxia and, in particular, address the question of whether this resistance is the result of the physicochemical free radical mechanism that produces resistance to radiation killing of cells in vitro. We conclude that a major part of the resistance, though perhaps not all, is the result of the physicochemical free radical mechanism of the oxygen effect in sensitizing cells to ionizing radiation. However, in modeling studies used to evaluate the effect of fractionated irradiation on tumor response, it is essential to consider the fact that the tumor cells are at a wide range of oxygen concentrations, not just at the extremes of oxygenated and hypoxic. Prolonged hypoxia of the tumor tissue also leads to necrosis, and necrotic regions are also characteristic of solid tumors. These two characteristics--hypoxia and necrosis--represent clear differences between tumors and normal tissues and are potentially exploitable in cancer treatment. We discuss strategies for exploiting these differences. One such strategy is to use drugs that are toxic only under hypoxic conditions. The second strategy is to take advantage of the selective induction under hypoxia of the hypoxia-inducible factor (HIF)-1. Gene therapy strategies based on this strategy are in development. Finally, tumor hypoxia can be exploited using live obligate anaerobes that have been genetically engineered to express enzymes that can activate nontoxic prodrugs into toxic chemotherapeutic agents.

    View details for DOI 10.1016/S0076-6879(07)35015-5

    View details for Web of Science ID 000251162300015

    View details for PubMedID 17998060

  • Hypoxia: Importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy INTERNATIONAL JOURNAL OF RADIATION BIOLOGY Tatum, J. L., Kelloff, G. J., Gillies, R. J., Arbeit, J. M., Brown, J. M., Chao, K. S., Chapman, J. D., Eckelman, W. C., Fyles, A. W., Giaccia, A. J., Hill, R. P., Koch, C. J., Krishna, M. C., Krohn, K. A., Lewis, J. S., Mason, R. P., Melillo, G., Padhani, A. R., Powis, G., Rajendran, J. G., Reba, R., Robinson, S. P., Semenza, G. L., Swartz, H. M., Vaupel, P., Yang, D., Croft, B., Hoffman, J., Liu, G., Stone, H., Sullivan, D. 2006; 82 (10): 699-757


    The Cancer Imaging Program of the National Cancer Institute convened a workshop to assess the current status of hypoxia imaging, to assess what is known about the biology of hypoxia as it relates to cancer and cancer therapy, and to define clinical scenarios in which in vivo hypoxia imaging could prove valuable.Hypoxia, or low oxygenation, has emerged as an important factor in tumor biology and response to cancer treatment. It has been correlated with angiogenesis, tumor aggressiveness, local recurrence, and metastasis, and it appears to be a prognostic factor for several cancers, including those of the cervix, head and neck, prostate, pancreas, and brain. The relationship between tumor oxygenation and response to radiation therapy has been well established, but hypoxia also affects and is affected by some chemotherapeutic agents. Although hypoxia is an important aspect of tumor physiology and response to treatment, the lack of simple and efficient methods to measure and image oxygenation hampers further understanding and limits their prognostic usefulness. There is no gold standard for measuring hypoxia; Eppendorf measurement of pO(2) has been used, but this method is invasive. Recent studies have focused on molecular markers of hypoxia, such as hypoxia inducible factor 1 (HIF-1) and carbonic anhydrase isozyme IX (CA-IX), and on developing noninvasive imaging techniques.This workshop yielded recommendations on using hypoxia measurement to identify patients who would respond best to radiation therapy, which would improve treatment planning. This represents a narrow focus, as hypoxia measurement might also prove useful in drug development and in increasing our understanding of tumor biology.

    View details for DOI 10.1080/09553000601002324

    View details for Web of Science ID 000242762100001

    View details for PubMedID 17118889

  • Use of three-dimensional tissue cultures to model extravascular transport and predict in vivo activity of hypoxia-targeted anticancer drugs JOURNAL OF THE NATIONAL CANCER INSTITUTE Hicks, K. O., Pruijn, F. B., Secomb, T. W., Hay, M. P., Hsu, R., Brown, J. M., Denny, W. A., Dewhirst, M. W., Wilson, W. R. 2006; 98 (16): 1118-1128


    Because of the inefficient vasculature of solid tumors, anticancer drugs must penetrate relatively long distances through the extravascular compartment. The requirement for such diffusion may limit their activity, especially that of hypoxia-targeted drugs. We tested whether a three-dimensional pharmacokinetic/pharmacodynamic (PK/PD) model based on a representative mapped tumor microvascular network could predict the therapeutic activity of anticancer drugs in mouse xenograft tumors.Diffusion coefficients of the hypoxia-activated anticancer drug tirapazamine (TPZ) and of 15 TPZ analogs were estimated by measuring their transport through HT29 colon cancer multicellular layers (MCLs). Anoxic cytotoxic potency (by clonogenic assay) and metabolism of the TPZ analogs were measured in HT29 cell suspensions, and their plasma pharmacokinetics was measured in CD-1 nude mice. This information was used to create a spatially resolved PK/PD model for the tumor microvascular network. Model predictions were compared with actual hypoxic cell kill as measured by clonogenic assays on HT29 xenograft tumors 18 hours after treatment with each TPZ analog.Modeling TPZ transport in the tumor microvascular network showed substantial drug depletion in the most hypoxic regions, with predicted maximum cell kill of only 3 logs, compared with more than 10 logs if there were no transport impediment. A large range of tissue diffusion coefficients (0.027 x 10(-6)-1.87 x 10(-6) cm2/s) was observed for the TPZ analogs. There was a strong correlation between model-predicted and measured hypoxic cell kill (R2 = 0.89) but a poor correlation when the model did not include extravascular transport (R2 = 0.32).Extravascular transport in tumors, and its consequences for tumor cell killing, can be predicted by measuring drug penetration through MCLs in vitro and modeling pharmacokinetics at each position in three-dimensional microvascular networks.

    View details for DOI 10.1093/jnci/djj306

    View details for Web of Science ID 000241720800009

    View details for PubMedID 16912264

  • The RAD6/BRE1 histone modification pathway in saccharomyces confers radiation resistance through a RAD51-dependent process that is independent of RAD18 GENETICS Game, J. C., Williamson, M. S., Spicakova, T., Brown, J. M. 2006; 173 (4): 1951-1968


    We examine ionizing radiation (IR) sensitivity and epistasis relationships of several Saccharomyces mutants affecting post-translational modifications of histones H2B and H3. Mutants bre1Delta, lge1Delta, and rtf1Delta, defective in histone H2B lysine 123 ubiquitination, show IR sensitivity equivalent to that of the dot1Delta mutant that we reported on earlier, consistent with published findings that Dot1p requires H2B K123 ubiquitination to fully methylate histone H3 K79. This implicates progressive K79 methylation rather than mono-methylation in IR resistance. The set2Delta mutant, defective in H3 K36 methylation, shows mild IR sensitivity whereas mutants that abolish H3 K4 methylation resemble wild type. The dot1Delta, bre1Delta, and lge1Delta mutants show epistasis for IR sensitivity. The paf1Delta mutant, also reportedly defective in H2B K123 ubiquitination, confers no sensitivity. The rad6Delta, rad51null, rad50Delta, and rad9Delta mutations are epistatic to bre1Delta and dot1Delta, but rad18Delta and rad5Delta show additivity with bre1Delta, dot1Delta, and each other. The bre1Delta rad18Delta double mutant resembles rad6Delta in sensitivity; thus the role of Rad6p in ubiquitinating H2B accounts for its extra sensitivity compared to rad18Delta. We conclude that IR resistance conferred by BRE1 and DOT1 is mediated through homologous recombinational repair, not postreplication repair, and confirm findings of a G1 checkpoint role for the RAD6/BRE1/DOT1 pathway.

    View details for DOI 10.1534/genetics.106.057794

    View details for Web of Science ID 000240620100010

    View details for PubMedID 16783014

    View details for PubMedCentralID PMC1569736

  • Mature results from a randomized phase II trial of cisplatin plus 5-fluorouracil and radiotherapy with or without tirapazamine in patients with resectable stage IV head and neck squamous cell carcinomas CANCER Le, Q. T., Taira, A. I., Budenz, S., Dorie, M. J., Goffinet, D. R., Fee, W. E., Goode, R., Bloch, D., Koong, A., Brown, J. M., Pinto, H. A. 2006; 106 (9): 1940-1949


    The objective of this article was to report the results from a randomized trial that evaluated the efficacy and toxicity of adding tirapazamine (TPZ) to chemoradiotherapy in the treatment of patients with head and neck squamous cell carcinomas (HNSCC).Sixty-two patients with lymph node-positive, resectable, TNM Stage IV HNSCC were randomized to receive either 2 cycles of induction chemotherapy (TPZ, cisplatin, and 5-fluorouracil [5-FU]) followed by simultaneous chemoradiotherapy (TPZ, cisplatin, and 5-FU) or to receive the same regimen without TPZ. Patients who did not achieve a complete response at 50 Grays underwent surgical treatment. Stratification factors for randomization included tumor site, TNM stage, and median tumor oxygen tension. The primary endpoint was complete lymph node response.The addition of TPZ resulted in increased hematologic toxicity. There was 1 treatment-related death from induction chemotherapy. The complete clinical and pathologic response rate in the lymph nodes was 90% and 74% for the standard treatment arm and the TPZ arm, respectively (P = .08) and 89% and 90% at the primary site in the respective treatment arms (P = .71). The 5-year overall survival rate was 59%, the cause-specific survival rate was 68%, the rate of freedom from recurrence was 69%, and the locoregional control rate was 77% for the entire group. There was no difference with regard to any of the outcome parameters between the 2 treatment arms. The significant long-term toxicity rate also was found to be similar between the 2 arms.The addition of TPZ increased hematologic toxicity but did not improve outcomes in patients with resectable, Stage IV HNSCC using the protocol administered this small randomized study. The combination of induction and simultaneous chemoradiotherapy resulted in excellent survival in these patients.

    View details for DOI 10.1002/cncr.21785

    View details for Web of Science ID 000237187400010

    View details for PubMedID 16532436

  • Global analysis of gene function in yeast by quantitative phenotypic profiling MOLECULAR SYSTEMS BIOLOGY Brown, J. A., Sherlock, G., Myers, C. L., Burrows, N. M., Deng, C., Wu, H. I., McCann, K. E., Troyanskaya, O. G., Brown, J. M. 2006; 2


    We present a method for the global analysis of the function of genes in budding yeast based on hierarchical clustering of the quantitative sensitivity profiles of the 4756 strains with individual homozygous deletion of nonessential genes to a broad range of cytotoxic or cytostatic agents. This method is superior to other global methods of identifying the function of genes involved in the various DNA repair and damage checkpoint pathways as well as other interrogated functions. Analysis of the phenotypic profiles of the 51 diverse treatments places a total of 860 genes of unknown function in clusters with genes of known function. We demonstrate that this can not only identify the function of unknown genes but can also suggest the mechanism of action of the agents used. This method will be useful when used alone and in conjunction with other global approaches to identify gene function in yeast.

    View details for DOI 10.1038/msb4100043

    View details for Web of Science ID 000243245400005

    View details for PubMedID 16738548

    View details for PubMedCentralID PMC1681475

  • Identification of mitogen-activated protein kinase signaling pathways that confer resistance to endoplasmic reticulum stress in Saccharomyces cerevisiae MOLECULAR CANCER RESEARCH Chen, Y. J., Feldman, D. E., Deng, C. C., BROWN, J. A., De Giacomo, A. F., Gaw, A. F., Shi, G. Y., Le, Q. T., Brown, J. M., Koong, A. C. 2005; 3 (12): 669-677


    Hypoxia activates all components of the unfolded protein response (UPR), a stress response initiated by the accumulation of unfolded proteins within the endoplasmic reticulum (ER). Our group and others have shown previously that the UPR, a hypoxia-inducible factor-independent signaling pathway, mediates cell survival during hypoxia and is required for tumor growth. Identifying new genes and pathways that are important for survival during ER stress may lead to the discovery of new targets in cancer therapy. Using the set of 4,728 homozygous diploid deletion mutants in budding yeast, Saccharomyces cerevisiae, we did a functional screen for genes that conferred resistance to ER stress-inducing agents. Deletion mutants in 56 genes showed increased sensitivity under ER stress conditions. Besides the classic UPR pathway and genes related to calcium homeostasis, we report that two additional pathways, including the SLT2 mitogen-activated protein kinase (MAPK) pathway and the osmosensing MAPK pathway, were also required for survival during ER stress. We further show that the SLT2 MAPK pathway was activated during ER stress, was responsible for increased resistance to ER stress, and functioned independently of the classic IRE1/HAC1 pathway. We propose that the SLT2 MAPK pathway is an important cell survival signaling pathway during ER stress. This study shows the feasibility of using the yeast deletion pool to identify relevant mammalian orthologues of the UPR.

    View details for DOI 10.1158/1541-7786.MCR-05-0181

    View details for Web of Science ID 000234499800003

    View details for PubMedID 16380504

  • A noninvasive approach for assessing tumor hypoxia in xenografts: Developing a urinary marker for hypoxia CANCER RESEARCH Nelson, D. W., Cao, H. B., Zhu, Y. H., Sunar-Reeder, B., Choi, C. Y., Faix, J. D., Brown, J. M., Koong, A. C., Giaccia, A. J., Le, Q. T. 2005; 65 (14): 6151-6158


    Tumor hypoxia modifies the efficacy of conventional anticancer therapy and promotes malignant tumor progression. Human chorionic gonadotropin (hCG) is a glycoprotein secreted during pregnancy that has been used to monitor tumor burden in xenografts engineered to express this marker. We adapted this approach to use urinary beta-hCG as a secreted reporter protein for tumor hypoxia. We used a hypoxia-inducible promoter containing five tandem repeats of the hypoxia-response element (HRE) ligated upstream of the beta-hCG gene. This construct was stably integrated into two different cancer cell lines, FaDu, a human head and neck squamous cell carcinoma, and RKO, a human colorectal cancer cell line. In vitro studies showed that tumor cells stably transfected with this plasmid construct secrete beta-hCG in response to hypoxia or hypoxia-inducible factor 1alpha (HIF-1alpha) stabilizing agents. The hypoxia responsiveness of this construct can be blocked by treatment with agents that affect the HIF-1alpha pathways, including topotecan, 1-benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole (YC-1), and flavopiridol. Immunofluorescent analysis of tumor sections and quantitative assessment with flow cytometry indicate colocalization between beta-hCG and 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) and beta-hCG and pimonidazole, two extrinsic markers for tumor hypoxia. Secretion of beta-hCG from xenografts that contain these stable constructs is directly responsive to changes in tumor oxygenation, including exposure of the animals to 10% O2 and tumor bed irradiation. Similarly, urinary beta-hCG levels decline after treatment with flavopiridol, an inhibitor of HIF-1 transactivation. This effect was observed only in tumor cells expressing a HRE-regulated reporter gene and not in tumor cells expressing a cytomegalovirus-regulated reporter gene. The 5HRE beta-hCG reporter system described here enables serial, noninvasive monitoring of tumor hypoxia in a mouse model by measuring a urinary reporter protein.

    View details for Web of Science ID 000230633400024

    View details for PubMedID 16024616

  • Multiple endonucleases function to repair covalent topoisomerase I complexes in Saccharomyces cerevisiae GENETICS Deng, C. C., BROWN, J. A., You, D. Q., Brown, J. M. 2005; 170 (2): 591-600


    Topoisomerase I plays a vital role in relieving tension on DNA strands generated during replication. However if trapped by camptothecin or other DNA damage, topoisomerase protein complexes may stall replication forks producing DNA double-strand breaks (DSBs). Previous work has demonstrated that two structure-specific nucleases, Rad1 and Mus81, protect cells from camptothecin toxicity. In this study, we used a yeast deletion pool to identify genes that are important for growth in the presence of camptothecin. In addition to genes involved in DSB repair and recombination, we identified four genes with known or implicated nuclease activity, SLX1, SLX4, SAE2, and RAD27, that were also important for protection against camptothecin. Genetic analysis revealed that the flap endonucleases Slx4 and Sae2 represent new pathways parallel to Tdp1, Rad1, and Mus81 that protect cells from camptothecin toxicity. We show further that the function of Sae2 is likely due to its interaction with the endonuclease Mre11 and that the latter acts on an independent branch to repair camptothecin-induced damage. These results suggest that Mre11 (with Sae2) and Slx4 represent two new structure-specific endonucleases that protect cells from trapped topoisomerase by removing topoisomerase-DNA adducts.

    View details for DOI 10.1534/genetics.104.028795

    View details for Web of Science ID 000230441900010

    View details for PubMedID 15834151

    View details for PubMedCentralID PMC1450417

  • Opinion - The role of apoptosis in cancer development and treatment response NATURE REVIEWS CANCER Brown, J. M., Attardi, L. D. 2005; 5 (3): 231-237


    The inactivation of programmed cell death, or apoptosis, is central to the development of cancer. This disabling of apoptotic responses might be a major contributor both to treatment resistance and to the observation that, in many tumours, apoptosis is not the main mechanism for the death of cancer cells in response to common treatment regimens. Importantly, this suggests that other modes of cell death are involved in the response to therapy.

    View details for DOI 10.1038/nrc1570

    View details for Web of Science ID 000227302700017

    View details for PubMedID 15738985

  • Genome-wide identification of genes conferring resistance to the anticancer agents cisplatin, oxaliplatin, and mitomycin C CANCER RESEARCH Wu, H. I., BROWN, J. A., Dorie, M. J., Lazzeroni, L., Brown, J. M. 2004; 64 (11): 3940-3948


    Cisplatin is a crucial agent in the treatment of many solid tumors, yet many tumors have either acquired or intrinsic resistance to the drug. We have used the homozygous diploid deletion pool of Saccharomyces cerevisiae, containing 4728 strains with individual deletion of all nonessential genes, to systematically identify genes that when deleted confer sensitivity to the anticancer agents cisplatin, oxaliplatin, and mitomycin C. We found that deletions of genes involved in nucleotide excision repair, recombinational repair, postreplication repair including translesional synthesis, and DNA interstrand cross-link repair resulted in sensitivity to all three of the agents, although with some differences between the platinum drugs and mitomycin C in the spectrum of required translesional polymerases. Putative defective repair of oxidative damage (imp2'Delta strain) also resulted in sensitivity to platinum and oxaliplatin, but not to mitomycin C. Surprisingly in light of their different profiles of clinical activity, cisplatin and oxaliplatin have very similar sensitivity profiles. Finally, we identified three novel genes (PSY1-3, "platinum sensitivity") that, when deleted, demonstrate sensitivity to cisplatin and oxaliplatin, but not to mitomycin C. Our results emphasize the importance of multiple DNA repair pathways responsible for normal cellular resistance to all three of the agents. Also, the similarity of the sensitivity profiles of the platinum agents with that of the known DNA interstrand cross-linking agent mitomycin C, and the importance of the gene PSO2 known to be involved in DNA interstrand cross-link repair strongly suggests that interstrand cross-links are important toxic lesions for cisplatin and oxaliplatin, at least in yeast.

    View details for Web of Science ID 000221727300033

    View details for PubMedID 15173006

  • Exploiting tumour hypoxia in cancer treatment NATURE REVIEWS CANCER Brown, J. M., William, W. R. 2004; 4 (6): 437-447

    View details for DOI 10.1038/nrc1367

    View details for Web of Science ID 000221759300013

    View details for PubMedID 15170446

  • Selective potentiation of the hypoxic cytotoxicity of tirapazamine by its 1-N-oxide metabolite SR 4317 CANCER RESEARCH Siim, B. G., Pruijn, F. B., Sturman, J. R., Hogg, A., Hay, M. P., Brown, J. M., Wilson, W. R. 2004; 64 (2): 736-742


    Tirapazamine (TPZ), a bioreductive drug with selective toxicity for hypoxic cells in tumors, is currently in Phase III clinical trials. It has been suggested to have a dual mechanism of action, both generating DNA radicals and oxidizing these radicals to form DNA breaks; whether the second (radical oxidation) step is rate-limiting in cells is not known. In this study we exploit the DNA radical oxidizing ability of the 1-N-oxide metabolite of TPZ, SR 4317, to address this question. SR 4317 at high, but nontoxic, concentrations potentiated the hypoxic (but not aerobic) cytotoxicity of TPZ in all four of the human tumor cell lines tested (HT29, SiHa, FaDu, and A549), thus providing a 2-3-fold increase in the hypoxic cytotoxicity ratio. In potentiating TPZ, SR 4317 was 20-fold more potent than the hypoxic cell radiosensitizers misonidazole and metronidazole but was less potent than misonidazole as a radiosensitizer, suggesting that the initial DNA radicals from TPZ and radiation are different. SR 4317 had favorable pharmacokinetic properties in CD-1 nude mice; coadministration with TPZ provided a large increase in the SR 4317 plasma concentrations relative to that for endogenous SR 4317 from TPZ. It also showed excellent extravascular transport properties in oxic and anoxic HT29 multicellular layers (diffusion coefficient 3 x 10(-6) cm(2)s(-1), with no metabolic consumption). Coadministration of SR 4317 (1 mmol/kg) with TPZ at a subtherapeutic dose (0.133 mmol/kg) significantly enhanced hypoxic cell killing in HT29 tumor xenografts without causing oxic cell killing, and the combination at its maximum tolerated dose was less toxic to hypoxic cells in the retina than was TPZ alone at its maximum tolerated dose. This study demonstrates that benzotriazine mono-N-oxides have potential use for improving the therapeutic utility of TPZ as a hypoxic cytotoxin in cancer treatment.

    View details for Web of Science ID 000188399300041

    View details for PubMedID 14744792

  • DNA-targeted 1,2,4-benzotriazine 1,4-dioxides: Potent analogues of the hypoxia-selective cytotoxin tirapazamine JOURNAL OF MEDICINAL CHEMISTRY Hay, M. P., Pruijn, F. B., Gamage, S. A., Liyanage, H. D., Kovacs, M. S., Patterson, A. V., Wilson, W. R., Brown, J. M., Denny, W. A. 2004; 47 (2): 475-488


    Tirapazamine (TPZ, 1,2,4-benzotriazin-3-amine 1,4-dioxide) is a bioreductive hypoxia-selective cytotoxin, currently in phase II/III clinical trials in combination with radiotherapy and with cisplatin-based chemotherapy. We have prepared a series of 1,2,4-benzotriazine 1,4-dioxide (BTO) analogues of TPZ where a DNA-targeting chromophore is attached at the 3-position via a flexible linker. DNA binding affinity was modified through variation of the chromophore or the pK(a) of the linker chain. The association constants (K(DNA)) for calf thymus DNA ranged from 1 x 10(2) to 5.6 x 10(5) M(-1) (ionic strength of 0.01 M). DNA binding affinity was dependent on the presence of a positive charge, either in the linker chain or in the chromophore, and (for a series of 4-acridine carboxamide chromophore analogues) correlated strongly with linker chain pK(a). The efficacy of these BTOs in killing aerobic and hypoxic mouse SCCVII tumor cells in vitro was determined by clonogenic survival. Cytotoxicity was measured as the concentration required to reduce plating efficiency to 10% of controls (C(10)), and the hypoxic cytotoxicity ratio (HCR) for each BTO was calculated as C(10)(aerobic)/C(10)(hypoxic). BTOs bearing a positive charge showed increased hypoxic cytotoxicity (1.5-56-fold) compared to TPZ and mostly modest HCRs (8-51), but some excellent (>167 and 400) values. There was a strong correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. Cytotoxicity in HT-29 human colon carcinoma cells, determined using IC(50) assays, showed similar relationships with a correlation between K(DNA) and hypoxic cytotoxicity but no correlation between K(DNA) and HCR. In this cell line, a higher proportion of compounds (7 of 11) had HCRs greater than or equal to that of TPZ. The data confirm that DNA targeting is a useful concept for increasing potency while maintaining hypoxic selectivity and provide a direction for the further development of DNA-targeted analogues of TPZ.

    View details for DOI 10.1021/jm030399c

    View details for Web of Science ID 000187941600019

    View details for PubMedID 14711317

  • Integrating phenotypic and expression profiles to map arsenic-response networks GENOME BIOLOGY Haugen, A. C., Kelley, R., Collins, J. B., Tucker, C. J., Deng, C. C., Afshari, C. A., Brown, J. M., Ideker, T., Van Houten, B. 2004; 5 (12)


    Arsenic is a nonmutagenic carcinogen affecting millions of people. The cellular impact of this metalloid in Saccharomyces cerevisiae was determined by profiling global gene expression and sensitivity phenotypes. These data were then mapped to a metabolic network composed of all known biochemical reactions in yeast, as well as the yeast network of 20,985 protein-protein/protein-DNA interactions.While the expression data unveiled no significant nodes in the metabolic network, the regulatory network revealed several important nodes as centers of arsenic-induced activity. The highest-scoring proteins included Fhl1, Msn2, Msn4, Yap1, Cad1 (Yap2), Pre1, Hsf1 and Met31. Contrary to the gene-expression analyses, the phenotypic-profiling data mapped to the metabolic network. The two significant metabolic networks unveiled were shikimate, and serine, threonine and glutamate biosynthesis. We also carried out transcriptional profiling of specific deletion strains, confirming that the transcription factors Yap1, Arr1 (Yap8), and Rpn4 strongly mediate the cell's adaptation to arsenic-induced stress but that Cad1 has negligible impact.By integrating phenotypic and transcriptional profiling and mapping the data onto the metabolic and regulatory networks, we have shown that arsenic is likely to channel sulfur into glutathione for detoxification, leads to indirect oxidative stress by depleting glutathione pools, and alters protein turnover via arsenation of sulfhydryl groups on proteins. Furthermore, we show that phenotypically sensitive pathways are upstream of differentially expressed ones, indicating that transcriptional and phenotypic profiling implicate distinct, but related, pathways.

    View details for Web of Science ID 000225460600007

    View details for PubMedID 15575969

    View details for PubMedCentralID PMC545798

  • Comment on "Tumor response to radiotherapy regulated by endothelial cell apoptosis" (II) SCIENCE Brown, M., Bristow, R., Glazer, P., Hill, R., McBride, W., McKenna, G., Muschel, R. 2003; 302 (5652)

    View details for PubMedID 14671275

  • Apoptosis genes and resistance to cancer therapy - What do the experimental and clinical data tell us? CANCER BIOLOGY & THERAPY Brown, M., Wilson, G. 2003; 2 (5): 477-490


    The dominant paradigm in cancer treatment is that cancer cells die from the common pathway of apoptosis subsequent to DNA damage by anticancer agents and that cells resistant to apoptosis are resistant to therapy. In this review we trace the logic that brought about this view and discuss whether the clinical and experimental data that have now accumulated over the past decade support the position. We show that there is support for the apoptosis model only for certain malignancies of hematopoietic origin. For the majority of cancers (which are of epithelial origin), even though some may exhibit apoptosis after treatment, there is little or no support for the concept that apoptosis, and the genes that govern it, determine the response to therapy. In general, whether apoptosis matters for overall tumor response depends on how soon after treatment apoptosis occurs. If it occurs early (within 4 to 6 hours after treatment), it is likely to be important for determining the overall response of the cell or tumor. This is the case for some tumors of myeloid and lymphoid origin. On the other hand, if apoptosis occurs long after exposure, 24-48 hrs, and usually after mitosis, then it is unlikely to be the determinant of cytotoxicity, and modifying it is unlikely to affect overall cell killing. This is the situation for most, if not all, tumors of epithelial or mesenchymal origin. What then causes cancer cells to die following treatment with anticancer agents? The evidence from ionizing radiation is clear: DNA damage leading to chromosome breaks. We argue that the situation is similar for most anticancer drugs. Indeed inability to repair the DNA damage produced by these agents, and the levels of the proteins involved in these repair processes, may explain the treatment sensitivity of some cancers. Further clinical studies in this area are needed.

    View details for Web of Science ID 000187076300003

    View details for PubMedID 14614312

  • Evaluation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) as an intrinsic marker of tumor hypoxia in U87 MG human glioblastoma: In vitro and xenograft studies INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Vordermark, D., Brown, J. M. 2003; 56 (4): 1184-1193


    The transcription factor subunit hypoxia-inducible factor-1alpha (HIF-1alpha) is a key regulatory element of the hypoxic response of cells. High protein levels have been linked to poor prognosis in several tumor types, and HIF-1alpha has been suggested as a potential endogenous marker of tumor hypoxia and associated radioresistance.HIF-1alpha expression following in vitro hypoxia was measured in U87 MG glioblastoma cells by Western blot and flow cytometry. Cell suspensions from U87 MG xenograft tumors grown in SCID mice were assayed by flow cytometry for HIF-1alpha and for pimonidazole as a reference hypoxia marker. After 1 h, 6 h, and 18 h of in vitro hypoxia, a constant increase in HIF-1alpha protein levels with decreasing oxygen concentrations between 20% and <0.02% was observed by both Western blot and flow cytometry, correlating with the pattern of pimonidazole labeling after in vitro hypoxia. In U87 MG xenograft tumors, flow-cytometric analysis of HIF-1alpha and pimonidazole showed a significant correlation of the two markers, but distinction of a HIF-1alpha-positive population was affected by a low dynamic range of the signal. As in published studies for HIF-1alpha and the hypoxic marker EF5, the colocalization of HIF-1alpha and pimonidazole in double-staining experiments was low.While the in vitro data in U87 MG human glioblastoma cells support the use of HIF-1alpha as an endogenous hypoxia marker, comparison with the standard pimonidazole makes its application to clinical material appear questionable.

    View details for DOI 10.1016/S0360-3016(03)00289-X

    View details for Web of Science ID 000183937500036

    View details for PubMedID 12829158

  • Comparison of the comet assay and the oxygen microelectrode for measuring tumor oxygenation in head-and-neck cancer patients Annual Meeting on Radiation Research Le, Q. T., Kovacs, M. S., Dorie, M. J., Koong, A., Terris, D. J., Pinto, H. A., Goffinet, D. R., Nowels, K., Bloch, D., Brown, J. M. ELSEVIER SCIENCE INC. 2003: 375–83


    To compare the Eppendorf PO2 histograph and the alkaline comet assay as methods of measuring tumor hypoxia in patients with head-and-neck squamous cell carcinomas.As part of a larger clinical trial, 65 patients with head-and-neck squamous cell carcinoma nodal metastasis underwent tumor oxygenation measurements with Eppendorf PO2 histographs and comet assays, performed on fine-needle aspirates at 1 and 2 min after 5 Gy. Fifty-four patients had sufficient tumor cells for comet analysis at 1 min and 26 at both 1 and 2 min. Individual cells were examined for DNA single-strand breaks by alkaline gel electrophoresis, and the distribution of values was quantified using median tail moment (MTM). Nonirradiated tumor cells from pretreatment fine-needle aspirates received 5 Gy in vitro to establish the oxygenated response.There was a significant correlation between the 1- and 2-min MTM (slope = 0.77 +/- 0.03). There was no relationship between DNA damage in tumor cells irradiated in vitro and in vivo. No correlation was found between Eppendorf PO2 measurements and comet MTM. There was a statistically significant correlation between the treatment response in the node studied and comet MTMs, whereas no correlation was observed between treatment response and Eppendorf measurements.Comet assays are reproducible, as shown by biopsies at 1 and 2 min. Intertumor variation in the MTM is not a result of intrinsic radiosensitivity but of tumor hypoxia. There was no correlation between Eppendorf PO2 measurements and comet MTM. Comet assays were better than Eppendorf in predicting treatment response as an end point for short-term outcome. Longer follow-up is needed to determine the role of the comet assay as a predictor for locoregional tumor control and survivals.

    View details for DOI 10.1016/S0360-3016(02)04503-0

    View details for Web of Science ID 000182861500010

    View details for PubMedID 12738312

  • Improved potency of the hypoxic cytotoxin tirapazamine by DNA-targeting BIOCHEMICAL PHARMACOLOGY Delahoussaye, Y. M., Hay, M. P., Pruijn, F. B., Denny, W. A., Brown, J. M. 2003; 65 (11): 1807-1815


    To improve the potency of the hypoxic cytotoxin tirapazamine (TPZ), we have constructed an analog, SN26955, with the TPZ moiety attached to an acridine chromophore to target the drug to DNA. The underlying reason for this is our previous finding that the hypoxic cytotoxicity of TPZ is a result of its ability to produce DNA double-strand breaks, whereas many of the toxicities of the drug in clinical use are likely the result of its metabolism in the cytoplasm and effects on mitochondria. We found that the DNA-targeted TPZ analog was more potent than TPZ in killing hypoxic cells by 1-2 orders of magnitude, yet it retained the hypoxic selectivity for cell killing of TPZ. We show that SN26955 is only active in producing DNA damage when it is enzymatically reduced while bound to, or in close association with, the DNA. We also show that it has a different cofactor dependence than TPZ for reduction leading to DNA double-strand breaks, suggesting the involvement of a different reductase for production of the lethal lesion than for TPZ. These results show the promise of DNA-targeting of TPZ to produce a DNA compound with greater clinical efficacy than TPZ itself.

    View details for DOI 10.1016/S0006-2952(03)00199-0

    View details for Web of Science ID 000183429900006

    View details for PubMedID 12781332

    View details for PubMedCentralID PMC2941974

  • Similar radiation sensitivities of acutely and chronically hypoxic cells in HT 1080 fibrosarcoma xenografts RADIATION RESEARCH Vordermark, D., Menke, D. R., Brown, J. M. 2003; 159 (1): 94-101


    It has been suggested that chronically hypoxic tumor cells may be more radiosensitive than acutely hypoxic or even aerobic cells. In the present study we have used the fact that chronically, but not acutely, hypoxic cells that are transformed with a vector containing an enhanced green fluorescent protein (EGFP) driven by a hypoxia-responsive promoter become green (high EGFP) at low oxygen concentrations and can be viably sorted from transplanted tumors in vitro. We showed that the fluorescence of HT 1080 human fibrosarcoma cells stably transfected with this vector increases constantly with decreasing O2 concentrations (<2%, longer than 1 h, half maximum approximately 0.2% for longer than 8 h), and that cells subjected to repeated cycles of hypoxia/reoxygenation (simulating acutely hypoxic cells) showed only background fluorescence. To test the radiosensitivity of acutely and chronically hypoxic cells in tumors, we isolated high-EGFP ("chronically hypoxic") and low-EGFP cells (containing both acutely hypoxic and aerobic cells) from HT 1080 xenograft tumors by fluorescence-activated cell sorting (FACS), immediately after in situ treatment with 20 Gy (ambient or clamped), and plated the cells to determine clonogenic survival in vitro. We found that the survival of high-EGFP cells after irradiation was not affected by clamping, suggesting that all, or almost all, of these cells were fully (chronically) hypoxic. Also, the survival of the low-EGFP cells irradiated under clamped conditions (acutely hypoxic cells) was not significantly different from that of the high-EGFR cells (chronically hypoxic) cells irradiated under nonclamped (or clamped) conditions. We therefore conclude that, at least in this tumor model, the radiation sensitivity of chronically hypoxic cells is similar to that of the acutely hypoxic cells.

    View details for Web of Science ID 000180260800010

    View details for PubMedID 12492372

  • Tirapazamine: A hypoxia-activated topoisomerase II poison CANCER RESEARCH Peters, K. B., Brown, J. M. 2002; 62 (18): 5248-5253


    Tirapazamine (TPZ), a hypoxia-selective cytotoxin, has demonstrated activity in cancer clinical trials. Under hypoxic conditions, TPZ is reduced to a radical that leads to DNA double-strand breaks (DSBs), single-strand breaks, and base damage. A previous finding of an association of the DSBs with protein led us to investigate the involvement of topoisomerase II (topo II) in their formation. Nuclear extracts from human lung cancer cells treated with either the topo II poison etoposide or TPZ under hypoxic conditions had markedly reduced topo II activity as judged by an inability to convert kinetoplast DNA from the catenated to the decatenated form. Because topo II poisons, such as etoposide, cause DNA DSBs, we hypothesized that pretreatment of cells with merbarone or aclarubicin, known catalytic inhibitors of topo II, would abrogate DNA DSBs caused by topo II. Cells pretreated with these catalytic inhibitors abrogated both DNA DSBs and cell kill induced by etoposide or by TPZ. Etoposide- and TPZ-mediated DSBs were also greatly reduced in a small cell lung cancer cell line with low levels of nuclear topo IIalpha. We also showed that topo IIalpha becomes covalently bound to DNA after TPZ treatment under hypoxic conditions, and that the cleavable complexes formed by TPZ are more stable over time than those formed by etoposide. Taken together, these data suggest that TPZ exerts its cytotoxic effect at least in part through poisoning topo II. Because TPZ is activated only under hypoxic conditions, which are characteristic of solid tumors, these data implicate TPZ as a tumor-specific topo II poison.

    View details for Web of Science ID 000178066400022

    View details for PubMedID 12234992

  • Tumor microenvironment and the response to anticancer therapy CANCER BIOLOGY & THERAPY Brown, J. M. 2002; 1 (5): 453-458


    Human solid tumors are invariably less well oxygenated than normal tissues. This leads to resistance to radiotherapy and anticancer chemotherapy, as well as predisposing for increased tumor metastases. Prolonged hypoxia of the tumor tissue also leads to necrosis, and necrotic regions are also characteristic of solid tumors. These two characteristics-hypoxia and necrosis-represent clear differences between tumors and normal tissues and are potentially exploitable in cancer treatment. This review focuses on the phenomenon of tumor hypoxia and how hypoxia and its accompanying necrosis can be exploited in therapy. One such strategy is to use drugs that are toxic only under hypoxic conditions, and the first drug of this class to enter clinical testing, tirapazamine, is showing considerable promise. The second way to exploit hypoxia is to take advantage of the selective induction under hypoxia of the transcription factor HIF-1 (hypoxia-inducible factor 1). Gene therapy strategies based on this are in development. Finally, tumor hypoxia can be exploited using live obligate anaerobes that have been genetically engineered to express enzymes that can activate non-toxic prodrugs into toxic chemotherapeutic agents.

    View details for Web of Science ID 000180996500002

    View details for PubMedID 12496469

  • Preferential expression of a mutant allele of the amplified MDR1 (ABCB1) gene in drug-resistant variants of a human sarcoma GENES CHROMOSOMES & CANCER Chen, G. K., Lacayo, N. J., Duran, G. E., Wang, Y., Bangs, C. D., Rea, S., Kovacs, M., Cherry, A. M., Brown, J. M., Sikic, B. I. 2002; 34 (4): 372-383


    Activation of the MDR1 (ABCB1) gene is a common event conferring multidrug resistance (MDR) in human cancers. We investigated MDR1 activation in MDR variants of a human sarcoma line, some of which express a mutant MDR1, which facilitated the study of allelic gene expression. Structural alterations of MDR1, gene copy numbers, and allelic expression were analyzed by cytogenetic karyotyping, oligonucleotide hybridization, Southern blotting, polymerase chain reaction, and DNA heteroduplex assays. Both chromosome 7 alterations and several cytogenetic changes involving the 7q21 locus are associated with the development of MDR in these sarcoma cells. Multistep-selected cells and their revertants contain three- to six-fold MDR1 gene amplification compared with that of the drug-sensitive parental cell line MES-SA and single-step doxorubicin-selected mutants. MDR1 gene amplification precedes the emergence of a mutant allele in cells that were coselected with doxorubicin and a cyclosporin inhibitor of P-glycoprotein (P-gp). Allele-specific oligonucleotide hybridization showed that the endogenous mutant allele was present as a single copy, with multiple copies of the normal allele. Reselection of revertant cells with doxorubicin in either the presence or the absence of the P-gp inhibitor resulted in exclusive reexpression of the mutant MDR1 allele, regardless of the presence of multiple wild-type MDR1 alleles. These data provide new insights into how multiple alleles are regulated in the amplicon of drug-resistant cancer cells and indicate that increased expression of an amplified gene can result from selective transcription of a single mutant allele of the gene.

    View details for DOI 10.1002/gcc.10067

    View details for PubMedID 12112526

  • Transcriptional response of Saccharomyces cerevisiae to DNA-damaging agents does not identify the genes that protect against these agents PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Birrell, G. W., BROWN, J. A., Wu, H. I., Giaever, G., Chu, A. M., Davis, R. W., Brown, J. M. 2002; 99 (13): 8778-8783


    The recent completion of the deletion of all of the nonessential genes in budding yeast has provided a powerful new way of determining those genes that affect the sensitivity of this organism to cytotoxic agents. We have used this system to test the hypothesis that genes whose transcription is increased after DNA damage are important for the survival to that damage. We used a pool of 4,627 diploid strains each with homozygous deletion of a nonessential gene to identify those genes that are important for the survival of yeast to four DNA-damaging agents: ionizing radiation, UV radiation, and exposure to cisplatin or to hydrogen peroxide. In addition we measured the transcriptional response of the wild-type parental strain to the same DNA-damaging agents. We found no relationship between the genes necessary for survival to the DNA-damaging agents and those genes whose transcription is increased after exposure. These data show that few, if any, of the genes involved in repairing the DNA lesions produced in this study, including double-strand breaks, pyrimidine dimers, single-strand breaks, base damage, and DNA cross-links, are induced in response to toxic doses of the agents that produce these lesions. This finding suggests that the enzymes necessary for the repair of these lesions are at sufficient levels within the cell. The data also suggest that the nature of the lesions produced by DNA-damaging agents cannot easily be deduced from gene expression profiling.

    View details for DOI 10.1073/pnas.132275199

    View details for Web of Science ID 000176478200057

    View details for PubMedID 12077312

    View details for PubMedCentralID PMC124375

  • Estimating DNA repair by sequential evaluation of head and neck tumor radiation sensitivity using the comet assay 5th International Conference on Head and Neck Cancer of the International-Society-of-Head-Neck-Cancer Terris, D. J., Ho, E. Y., Ibrahim, H. Z., Dorie, M. J., Kovacs, M. S., Le, Q. T., Koong, A. C., Pinto, H. A., Brown, J. M. AMER MEDICAL ASSOC. 2002: 698–702


    The alkaline comet assay is a microelectrophoretic technique for detecting single-strand DNA breaks, and may be used as an indirect measure of hypoxia by determining the radiation sensitivity of individual cells.To assess the ability of the comet assay to estimate the rate of DNA repair after irradiation in patients with head and neck cancer.The comet assay was used to evaluate DNA damage in fine-needle aspirates of lymph nodes containing metastatic squamous cell carcinoma in patients with head and neck cancer 1, 2, and 3 minutes after treatment with 500 rad (5 Gy) of irradiation. The amount of DNA damage (measured as the "tail moment" of the comet) is proportional to the number of DNA single-strand breaks after irradiation, which in turn depends on the oxygen concentration in each cell.The mean +/- SD of the median tail moment of the 1-minute postirradiation comets was 29.4 +/- 14.2 (n = 27). After 2 minutes, the mean median tail moment decreased to 25.4 +/- 13.6 (n = 25), representing a mean decrease of 11.9% in those patients with both 1- and 2-minute comet assays. Assuming a linear rate of repair, this decrease in DNA damage corresponds to a repair half-life of 4.2 minutes. A 3-minute assay was also performed on samples from a smaller number of patients (n = 9), with a mean value not significantly different from that of the 2-minute assay of the samples from this group.The comet assay is a promising tool for evaluating radiation sensitivity in individual cells. The rate of DNA repair early after irradiation is consistent with data in the literature.

    View details for Web of Science ID 000176264300013

    View details for PubMedID 12049567

  • The 93rd Annual Meeting of the American Association for Cancer Research. 6-10 April 2002, San Francisco, California, USA. Expert opinion on pharmacotherapy Brown, J. M., Siemann, D. W. 2002; 3 (5): 629-632


    The American Association for Cancer Research held its 93rd Annual Meeting on 6 - 10 April 2002. This was an enormous meeting with approximately 14,000 registrants and some 5750 abstracts, the vast majority of which were presented as posters. Those abstracts judged to be of particular significance or interest were presented as 15-min platform presentations in minisymposia, of which there were 36 during the meeting. In addition to this, there was a full day of 15 educational sessions (each with three to four speakers), 11 award lectures, 47 early morning Meet-the-Expert Sunrise Sessions. There was a mind-boggling array of choices for those interested in cancer research. This meeting was the largest ever convened on a single disease. We will endeavour to provide some of the highlights of this exciting meeting.

    View details for PubMedID 11996641

  • Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis GENE THERAPY Liu, S. C., Minton, N. P., Giaccia, A. J., Brown, J. M. 2002; 9 (4): 291-296


    A major obstacle in cancer gene therapy is selective tumor delivery. Previous studies have suggested that genetically engineered anaerobes of the genus Clostridium might be gene therapy vectors because of their ability to proliferate selectively in the hypoxic/necrotic regions common to solid tumors. However, the tumor colonization efficiency of the strain previously used was insufficient to produce any antitumor effect. Here we describe for the first time the successful transformation of C. sporogenes, a clostridial strain with the highest reported tumor colonization efficiency, with the E. coli cytosine deaminase (CD) gene and show that systemically injected spores of these bacteria express CD only in the tumor. This enzyme can convert the nontoxic prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU). Furthermore, systemic delivery of 5-FC into mice previously injected with CD-transformed spores of C. sporogenes produced greater antitumor effect than maximally tolerated doses of 5-FU. Since most human solid tumors have hypoxic and necrotic areas this vector system has considerable promise for tumor-selective gene therapy.

    View details for DOI 10.1038/sj/gt/3301659

    View details for Web of Science ID 000174314000007

    View details for PubMedID 11896468

  • Hypoxia-inducible regulation of a prodrug-activating enzyme for tumor-specific gene therapy NEOPLASIA Shibata, T., Giaccia, A. J., Brown, J. M. 2002; 4 (1): 40-48


    Previous studies have suggested that tumor hypoxia could be exploited for cancer gene therapy. Using hypoxia-responsive elements derived from the human vascular endothelial growth factor gene, we have generated vectors expressing a bacterial nitroreductase (NTR) gene that can activate the anticancer prodrug CB1954. Stable transfectants of human HT1080 tumor cells with hypoxia-inducible vectors were established with G418 selection. Hypoxic induction of NTR protein correlated with increased sensitivity to in vitro exposure of HT1080 cells to the prodrug. Growth delay assays were performed with established tumor xenografts derived from the same cells to detect the in vivo efficacy of CB1954 conversion to its cytotoxic form. Significant antitumor effects were achieved with intraperitoneal injections of CB1954 both in tumors that express NTR constitutively or with a hypoxia-inducible promoter. In addition, respiration of 10% 02 increased tumor hypoxia in vivo and enhanced the antitumor effects. Taken together, these results demonstrate that hypoxia-inducible vectors may be useful for tumor-selective gene therapy, although the problem of delivery of the vector to the tumors, particularly to the hypoxic cells in the tumors, is not addressed by these studies.

    View details for Web of Science ID 000173412000006

    View details for PubMedID 11922390

  • Metabolism of tirapazamine by multiple reductases in the nucleus BIOCHEMICAL PHARMACOLOGY Delahoussaye, Y. M., Evans, J. W., Brown, J. M. 2001; 62 (9): 1201-1209


    Tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-di-N-oxide, SR4233, Tirazone), a bioreductive drug currently in clinical trials, is selectively toxic to hypoxic cells commonly found in solid tumors. The toxicity results from the intracellular metabolism of TPZ to a highly toxic radical. When oxygen levels are low, the TPZ radical reacts with cellular molecules, producing DNA damage and cell death. The much lower toxicity towards aerobic cells results from the back-oxidation of the TPZ radical by oxygen. A major unresolved aspect of the mechanism of TPZ is the identity of the reductase(s) in the cell responsible for activating the drug to its toxic form. We have studied both the metabolism of the drug using HPLC and the formation of the TPZ radical with a fluorescence assay using dihydrorhodamine 123. We also measured DNA double- and single-strand breaks produced by TPZ, using the comet assay. We demonstrated that multiple reductases in the nucleus metabolize TPZ under hypoxia. Using the cofactor dependence of the reductases for metabolizing TPZ and of the DNA damage caused by TPZ, we show that DNA single-strand breaks after TPZ metabolism are probably caused by the most abundant source of reductase in the nucleus. DNA double-strand breaks, on the other hand, are formed by TPZ metabolism by an unknown nuclear reductase that requires only NADPH for its activity. This study is the first to characterize multiple nuclear reductases capable of activating TPZ.

    View details for Web of Science ID 000172061800006

    View details for PubMedID 11705453

  • Green fluorescent protein is a suitable reporter of tumor hypoxia despite an oxygen requirement for chromophore formation NEOPLASIA Vordermark, D., Shibata, T., Brown, J. M. 2001; 3 (6): 527-534


    The oxygen requirement for chromophore formation potentially limits the use of green fluorescent protein as a reporter under hypoxic conditions. In the light of this, the applicability of a hypoxia-responsive enhanced green fluorescent protein (EGFP)-based system to the measurement of tumor hypoxia was tested in human HT 1080 fibrosarcoma cells stably transfected with a destabilized EGFP vector containing the hypoxia-responsive 5HRE-hCMVmp promoter or, as a positive control, the strong constitutive CMV promoter. After various schedules of hypoxia and reoxygenation, EGFP fluorescence of live cells was assessed by flow cytometry, and protein levels were analyzed by Western blot. Fluorescence of CMV promoter positive control cells dropped to 38+/-5% of aerobic levels after 12 hours at <0.02% oxygen, but was unaffected by higher oxygen concentrations. Following 12 hours at <0.02% oxygen, cells transfected with the hypoxia-responsive vector exhibited maximum fluorescence after 4 hours of subsequent reoxygenation, reaching 68+/-2% of the levels in CMV promoter controls under aerobic conditions. With such reoxygenation, these cells exhibited a constant increase in fluorescence between 2% and <0.02% oxygen. EGFP chromophore formation is only affected by near-anoxic oxygen concentrations. The correlation of fluorescence and oxygen concentration is restored by a 4-hour reoxygenation period due to oxidation of pre-synthesized EGFP and a delayed increase in EGFP protein synthesis.

    View details for Web of Science ID 000172636900008

    View details for PubMedID 11774035

  • A genome-wide screen in Saccharomyces cerevisiae for genes affecting UV radiation sensitivity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Birrell, G. W., Giaever, G., Chu, A. M., Davis, R. W., Brown, J. M. 2001; 98 (22): 12608-12613


    The recent completion of the deletion of essentially all of the ORFs in yeast is an important new resource for identifying the phenotypes of unknown genes. Each ORF is replaced with a cassette containing unique tag sequences that allow rapid parallel analysis of strains in a pool by using hybridization to a high-density oligonucleotide array. We examined the utility of this system to identify genes conferring resistance to UV irradiation by using a pool of 4,627 individual homozygous deletion strains (representing deletions of all nonessential genes). We identified most of the nonessential genes previously shown to be involved in nucleotide excision repair, in cell cycle checkpoints, in homologous recombination, and in postreplication repair after UV damage. We also identified and individually confirmed, by replacing the genes, three new genes, to our knowledge not previously reported to confer UV sensitivity when deleted. Two of these newly identified genes have human orthologs associated with cancer, demonstrating the potential of this system to uncover human genes affecting sensitivity to DNA-damaging agents and genes potentially involved in cancer formation.

    View details for Web of Science ID 000171806100058

    View details for PubMedID 11606770

  • Inhibition of DNA replication by tirapazamine CANCER RESEARCH Peters, K. B., Wang, H. Y., Brown, J. M., Iliakis, G. 2001; 61 (14): 5425-5431


    Tirapazamine (TPZ) is a hypoxia-selective cytotoxin that is currently being examined in Phase II and III clinical trials in combination with radiotherapy and cisplatin-based chemotherapy. Reductases convert TPZ to a cytotoxic radical that produces DNA damage under hypoxic conditions. Because one or more of the enzymes responsible for the bioactivation of TPZ is/are thought to be at or near the nuclear matrix, we hypothesized that TPZ may have a major affect on DNA replication, a process that is known to occur predominantly at the nuclear matrix. To assess the effect of TPZ on DNA replication, we measured the incorporation of radioactive thymidine into DNA of HCT116 human colon cancer cells and HeLa cells. We show that incorporation of radioactive thymidine is dramatically inhibited in cells that are pretreated with TPZ under hypoxic conditions. TPZ-induced inhibition of DNA synthesis was much greater than that produced by more toxic doses of ionizing radiation. We used the SV40-based in vitro DNA replication assay to study the mechanism of inhibition of DNA synthesis in cells treated with TPZ. Using this assay, we show that extracts prepared from cells treated with TPZ under hypoxic conditions had only 25-50% of the DNA replication activity measured in control cells. This reduction in DNA replication activity was associated with a reduction in levels of replication protein A (RPA) in cytoplasmic extracts used for the in vitro DNA replication assay and could be overcome by addition of recombinant human RPA. Furthermore, we show by indirect immunofluorescence that TPZ leads to a localization of the p34 subunit of RPA (RPA2) to small subnuclear foci. These results show that TPZ dramatically inhibits DNA replication and that the mechanism of inhibition, at least in part, involves changes in RPA that alter its cellular localization.

    View details for PubMedID 11454687

  • Apoptosis: mediator or mode of cell killing by anticancer agents? DRUG RESISTANCE UPDATES Brown, J. M., Wouters, B. G. 2001; 4 (2): 135-136

    View details for DOI 10.1054/drup.2001.0193

    View details for Web of Science ID 000172933600008

    View details for PubMedID 11512523

  • Therapeutic targets in radiotherapy 1st International Conference on Translational Research and Pre-Clinical Strategies in Clinical Radio-Oncology (ICTR 2000) Brown, J. M. ELSEVIER SCIENCE INC. 2001: 319–26


    Enormous progress has been made in the past 5 years in our understanding of the gene products governing the response of mammalian cells to ionizing radiation. Many of these are potential targets for enhancing the effectiveness of radiotherapy. However, a major barrier to such efforts is the requirement for a preferential effect on tumor vs. normal cells. Such a requirement can only come about by exploiting a known difference between tumor and normal cells.This review highlights three differences between tumor and normal cells that are being exploited with fractionated radiotherapy.The three strategies to enhance preferentially tumor response to radiotherapy are inhibition of ras activity using farnesyltransferase inhibitors (FTIs), inhibition of epidermal growth factor receptors (EGFRs), and the use of drugs that preferentially kill hypoxic cells. Each of these strategies exploits a known difference between at least some tumors and their surrounding normal tissues, and each has shown encouraging results when combined with fractionated radiation in preclinical studies.For each of the three strategies to enhance preferentially the sensitivity of cancers, the preclinical and early clinical data are promising for their successful application in radiotherapy.

    View details for Web of Science ID 000166707700004

    View details for PubMedID 11173124

  • An investigation of the molecular basis for the synergistic interaction of tirapazamine and cisplatin 41st Annual Meeting of the American-Society-for-Therapeutic-Radiology-and-Oncology (ASTRO 99) Goldberg, Z., Evans, J., Birrell, G., Brown, J. M. ELSEVIER SCIENCE INC. 2001: 175–82


    To determine if the observed tirapazamine (TPZ)-cisplatin synergistic cell kill was mediated at the cellular level by impairment of upregulation of key proteins involved in repair of DNA interstrand crosslinks. Cisplatin sensitivity has been shown to be dependent on the expression of the two DNA repair proteins ERCC1 and XPA. ERCC1 expression has also been shown to be upregulated by cisplatin exposure. Therefore, these studies were undertaken to determine if hypoxia-activated TPZ pretreatment inhibited the cells' normal protective response to cisplatin via inhibiting the upregulation of ERCC1 and/or XPA expression.Two different cell lines, one cisplatin sensitive and one cisplatin resistant, were treated with TPZ, cisplatin, both drugs together (which results in additive cytotoxicity), or TPZ followed by cisplatin (which results in synergistic cytotoxicity). All cells were exposed to 1 h of hypoxia to bioactivate the TPZ. Expression of ERCC1 and XPA were evaluated at the mRNA and protein level at 24 or 48 h after drug exposure.In the cisplatin-sensitive non-small-cell lung cancer cell line, ERCC1 expression at the mRNA or protein level was not significantly altered in any of the treatment groups. In the cisplatin-resistant ovarian cancer cell line, ERCC1 expression was upregulated by TPZ, but not by cisplatin alone. The change in protein expression was less pronounced than the change in mRNA level. XPA expression was not significantly changed from baseline in either cell line at the mRNA or protein level.In contrast to reports in the literature, this study did not demonstrate cisplatin inducing its own repair by upregulating the DNA crosslink repair proteins ERCC1 or XPA. Therefore, the TPZ-cisplatin synergism cannot be mediated through hypoxia-activated TPZ inhibiting this cellular protective response. TPZ alone, however, was able to alter repair protein expression, which may play a role in mediating its cytotoxicity.

    View details for Web of Science ID 000166317300022

    View details for PubMedID 11163512

  • Mitochondrial dysfunction after aerobic exposure to the hypoxic cytotoxin tirapazamine CANCER RESEARCH Wouters, B. G., Delahoussaye, Y. M., Evans, J. W., Birrell, G. W., Dorie, M. J., Wang, J. L., MacDermed, D., Chiu, R. K., Brown, J. M. 2001; 61 (1): 145-152


    Tirapazamine (TPZ) is a bioreductive drug that exhibits a high degree of selective toxicity toward hypoxic cells, and at doses that are used clinically, little or no cell killing is observed in aerobic cells. Nonetheless, the effects of TPZ on aerobic tissues are still responsible for the dose limitations on the clinical administration of this drug. Clinical side effects include fatigue, muscle cramping, and reversible ototoxicity. We have investigated TPZ-induced changes in the mitochondria in aerobically exposed cells as a potential mediator of these side effects. Our data show that aerobic administration of TPZ at clinically relevant doses results in a profound loss in the mitochondrial membrane potential (MMP). We show that loss in the MMP occurs in a variety of cell lines in vitro and also occurs in muscle tissues in vivo. The loss in MMP is temporary because recovery occurs within 2 h. TPZ is directly metabolized within mitochondria to a DNA-damaging form, and this metabolism leads to both the cell-killing effects of TPZ on aerobic cells at high doses and to the loss in MMP at clinically relevant doses. Using cell lines derived from genetically modified mice with a targeted deletion in manganese superoxide dismutase, we have further distinguished the phenotypic effects of TPZ in mitochondria at high toxic doses versus those at clinically relevant doses. We have investigated several potential mechanisms for this TPZ-induced loss in MMP. Our results indicate no change in the rate of cellular respiration in TPZ-treated cells. This implies that the loss in MMP results from an inability of the inner mitochondrial membrane to sustain a potential across the membrane after TPZ treatment. Incubation of cells with an inhibitor of the mitochondrial permeability transition suggests that the loss of MMP may result from the regulated opening of a large mitochondria channel.

    View details for Web of Science ID 000166436000027

    View details for PubMedID 11196153

  • Pancreatic tumors show high levels of hypoxia 36th Annual Meeting of the American-Society-of-Clinical-Oncology Koong, A. C., Mehta, V. K., Le, Q. T., Fisher, G. A., Terris, D. J., Brown, J. M., Bastidas, A. J., Vierra, M. ELSEVIER SCIENCE INC. 2000: 919–22


    Because of the dismal outcomes of conventional therapies for pancreatic carcinomas, we postulated that hypoxia may exist within these tumors.Seven sequential patients with adenocarcinomas of the pancreas consented to intraoperative measurements of tumor oxygenation using the Eppendorf (Hamburg, Germany) polargraphic electrode.All 7 tumors demonstrated significant tumor hypoxia. In contrast, adjacent normal pancreas showed normal oxygenation.Tumor hypoxia exists within pancreatic cancers.

    View details for Web of Science ID 000165238800002

    View details for PubMedID 11072146

  • An early oxygen-dependent step is required for dexamethasone-induced apoptosis of immature mouse thymocytes JOURNAL OF IMMUNOLOGY Torres-Roca, J. F., Tung, J. W., Greenwald, D. R., Brown, J. M., Herzenberg, L. A., Herzenberg, L. A., Katsikis, P. D. 2000; 165 (9): 4822-4830


    The roles of oxygen and reactive oxygen intermediates in apoptosis are unclear at present. Although oxygen and reactive oxygen intermediates are not required for the execution of apoptosis, oxygen may be involved in at least some forms of apoptosis. In this study we show that dexamethasone (Dex)-induced apoptosis of immature mouse thymocytes is completely inhibited by hypoxic culture. In contrast, anti-CD95 thymocyte apoptosis is unaffected by hypoxia, indicating the existence of two forms of thymocyte apoptosis: an oxygen-dependent pathway (Dex induced) and an oxygen-independent pathway (anti-CD95 induced). Furthermore, hypoxia inhibited mitochondrial permeability transition (PT) in Dex-treated, but not in anti-CD95-treated, thymocytes, suggesting that the oxygen-sensitive step is upstream of mitochondria. Both Dex- and anti-CD95-induced PT and apoptosis were dependent on activation of IL-converting enzyme-like protease, as PT and apoptosis were inhibited by preincubation with Cbz-Val-Ala-Asp-fluoromethyl ketone, an irreversible inhibitor of IL-converting enzyme-like proteases. In addition, hypoxia inhibited the activation by Dex of caspase-3 (CPP32)-like proteases. Our data show that the private signaling pathways of Dex (oxygen dependent) and anti-CD95 (oxygen independent) both converge upstream of mitochondrial changes. The oxygen-dependent step in Dex-induced apoptosis lies upstream of caspase-3-like protease activation. Our observations support a model of apoptosis signaling in which independent pathways (oxygen dependent and oxygen independent) particular to each stimuli converge at a central point in the apoptotic cascade.

    View details for Web of Science ID 000090076000009

    View details for PubMedID 11046005

  • Exploiting the hypoxic cancer cell: mechanisms and therapeutic strategies MOLECULAR MEDICINE TODAY Brown, J. M. 2000; 6 (4): 157-162


    Human solid tumours are considerably less well oxygenated than normal tissues. This leads to resistance to radiotherapy and anticancer chemotherapy, as well as predisposing to increased tumour metastases. However, tumour hypoxia can be exploited in cancer treatment. One such strategy is to use drugs that are toxic only under hypoxic conditions, and the first drug of this class to enter clinical testing, tirapazamine, is showing considerable promise. The second way to exploit hypoxia is to take advantage of the selective induction of the transcription factor hypoxia-inducible factor 1 (HIF-1) under hypoxic conditions; gene therapy strategies based on this are in development.

    View details for Web of Science ID 000086266500007

    View details for PubMedID 10740254

  • Development of a hypoxia-responsive vector for tumor-specific gene therapy GENE THERAPY Shibata, T., Giaccia, A. J., Brown, J. M. 2000; 7 (6): 493-498


    We are developing new gene therapy vectors whose expression is selectively activated by hypoxia, a unique feature of human solid tumors. As vascular endothelial growth factor (VEGF) is upregulated by hypoxia, such regulatory mechanisms would enable us to achieve hypoxia-inducible expression of therapeutic genes. Constructs with five copies of hypoxia-responsive elements (HREs) derived from the 5'-untranslated region (UTR) of the human VEGF showed excellent transcriptional activation at low oxygen tension relevant to tumor hypoxia. In an attempt to achieve higher responsiveness, various combinations of HREs and promoters were examined. In addition, we also investigated whether the 3' UTR of the VEGF gene would confer increased post-transcriptional mRNA stability under hypoxic conditions. However, despite increases in the hypoxic/aerobic ratio of luciferase activity, gene expression with 3' UTR was lower due to mRNA destabilization by AU-rich elements (AREs). Thus, we found no benefit from the inclusion of the 3' UTR in our vectors. Of all the vectors tested, the combination of 5HRE and a CMV minimal promoter exhibited hypoxia responsiveness (over 500-fold) to the similar level to the intact CMV promoter. We propose that this vector would be useful for tumor selective gene therapy.

    View details for Web of Science ID 000085785500006

    View details for PubMedID 10757022

  • In vivo H-1 MR spectroscopy of human head and neck lymph node metastasis and comparison with oxygen tension measurements AMERICAN JOURNAL OF NEURORADIOLOGY Star-Lack, J. M., Adalsteinsson, E., Adam, M. F., Terris, D. J., Pinto, H. A., Brown, J. M., Spielman, D. M. 2000; 21 (1): 183-193


    Current diagnostic methods for head and neck metastasis are limited for monitoring recurrence and assessing oxygenation. 1H MR spectroscopy (1H MRS) provides a noninvasive means of determining the chemical composition of tissue and thus has a unique potential as a method for localizing and characterizing cancer. The purposes of this investigation were to measure 1H spectral intensities of total choline (Cho), creatine (Cr), and lactate (Lac) in vivo in human lymph node metastases of head and neck cancer for comparison with normal muscle tissue and to examine relationships between metabolite signal intensities and tissue oxygenation status.Volume-localized Lac-edited MRS at 1.5 T was performed in vivo on the lymph node metastases of 14 patients whose conditions were untreated and who had primary occurrences of squamous cell carcinoma. MRS measurements were acquired also from the neck muscle tissue of six healthy volunteers and a subset of the patients. Peak areas of Cho, Cr, and Lac were calculated. Tissue oxygenation (pO2) within the abnormal lymph nodes was measured independently using an Eppendorf polarographic oxygen electrode.Cho:Cr ratios were significantly higher in the nodes than in muscle tissue (node Cho:Cr = 2.9 +/- 1.6, muscle Cho:Cr = 0.55 +/- 0.21, P = .0006). Lac was significantly higher in cancer tissue than in muscle (P = .01) and, in the nodes, showed a moderately negative correlation with median pO2 (r = -.76) over a range of approximately 0 to 30 mm Hg. Nodes with oxygenation values less than 10 mm Hg had approximately twice the Lac signal intensity as did nodes with oxygenation values greater than 10 mm Hg (P = .01). Cho signal intensity was not well correlated with pO2 (r = -.46) but seemed to decrease at higher oxygenation levels (>20 mm Hg).1H MRS may be useful for differentiating metastatic head and neck cancer from normal muscular tissue and may allow for the possibility of assessing oxygenation. Potential clinical applications include the staging and monitoring of treatment.

    View details for PubMedID 10669248

  • Genetically modified clostridium for gene therapy of tumors. Methods in molecular medicine Fox, M. E., LEMMON, M. J., Giaccia, A. J., Minton, N. P., Brown, J. M. 2000; 35: 413-418


    Many murine and human tumors contain hypoxic or necrotic regions in which the oxygen tension is abnormally low. For example,>50% of primary tumors of the breast, cervix, and head and neck contain areas that are hypoxic. Because hypoxic regions are not present in normal tissue, this provides the potential for selectively targeting gene therapy to tumor cells.

    View details for DOI 10.1385/1-59259-086-1:413

    View details for PubMedID 21390820

  • Hypoxic cytotoxic agents: a new approach to cancer chemotherapy. Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy Brown, J. M. 2000; 3 (1): 7–13


    Many of the cells in solid tumors are hypoxic. This makes them resistant to killing by both radiotherapy and most commonly used anticancer drugs. The resistance towards anticancer drugs is because hypoxia induces a non-proliferative state as well as the fact that the hypoxic cells are furthest from the blood vessels in the tumor (and hence the source of drug). However, a new class of drugs - hypoxic cytotoxins can, by killing these hypoxic cells, not only overcome this resistance, but also exploit hypoxia thereby converting it into a therapeutic advantage. The first drug to be tested in the clinic purely for its activity towards hypoxic cells, tirapazamine, is described. Copyright 2000 Harcourt Publishers Ltd.

    View details for PubMedID 11498360

  • The hypoxic cell: A target for selective cancer therapy - Eighteenth Bruce F. Cain Memorial Award lecture CANCER RESEARCH Brown, J. M. 1999; 59 (23): 5863-5870


    It has been appreciated for more than 50 years that very low levels of oxygenation, or hypoxia, both protect cells from killing by X-irradiation and are present in solid tumors but not in normal tissues. Until recently, however, there has been no definitive proof that hypoxia in human tumors contributes to radiotherapy treatment failure. We now know that hypoxia in solid tumors is not only a major problem for radiation therapy but also leads to resistance to most anticancer drugs and, importantly, appears to accelerate malignant progression and increase metastasis. To date, efforts to overcome the problem of hypoxia have had only limited success. However, the recent development of new drugs that are nontoxic until they are activated in the hypoxic cell opens a new era. The first of these new drugs to be tested clinically, tirapazamine, a drug that is highly toxic to hypoxic but not aerobic cells, has already demonstrated efficacy in selective potentiation of cisplatin in randomized Phase III trials with non-small cell lung cancer. The unique presence of hypoxic cells in human tumors provides an important target for selective cancer therapy.

    View details for Web of Science ID 000084296800002

    View details for PubMedID 10606224

  • A p53 and apoptotic independent role for p21(waf1) in tumour response to radiation therapy ONCOGENE Wouters, B. G., Denko, N. C., Giaccia, A. J., Brown, J. M. 1999; 18 (47): 6540-6545


    Loss of p21 in human cancer cells results in checkpoint failure, induction of polyploidy and subsequent apoptosis following DNA damage. Tumours in immunodeficient mice derived from cells lacking p21 are also more sensitive to ionizing radiation than their wild-type counterparts. Abrogation of p53 in the p21+/+ parental cells results in an in vitro phenotype that is indistinguishable from that of the p21 knockout cells. Thus, the in vitro phenotype resulting from loss of p21 is consistent with its well-established role in the p53/p21 damage response pathway. However, despite the similar in vitro phenotype, p21+/+ cells with abrogated p53 show no evidence of the sensitivity observed in the p21-/- cells when grown as tumours in immunodeficient mice. The increased radio-sensitization stabilization of p21-/- tumours is also unrelated to the increase in apoptosis observed in these tumours following radiation treatment. Apoptosis in the p21-/- tumours was significantly reduced by expression of bcl-2 without any corresponding change in the overall response of the tumour. Similarly, abrogation of p53 in the p21+/+ tumours substantially increased radiation-induced apoptosis within the tumours without increasing their radiation sensitivity. Dissociation of these in vivo and in vitro phenotypes indicates that p21 participates in a novel in vivo specific damage response pathway that is distinct from its role in the p53 pathway, and therefore that it may be an effective therapeutic target for cancer therapy.

    View details for Web of Science ID 000083709200017

    View details for PubMedID 10597257

  • DNA damage measured by the comet assay in head and neck cancer patients treated with tirapazamine. Neoplasia Dorie, M. J., Kovacs, M. S., Gabalski, E. C., Adam, M., Le, Q. T., Bloch, D. A., Pinto, H. A., Terris, D. J., Brown, J. M. 1999; 1 (5): 461-467


    Tirapazamine (TPZ) [3-amino-1,2,4-benzotriazine 1,4-dioxide, SR4233, WIN 59075, and Tirazone] is a novel anticancer drug that is selectively activated by the low oxygen environment in solid tumors. By killing the radioresistant hypoxic cells, TPZ potentiates the antitumor efficacy of fractionated irradiation of transplanted tumors in mice. As this cell kill is closely correlated with TPZ-induced DNA damage, we investigated whether human head and neck cancers would show DNA damage similar to that seen in mouse tumors following TPZ administration. TPZ-induced DNA damage in both transplanted tumors in mice and in neck nodes of 13 patients with head and neck cancer was assessed using the alkaline comet assay on cells obtained from fine-needle aspirates. The oxygen levels of the patients' tumors were also measured using a polarographic oxygen electrode. Cells from the patients' tumors showed DNA damage immediately following TPZ administration that was comparable to, or greater than, that seen with transplanted mouse tumors. The heterogeneity of DNA damage in the patients' tumors was greater than that of individual mouse tumors and correlated with tumor hypoxia. The similarity of TPZ-induced DNA damage in human and rodent tumors suggests that tirapazamine should be effective when added to radiotherapy or to cisplatin-based chemotherapy in head and neck cancers.

    View details for PubMedID 10933062

  • Oxygen tension measurements of tumors growing in mice INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Adam, M. F., Dorie, M. J., Brown, J. M. 1999; 45 (1): 171-180


    Clinical studies using the Eppendorf histograph have shown that patients whose tumors have a low pO2 have worse local control after radiotherapy, and have higher metastatic rates. Because preclinical studies of methods of overcoming, or exploiting, hypoxia generally use transplanted tumors in mice, we have compared the oxygenation of mouse tumors with human tumors to determine the appropriateness of the transplanted mouse model for such preclinical studies.We evaluated the oxygenation status of subcutaneous (s.c.) tissue and of 12 intradermally (i.d.)- and 7 s.c.-growing mouse or human transplanted tumors in mice using the Eppendorf histograph, and compared the values obtained with measurements of human head and neck nodes.The normal tissue pO2 profile of air-breathing mice showed a nearly Gaussian distribution (38.2+/-14.9 mmHg). Breathing 10% O2 or carbogen resulted in dramatic changes in normal tissue oxygenation. Tumors growing intradermally in the back of air-breathing mice were extremely hypoxic and resistant to expected changes in oxygenation (carbogen breathing, size, and use of anesthetics). Tumors growing s.c. in the foot showed higher oxygen profiles with marked changes in oxygenation when exposing the animals to different levels of oxygen. However, the oxygenation of the mouse tumors transplanted in either site was only a fraction of that of the majority of human tumors.Experimental mouse tumors are markedly hypoxic, with median values of 10-20% of those of human tumors. Hence, mouse tumors are probably good models for the most hypoxic human tumors that respond poorly to radiotherapy; however, caution has to be exercised in extrapolating data from mouse to man.

    View details for PubMedID 10477021

  • Managed care, consolidation among health care providers, and health care: evidence from mammography RAND JOURNAL OF ECONOMICS Baker, L. C., Brown, M. L. 1999; 30 (2): 351-U2


    We discuss the effects of managed care on the structure of the health care delivery system, focusing on managed-care-induced consolidation among health care providers. We empirically investigate the relationship between HMO market share and mammography providers. We find evidence of consolidation: increases in HMO activity are associated with reductions in the number of mammography providers and with increases in the number of services produced by remaining providers. We also find that increases in HMO market share are associated with reductions in costs for mammography and with increases in waiting times for appointments, but not with worse health outcomes.

    View details for Web of Science ID 000080841000009

    View details for PubMedID 10558503

  • Cisplatin anti-tumour potentiation by tirapazamine results from a hypoxia-dependent cellular sensitization to cisplatin BRITISH JOURNAL OF CANCER Kovacs, M. S., Hocking, D. J., Evans, J. W., Slim, B. G., Wouters, B. G., Brown, J. M. 1999; 80 (8): 1245-1251


    Tirapazamine (TPZ) is a new anticancer drug that is activated specifically at the low oxygen level typically found in solid tumours. It exhibits preferential cytotoxicity towards hypoxic cells and has been shown in preclinical studies with transplanted tumours and in phase II and III clinical trials to potentiate the anti-tumour efficacy of cisplatin without increasing its systemic toxicity. At present, the mechanism for this potentiation is unknown. Here we show that there is a schedule-dependent enhancement of cisplatin cytotoxicity by TPZ for cells in vitro that is similar to that seen with transplanted murine tumours. This cisplatin potentiation depends on the TPZ exposure being at oxygen concentrations below 1%, which are typical of many cells in tumours but not in normal tissues. Also, the interaction between TPZ and cisplatin does not occur in cells mutant in ERCC4, a protein essential for repair of DNA interstrand cross-links. Incubation of the cells with TPZ under hypoxia prior to cisplatin treatment increases cisplatin-induced DNA interstrand cross-links with kinetics suggesting that TPZ inhibits or delays repair of the DNA cross-links. In conclusion, we show that the tumour-specific potentiation of cisplatin cytotoxicity is likely the result of an interaction between TPZ and cisplatin at the cellular level that requires the low oxygen levels typical of those in solid tumours. The mechanism of the interaction appears to be through a potentiation of cisplatin-induced DNA interstrand cross-links, possibly as a result of a diminished or delayed repair of these lesions

    View details for Web of Science ID 000080790900019

    View details for PubMedID 10376978

  • Apoptosis, p53, and tumor cell sensitivity to anticancer agents CANCER RESEARCH Brown, J. M., Wouters, B. G. 1999; 59 (7): 1391-1399


    A widely held tenet of present day oncology is that tumor cells treated with anticancer agents die from apoptosis, and that cells resistant to apoptosis are resistant to cancer treatment. We suggest, in this review, that this tenet may need to be reexamined for human tumors of nonhematological origin, for two principal reasons: (a) cell killing has often been assessed in short term assays that are more influenced by the rate, than the overall level, of cell killing. This has tended to underestimate cell killing for cells not susceptible to apoptosis or having mutant p53; and (b) conclusions from experiments with normal cells transformed with dominant oncogenes have often been extrapolated to tumor cells. This does not take into account the fact that tumor cells have invariably undergone selection to an apoptotically resistant phenotype. In this review, we examine the impact of these two factors with particular emphasis on the influence of mutations in p53 on the sensitivity of tumor cells to DNA-damaging agents. We find that because wild-type p53 predisposes cells to a more rapid rate of cell death after DNA damage, particularly with normal or minimally transformed cells, that short-term assays have led to the conclusion that mutations in p53 confer resistance to genotoxic agents. On the other hand, if clonogenic survival is used to assess killing in cells derived from actual solid human tumors, then apoptosis and the genes controlling it, such as p53 and bcl-2, appear to play little or no role in the sensitivity of these cells to killing by anticancer drugs and radiation.

    View details for Web of Science ID 000079527800002

    View details for PubMedID 10197600

  • Tissue oxygen distribution in head and neck cancer patients HEAD AND NECK-JOURNAL FOR THE SCIENCES AND SPECIALTIES OF THE HEAD AND NECK Adam, M. F., Gabalski, E. C., Bloch, D. A., Oehlert, J. W., Brown, J. M., Elsaid, A. A., Pinto, H. A., Terris, D. J. 1999; 21 (2): 146-153


    The importance of hypoxia in limiting the sensitivity of tumor cells to ionizing radiation has long been known.We evaluated the tissue oxygenation status with a polarographic needle electrode system in 37 patients with malignancies of the head and neck and correlated the pO2 of 25 patients with treatment outcome.Sixteen tumors contained areas of severe hypoxia, defined by pO2 values below 2.5 mm Hg. Tumor oxygenation parameters were not correlated with hemoglobin, age, and history of tobacco use. There were no subcutaneous PO2 values below 10 mm Hg (ie, no areas of moderate or severe hypoxia), whereas this degree of hypoxia was commonly found in the tumors. Though not statistically significant, hypoxic tumors showed trends for poorer treatment outcome.Our data demonstrate a great interindividual variability in the oxygenation of head and neck cancers and appears unassociated with clinical parameters. The method is capable of identifying patients with poorly oxygenated tumors, thereby providing important information for selecting patients who might need customized therapy designed to kill hypoxic tumor cells. Hypoxic tumors show a consistent trend for poor treatment outcome.

    View details for PubMedID 10091983

  • Comparison of the effectiveness of tirapazamine and carbogen with nicotinamide in enhancing the response of a human tumor xenograft to fractionated irradiation RADIATION ONCOLOGY INVESTIGATIONS El-Said, A., Menke, D., Dorie, M. J., Brown, J. M. 1999; 7 (3): 163-169


    The goal of this study was to compare, with a human tumor xenograft, two different strategies for increasing tumor response to fractionated irradiation, namely, oxygenating the hypoxic tumor cells with carbogen and nicotinamide, or killing these cells with the hypoxic cytotoxin, tirapazamine (TPZ). We used the human hypopharyngeal squamous cell carcinoma cell line FaDu implanted in immune-deficient SCID mice and assessed its response to radiation by cell survival and by growth delay. The tumors were irradiated either once or twice daily with 2 or 2.5 Gy/fraction with either TPZ (0.08 mmol/kg) or nicotinamide (1,000 mg/kg) with carbogen breathing. We also tested the effect of giving TPZ on alternate days, or daily during the first half of the course, the second half, or for the whole course of radiation. We found that adding TPZ or nicotinamide with carbogen to the fractionated radiation regimen enhanced the response of the human xenograft. The enhancement was somewhat greater (though not significantly so) for TPZ, especially when given with each radiation dose. In conclusion, adding TPZ, or nicotinamide plus carbogen, to fractionated irradiation enhanced the response of this human tumor xenograft to fractionated irradiation. Consistent with theoretical modeling, there was a greater enhancement of the radiation response of the tumor when TPZ was given with each radiation dose than when given with only half of the radiation doses.

    View details for Web of Science ID 000082410500005

    View details for PubMedID 10406058

  • Tirapazamine: a new drug producing tumor specific enhancement of platinum-based chemotherapy in non-small-cell lung cancer. Annals of oncology Wouters, B. G., Wang, L. H., Brown, J. M. 1999; 10: S29-33


    Tirapazamine (TPZ), a new anti-cancer drug activated to a toxic free radical under hypoxic conditions, produces a tumor specific potentiation of cell kill by cisplatin. In the present study we discuss the mechanism and clinical potential of this effect, as well as investigate the influence of p53 mutations on the activity of TPZ.For in vitro experiments we have used mouse SCCVII tumor cells, minimally transformed mouse embryo fibroblasts (MEFs) from wild-type and p53 knockout mice, and several human NSCLC cell lines. For in vivo experiments we have used RIF-1 tumors implanted subcutaneously into C3H mice.Prior injection of TPZ into tumor-bearing mice markedly potentiated tumor cell kill by cisplatin, but produced no effect on systemic toxicity. The maximum potentiation occurred when TPZ was injected two to three hours prior to cisplatin administration. Experiments performed with cells in vitro showed a similar synergistic interaction between the two drugs when cells were exposed to TPZ under hypoxic conditions prior to exposure to cisplatin. Experiments with MEFs from either p53 wild-type or p53-knockout mice showed no influence of p53 on the sensitivity of cells to killing by TPZ under hypoxia. A similar lack of influence of p53 on the toxicity to TPZ was obtained for a panel of NSCLC cell lines.TPZ is a novel anticancer drug that produces tumor selective potentiation of cisplatin and carboplatin in both pre-clinical and clinical studies. The fact that the drug produces no potentiation of the systemic side effects of these drugs, or of other anticancer drugs used in combination with platinum in NSCLC, suggests that TPZ could become a useful agent in the treatment of lung cancer.

    View details for PubMedID 10582136

  • Pretreatment and midtreatment measurement of oxygen tension levels in head and neck cancers LARYNGOSCOPE Gabalski, E. C., Adam, M., PINTO, H., Brown, J. M., Bloch, D. A., Terris, D. J. 1998; 108 (12): 1856-1860


    Considerable evidence exists to suggest that tumor hypoxia results in radioresistance. Historically, it has been difficult to assess tumor oxygen tension levels reliably. These levels can now be assessed in head and neck malignancies using the Eppendorf pO2 histograph, which uses a fine-needle electrode and a computerized micromanipulator. This technology was used to compare the pretreatment tumor oxygen tension level in lymph node metastases of patients with head and neck cancer to measurements taken during nonsurgical treatment after a partial response had been achieved.Prospective study.Oxygen tension levels were measured in the cervical lymph nodes of 10 patients with biopsy-proven head and neck squamous cell carcinoma and cervical metastases who were being treated with nonsurgical management. These levels were obtained using the Eppendorf pO2 histograph system. Measurements were taken before the start of treatment and were repeated when the size of the cervical metastatic node had decreased by 50%. Normal subcutaneous tissue was measured during the same session. The mean and median pO2 levels, as well as the percentage of measurements with pO2 less than 5 mm Hg were determined.A mean of 72.6 measurements per session was taken from each lymph node. The median tumor pO2 measurement fell from a mean (+/-SD) of 13.9+/-8.0 mm Hg to 7.3+/-9.9 mm Hg. Even more dramatic, however, was the substantial increase in the percentage of values less than 5 mm Hg, a rise from 29% to 52%.While there is variability both in the pretreatment oxygenation of head and neck cervical metastases and in the change in tumor oxygen tension during treatment, there appears to be a decrease in the overall oxygenation of the tumors. The dramatic increase in very low oxygen measurements may reflect selective survival of radioresistant or chemoresistant hypoxic tumor cells. Cells at the very low level would be expected to be radiobiologically hypoxic (resistant to radiation-induced cell kill).

    View details for PubMedID 9851504

  • Tirapazamine: laboratory data relevant to clinical activity ANTI-CANCER DRUG DESIGN Brown, J. M., Wang, L. H. 1998; 13 (6): 529-539


    Tirapazamine (TPZ, SR 4233, WIN 59075, 3-amino-1,2,4-benzotriazine 1,4-dioxide, Tirazone) is the lead compound in the benzotriazine di-N-oxide class of bioreductive anticancer agents. Extensive preclinical testing has established that the mechanism for the selective toxicity towards hypoxic cells is the result of a one-electron reduction of the parent molecule to a free radical species that interacts with DNA to produce single- and double-strand breaks and lethal chromosome aberrations. It has also shown activity when combined with fractionated irradiation and when combined with some chemotherapy agents, particularly cisplatin and carboplatin. In this review we address those questions about the drug that are most relevance to the clinical use of the compound. In particular we review the evidence for the mechanism of action of the drug, and also show that a large portion of the synergy seen in experimental tumors when TPZ is combined with cisplatin is the result of a cellular interaction between TPZ and cisplatin that depends on hypoxia. Also of relevance to clinical use is whether the toxicity of TPZ is cumulative such as occurs with nitroimidazoles, another class of hypoxia-activated agents. Such cumulative toxicity is not evident. Finally, we present an analysis based on the area under the curve for mice and humans that demonstrates that the doses being used in current Phase II radiotherapy protocols and Phase III chemotherapy protocols should be sufficient to produce clinical activity. We conclude that the preclinical data suggest that it is likely that TPZ will be active in the clinic, particularly when combined with cisplatin.

    View details for Web of Science ID 000077238500002

    View details for PubMedID 9755717

  • Exploiting tumour hypoxia and overcoming mutant p53 with tirapazamine Satellite Symposium on Colorectal Cancer, Lung Cancer - Hopes and Reality, at the ECCO 9 Conference Brown, J. M. NATURE PUBLISHING GROUP. 1998: 12–14


    Human solid tumours are composed of a significant proportion of hypoxic cells, i.e. cells with oxygen levels lower than those of normal tissues. Tumour hypoxic cells have been shown to have a negative impact on the response of solid tumours to radiation therapy and chemotherapy. However, these low cellular oxygen levels can be exploited by a drug that is specifically activated to a cytotoxic metabolite at these low levels. Tirapazamine is a novel bioreductive agent with selective cytotoxicity to hypoxic tumour cells, irrespective of their p53 status or apoptotic response, and acts synergistically with cisplatin. This potentiation is dependent on an interaction that can only take place in a hypoxic environment, resulting in a significant sensitization of the cells to cisplatin cell killing, with no increase in the systemic toxicity of cisplatin. Thus, the low cellular oxygen levels common in solid tumours can be turned from disadvantage to advantage using the hypoxia-selective cytotoxic drug tirapazamine.

    View details for Web of Science ID 000074376300004

    View details for PubMedID 9647614

  • Tirapazamine is metabolized to its DNA-damaging radical by intranuclear enzymes CANCER RESEARCH Evans, J. W., Yudoh, K., Delahoussaye, Y. M., Brown, J. M. 1998; 58 (10): 2098-2101


    Tirapazamine (TPZ), a new anticancer drug that is currently in Phase II and III clinical trials, has a unique mechanism of action. Its cytotoxicity is selective for hypoxic cells in solid tumors and results from DNA damage produced by a free radical, which is generated by enzymatic reduction of the parent molecule. However, there is no agreement as to which enzyme(s) are involved. Here, we have measured both DNA damage and TPZ metabolism in A549 human lung cancer cells and in isolated nuclei derived from the cells. We show that, although the nuclei metabolize TPZ at a rate that is only 20% of that of whole cells, they have levels of DNA damage that are similar to those of the cells. We also show that TPZ radicals that are formed outside nuclei do not contribute to intranuclear DNA damage. Thus, essentially all of the DNA damage from TPZ results from radicals generated within the nucleus, and the 80% of the drug metabolism that occurs in the cytoplasm is probably irrelevant for the activity of this drug in killing hypoxic cells.

    View details for Web of Science ID 000073746700013

    View details for PubMedID 9605751

  • The unique physiology of solid tumors: Opportunities (and problems) for cancer therapy CANCER RESEARCH Brown, J. M., Giaccia, A. J. 1998; 58 (7): 1408-1416


    The physiology of solid tumors differs from that of normal tissues in a number of important aspects, the majority of which stem from differences between the two vasculatures. Compared with the regular, ordered vasculature of normal tissues, blood vessels in tumors are often highly abnormal, distended capillaries with leaky walls and sluggish flow. Tumor growth also requires continuous new vessel growth, or angiogenesis. These physiological differences can be problems for cancer treatment; for example, hypoxia in solid tumors leads to resistance to radiotherapy and to some anticancer drugs. However, these differences can also be exploited for selective cancer treatment. Here we review four such areas that are under active investigation: (a) hypoxia-selective cytotoxins take advantage of the unique low oxygen tension in the majority of human solid tumors. Tirapazamine, a drug in the final stages of clinical trials, is one of the more promising of these agents; (b) leaky tumor blood vessels can be exploited using liposomes that have been sterically stabilized to have a long intravascular half-life, allowing them to selectively accumulate in solid tumors; (c) the tumor microenvironment is a stimulus to angiogenenesis, and inhibition of angiogenesis can be a powerful anticancer therapy not susceptible to acquired drug resistance; and (d) we discuss attempts to use gene therapy activated either by the low oxygen environment or by necrotic regions of tumors.

    View details for Web of Science ID 000072780200020

    View details for PubMedID 9537241

  • Loss of p21(Waf1/Cip1) sensitizes tumors to radiation by an apoptosis-independent mechanism CANCER RESEARCH Wouters, B. G., Giaccia, A. J., Denko, N. C., Brown, J. M. 1997; 57 (21): 4703-4706


    Cellular checkpoints are important mediators of the response of normal cells following genotoxic damage, and interruption of these checkpoints is a common feature of many solid tumors. Although the effects of loss in checkpoint function in tumor cells are well understood in terms of cell cycle control, there is little information on their role in determining treatment efficacy in vivo. We have examined both the in vitro and in vivo responses of isogenic lines differing only in the p53-transactivated checkpoint gene, p21Waf1/Cip1. When assayed in vitro, loss of p21 in human colon tumor cells results in a selective induction of apoptosis [Waldman, T., et al., Nature (Lond.), 381: 713-716, 1996.] but no difference in the clonogenic survival. However, when grown as xenografts and irradiated in situ, p21-deficient tumors were significantly more sensitive to radiation as assessed both by clonogenic survival and by regrowth of the tumors following treatment. These data indicate that loss of p21 results in increased sensitivity to killing by ionizing radiation that is independent of the induction of apoptosis and cell cycle arrest but that is specific to cells when they are grown as a solid tumor. These results have important implications for assessing both the genetic determinants of sensitivity to anticancer agents and efficacy of anticancer agents.

    View details for Web of Science ID A1997YD35800009

    View details for PubMedID 9354425

  • Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment GENE THERAPY LEMMON, M. J., VANZIJL, P., Fox, M. E., MAUCHLINE, M. L., Giaccia, A. J., Minton, N. P., Brown, J. M. 1997; 4 (8): 791-796


    A fundamental obstacle in gene therapy for cancer treatment is the specific delivery of an anticancer gene product to a solid tumor. Although several strategies exist to control gene expression once a vector is directly introduced into a tumor, as yet no systemic delivery system exists that specifically targets solid tumors. Nonpathogenic, obligate anaerobic bacteria of the genus Clostridium have been used experimentally as anticancer agents because of their selective growth in the hypoxic regions of solid tumors after systemic application. In this report we further describe a novel approach to cancer gene therapy in which genetically engineered clostridia are used as tumor-specific vectors for the delivery of antitumor genes. We have introduced into a strain of C. beijerinckii the gene for an E. coli nitroreductase known to activate the nontoxic prodrug CB 1954 to a toxic anticancer drug. Nitroreductase produced by these clostridia enhanced the killing of tumor cells in vitro by CB 1954, by a factor of 22. To demonstrate the specificity of this approach for tumor targeting, we intravenously injected the inactive spore form of C. beijerinckii, which upon transition to a reproductive state will express the E. coli nitroreductase gene. Nitroreductase activity was detectable in 10 of 10 tumors during the first 5 days after intravenous injection of inactive clostridial spores, indicating a rapid transition from spore to reproductive state. Tumors harboring clostridial spores which did not possess the E. coli nitroreductase gene were devoid of nitroreductase activity. Most importantly, E. coli nitroreductase protein was not found in a large survey of normal mouse tissues following intravenous injection of nitroreductase containing clostridia, strongly suggesting that obligate anaerobic bacteria such as clostridia can be utilized as highly specific gene delivery vectors for cancer therapy.

    View details for Web of Science ID A1997XP16200007

    View details for PubMedID 9338007

  • Tirapazamine-induced cytotoxicity and DNA damage in transplanted tumors: Relationship to tumor hypoxia CANCER RESEARCH Siim, B. G., Menke, D. R., Dorie, M. J., Brown, J. M. 1997; 57 (14): 2922-2928


    Tirapazamine (TPZ) is a hypoxia-selective bioreductive drug currently in Phases II and III clinical trials with both radiotherapy and chemotherapy. The response of tumors to TPZ is expected to depend both on the levels of reductive enzymes that activate the drug to a DNA-damaging and toxic species and on tumor oxygenation. Both of these parameters are likely to vary between individual tumors. In this study, we examined whether the enhancement of radiation damage to tumors by TPZ can be predicted from TPZ-induced DNA damage measured using the comet assay. DNA damage provides a functional end point that is directly related to cell killing and should be dependent on both reductive enzyme activity and hypoxia. We demonstrate that TPZ potentiates tumor cell kill by fractionated radiation in three murine tumors (SCCVII, RIF-1, and EMT6) and two human tumor xenografts (A549 and HT29), with no potentiation observed in a third xenograft (HT1080). Overall, there was no correlation of radiation potentiation and TPZ-induced DNA damage in the tumors, except that the nonresponsive tumor xenograft had significantly lower levels of DNA damage than the other five tumor types. However, there was a large tumor-to-tumor variability in DNA damage within each tumor type. This variability appeared not to result from differences in activity of the reductive enzymes but largely from differences in oxygenation between individual tumors, measured using fluorescent detection of the hypoxia marker EF5. The results, therefore, suggest that the sensitivity of individual tumors to TPZ, although not necessarily the response to TPZ plus radiation, might be assessed from measurements of DNA damage using the comet assay.

    View details for Web of Science ID A1997XL16200023

    View details for PubMedID 9230202

  • Adaptation of human tumor cells to tirapazamine under aerobic conditions - Implications of increased antioxidant enzyme activity to mechanism of aerobic cytotoxicity BIOCHEMICAL PHARMACOLOGY ELWELL, J. H., Siim, B. G., Evans, J. W., Brown, J. M. 1997; 54 (2): 249-257


    Tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-di-N-oxide, SR 4233, WIN 59075) is a bioreductive antitumor agent with a high selective toxicity for hypoxic cells. The selective hypoxic toxicity of TPZ results from the rapid reoxidation of the one-electron reduction product, the TPZ radical, in the presence of molecular oxygen with the concomitant production of superoxide radical. Under hypoxia the TPZ radical kills cells by causing DNA double-strand breaks and chromosome aberrations. However, the mechanism of aerobic cytotoxicity is still a matter of debate. In this study, we investigated the mechanism of aerobic cytotoxicity by adapting human lung adenocarcinoma A549 cells to aerobic TPZ exposure and characterizing the changes associated with drug resistance. The adapted cells were resistant to aerobic TPZ exposures (with dose-modifying factors of up to 9.2), although hypoxic sensitivity was largely unchanged. Relative to the parental A549 cell line, adaptation to continuous aerobic TPZ exposure resulted in increased levels of manganese superoxide dismutase (up to 9.4-fold), moderate increases in glutathione reductase (up to 2.1-fold), and loss of both quinone oxidoreductase (DT-diaphorase) activity and NADPH cytochrome P450 reductase activity. There was essentially no change in the activity of the cytoplasmic form of superoxide dismutase (CuZnSOD), catalase, or glutathione peroxidase. The increased activity of antioxidant enzymes in the resistant cell lines (in particular MnSOD) strongly suggests that reactive oxygen species are, in large part, responsible for the toxicity of TPZ under aerobic conditions, and is consistent with aerobic and hypoxic drug cytotoxicity resulting from different mechanisms.

    View details for Web of Science ID A1997XR39600005

    View details for PubMedID 9271329

  • Cells at intermediate oxygen levels can be more important than the ''hypoxic fraction'' in determining tumor response to fractionated radiotherapy RADIATION RESEARCH Wouters, B. G., Brown, J. M. 1997; 147 (5): 541-550


    The presence of hypoxic cells in human tumors is thought to be one of the principal reasons for the failure of radiation therapy. Intensive laboratory and clinical efforts to overcome tumor hypoxia have focused on oxygenating, radiosensitizing or killing the maximally radioresistant fraction of tumor cells. This "hypoxic fraction" dominates the single-dose radiation response, irrespective of the oxygenation status of the remainder of the tumor cell population. However, at doses that are typical of those delivered in a daily radiotherapy protocol, we show that the tumor response is highly dependent upon the cells at oxygen levels intermediate between fully oxygenated and hypoxic (0.5-20 mm Hg). For most tumors, these cells are more important than the radiobiologically hypoxic cells in determining treatment outcome after 30 fractions of 2 Gy. We also show that under conditions of diffusion-limited hypoxia, the impact of full reoxygenation between fractions is much smaller than previously realized. Together, the results imply that tumor hypoxia plays a more significant role in determining the outcome of fractionated radiotherapy than previous measurements and assumptions of hypoxic fractions have indicated. Therefore, the concept of a hypoxic fraction in human tumors is less meaningful when pertaining to a fractionated radiotherapy regimen, and should not be expected to be useful for predicting tumor responses in the clinic. This implies the need to characterize tumor oxygenation in a manner that reflects the true oxygenation status of all the tumor cells, not just the ones most refractory to the effects of ionizing radiation. Furthermore, effective therapeutic agents must have the ability to specifically sensitize or kill those cells at intermediate levels of oxygen in addition to the radiobiologically hypoxic cells.

    View details for Web of Science ID A1997WY05300003

    View details for PubMedID 9146699

  • Baroreflex dysfunction after nasopharyngectomy and bilateral carotid isolation ARCHIVES OF OTOLARYNGOLOGY-HEAD & NECK SURGERY Lee, H. T., Brown, J., Fee, W. E. 1997; 123 (4): 434-437


    Baroreflex dysfunction (BRD) is an uncommon but perplexing clinical entity that occurs after an operation performed in the head and neck. Cases of BRD have occasionally been reported after bilateral carotid endarterectomies and in rare brain-stem tumors. We describe, for the first time to our knowledge, BRD in a patient after nasopharyngectomy and bilateral carotid isolation for recurrent nasopharyngeal carcinoma.

    View details for Web of Science ID A1997WT84900014

    View details for PubMedID 9109794

  • Stable translocations detected by fluorescence in situ hybridization: A rapid surrogate end point to evaluate the efficacy of a potentiator of tumor response to radiotherapy CANCER RESEARCH Kovacs, M. S., Yudoh, K., Evans, J. W., Menke, D., Brown, J. M. 1997; 57 (4): 672-677


    Testing potential modifiers of the response of tumors to radiation therapy requires large, expensive, and time-consuming clinical trials. It would, therefore, be of value to have a rapid surrogate end point of tumor response that could be used to evaluate such modifiers. We here propose that radiation-induced stable chromosome translocations measured by fluorescence in situ hybridization (FISH) could fulfill this purpose. The assay requires that the ratio of nonlethal stable translocations to lethal dicentric aberrations be unity and not change with radiation dose and that radiation-induced stable translocations remain in the tumor cell population essentially indefinitely after irradiation. We have tested these assumptions with four human tumor cell lines in vitro at doses of 1-5 Gy and found them to be valid. We also modified the response to fractionated irradiation of a human tumor xenograft in three different ways and quantitated the tumor response using clonogenic cell survival and using the FISH stable translocation assay. Both assays gave similar values for the extent of radiation modification. These data suggest that this assay could allow clinical evaluation of potential radiation sensitizers with fewer patients and in shorter times than is the case with conventional clinical trials.

    View details for Web of Science ID A1997WH81500022

    View details for PubMedID 9044844

  • Modification of the antitumor activity of chemotherapeutic drugs by the hypoxic cytotoxic agent tirapazamine CANCER CHEMOTHERAPY AND PHARMACOLOGY Dorie, M. J., Brown, J. M. 1997; 39 (4): 361-366


    Preclinical studies were performed to examine the interaction of the hypoxic cell toxin tirapazamine (TPZ), a benzotriazine di-N-oxide, with several chemotherapeutic agents, including carboplatin, cyclophosphamide, doxorubicin, etoposide, 5-fluorouracil (5-FU), taxol, and navelbine.The modification by TPZ of the antitumor drug activity and the effect of schedule were determined with an in vivo/in vitro clonogenic assay using well-established RIF-1 murine tumors transplanted into C3H mice.Additive, or greater than additive, tumor cell killing was observed when TPZ was combined with carboplatin, cyclophosphamide, doxorubicin, etoposide, 5-FU and taxol. With the exception of 5-FU there were only small, or no, enhancements of the systemic toxicities of the drugs by TPZ. The greatest enhancement of antitumor activity was with carboplatin, with the maximum effectiveness when TPZ was given 2-3 h before the carboplatin. The activity of cyclophosphamide, doxorubicin, etoposide and taxol were most enhanced when TPZ was given 24 h before the drug. Additional investigations with three-drug combination treatments using cisplatin and TPZ with either etoposide or navelbine indicated a substantial therapeutic gain from the addition of TPZ.The data for each of the drugs tested in combination with TPZ, with the exception of 5-FU, indicate that potential clinical benefit may be obtained from therapies combining TPZ with conventional chemotherapy.

    View details for Web of Science ID A1997WE05600012

    View details for PubMedID 9025778

  • NCI's anticancer drug screening program may not be selecting for clinically active compounds ONCOLOGY RESEARCH Brown, J. M. 1997; 9 (5): 213-215


    To discover new anticancer drugs the U.S. National Cancer Institute (NCI) currently screens roughly 10,000 compounds annually against a panel of 60 human tumor cell lines. Drug activity is determined using a 2-day growth inhibition assay with the cells maintained in contact with the drugs for the 2-day period. Analyses of the more than 60,000 compounds screened to date against this panel of cell lines have revealed surprisingly detailed correlations between drug activities and the molecular genotypes and phenotypes of the cells. For example, most of the drugs, particularly those active in the clinic, are most active against p53 wild-type cells. This commentary points out, however, that these patterns of activity, and by implication the compounds identified as active, may not be relevant to tumor response in vivo. This is because tumor response is determined by tumor cell kill, and there is no reason to suppose that drug-induced short-term growth inhibition should correlate with drug-induced cell kill, usually measured by the ability of the cell to divide indefinitely to form a colony. We have tested a subset of the panel of NCI cell lines for cell kill by clonogenic assay using cisplatin and mitomycin C and have found only a weak and nonsignificant correlation between cell kill and activity determined by the NCI using the growth inhibition assay. It is concluded that a more extensive comparison of the 2-day growth inhibition and clonogenic assays should be made, as well as a test of which is more relevant to the response of human tumor xenografts.

    View details for Web of Science ID A1997XV83100001

    View details for PubMedID 9306428

  • The DNA damage response in DNA-dependent protein kinase-deficient SCID mouse cells: Replication protein A hyperphosphorylation and p53 induction PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fried, L. M., Koumenis, C., Peterson, S. R., GREEN, S. L., VANZIJL, P., ALLALUNISTURNER, J., Chen, D. J., Fishel, R., Giaccia, A. J., Brown, J. M., Kirchgessner, C. U. 1996; 93 (24): 13825-13830


    Severe combined immunodeficient (SCID) mice display an increased sensitivity to ionizing radiation compared with the parental, C.B-17, strain due to a deficiency in DNA double-strand break repair. The catalytic subunit of DNA-dependent protein kinase (DNA-PKCS) has previously been identified as a strong candidate for the SCID gene. DNA-PK phosphorylates many proteins in vitro, including p53 and replication protein A (RPA), two proteins involved in the response of cells of DNA damage. To determine whether p53 and RPA are also substrates of DNA-PK in vivo following DNA damage, we compared the response of SCID and MO59J (human DNA-PKcs-deficient glioblastoma) cells with their respective wild-type parents following ionizing radiation. Our findings indicate that (i) p53 levels are increased in SCID cells following ionizing radiation, and (ii) RPA p34 is hyperphosphorylated in both SCID cells and MO59J cells following ionizing radiation. The hyperphosphorylation of RPA p34 in vivo is concordant with a decrease in the binding of RPA to single-stranded DNA in crude extracts derived from both C.B-17 and SCID cells. These results suggest that DNA-PK is not the only kinase capable of phosphorylating RPA. We conclude that the DNA damage response involving p53 and RPA is not associated with the defect in DNA repair in SCID cells and that the physiological substrate(s) for DNA-PK essential for DNA repair has not yet been identified.

    View details for Web of Science ID A1996VV46700068

    View details for PubMedID 8943020

  • Tirapazamine-induced DNA damage measured using the comet assay correlates with cytotoxicity towards hypoxic tumour cells in vitro BRITISH JOURNAL OF CANCER Siim, B. G., VANZIJL, P. L., Brown, J. M. 1996; 73 (8): 952-960


    Tirapazamine (SR 4233), a bioreductive drug selectively toxic towards hypoxic cells, is presently in phase II clinical trials. Since it would not be expected that all tumours would respond equally to the drug, we are exploring ways of predicting the response of individual tumours. In this study we have tested whether the comet assay, which measures DNA damage in individual cells, can provide a simple, surrogate end point for cell killing by tirapazamine. We examined the relationship between the cytotoxicity of tirapazamine under hypoxic conditions and tirapazamine-induced DNA strand breaks in murine (SCCVII, EMT6, RIF-1) and human (HT1080, A549, HT29) tumour cell lines. These results were compared with the relationship between tirapazamine cytotoxicity and another measure of the ability of cells to metabolise tirapazamine; high-performance liquid chromatography (HPLC) analysis of tirapazamine loss or formation of the two electron reduction product SR 4317. The correlation between the hypoxic cytotoxic potency of tirapazamine and DNA damage was highly significant (r = 0.905, P = 0.013). A similar correlation was observed for hypoxic potency and tirapazamine loss (r = 0.812, P = 0.050), while the correlation between hypoxic potency and SR 4317 formation was not significant (r = 0.634, P = 0.171). The hypoxic cytotoxicity of tirapazamine in vitro can therefore be predicted by measuring tirapazamine-induced DNA damage using the comet assay. This approach holds promise for predicting the response of individual tumours to tirapazamine in the clinic.

    View details for Web of Science ID A1996UE60600012

    View details for PubMedID 8611431

  • Anaerobic bacteria as a delivery system for cancer gene therapy: In vitro activation of 5-fluorocytosine by genetically engineered clostridia GENE THERAPY Fox, M. E., LEMMON, M. J., MAUCHLINE, M. L., Davis, T. O., Giaccia, A. J., Minton, N. P., Brown, J. M. 1996; 3 (2): 173-178


    Certain species of anaerobic bacteria have been shown to localise and germinate specifically in the hypoxic regions of tumours, resulting in tumour lysis. We propose an innovative approach to cancer gene therapy in which genetically engineered anaerobic bacteria of the genus Clostridium are used to achieve tumour-specific gene delivery. Our strategy involves enzyme/prodrug therapy, in which the Escherichia coli enzyme cytosine deaminase is used to convert the non-toxic prodrug 5-fluorocytosine to the active chemotherapeutic agent 5-fluorouracil. The E. coli gene encoding cytosine deaminase has been cloned into a clostridial expression vector and transformed into Clostridium beijerinckii, resulting in constitutive expression of cytosine deaminase and significant levels of active enzyme in the bacterial medium. When added to an in vitro clonogenic survival assay, supernatant from clostridia expressing cytosine deaminase increased the sensitivity of murine EMT6 carcinoma cells to 5-fluorocytosine approximately 500-fold. This high level of prodrug activation, combined with the specificity of clostridia for hypoxic regions of tumours, indicates a potential use in cancer gene therapy.

    View details for Web of Science ID A1996TZ78700010

    View details for PubMedID 8867865

  • Induction and repair of chromosome aberrations in scid cells measured by premature chromosome condensation RADIATION RESEARCH Evans, J. W., Liu, X. F., Kirchgessner, C. U., Brown, J. M. 1996; 145 (1): 39-46


    Severe combined immunodeficient (scid) murine cells, which are defective in both repair of DNA double-strand breaks and V(D)J recombination, are deficient in DNA-dependent protein kinase (DNA-PK), a protein which forms an activated complex with the DNA end-binding Ku proteins (p80 and p70) upon association with damaged DNA. Xrs 5 cells are deficient in the Ku p80 protein and also fail to form an active DNA-PK repair complex. Since both scid and xrs cells are defective in the same protein complex, we compared the kinetics of chromosome repair in scid cells to results published previously for xrs 5 cells. C.B-17 cells, scid cells and scid cells complemented with a single human chromosome 8 were irradiated with 6 Gy and allowed to repair from 0-24 h before fusion to HeLa cells for chromosome condensation. Breaks and dicentrics were visualized by fluorescence in situ hybridization. All cells had the same initial amount of chromosome damage, but scid cells had a slower rate of rejoining, more unrejoined breaks and more dicentrics than C.B-17 and scid cells with human chromosome 8. The scid cells appear to respond differently than xrs 5 cells, despite both cells lacking an essential component of the same DNA repair complex.

    View details for Web of Science ID A1996TM49100006

    View details for PubMedID 8532835

  • Hypoxia-Specific Cytotoxins in Cancer Therapy. Seminars in radiation oncology Brown, J. M., Siim, B. G. 1996; 6 (1): 22–36


    Hypoxic-specific cytotoxins are a new, and as yet clinically untested, mode of treatment of solid tumors. If they can be given at high enough concentrations and sufficiently often, they should prove extremely effective both in combination with standard radiotherapy and also with certain chemotherapeutic drugs. It is likely that their optimum use will turn hypoxic cells in solid tumors from a therapeutic disadvantage to an advantage. In this report, we review the rationale for the use of hypoxia- cytotoxins, including both the theoretical basis for combining them with fractionated radiation and the preclinical results that have been obtained to date combing these agents with fractionated radiation. We also discuss the three major classes of bioreductive drugs, including the quinones (mitomycin C, porfiromycin, and E09), notroaromatic compounds (including RB6145 and various deoxyribonucleic acid [DNA] targeted aromatics), and finally the n-oxides of which tirapazamine is the lead compound. We also briefly discuss new approaches to bioreductive drug development. The best ways to use these agents are also covered. These include using them in combination with radiation, in combination with chemotherapy, and in combination with agents that increase tumor hypoxia. Finally, the importance of the selection of patients for clinical trials is illustrated by showing how dramatically the number of patients for clinical trials is illustrated by showing how dramatically the number of patients in a clinical trial has to increase to obtain statistical significance for a procedure targeted towards hypoxic cells if some of the patients in the trials have well-oxygenated tumors.

    View details for PubMedID 10717159

  • DNA-DEPENDENT KINASE (P350) AS A CANDIDATE GENE FOR THE MURINE SCID DEFECT SCIENCE Kirchgessner, C. U., Patil, C. K., Evans, J. W., Cuomo, C. A., Fried, L. M., Carter, T., Oettinger, M. A., Brown, J. M. 1995; 267 (5201): 1178-1183


    Severe combined immunodeficient (SCID) mice are deficient in a recombination process utilized in both DNA double-strand break repair and in V(D)J recombination. The phenotype of these mice involves both cellular hypersensitivity to ionizing radiation and a lack of B and T cell immunity. The catalytic subunit of DNA-dependent protein kinase, p350, was identified as a strong candidate for the murine gene SCID. Both p350 and a gene complementing the SCID defect colocalize to human chromosome 8q11. Chromosomal fragments expressing p350 complement the SCID phenotype, and p350 protein levels are greatly reduced in cells derived from SCID mice compared to cells from wild-type mice.

    View details for Web of Science ID A1995QJ23700041

    View details for PubMedID 7855601



    The purpose of the present investigation was to determine whether chromosome aberrations scored by premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) can predict the radiosensitivity of human cell lines, thereby providing a possible means of assessing the in situ radiosensitivity of normal tissues and the radiocurability of individual human cancers.We used four cells lines of different radiosensitivity: normal human fibroblasts (AG1522), ataxia-telangiectasia fibroblasts (AT2052), a human fibrosarcoma cell line (HT1080), and a human melanoma cell line (melanoma 903). These were irradiated in plateau phase with a range of doses and assessed both for clonogenic cell survival and for aberrations in a single chromosome (number 4) immediately after, and 24 h after irradiation.The initial number of breaks in chromosome 4 was proportional to irradiation dose and was identical for all the different human cell lines, irrespective of radiosensitivity. On the other hand, the number of chromosome 4 breaks remaining 24 h after irradiation reflected the radiosensitivity of the cells such that the relationship between residual chromosome aberrations and cell survival was the same for the different cell lines.These results suggest that the scoring of chromosome aberrations in interphase using FISH with PCC holds considerable promise for predicting the radiosensitivity of normal and tumor tissues in situ.

    View details for Web of Science ID A1994PT59800016

    View details for PubMedID 7961021



    This study was conducted to assess the ability of computerized pO2 histography to measure changes in tumor oxygenation produced by low oxygen breathing.Female syngeneic C3H/Km mice bearing SCC VII/St carcinomas were used in these experiments. Changes in tumor oxygenation produced by the mice breathing 10% oxygen were assessed with computerized pO2 histography, 3H-misonidazole binding, and the paired survival curve assay of radiosensitivity.The hypoxic cell fraction of the tumors in mice breathing 10% oxygen was 3.1 times higher than that of tumors in mice breathing normal air determined by an in vivo-in vitro clonogenic assay. Binding of radiolabeled misonidazole to the tumors in mice breathing 10% oxygen was also significantly higher than that to tumors in mice breathing normal air (p < 0.05). In addition, oxygen tension histograms for normal tissue showed a dramatic shift to a lower oxygen tension when the mice were breathing 10% oxygen. However, under identical conditions, there was only a minimal shift in the oxygen tension of tumor tissue. Although the number of oxygen tension readings in the relatively oxic class in tumor tissue was lower when the mice were breathing 10% oxygen than when breathing normal air, there was not a significant decrease in the median pO2 value for the tumor. The number of pO2 readings lower than 5 mmHg in the tumor was not affected by the 10% oxygen breathing.These findings indicate that increases in radiobiological hypoxic fraction produced by lower blood oxygen levels may not correlate well with the results of polarographic measurements of tumor pO2 levels.

    View details for Web of Science ID A1994PK07200013

    View details for PubMedID 7928462



    Determining the volumes of peaks in 2D NMR spectra can be prohibitively difficult in cases of overlapping, broad lines. Deconvolution and parameter estimation can be attempted on either the time-domain or the frequency-domain data. We present a method of estimating spectral parameters from frequency-domain data, using a combination of Lorentzian and Gaussian lineshapes for reference lines. This approach combines a previously published method of projecting the data on a linear space spanned by reference lines with a nonlinear least-squares fitting algorithm. Comparison of this method with other published methods of frequency-domain deconvolution shows that it is both more precise and more accurate when estimating 2D volumes.

    View details for Web of Science ID A1994PE35200005

    View details for PubMedID 7919951

  • REOXYGENATION AND REHYPOXIATION IN THE SCCVII MOUSE-TUMOR 8th International Conference on Chemical Modifiers of Cancer Treatment Kim, I. H., Brown, J. M. ELSEVIER SCIENCE INC. 1994: 493–97


    To test the hypothesis that, following preferential killing of tumor hypoxic cells, the fraction of hypoxic cells in the tumor will reestablish itself to pretreatment levels (rehypoxiation) with the same kinetics as for reoxygenation.Mouse squamous cell carcinoma VII (SCCVII) tumors were treated with a single dose of 10 Gy or a single dose of the bioreductive hypoxic cell cytotoxin, tirapazamine (SR 4233, 0.2 mmol/kg), which preferentially kills hypoxic cells within the tumor. Hypoxic fractions were determined by the paired survival curve technique using the in vivo-in vitro clonogenic assay 0-24 h after treatment.Immediately after irradiation with 10 Gy, the hypoxic fraction of the tumors increased to 80% and rapidly returned to pretreatment levels 3-6 h later. Within 1 h of injecting tirapazamine, the hypoxic fraction fell to 0.57% (about 7% of pretreatment levels) and returned to pretreatment levels 3-5 h later.The return to pretreatment levels of hypoxia among tumor cells surviving a single dose of radiation (reoxygenation) and of the hypoxic cell toxin tirapazamine (rehypoxiation) was rapid and occurred with similar kinetics for the two processes. These data support the hypothesis that reoxygenation and rehypoxiation are different manifestations of the same phenomenon and result from fluctuating tumor blood flow which creates acute hypoxia.

    View details for Web of Science ID A1994NQ84300015

    View details for PubMedID 8005805



    This review describes molecular cytogenetic approaches to genetic analysis including fluorescence in situ hybridization, primed in situ labeling and comparative genomic hybridization. It also summarizes the applications of this technology to physical mapping, cancer diagnosis and prognostication and in radiation biology.

    View details for Web of Science ID A1994MY44100001

    View details for PubMedID 8146269



    To develop a molecular strategy of increasing cytotoxicity of chronically hypoxic cells by inhibiting Glucose Regulated Protein 78 kDal (GRP78) induction.A mutant nonGRP78 inducing cell line (78WO) was developed from its parent (DG44) by overexpressing antisense GRP78 mRNA. Following exposure to varying durations of hypoxia, Northern and Western blot analysis were used to characterize the amount of GRP78 expression both at the RNA and protein level. Hypoxia was achieved by placing cells in specially designed hypoxic chambers which were subjected to successive rounds of evacuation and flushing with 95% CO2/N2 to reduce the oxygen in the environment to 0.02% oxygen. After treatment with hypoxia, cells were assayed for colony forming ability.GRP78 mRNA and protein induction following exposure to hypoxia was 3-4 fold lower in the 78WO cell line than in the parental DG44 cell line. Furthermore, it was observed that there was no difference in the cytotoxicity of 78WO and DG44 cells after 10 h of hypoxia. However, after 15 h of hypoxia, the survival of 78WO cells decreased by 1 log and after 20 h of hypoxia, the survival of 78WO decreased by another log.These results show that stress protein induction is important for cellular survival to chronic hypoxia and that inhibition of GRP78 induction may represent a novel therapeutic strategy by selectively sensitizing chronically hypoxic cells within solid tumors.

    View details for Web of Science ID A1994NA83600012

    View details for PubMedID 8113109



    We have used fluorescence in situ hybridization with whole-chromosome probes for human chromosomes 1, 4, 8 and 13 to investigate the extent to which the induction of damage and its repair after exposure to ionizing radiation is distributed randomly among these chromosomes. All the studies were performed with AG1522 human fibroblasts irradiated with 6 Gy and maintained in a nondividing state for at least 6 h after irradiation except for the measurements of initial damage. The extent of initial damage was determined by fusion of the cells immediately after irradiation with metaphase HeLa cells to obtain premature chromosome condensation (PCC). Breaks and exchanges were also scored by PCC 24 h after irradiation and in metaphase spreads at the first division after irradiation. The data obtained were consistent with random breakage and repair in these chromosomes. Comparing PCC 24 h after irradiation with first metaphase, there was a deficit in aberrations at metaphase, particularly in unrejoined breaks, implying loss or slowing of cells containing aberrations prior to the first division. An analysis of dicentrics and translocations in chromosome 4 at first and in subsequent divisions showed that there was an equal number of dicentrics and translocations at first metaphase with loss of dicentrics, but no loss of translocations in subsequent divisions. These data are supportive of the hypothesis tht the total number of chromosome aberrations in cells can be estimated from a single chromosome pair.

    View details for Web of Science ID A1994MN70900006

    View details for PubMedID 8265786

  • TUMOR HYPOXIA - THE PICTURE HAS CHANGED IN THE 1990S Radiation Research 93 - Annual Meeting of the Association-for-Radiation-Research Brown, J. M., Giaccia, A. J. TAYLOR & FRANCIS LTD. 1994: 95–102


    Since the 1950s, the presence of hypoxic cells in human tumours has been widely regarded as a problem, and a variety of strategies have been developed and tested, both in experimental and clinical studies, to overcome this perceived problem. One of these strategies was the development of bioreductive cytotoxins--drugs which in themselves were relatively innocuous, but when metabolized under hypoxic conditions, became highly cytotoxic, thereby preferentially killing the hypoxic cells. Modelling studies and experimental data with newly developed hypoxic cytotoxins, such as SR 4233 (tirapazamine) and RSU 1069, have led to the realization not only that it is better to kill hypoxic cells in tumours than to radiosensitize or oxygenate them, but also that with these bioreductive cytotoxins hypoxic cells in tumours can be an advantage in cancer therapy. However, to realize the advantage of adding the drug with each radiation dose, the tumour must undergo a process analogous to reoxygenation, which we have termed 'rehypoxiation', by which hypoxic cells are regenerated after each dose of the hypoxic cytotoxin. In addition, we also discuss the fact that hypoxia is a cellular stress which activates many new genes. The activation of these genes will be a major focus for research in coming years and will undoubtedly lead to new approaches in cancer detection and treatment. In summary, the 1990s are bringing a fundamental change in our perception of tumour hypoxia, from a position of being a problem to that of being a solution in cancer treatment.

    View details for Web of Science ID A1994NE75800013

    View details for PubMedID 7905916



    This study was undertaken to compare in a fractionated regimen, with clinically relevant radiation doses, two radiation response modifiers that function by different mechanisms: SR 4233, a bioreductive agent toxic to hypoxic cells, and nicotinamide with carbogen, a combination that has been shown to improve tumor oxygenation.Cell survival assays were used to examine the response of three different tumors: KHT, RIF-1 and SCCVII/St in C3H/Km mice. Regrowth delay studies were also performed with the RIF-1 tumor. A fractionated irradiation schedule, consisting of twice daily 2.5 Gy treatments was investigated with and without drug pretreatment. SR 4233 was given IP at 0.12 mmol/kg one half hour before each irradiation. Nicotinamide (250, 500, 1000 mg/kg) was given IP 1 h before each irradiation with carbogen exposure 5 min prior to and during the irradiation.Both treatment strategies enhanced the response of all three tumors to the fractionated radiation regimen. However, for two of the tumors (KHT and SCCVII), SR 4233 produced a significantly greater enhancement than did the combination of nicotinamide + carbogen. For the RIF-1 tumor (which has the lowest hypoxic fraction of the three), the response was comparable for the two modalities. For nicotinamide + carbogen, there was no significant change in the radiation enhancement at nicotinamide doses between 250 and 1000 mg/kg.Adding the bioreductive cytotoxin SR 4233 or nicotinamide + carbogen to fractionated irradiation enhances the response of the three transplanted tumors used in this study to fractionated irradiation. The radiation enhancement was significantly greater, however, for SR 4233 for two of the tumors with comparable results in the third. The data are consistent with the prediction that killing tumor hypoxic cells can produce a similar or greater enhancement of the efficacy of fractionated radiation in enhancing tumor response than either oxygenating or radiosensitizing these cells.

    View details for Web of Science ID A1994MP35300019

    View details for PubMedID 8270435

  • COMPLEMENTATION OF THE RADIOSENSITIVE PHENOTYPE IN SEVERE COMBINED IMMUNODEFICIENT MICE BY HUMAN CHROMOSOME-8 CANCER RESEARCH Kirchgessner, C. U., TOSTO, L. M., Biedermann, K. A., Kovacs, M., Araujo, D., Stanbridge, E. J., Brown, J. M. 1993; 53 (24): 6011-6016


    Severe combined immunodeficient (scid) C.B-17 mice are deficient in variable (diversity) joining region recombination, the process of assembling the immunoglobulin and T-cell receptor genes from gene segments, thereby creating much of the enormous diversity of antigen-binding capacity, scid mice are also sensitive to ionizing radiation, as a result of their deficiency in double-strand break repair. Here we report the complementation of the radiation-sensitive scid phenotype by transferring human chromosome 8 into scid cells. Somatic cell hybrids were generated by fusing scid cells with human HT-1080 cells, resulting in radioresistant hybrids with several human chromosomes. One of the identified human chromosomes in the radioresistant scid cell line 4.61, which retains only two human chromosomes, is a rearranged 8/21 translocation. Proof that chromosome 8 confers the complementation was achieved by transferring only human chromosome 8 into scid cells by microcell-mediated chromosome transfer (scid/hu8 cell line). The presence of chromosome 8 in our scid/hu8 cell line was monitored by fluorescence in situ hybridization and polymerase chain reaction. We demonstrated the radioresistance of this hybrid not only to high dose rate but also to low dose rate radiation. We also showed that transference of human chromosome 8 to scid cells fully complements the DNA double-strand break repair deficiency and the high sensitivity of scid cells to radiation-induced chromosome aberrations. Mapping the scid gene to human chromosome 8 is an important first step in cloning the scid gene, which will enhance our understanding of double-strand break repair pathways in humans.

    View details for Web of Science ID A1993ML58900032

    View details for PubMedID 8261415



    Tirapazamine (SR 4233), a benzotriazine di-N-oxide, a potent and selective killer of hypoxic cells, is currently in Phase I clinical trials with the expectation that it will be combined with radiation therapy. However, because of the likelihood that hypoxic tumor cells may also be resistant to some commonly used chemotherapeutic agents, we have tested tirapazamine in combination with cisplatin (c-DDP) in the mouse RIF-1 tumor. A large, schedule-dependent enhancement of tumor cell killing was observed both in vivo and in vitro, with a maximal response observed when the SR 4233 was given 2-3 h before c-DDP. Assay of serum blood urea nitrogen levels following treatment with these two drugs indicates that SR 4233 does not enhance the kidney damage which can result from high doses of c-DDP. Leukopenia induced by the two drugs in combination was equal to that predicted from an additive effect of the responses to the individual drugs. Also, there was no change in the systemic toxicity of c-DDP (as judged by 50% lethal dose) when SR 4233 was combined with c-DDP at a dose and timing that produced the maximum tumor interaction. These observations point to a promising new combination therapy with considerable therapeutic advantage.

    View details for Web of Science ID A1993LZ63700034

    View details for PubMedID 8402639



    A long-standing controversy in radiation cytogenetics is the precise mechanism for the formation of chromosome exchanges. The classical breakage-and-reunion hypothesis suggested by Stadler and developed by Sax was challenged in 1959 by Revell, who proposed that radiation produced an "unstable lesion" which could interact with another such lesion to form an exchange. A difference between the predictions of these two theories is that the breakage-and-reunion hypothesis would allow the two broken ends of a chromosome to join with contemporary broken ends in different chromosomes, thereby producing nonreciprocal exchanges involving more than two chromosomes. This would not occur according to the Revell theory, which demands pairwise commital to the exchange. The ability to "paint" a whole chromosome using fluorescence in situ hybridization allows a discrimination between reciprocal and nonreciprocal chromosome exchanges. We scored metaphases in AG1522 human fibroblasts irradiated in G1 phase with 6 Gy and hybridized to chromosomes 1, 4, or 8. Of the complete exchanges involving one of these chromosomes, 26% were found to possess either one or three centromeres in the exchange pair. Since we could rule out any significant contribution of complex exchanges (i.e., those involving more than one break per chromosome), these one- and three-centromere exchange pairs must have arisen from a nonreciprocal exchange. Because an equal number of nonreciprocal exchanges would be expected to have a total of two centromeres, this suggests that approximately 50% of all exchanges at this dose were nonreciprocal. These data support the breakage-and-reunion hypothesis and are incompatible with the standard form of the exchange hypothesis of Revell.

    View details for Web of Science ID A1993MD36000010

    View details for PubMedID 8210341



    Telomeric DNA is composed of highly conserved sequences which are present at the termini of chromosomes as well as at intrachromosomal locations. Here, we studied a Chinese hamster ovary (CHO) cell line, BL-10, with highly stable amplified telomeric DNA at the termini as well as at intrachromosomal locations. We show that intrachromosomal or interstitial telomeric sites in this cell line and in another CHO cell line, HA-I, are radiosensitive in that they are more prone to breakage than would be expected based on the percentage of the genome composed of telomeric sequences. The frequency of breakage at interstitial telomeric sites is 4.3 to 8.3 times higher than that in the CHO genome overall. These conclusions are reached by both conventional cytogenetic analysis of two CHO cell lines which have the same survival rates after exposure to ionizing radiation, and by use of double fluorescence in situ hybridization (FISH) with a pan-telomere-specific probe and a CHO chromosome-specific library in the same metaphase cells after irradiation.

    View details for Web of Science ID A1993LU38000002

    View details for PubMedID 7691162



    SCCVII carcinomas and RIF-1 sarcomas were transplanted and grown in the unirradiated (control tumors) or preirradiated tissue of mice. Tumor oxygenation was measured when the tumors were in the size range 50-150 mm3 using three different methods. The tumors growing in tissues preirradiated with 20 Gy were significantly more hypoxic than the control tumors for both tumor types. Radiobiologically hypoxic fractions of these tumors were approximately 10 times higher than those of the controls. The relative [3H]misonidazole binding of the tumors transplanted into irradiated tissue was approximately five times (P < 0.01) higher in SCCVII tumors or approximately two times (P < 0.05) higher in RIF-1 tumors than that of the controls. The pO2 histographs measured directly by a polarographic microelectrode were more hypoxic (P < 0.001) and changed from bimodal to unimodal in pattern in the tumors transplanted into irradiated tissue compared to those of the controls. The increase of the relative [3H]misonidazole binding in the tumors transplanted into irradiated tissue compared to that of the controls was similar to the increase of the cumulative frequency of pO2 below 10.0 mm Hg for both the SCCVII and the RIF-1 tumors. The data suggest that the majority of tumor cells with pO2 values below 5.0 mm Hg were clonogenic in the SCCVII tumors while only a small portion of tumor cells with pO2 below 5.0 mm Hg were clonogenic in the control RIF-1 tumors. Increased hypoxia caused by preirradiation of the tumor bed is a useful model in evaluating the efficacy of various treatments affected by tumor oxygenation levels. The model is also useful in evaluating techniques for measuring oxygen levels.

    View details for Web of Science ID A1993MA35100018

    View details for PubMedID 8378534



    SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide, WIN 59075, tirapazamine) is the lead compound in a new class of bioreductive anticancer drugs, the benzotriazine di-N-oxides. It is currently undergoing Phase I clinical testing. The preferential tumour cell killing of SR 4233 is a result of its high specific toxicity to cells at low oxygen tensions. Such hypoxic cells are a common feature of solid tumours, but not normal tissues, and are resistant to cancer therapies including radiation and some anticancer drugs. The killing of these tumour cells by SR 4233, particularly when given on multiple occasions, can increase total tumour cell killing by fractionated irradiation by several orders of magnitude without increasing toxicity to surrounding normal tissues. Topics covered in this review include the rationale for developing a hypoxic cytotoxic agent, the cytotoxicity of SR 4233 as a function of oxygen concentration, the mechanism of action of the drug and its intracellular target and the in vivo evidence that the drug may be useful as an adjunct both to radiotherapy and chemotherapy. Finally, the major unanswered questions on the drug are outlined.

    View details for Web of Science ID A1993LF31900001

    View details for PubMedID 8512801

  • CHARACTERIZATION OF THE DNA DOUBLE STRAND BREAK REPAIR DEFECT IN SCID MICE CANCER RESEARCH Chang, C., Biedermann, K. A., Mezzina, M., Brown, J. M. 1993; 53 (6): 1244-1248


    The scid mutation in CB-17 mice confers a profound immunodeficiency, resulting from an inability to rearrange immunoglobulin and T-cell receptor genes during lymphocyte development. Moreover, we and others have recently demonstrated in these scid mice a hypersensitivity to the lethal effects of ionizing radiation and a defect in DNA double strand break rejoining. In this report, we further characterize the radiosensitivity and repair defect in cells from scid mice. In order to determine whether scid cells were specifically sensitive to agents that produce double strand breaks, restriction enzymes RsaI and Sau3AI were introduced into scid and parental C.B-17 cells by electroporation. scid cells were 2-fold more sensitive than C.B-17 cells to both the blunt and the staggered end cuts produced by these restriction enzymes. However, the scid cells proficiently ligated both staggered and blunt ends of transfected plasmids. To determine whether the extent of DNA rejoining in scid cells was dependent on the initial dose of gamma-rays, final levels of DNA double strand break rejoining in scid and C.B-17 cells were quantitated by asymmetric field inversion gel electrophoresis. The results indicate an apparent difference in repair levels dependent on the dose of gamma-rays, ranging from 75% rejoining at 10 Gy to 40% rejoining at 50 Gy. In contrast, > 90% rejoining was observed in control C.B-17 cells at all doses. Delineating the links between these aberrant recombinational events, abnormal V(D)J recombination, and double strand break repair defects, will aid in the understanding of the basic mechanisms involved in these processes.

    View details for Web of Science ID A1993KR29700008

    View details for PubMedID 8443804



    Korbelik and Skov (Radiat. Res. 119, 145-156, 1989) have reported that cis-diamminedichloroplatinum (II) (cisplatin) shows substantial preferential radiosensitization of hypoxic cells in vitro at low radiation doses (1-4 Gy), and that the interaction seen with low doses of radiation is greatly diminished at high radiation doses. If such an interaction occurred with fractionated irradiation in vivo, it would be extremely important to radiation therapy, since the sensitizer enhancement ratios achievable in the low-dose region are higher than those achievable with current hypoxic cell radiosensitizers. We have tested this possibility in an experimental mouse tumor using fractionated irradiation under conditions in which the response of the tumor was determined by either its aerobic or its hypoxic cells. RIF-1 tumors were irradiated with 10 fractions of 1-4 Gy every 12 h with cisplatin given either as 12 mg/kg once before the first radiation dose or as 1.2 mg/kg at various times prior to each radiation dose. The tumors were irradiated with or without a clamp applied 2-3 min before each radiation dose. The effectiveness of the treatments was assayed by regrowth delay. Cisplatin caused a similar regrowth delay when used alone in both clamped and nonclamped tumors and produced a similar additive or supra-additive interaction when used with the 10 fractionated radiation schedule whether the tumors were hypoxic or aerobic. Our data suggest that cisplatin does not show any preferential radiosensitization of hypoxic cells with low-dose multifraction irradiation in this tumor, although a clear schedule-dependent interaction between the drug and radiation was seen for both aerobic and hypoxic tumors.

    View details for Web of Science ID A1993KN25000015

    View details for PubMedID 8438067



    SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is an anti-tumour agent that has a highly selective toxicity to hypoxic cells. In this study we delineate the role of several different bioreductive enzymes in the metabolism of SR 4233 by two tumour cell lines HT 1080 (human fibrosarcoma) and SCCVII (mouse carcinoma). Enzyme kinetics demonstrates similar KM of HT 1080 and SCCVII cell sonicates and differing Vmax. Among all cofactors tested, NADPH was the most important one in reducing SR 4233 by both tumour cell sonicates. NADH was the second most important cofactor while hypoxanthine and N-methylnicotinamide were less involved in the reduction of SR 4233. Carbon monoxide inhibited the reduction by about 60% suggesting that cytochrome P-450 may play a major role in the reduction of SR 4233 under hypoxia in both SCCVII and HT 1080 cells. DT diaphorase is also involved, particularly in HT 1080 cells, in this drug reduction. The level of functional cytochrome P-450, cytochrome P-450 reductase activity and DT diaphorase activity in both cell lines were assayed. These enzyme levels were all higher in SCCVII cells than in HT 1080 cells. This result correlated the higher Vmax of SR 4233 reduction in SCCVII cells than in HT 1080 cells.

    View details for Web of Science ID A1993KK59600020

    View details for PubMedID 8431360



    We have investigated the feasibility of administration of an effective dose of the hypoxic cytotoxin, SR 4233, Monday-Friday daily for 6 weeks. From a thorough hematological, histopathological and clinical chemistry evaluation throughout the course and during a 3-week recovery period, we conclude that daily administration of a radiopotentiating dose of SR 4233 in mice is well tolerated and that bone marrow suppression is likely to be the dose-limiting toxicity.

    View details for Web of Science ID A1993KN76200013

    View details for PubMedID 8438092



    We have used the techniques of premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) with a library for human chromosome 4 to analyze the rate of rejoining of chromosome breaks and development of exchange aberrations in AG1522 human fibroblasts. AG1522 cells were irradiated in plateau phase with 10 Gy and fused with mitotic HeLa cells either immediately after irradiation or at intervals up to eight days later. The slides were then hybridized with the chromosome 4 library and unrejoined breaks and exchange events (visualized as bicolor chromosomes) scored in these cells. At the earliest time point after irradiation, the number of exchange events in the irradiated cells was low, but increased with kinetics similar to that of the joining of the breaks. Furthermore, when we analyzed those cells which had exchange events for their distribution, almost all of the cells initially contained one exchange event (1 bicolor chromosome). As time progressed, the number of cells containing exchanges with two exchange events per cell increased as the number with one exchange event per cell decreased. Extrapolation of the number of exchange events to zero time (with an estimate of 20 min for the fusion and condensation times) gave a value consistent with zero exchanges at zero time after irradiation. In a separate experiment, we also scored AG 1522 cells at the first metaphase after a dose of 6 Gy and were able to show that as many as 50% of the complete exchanges were non-reciprocal in nature, that is, the two broken ends of a single break in chromosome 4 joined to two different chromosomes. These data support the classical breakage-and-reunion model rather than the Revell Exchange Theory of exchange formation.

    View details for Web of Science ID A1993MJ11200005

    View details for PubMedID 8223502



    The bioreductive cytotoxic agent SR 4233 (1,2,4-benzotriazine 3-amine 1,4-dioxide) has been shown to markedly potentiate the cell killing of mouse tumours when combined with fractionated radiation therapy. Differential metabolism under oxic compared to hypoxic conditions results in SR 4233 exhibiting selective cytotoxicity to hypoxic cells. This is thought to result from the production of a cytotoxic free radical which is generated predominantly in the absence of oxygen. We have examined a way of enhancing the effectiveness of this antitumour agent in vivo by artificially increasing the hypoxic fraction of tumours by hypoxic breathing. Mice are placed in a chamber containing 10% Oxygen 90% Nitrogen for 1 h after each administration of SR 4233. Our results in the SCCVII tumour model indicate that this manoeuvre results in a 10-fold increase in antitumour effectiveness of SR 4233 when administered in a fractionated regime with radiotherapy (8 x 2.5 Gy and 0.08 mmol kg-1), but not when a single treatment regime (1 x 20 Gy and 0.3 mmol kg-1) is used. Mathematical modelling of this effect is used to illustrate this phenomenon and can be used to predict the dependence of this type of therapy on the modification of tumour oxygenation.

    View details for Web of Science ID A1992KC34300011

    View details for PubMedID 1457345



    The effect of ionising radiation on the regulation of gene and protein expression is complex. This study focuses on the translational regulational of the epsilon isoform of protein kinase C by ionising radiation. We found that protein kinase C epsilon is rapidly increased in the human lung adenocarcinoma cell line A549 following irradiation. Western blots showed increased accumulation of this protein at doses as low as 75 cGy after 15 min post irradiation. Maximal induction (11-fold over unirradiated cells) of PKC epsilon occurred at 150 cGy within 1 h after treatment by X-rays in A549 cells. The increased levels of PKC epsilon protein after X-rays does not require de novo protein or RNA synthesis, suggesting that this increase is post-translationally controlled. In contrast to A549 cells PKC epsilon levels in the large cell lung carcinoma cell line NCI H661 were not induced by radiation. In the small cell lung carcinoma cell line NCI N417, PKC epsilon was also not induced but a higher molecular weight PKC epsilon protein, suggestive of phosphorylation, appeared at 2 h after irradiation. The variation in induction or phosphorylation of PKC epsilon by ionising radiation in the cell lines tested in this study suggested that no clear correlation existed between intrinsic radiation sensitivity and PKC epsilon induction. To determine whether PKC epsilon does play a role in cell survival to irradiation, we used the protein kinase inhibitor staurosporin to decrease PKC activity and found that staurosporin sensitised cells to killing by ionising radiation. Pulsed field gel electrophoresis, however, indicated that DNA double-strand break repair was not decreased, suggesting that PKC epsilon is modifying the fidelity of rejoining and not the overall magnitude of repair. The regulation of PKC by ionising radiation will be discussed with respect to the biological consequences of gene induction by DNA damage agents.

    View details for Web of Science ID A1992JW71300014

    View details for PubMedID 1329908



    3-Amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) is a bioreductive anticancer drug which has a high selective toxicity to hypoxic cells. We have characterized the DNA and chromosome damage in wild-type Chinese hamster ovary (CHO) cells and mutant XR-1 cells after exposure to SR 4233 under hypoxia and compared it to the damage produced by ionizing radiation (gamma-rays). Using the technique of pulsed field gel electrophoresis, we found that the kinetics of rejoining of DNA double-strand breaks in CHO cells after treatment with SR 4233 was concentration dependent, varying from 95% (less than 50 microM) to 10% (200 microM) by 24 h. This contrasted with the dose-independent kinetics exhibited in cells after gamma-ray exposure. XR-1 cells were deficient in rejoining double-strand breaks produced by either SR 4233 or gamma-rays. XR-1 cells were 2-fold more sensitive than wild-type CHO cells to SR 4233 but were 10-fold more sensitive than CHO to gamma-rays. These results suggested that DNA double-strand breaks are involved in hypoxic cell killing by SR 4233, but the specific type of lesion produced is not identical with that causing cell killing by gamma-rays. To further investigate this, we measured chromosome breaks in CHO cells by premature chromosome condensation after equitoxic doses of SR 4233 under hypoxia and gamma-rays. SR 4233 produced lower initial but similar final (after 6 h of repair) numbers of chromosome breaks compared to gamma-rays at equitoxic doses. These results suggest that, at low doses, chromosome breaks can entirely account for hypoxic cell killing by SR 4233 and that chromosome breaks produced by SR 4233 are more damaging and/or more difficult to repair than those produced by gamma-rays.

    View details for Web of Science ID A1992JJ83700027

    View details for PubMedID 1643639



    The success of a predictive assay for radiotherapy relies on the use of one or more tumor cell traits that equate with tumor radioresistance or radiosensitivity. These traits can be divided into intrinsic (genetic) and extrinsic (epi-genetic) factors. Most probably, a tumor's response to radiotherapy will be influenced by both of these sets of traits. Radiobiological analysis of cultured cells derived from explanted tumors of head and neck patients has shown that in vitro survival of tumor cells is not the only factor affecting tumor radiocurability. Two possible reasons are the high degree of selection involved in growing the cells in vitro and the inability to assess the contribution of the cell-cell contact effect with cultured cells. A possible means of overcoming both of these problems would be an assessment of the radiosensitivity of the cell population immediately after removal from the tumor. Since a good correlation exists between intrinsic cellular radioresistance and DNA double-strand break repair (DSBR) as assayed by the Neutral Elution technique [21], we have investigated the feasibility of using asymmetric field inversion gel electrophoresis (AFIGE) in identifying resistant tumor cells in vitro. AFIGE has several advantages over neutral elution in that it is faster (approximately 60-80 samples can be run on the same agarose gel) and, most importantly, one can visualize DNA damage and repair by staining the DNA with ethidium bromide.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1992JN42500004

    View details for PubMedID 1410578


    View details for Web of Science ID A1992JE49200001

    View details for PubMedID 1386679



    The correlation between mean arterial blood pressure (MABP) and vascular perfusion in SCC-VII/St tumors in mice was compared following administration of three vasoactive drugs: flavone acetic acid (200 mg/kg), hydralazine (5 mg/kg), or nicotinamide (500, 750, and 1000 mg/kg). MABP was measured by the direct method in unanesthetized, unrestrained mice bearing a carotid catheter. Vascular perfusion of the tumor was measured using the 86RbCl extraction method. Body temperature was maintained at 36 degrees to 37 degrees C after drug administration when necessary. All three drugs reduced MABP from a control value of 125 +/- 2 (s.e.) mm Hg in mice without tumors. Flavone acetic acid at this dose had the least effect on blood pressure, with a minimum of 86% of control values at 10 to 20 min, and a return to control values by 1 hr. However, it produced a profound reduction in tumor perfusion that lasted more than 48 hr. Hydralazine and nicotinamide reduced blood pressure to minima between 55% and 69% of control values within 30 min, followed by a gradual return toward control values by about 8 hr. The reduction in tumor perfusion by hydralazine paralleled its effect on blood pressure. However, nicotinamide produced a transitory, although not statistically significant, increase in tumor perfusion at the highest dose given. These data demonstrate that tumor blood flow modification by drugs is not necessarily the result of changes in MABP, and blood pressure changes alone do not inevitably lead to changes in tumor perfusion.

    View details for Web of Science ID A1992GU93200011

    View details for PubMedID 1530755



    As further work on tumor oxygenation results in the development of agents capable of modulating hypoxic cell radiosensitivity, and chemotherapeutic agents capable of targeting hypoxic cells, knowledge of relative tumor oxygenation takes on greater importance. The pO2 Histograph was used to characterize the oxygen tension of a murine sarcoma (RIF1) and a murine carcinoma (SCCVII), each with different hypoxic fractions. These tumors were studied sequentially to assess the suitability of the Histograph both as a research tool and, ultimately, as a clinical monitor of tumor hypoxia.

    View details for Web of Science ID A1992BX33D00015

    View details for PubMedID 1288122



    The glutathione S-transferases (GST's) are cytosolic dimeric proteins that are composed of three family members, alpha, pi, and mu, and a fourth microsomal member. These four family members are primarily involved in cellular detoxification of xenobiotics and hydroperoxides. Recently, a strong correlation has been found between the overexpression of GST's and resistance to chemotherapeutic drugs. In comparison to chemotherapy, little is known about the role of GST's in the cellular response to ionizing radiation. To determine which GST's may be involved in this response, we have identified Chinese hamster ovary cell lines that possess different levels of alpha and pi GST isozyme activity. The survival of these cell lines to ionizing radiation was similar to that of wild-type Chinese hamster ovary-KI cells from which they were derived. Although differences in GST levels did not affect ionizing radiation sensitivity per se, we found that ionizing radiation decreased the amount of cytosolic pi GST without affecting alpha GST levels. Taken together, these data suggest that GST's are involved in the cellular response against oxidative stress generated by ionizing radiation.

    View details for Web of Science ID A1992HJ37700028

    View details for PubMedID 1544849


    View details for Web of Science ID A1992BX33D00018

    View details for PubMedID 1288125



    Cells exposed to hypoxia increase their synthesis of a specific set of proteins called oxygen regulated proteins. Recently, three of these proteins have been identified as hemoxygenase, Glucose Regulated Protein 78 kilodaltons and Glucose Regulated Protein 94 kilodaltons. In contrast, reoxygenation from hypoxic conditions increases the synthesis of the heat shock proteins. Although the molecular signals required for regulation of both sets of proteins by hypoxia and reoxygenation are still under investigation, it is known that their expression is regulated at the transcriptional level. This finding suggests that these stresses work either singularly or together to control the activation of nuclear transcription factors which bind distinct regulatory sequences in the promoter region of these genes. One possible nuclear transcription factor which could act as a transcriptional regulator for both hypoxia and reoxygenation gene transcription is the heat shock transcription factor. In this report, we focused on the kinetics of HSF activation by hypoxia in normal and tumor cell lines of murine and human origins. In cell culture, both the normal diploid cell line AG1522 and the tumor cell line JSQ-3 possess the same kinetics of HSF activation (binding to the heat shock element) by hypoxia, with maximal induction at or after 3 hr. We have also shown that the activation of HSF occurs in the SCCVII tumor in vivo without clamping, but not in SCCVII cells grown in monolayers. When SCCVII tumors are dissociated and allowed to reoxygenate in cell culture, HSF binding decreased in 5 hr, and was undetectable after 18 hr. Furthermore, one human tumor biopsy tested for the presence of hypoxia by both the pO2 histograph (Eppendorf, Germany) and HSF binding showed good agreement for both techniques. These results suggest that HSF binding may be a useful marker for monitoring the tumor hypoxia.

    View details for Web of Science ID A1992JD82800027

    View details for PubMedID 1618682



    One approach to understanding the mechanism of selective hypoxic cell killing by the benzotriazine-di-N-oxide, SR 4233, is to characterize cell lines that exhibit increased resistance to killing by this drug. The Chinese Hamster Ovary cell line BL-10 was originally isolated on the basis of its hypersensitivity to killing by bleomycin. It is 2.7-fold more resistant to hypoxic cell killing by SR 4233 than wild-type CHO on comparison of D0's. However, both BL-10 and CHO possess the same sensitivity to killing by SR 4233 under aerobic conditions. We have excluded the explanation that differential metabolism of SR 4233 is responsible for its increased survival as both BL-10 and CHO produce the two-electron product SR 4317 at the same rate (3 nmoles/hr/10(6) cells). Analysis of free radical production, DNA double-strand break induction, and glutathione (GSH) levels suggested that the resistance of BL-10 to killing by SR 4233 might result from increased intracellular radical scavenger pathways. Using buthionine sulfoximine (BSO) to decrease cellular GSH levels, we found a marked increase in the sensitivity of BL-10 cells to SR 4233 killing under hypoxia, but a much smaller increase in the sensitivity of CHO cells. Taken together, these data imply that the high GSH levels in BL-10 cells is responsible for its resistance to SR 4233 cytotoxicity.

    View details for Web of Science ID A1992HJ37700011

    View details for PubMedID 1544836



    No method of predicting the radiation sensitivity of individual human tumors is presently available, and recently published data show that other factors, in addition to the intrinsic radiosensitivity of the tumor cells, may play a role in the in vivo response of human tumors. Since these factors likely involve the tumor milieu (e.g., cell-cell contact and tumor hypoxia), an in situ assay of radiosensitivity is required. Although an analysis based on chromosome damage is the only suitable assay that would fit the requirements of sensitivity and speed of analysis, conventional examination of chromosome damage is impractical. By allowing the visualization of chromosomes in interphase cells, the technique of premature chromosome condensation (PCC) overcomes the need to culture the tumor cells in vitro, but the technical problem remains of counting a small excess number of breaks over the often large pretreatment chromosome number. We demonstrate here that the combination of fluorescence in situ hybridization (FISH) with PCC enormously simplifies the problem by focusing the analysis on a single chromosome. It also allows exchange aberrations to be scored easily. We demonstrate that the FISH technology may also be used to estimate radiation sensitivity from stable reciprocal translocations in metaphase identified by combining whole chromosome painting with a second color hybridization to the repeat sequences common to the centromeres. Since the frequency of stable translocations should correlate with initial chromosome damage, and since these translocations are not preferentially lost from the irradiated tumor cell population by cell death, an estimate of tumor cell killing following 1-5 dose fractions should be possible. Each of these two methods has its advantages, and a careful study of the two should establish which is superior for routine use to determine tumor radiosensitivity in situ.

    View details for Web of Science ID A1992JP78400012

    View details for PubMedID 1526866

  • Fluorescence in situ hybridization: an improved method of quantitating chromosome damage and repair. BJR supplement / BIR Brown, J. M., Evans, J. W. 1992; 24: 61-64

    View details for PubMedID 1290714



    SR 4233 (1,2,4-benzotriazine-3-amine 1,4-dioxide) will soon be entering Phase I clinical trials as a new bioreductive cytotoxic agent for the treatment of solid tumors in combination with fractionated radiotherapy. We have selected 3 from over 50 analogues of SR 4233 which showed particular promise as second generation bioreductive antitumor agents. These compounds, when compared to SR 4233, have higher hypoxic toxicity and comparable or higher oxic to hypoxic cytotoxicity ratios in vitro and similar animal toxicity. We have compared the effectiveness of these three compounds with SR 4233 in two tumor systems and have examined some pharmacokinetic properties. The results show that replacement of the amino group at the 3-position of SR 4233 with either a hydrogen or an N,N-dialkylaminoalkylamino group shortens the half-life of these compounds in the blood because of the combined effects of partition coefficients, basicity, and higher reactivity. SR 4754 and SR 4755, the N,N-dialkylaminoalkylamino derivatives, exhibited shorter plasma half-lives than SR 4233 but exhibited lower anti-tumor activity than SR 4233 based on equal mouse toxicity in a fractionated regimen. SR 4482, with the hydrogen substitution and very high electron affinity, possessed a very short blood half life yet retained similar anti-tumor activity as SR 4233.

    View details for Web of Science ID A1992HJ37700016

    View details for PubMedID 1544841



    We studied the frequency of translocations in peripheral blood lymphocytes of patients with Hodgkin's disease to determine the extent of chromosome changes induced by radiation or radiation and chemotherapy. Comparisons were made to patients with second cancers to determine if this population is more susceptible to the effects of treatment. Group one included six patients with newly diagnosed Hodgkin's disease who were treated with radiation only. Group two included Hodgkin's disease patients who were treated 12-24 years previously and have been continuously free of disease. Five of these patients were treated with radiation only and five patients received radiation and mechlorethaminehydrochloride, oncovin, procarbazine, prednisone (MOPP) chemotherapy for six cycles. Group three included three patients who developed a second cancer after successful treatment for Hodgkin's disease. Two of these patients had a sarcoma within the radiation field and one had breast cancer. Metaphase spreads were obtained from cultured lymphocytes and hybridized with a chromosome 4 specific probe. After fluorescein staining, approximately 1000 metaphases were scored per patient. In group one only one patient in six demonstrated translocations in chromosome 4 before treatment for a mean frequency of .0009. After treatment the frequency of translocations increased to a mean of .016 (p = .036) (range .006-.034). Group two patients treated with radiation only had a mean translocation frequency of .012 (range .004-.022) in comparison to the radiation/mechlorethaminehydrochloride, oncovin, procarbazine, prednisone chemotherapy treated patients who demonstrated a mean frequency of .016 (p = .425) (range .0009-.023). The third group of second cancer patients showed inconsistent translocation frequencies of .002, .020, and .035. Of these patients, the one who demonstrated the greatest frequency of translocations (.035) was treated with mechlorethaminehydrochloride, oncovin, procarbazine, prednisone/adriamycin, bleomycin, vinblastine, decadron) and radiation. Our data demonstrates a statistically significant increase in translocations detected after radiation. When compared to combined modality therapy a greater mean frequency of translocations is observed over radiation alone; however, this was not statistically significant. In the three patients who developed second cancers in our series we saw no consistent increase in translocation frequency compared to Hodgkin's disease patients who did not develop a second cancer.

    View details for Web of Science ID A1992JY08900021

    View details for PubMedID 1429098


    View details for Web of Science ID A1991JM20900072

    View details for PubMedID 1785829



    There is a growing interest in the exploitation of hypoxia in solid tumors for therapeutic gain by the use of hypoxic cytotoxins and other agents. Tumor hypoxia can be greatly increased in a number of animal tumor models with the vasoactive drug hydralazine (HDZ), and in some cases this potentiates the effect of drugs that are selectively toxic to hypoxic cells. Our interest was to determine if HDZ would also increase tumor hypoxia in tumors growing in previously irradiated normal tissue- a situation such as might be found in the clinic with regrowing solid tumors after radiotherapy. SCCVII tumors in untreated mice were compared with tumors growing in a previously irradiated tissue with respect to their level of hypoxia in response to HDZ. HDZ increased tumor hypoxia in the tumors from unirradiated mice as measured by 14C-misonidazole binding. However, HDZ had little or no effect on tumor hypoxia in tumors growing in previously irradiated sites. We also showed that the pre-HDZ extent of hypoxia was higher in tumors growing in a previously irradiated tissue. This may in part explain the lack of effect of HDZ in these tumors. The lack of response of the tumors growing in irradiated sites suggests a limitation on the use of HDZ in combination with specific hypoxic cytotoxins or other chemotherapeutic drugs in the treatment of recurrent solid tumors. The data also show that if such tumors have an elevated hypoxic fraction relative to their counterparts growing in untreated sites, these tumors might be intrinsically more resistant to conventional radiotherapy, but, on the other hand, might be sensitive to bioreductive drugs and more likely to be radiosensitized by a hypoxic cell sensitizer.

    View details for Web of Science ID A1991GR72700005

    View details for PubMedID 1938551



    As a means to understand the fundamental mechanisms of bleomycin cell killing, we previously isolated 19 bleomycin-sensitive mutants which represent at least six genetically distinct complementation groups (T.D. Stamato, B. Peters, P. Patil, N. Denko, R. Weinstein, and A. Giaccia. Cancer Res., 47: 1588-1592, 1987). One class of mutants represented by the cell line BL-10 displays only hypersensitivity to killing by bleomycin in both acute (16 h) and chronic treatments but no sensitivity to killing by other DNA-damaging agents. Complementation studies between this mutant and human fibroblasts suggested that the human gene which corrects the defect of BL-10 rested on human chromosome 6. It has been reported that the gene for human glutathione S-transferase (GST) alpha also resides on chromosome 6. Measurements of selenium-independent peroxidase (alpha-GST + glutathione peroxidase) activity in wild-type Chinese hamster ovary (CHO) cells, using cumene hydrogen peroxide as a substrate, gave a value of 112 nmol of glutathione oxidized/min/mg protein compared with 88.1 nmol of glutathione oxidized/min/mg protein for BL-10. Measurement of the selenium-dependent peroxidase activity, using H2O2 as a substrate, resulted in 65.9 nmol of reduced glutathione oxidized/min/mg protein in CHO and 81.5 nmol of reduced glutathione oxidized/min/mg protein for BL-10. In other words, BL-10 cells did not exhibit a difference in their ability to metabolize both substrates in contrast to CHO cells. This indicates that BL-10 possesses little alpha-GST activity. Transfection of BL-10 cells with a mammalian expression vector containing the alpha-GST gene increases the survival of BL-10 to bleomycin and does not increase the bleomycin resistance of two other bleomycin mutants which lie in different genetic complementation groups. These data strongly implicate a role for alpha-GST in the resistance of cells to bleomycin.

    View details for Web of Science ID A1991GA54300054

    View details for PubMedID 1714344


    View details for Web of Science ID A1991GA96900036

    View details for PubMedID 1677976



    Using pulsed-field gel electrophoresis, we have measured the ability of two bleomycin-sensitive mutants, XR-1 and BL-10, to repair DNA double-strands breaks (DSB). XR-1 was originally isolated by its hypersensitivity to killing with ionizing radiation, but we have also shown that it is sensitive to killing with bleomycin. In contrast, BL-10 was isolated by its extreme sensitivity to killing with bleomycin, and it is not cross-sensitive to other DNA breaking agents. A 1-h treatment of bleomycin induces a similar number of DNA double-strand breaks in XR-1, BL-10 and CHO cells. However, XR-1 is unable to repair bleomycin-induced DNA double-strand breaks, whereas BL-10 possesses the same kinetics of repair as parental CHO. These data lead us to conclude that at least two mechanisms of killing exist for bleomycin; one of them is DNA DSB-dependent, and the other seems to be DNA DSB-independent.

    View details for Web of Science ID A1991FT58100002

    View details for PubMedID 1710776



    The technique of fusing mitotic cells to interphase cells, thereby producing condensation of the chromosomes of the interphase cell (so-called 'premature chromosome condensation' or PCC), has allowed detection of the initial number of chromosome breaks and their repair following ionising radiation. However, the difficulty and tedium of scoring all the chromosome fragments, as well as the inability to readily detect exchange aberrations, has limited the use of PCC. We describe here the use of the recently developed technique of fluorescence in situ hybridisation with whole chromosome libraries to stain individual human chromosomes (also called 'chromosome painting') with the PCC's and show that this overcomes most of the limitations with the analysis of PCC's. First, by focusing on a single chromosome, scoring of breaks in the target chromosome is easy and rapid and greatly expands the radiation dose range over which the PCC technique can be used. Second, it allows the easy recognition of exchange type aberrations. A number of new applications of this technology, such as predicting the radiosensitivity of human tumours in situ, are feasible.

    View details for Web of Science ID A1991FH59600011

    View details for PubMedID 2021536



    Nicotinamide has been shown in our laboratory and those of other investigators to be an effective radiosensitizer of a variety of mouse tumours, while producing little or no radiosensitization of normal tissues. Its mechanism of action is different from classical electron-affinic compounds and appears to be the result of improved tumour oxygenation. In this study we have synthesized 29 analogues of nicotinamide and benzamide and characterized them for their tumour radiosensitization and acute toxicity in mice. The data show that a wide range of additions to the nicotinamide and benzamide ring produce tumour radiosensitization similar to that produced by equimolar doses of misonidazole, but that substitutions of the amide tend to reduce radiosensitization. Other structure-activity relationships are evident. Although some compounds produce similar tumour radiosensitization to nicotinamide at equimolar doses, and are comparably low in acute toxicity, none appears sufficiently superior to supplant nicotinamide itself as a candidate for clinical trials. Thus these data provide evidence that nicotinamide, because of the extensive experience with its use in man, is likely to be the best drug in the benzamide-nicotinamide series for development as a radiosensitizer of human tumours.

    View details for Web of Science ID A1991FB70000013

    View details for PubMedID 1672362



    In order to understand in more detail the mechanism underlying the preferential hypoxic cytotoxicity of the benzotriazine N-oxide SR 4233, we have compared the hypoxic cytotoxicity of this drug to the rates of hypoxic metabolism in both DNA double strand break repair-competent and repair-deficient cell cultures. Rodent SCCVII cells and repair deficient, radiation sensitive cells (rodent XR-1, V-3, and human AT5BI) were most sensitive to SR 4233 under hypoxia with a lethal dose needed to kill 50% of cells (LD50) of less than 5 microM. SR 4233 was less cytotoxic to human AG 1522 (LD50 = 18 microM), CHO 4364 (LD50 = 25 microM) and human HT 1080 cells (LD50 = 33 microM). The sensitivities to SR 4233 were found to be inversely proportional to the rates of SR 4233 metabolism in repair-competent cells (R2 = 0.9). However, XR-1 and V-3 cells were more sensitive to SR 4233 than predicted by the metabolism rate. Thus, the toxicity by SR 4233 towards hypoxic cells appears to result from two mechanisms; the rate of drug metabolism and the ability to repair DNA double strand breaks.

    View details for Web of Science ID A1991FC08200009

    View details for PubMedID 2003976



    The present study describes a new way in which tumors may be made more sensitive to fractionated irradiation without affecting the sensitivity of surrounding normal tissues. It involves exploiting the cycling or intermittent hypoxia that occurs in at least some solid tumors, but not in normal tissues, using a new drug SR 4233, a benzotriazine di-N-oxide, which is rapidly metabolized in hypoxic cells to a product that kills these cells. Using this approach with a rodent tumor in a fractionated x-ray treatment regimen similar to that used in human radiotherapy, the addition of SR 4233 produced a large enhancement of the radiation response of the tumor with no change in the sensitivity of normal mouse skin. Under identical circumstances, there was no effect of the hypoxic cell radiosensitizer SR 2508, showing that SR 4233 with intermittent hypoxia was superior to a protocol which sensitized the hypoxic cells to doses of 2.5 Gy per fraction.

    View details for Web of Science ID A1991EZ90000011

    View details for PubMedID 1995531



    The presence of hypoxic cells in solid tumors has long been considered a problem in cancer treatment, particularly for radiation therapy but also for treatment with some anticancer drugs. Three general strategies are being actively explored to overcome the problem: oxygenating the tumor, sensitizing the hypoxic cells to radiation (or chemotherapy), or killing the hypoxic cells (with a hypoxic cell cytotoxin). In the present study, we have examined the impact of each of these three strategies on a standard radiation therapy regimen (30 doses of 2 Gy), using either of two major assumptions: full reoxygenation or no reoxygenation of the tumor cells. We demonstrate that a hypoxic cell cytotoxin can produce a level of tumor cell killing higher (by several orders of magnitude) than that produced by full oxygenation of a tumor or by use of an optimum hypoxic cell radiosensitizer, provided the cytotoxin kills more than approximately 50% of the hypoxic cells each time it is given. The only assumption that makes a difference is whether reoxygenation occurs: In the worst case (ie, no reoxygenation), the hypoxic cell cytotoxin performs only as well as an optimum radiosensitizer. The analysis shows that hypoxic cells in tumors can be a major therapeutic advantage. Therefore, we recommend that research efforts be concentrated on development of superior hypoxic cell cytotoxins rather than improved hypoxic cell radiosensitizers and that, in parallel, emphasis be placed on development of agents to increase hypoxia.

    View details for Web of Science ID A1991EW64900008

    View details for PubMedID 1988703



    C.B-17 severe combined immunodeficient (scid) mice carry the scid mutation and are severely deficient in both T cell- and B cell-mediated immunity, apparently as a result of defective V(D)J joining of the immunoglobulin and T-cell receptor gene elements. In the present studies, we have defined the tissue, cellular, and molecular basis of another characteristic of these mice: their hypersensitivity to ionizing radiation. Bone marrow stem cells, intestinal crypt cells, and epithelial skin cells from scid mice are 2- to 3-fold more sensitive when irradiated in situ than are congenic BALB/c or C.B-17 controls. Two independently isolated embryo fibroblastic scid mouse cell lines display similar hypersensitivities to gamma-rays. In addition, these cell lines are sensitive to cell killing by bleomycin, which also produces DNA strand breaks, but not by the DNA crosslinking agent mitomycin C or UV irradiation. Measurement of the rejoining of gamma-ray-induced DNA double-strand breaks by pulsed-field gel electrophoresis indicates that these animals are defective in this repair system. This suggests that the gamma-ray sensitivity of the scid mouse fibroblasts could be the result of reduced repair of DNA double-strand breaks. Therefore, a common factor may participate in both the repair of DNA double-strand breaks as well as V(D)J rejoining during lymphocyte development. This murine autosomal recessive mutation should prove extremely useful in fundamental studies of radiation-induced DNA damage and repair.

    View details for Web of Science ID A1991EY61700064

    View details for PubMedID 1996340



    Mammalian cells surviving exposure to the bioreductive, cytotoxic agent SR 4233 under hypoxic conditions are sensitized to X-irradiation under aerobic conditions (and in the absence of drug). Fits of both the single-hit, multi-target and linear-quadratic expressions to survival data, as well as direct measurement of surviving fractions after a dose of 2 Gy, indicate that the aerobic radiosensitization produced by SR 4233 can increase both the initial and final 'slopes' of the X-ray survival curve. The amount of radiosensitization produced, and whether the modification is principally in the slope or shoulder region of the survival curve, varies from cell line to cell line. Rodent cells are radiosensitized equally whether the drug treatment is given immediately before or after, the irradiation, but human cells are only sensitized for SR 4233 exposure administered before irradiation. Using rodent CHO cells, time-course experiments for SR 4233 and X-rays given in sequence, in which an interval of up to 2 h was interposed between the treatments, reveal different kinetics for the loss of radiosensitization depending on whether the hypoxic drug exposure was given before or after the aerobic irradiation. When SR 4233 treatment is given pre-irradiation, the radiosensitization effect persists for at least 2 h, but it does not when drug is given after irradiation. Taken together, the finding of a difference between rodent and human cells with respect to post-irradiation sensitization by SR 4233, and the differing time-course kinetics for this effect as a function of how the drug and radiation are sequenced, suggest that while SR 4233 behaves in a radiomimetic manner in most respects, there may be subtle differences in the nature of the lesions produced by the drug, the important cellular targets for this damage, and/or the cell's management of the damage.

    View details for Web of Science ID A1991EV61500011

    View details for PubMedID 1671059



    The benzotriazine SR 4233, in addition to preferential killing of hypoxic cells both in vitro and in vivo, also radiosensitizes aerobic cells in vitro if the cells are exposed to the drug under hypoxic conditions, either before or after irradiation. We have attempted to exploit this aerobic radiosensitization in vivo, by giving SR 4233 with the hypoxia inducing agent, hydralazine, after each radiation dose in a 8 x 2.5 Gy fractionated regime. The results show greater than additive cytotoxicity using both cell survival and regrowth delay as the endpoints of radiation response, but no radiosensitization in parallel groups treated with the hypoxic cell radiosensitizer SR 2508. The data are, therefore, consistent with radiosensitization of the tumor aerobic cells by the SR 4233 treatments. Significantly, the effect occurred with equal magnitude with or without hydralazine. Further, there was no radiosensitization to radiation induced leg contraction in the thighs of mice, a late responding normal tissue endpoint. The results, therefore, demonstrate a selective radiosensitization of tumors to a multifraction regime and suggest that SR 4233, or a close analog, may be useful in radiation therapy.

    View details for Web of Science ID A1991FA48000026

    View details for PubMedID 2020764



    The new bioreductive drug 3-amino-1,2,4-benzotriazine 1,4-dioxide (SR 4233) shows a high selective cytotoxicity for hypoxic cells, both in vitro and in tumors in vivo. In the present experiments, we have tested the hypothesis that this selective killing of hypoxic cells might be exploited by taking advantage of the fluctuating hypoxia in tumors by use of a multidose regimen of SR 4233 with multiple small doses of X-rays. We have tested four different transplantable mouse tumors using a standard fractionated protocol of 8 x 2.5 Gy in 4 days, using a well tolerated dose of SR 4233 given with each radiation dose. All of the tumors showed a substantial enhancement of cell killing by SR 4233 over that produced by radiation alone with dose-modifying factors of 1.5 to 3.0 for the different tumors. In all four tumors, the enhancement of cell killing was greater than that produced by a large dose of the hypoxic cell sensitizer SR 2508 given before each irradiation, thereby demonstrating the superiority of the approach of using a hypoxic cytotoxic agent rather than a radiosensitizer in fractionated radiation protocols. The data suggest that SR 4233 has considerable promise as an adjunct to standard radiotherapy.

    View details for Web of Science ID A1990EM16500002

    View details for PubMedID 2253217



    In situ hybridization of hamster/human hybrids with biotinylated human genomic DNA has revealed that human chromosomal DNA can integrate into the hamster genome and is not always cytologically detectable. This finding helps to explain why discordancy can arise in gene mapping by failing to recognize small pieces of foreign DNA in the rodent genome. Fluorescent in situ hybridization allows one to locate these fragments in rodent chromosomes visually and possibly to identify their chromosome of origin.

    View details for Web of Science ID A1990ED57700013

    View details for PubMedID 2078516



    We have continued our assessment of the ability of nicotinamide to sensitize tumours to radiation. Using the SCCVII carcinoma and estimating tumour response by either a regrowth delay or an in vivo/in vitro survival assay, it was found that a large single dose of nicotinamide (1000 mg/kg) increased radiation-induced tumour damage. This effect was observed regardless of whether the tumour was grown intramuscularly, subcutaneously or intradermally, or whether the nicotinamide was administered intraperitoneally, intravenously or orally. The enhancement was maximal when the drug was given between 30 min and 2 h prior to irradiation and resulted in enhancement ratios ranging from 1.1 to 1.7. Although the radiation response of tumours was dependent on tumour size, the radiation enhancement produced by nicotinamide was not. Utilizing the technique of labelling tumour cells with the fluorescent stain Hoechst 33342, we were able to identify the presence of both chronic and acutely hypoxic cells in this tumour model and obtained results suggesting that apart from reducing chronic hypoxia, nicotinamide may also have the ability to decrease the level of radioresistant acute hypoxia.

    View details for Web of Science ID A1990DJ01300006

    View details for PubMedID 2141694


    View details for Web of Science ID A1990CQ99300001

    View details for PubMedID 2406450



    During the past 3 years, our laboratory has identified and characterized the drug SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) as the lead compound in a series of benzotriazine di-N-oxides that are both potent and selective killers of hypoxic cells in vitro and in rodent tumors in vivo. Recently, we have identified a novel property of SR 4233: the ability of a pre- or post-irradiation drug treatment under hypoxic conditions to radiosensitize aerobic cells in culture. For the mouse cell lines RIF-1 and SCC VII in vitro, this radiosensitization took the form of a steepening of the slope of the acute dose radiation survival curve, although there was also reduced survival in the "shoulder region" of the curve. For both cell lines, the sensitization occurred whether the hypoxic drug exposure was given immediately before or after the irradiation under aerobic conditions. To determine whether radiosensitization could be demonstrated for RIF-1 and SCC VII mouse tumors in vivo, tumor-bearing animals were exposed to 4 daily dose fractions of 5 Gy of X rays either alone, or followed immediately by injections of SR 4233 and the vasoactive agent hydralazine, which increases tumor hypoxia and therefore can potentiate the effect of such hypoxiaspecific drugs. Although treatment with the SR 4233/hydralazine combination after each dose fraction reduced tumor cell survival to between 10(-5) and 10(-6), near the limits of resolution of the clonogenic survival assay, the effect appeared to be strictly additive, suggesting that with this fractionated protocol, aerobic radiosensitization could not be detected. This is likely to be a consequence of the exquisite direct cytotoxicity of the SR 4233 and hydralazine combination toward the hypoxic cells in tumors.

    View details for Web of Science ID A1990CK39500017

    View details for PubMedID 2298615


    View details for Web of Science ID A1989CB49100042

    View details for PubMedID 2573680



    Flavone acetic acid (FAA, NSC 347512) is a new anticancer drug currently undergoing clinical investigation. Although the precise mechanism for its broad spectrum of activity against transplanted murine solid tumors is unknown, it has been reported that FAA reduces tumor blood flow and produces hemorrhagic necrosis. We have confirmed this finding with the murine transplanted carcinoma SCCVII: 200 mg/kg FAA reduced tumor blood flow to 20-30% of normal for 1-2 days as determined by rubidium 86 extraction. In an attempt to exploit the tumor hypoxia produced by FAA, we have combined it with the novel bioreductive drug SR 4233, a benzotriazine dioxide with high selective toxicity for hypoxic cells. Marked enhancement of the antitumor effect of FAA (200 mg/kg) was observed when it was combined with SR 4233 (0.1 and 0.2 mmol/kg). This was seen using tumor cell survival, regrowth delay, and histological endpoints, with the best results obtained when the two agents were injected simultaneously. These data suggest that targeting bioreductive cytotoxic agents to tumors by producing tumor hypoxia may be a valid way of increasing the tumor cell killing of these agents.

    View details for Web of Science ID A1989AU13800031

    View details for PubMedID 2790784



    Nitroaromatic radiosensitizers are effective chemosensitizers in vitro and in vivo. We have used EMT6 tumour cells grown as multicellular spheroids to try and further understand the role that hypoxia plays in this process. Our results show that the cell killing produced in whole spheroids following a 1-h exposure to melphalan (L-PAM) was enhanced by a 3-h pre-exposure to 5 mM misonidazole (MISO), an enhancement ratio of 1.3-1.7 being obtained. Sequentially disaggregating spheroids we also found that both L-PAM toxicity and MISO chemosensitization were relatively constant as a function of depth within the spheroid. The binding of 14C-MISO to spheroid cells, measured by scintillation counting of disaggregated cells and by autoradiography analysis of sectioned spheroids, demonstrated that binding increased with depth. However, cells in the outer layers of the spheroid bound more 14C-MISO than expected with fully aerobic cells, while in the innermost viable cells the binding was less than that measured in cells which were fully radiobiologically hypoxic. This suggests that the majority of viable spheroid cells were at oxygen tensions intermediate between those found in either fully aerobic or radiobiologically hypoxic cells, yet their levels of oxygenation were sufficiently low for MISO chemosensitization to occur.

    View details for Web of Science ID A1989AB09800012

    View details for PubMedID 2748937



    SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is a bioreductive agent that selectively kills and radiosensitizes hypoxic mammalian cells in vitro and murine tumors in vivo. In an attempt to better understand the mechanism of action of the drug, and to determine whether a superior analog may exist, 15 benzotriazine-di-N-oxide analogs of SR 4233 have been evaluated to date for the following properties: hypoxic and aerobic toxicity toward CHO cells in vitro, drug-induced stimulation of oxygen consumption by incubation with respiration-inhibited cells, and acute LD50 evaluated in BALB/c mice. We noted several correlations between these biological properties of the drugs and some of their physicochemical characteristics. Both the hypoxic cytotoxicity and stimulation of oxygen consumption by respiration-inhibited cells were positively correlated with E1/2, the polarographic half-wave reduction potential, and a measure of electron affinity. The air-to-nitrogen differential cytotoxicity reached a maximum (corresponding to SR 4233) and then declined with increasing E1/2. The acute LD50 of each analog in mice decreased with increasing E1/2. One new compound, SR 4482, was found to be more toxic to hypoxic cells in vitro, but less toxic to mice, than SR 4233. It is similar in structure to SR 4233, but lacks any substituent in the 3-position of the triazine ring. This promising drug may represent a member of a new subseries of 1,2,4-benzotriazines with different structure-activity relationships.

    View details for Web of Science ID A1989U113200019

    View details for PubMedID 2703405



    Nicotinamide has been shown to sensitize tumors to radiation in preference to normal tissues. We have extended our studies to examine the mechanism responsible for this radiosensitization, using the EMT6 tumor model. Our results confirm that nicotinamide (1000 mg/kg) significantly enhances the radiation damage in this tumor when given as a single intraperitoneal injection 90 min before irradiation. The data also show that nicotinamide does not directly sensitize hypoxic cells to radiation either in vitro or in vivo. Excising tumors immediately after irradiation and exposing them to nicotinamide (7 mM) for 24 h similarly failed to increase the radiation damage, implying that nicotinamide does not inhibit the repair of radiation-induced potentially lethal damage. Nicotinamide did, however, produce a decrease in the binding of [14C]-misonidazole in tumors, consistent with a reduction in the degree of tumor hypoxia. There was also an increase in mean tumor cell fluorescence of Hoechst 33342 in nicotinamide-treated mice compared to that of controls, suggesting that the increase in tumor oxygenation was probably a consequence of an increase in tumor blood perfusion.

    View details for Web of Science ID A1989U220900011

    View details for PubMedID 2523079



    The second generation hypoxic cell radiosensitizers SR 2508 (etanidazole) and Ro 03-8799 (pimonidazole) are now undergoing Phase III clinical trials and are predicted from in vitro, animal, and clinical studies to be equivalent to an increase in the tolerable dose of Miso of 5-6 fold. Nonetheless, this will only produce an SER of the hypoxic cells of approximately 1.5 when given with each dose of a 24 fraction course of radiotherapy. There is ample opportunity, therefore, for a sufficiently improved third generation radiosensitizer to warrant the considerable time, effort, and money that would be involved for its clinical testing. Many groups are now involved in the synthesis and testing of drugs with the goal of producing this third generation sensitizer. The purpose of the present review is to summarize what we have learned from the many studies of new radiosensitizers and attempt to draw conclusions about the most fruitful directions for further work. One of the conclusions is that in vitro testing of radiosensitizers, essential though it is as a first screen, can lead to many false predictions. Fortunately, these in vitro systems tend to "overpredict" (i.e., produce false positives rather than false negatives) for in vivo activity. There is only one class of drugs, so far, (those that increase tumor oxygenation) which radiosensitize tumors in vivo, but not hypoxic cells in vitro. Another major conclusion is that the physico-chemical characteristics of drugs that have been shown to be important in enhancing radiosensitization or reducing toxicity (electron affinity, pKa, partition coefficient, DNA binding), have not been systematically optimized in any one compound. Systematic rational drug design is needed to achieve this. The search for new hypoxic cell radiosensitizers must not detract from the fact that a sensitizer of aerobic cells to low radiation doses is needed. A major challenge in achieving a useful drug for radiotherapy is the desirability for such a drug to be tumor specific. We present data which show that tumor hypoxia might be exploited to achieve this goal.

    View details for Web of Science ID A1989U113200021

    View details for PubMedID 2649468



    SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is a bioreductive agent which exhibits highly selective killing of hypoxic cells in a variety of mammalian cell lines in vitro and in murine tumors in vivo. The selective toxicity of the drug results from its one-electron reduction under hypoxic conditions to form a free radical intermediate capable of damaging DNA, through the formation of strand breaks. Using the neutral filter elution assay, SR 4233 was found to be more efficient at producing DNA double strand breaks in Chinese hamster ovary (CHO) cells than an equitoxic dose of gamma-rays. Drug and radiation sequencing experiments were also performed, with both cell survival and DNA strand break rejoining used as endpoints. As a result of these studies, we now describe two additional properties of SR 4233: (a) radiosensitization of aerobic cells in culture produced by hypoxic incubation with drug either before or after irradiation, and (b) the inhibition of subsequent rejoining of radiation-induced DNA double strand breaks after hypoxic pretreatment with drug. The magnitude of the radiosensitization produced did not vary for drug treatments which, when given alone, reduced cell survival over a range from 30% to 2%. The extent of DNA repair inhibition increased with increasing severity of the SR 4233 pretreatment, but was quite small for non-lethal drug exposures.

    View details for Web of Science ID A1989U113200017

    View details for PubMedID 2703404



    SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide) is presently undergoing investigation as an antitumor agent because of its high selective toxicity for hypoxic cells in vitro and in vivo. It has been found to be 15 to 200 times more toxic to hypoxic rodent and human cell lines than their normoxic counterparts. We investigated the toxicity of SR 4233 in primary cultures of hepatocytes under various oxygen tensions, ranging from 1% to 20% oxygen. The 50% lethal dose of SR 4233 was found to be 50 times lower in hepatocyte monolayers at 1% O2 versus 20% O2. Even at 4% O2, a concentration that prevails in the pericentral area of the liver under conditions of normal blood flow, SR 4233 was an order of magnitude more toxic than at 20% O2. All samples were analyzed for metabolites, and metabolism was found to be dependent on both the SR 4233 concentration and the oxygen tension. Formation of the major metabolite SR 4317 occurred to the greatest extent at the lowest oxygen concentration and the highest SR 4233 concentration. Very little metabolism occurred at 10 to 20% O2, which is in agreement with data in Chinese hamster ovary cells under aerobic conditions.

    View details for Web of Science ID A1989T082400029

    View details for PubMedID 2912562



    The metabolism of SR 4233 (3-amino-1,2,4-bentotriazine-1,4-dioxide), recently reported as highly toxic to hypoxic cells in vitro, was studied by using suspensions of Chinese hamster ovary cells. The rates of formation of two known reduction products, the 1-oxide and the unoxygenated 3-aminobenzotriazine, were measured in aerobic and hypoxic cell suspensions for drug treatments producing both hypoxic and aerobic cytotoxicity. Formation of the 1-oxide and a small amount of the 3-aminobenzotriazine occurred preferentially in hypoxic suspensions. These metabolites were relatively nontoxic to either aerobic or hypoxic cells, implying another mechanism of toxicity. The activation of SR 4233 by single electron transfer, hypothetically forming a toxic drug radical, was explored. Aerobic stimulation of oxygen consumption in respiration-inhibited cells and malondialdehyde release from aerobic cells in the presence of SR 4233 indicated the formation of active oxygen species during drug activation. Increased malondialdehyde release in hypoxic cells and its attenuation by the hydrogen donor, dimethylthiourea, implied the presence of an oxidizing radical. Unlike the nitroimidazole, misonidazole, hypoxic metabolism of SR 4233 did not deplete intracellular glutathione or result in increased binding of drug metabolites to cellular macromolecules. These results are consistent with macromolecular damage caused by an oxygen sensitive, nonbinding, drug-free radical intermediate with oxidizing properties as the mechanism of selective hypoxic toxicity of SR 4233.

    View details for Web of Science ID A1988Q735300008

    View details for PubMedID 3167847



    Nicotinamide has been shown to selectively enhance the radiation damage of tumors in preference to normal tissues. Our present study was an investigation into the mechanism responsible for this effect in the SCCVII/St tumor model grown on the backs of C3H/km mice. A large single injection of nicotinamide (1000 mg/kg), given intraperitoneally 60 minutes before whole body irradiation, significantly enhanced the radiation response of SCCVII tumors as measured by an in vivo/in vitro excision assay performed 24 hr following irradiation. It also gave rise to an almost 4-fold reduction in the binding of 14C-misonidazole, injected 1 hr after the nicotinamide and measured by scintillation counting of excised tumor material 24 hr later. This suggested that nicotinamide was decreasing the degree of tumor hypoxia. Attempts were made to correlate these results with nicotinamide-induced changes in tumor blood flow using the techniques of 133Xe clearance, 86RbCl extraction and Hoechst 33342 fluorescent labelling. Nicotinamide produced between a 30-40% increase in mean tumor cell fluorescence of Hoechst 33342, which was consistent with an increase in tumor blood flow. A similar response was obtained using the uptake of 86RbCl as the end point. However, no statistically significant difference was seen between the tumor blood flow of control and nicotinamide treated mice using the 133Xe clearance procedure. These results are discussed with respect to their clinical implications.

    View details for Web of Science ID A1988Q175500024

    View details for PubMedID 2971029



    SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is the lead compound in a series of benzotriazine di-N-oxides which exhibit high selective killing of hypoxic mammalian cells in vitro. Drug concentrations to produce equivalent levels of cell killing of SCC VII murine carcinoma cells under hypoxia were nearly 200-fold lower than under aerobic conditions. Following a one hour hypoxic incubation with drug, 20 microM SR 4233 killed 99.9% of SCC VII cells. The hypoxia-specific cytotoxicity of SR 4233 is due to bioreductive metabolism. For in vivo studies, pharmacokinetic measurements showed that drug concentrations well in excess of 20 microM were achievable in SCC VII tumors in mice for approximately one hour after a single injection of SR 4233. Under these conditions, cell killing was considerably enhanced in SCC VII tumors when SR 4233 was combined with a single X-ray dose of 20 Gy. The enhancement was seen whether SR 4233 was given for up to 2 h before or for up to an hour after radiation, and was comparable to the enhanced cell killing achievable with a single large dose of the radiosensitizer misonidazole. While this finding is consistent with the selective killing of at least some subset of hypoxic tumor cells by SR 4233, other interactions between the drug and radiation damage may contribute to the overall effect observed.

    View details for Web of Science ID A1988P605500006

    View details for PubMedID 3175048

  • CHEMICAL MODIFICATION OF RADIATION AND CHEMOTHERAPY AMERICAN JOURNAL OF CLINICAL ONCOLOGY-CANCER CLINICAL TRIALS Brown, J. M., Hall, E. J., Hirst, D. G., Kinsella, T. J., Kligerman, M. M., Mitchell, J. B., TRAVIS, E. J., Valeriote, F. 1988; 11 (3): 288-303

    View details for Web of Science ID A1988N875600006

    View details for PubMedID 3289362



    The mechanism of radiosensitization by diazenedicarboxylic acid bis(N),N-piperidide (SR 4077), a less toxic analog of diamide, was studied using Chinese hamster ovary cells. SR 4077 gave an average SER of 1.58 for postirradiation incubations of 0.5, 1.0, or 2.0 h. Intracellular GSH and protein thiols decreased rapidly following drug addition and GSSG increased. The GSH/GSSG ratio shifted to 1/1.6 after SR 4077 addition but returned to greater than 10/1 between 0.5 and 1.0 h. After 4 h, total intracellular GSH was only 58% of pretreatment level and extracellular GSSG increased. Protein thiols decreased to 18% of pretreatment values, recovered most rapidly between 0.5 and 1.0 h, and reached 87% of pretreatment level after 4 h. A decrease in DNA single-strand break repair as measured by alkaline filter elution rate over 0.5 h was seen, and the initial rate of repair was slower than in cells not treated with SR 4077. DNA double-strand break repair as measured by neutral filter elution rate was delayed during the first hour after irradiation when cells were treated with SR 4077. The times for maximum radiosensitization, GSH and protein thiol oxidation and recovery, and DNA strand break repair kinetics were closely linked. We propose that a protein thiol(s) required in repair processes was reversibly oxidized during SR 4077 treatment.

    View details for Web of Science ID A1988L915300014

    View details for PubMedID 3340738


    View details for Web of Science ID A1988R169200045

    View details for PubMedID 3200890



    Stable monolayers of contact-inhibited C3H 10T1/2 cells were used in multifraction radiation experiments to measure the oxygen enhancement ratio (OER) at low doses/fraction under conditions where cell cycle effects (repopulation, redistribution) were minimal. Consistent with there being a dose-dependent reduction in the OER at low doses, an extremely low OER of 1.34 was measured after 20 fractions of 1.7 Gy every 12 h. The sparing effects of fractionating radiation doses were not apparent for cells irradiated under hypoxic conditions (i.e., multifraction survivals were lower than acute single-dose values) until doses exceeding 15 Gy were reached. This result suggested a deficiency in the recovery from sublethal and/or potentially lethal damage might exist after hypoxic irradiations, thereby reducing the OER. The capacity to repair potentially lethal damage was found to be nearly the same after hypoxic as compared to aerobic irradiations. However, there was an apparent absence of sublethal damage repair by 10T1/2 cells between two hypoxic irradiations which could be a major contributing factor to the extremely low OER value measured in this multifraction schedule.

    View details for Web of Science ID A1987K476800013

    View details for PubMedID 3659293



    The objective of this study was to characterize the extent of and mechanisms involved in radiosensitization by 2-nitroimidazoles in multifraction schedules using low doses per fraction. For this purpose, contact-inhibited monolayers of C3H 10T1/2 cells were given 1.7 Gy every 12 h and plated 12 h after the last dose received to allow full repair of potentially lethal damage (PLD). Severe hypoxia was obtained by a 1-h gassing procedure at room temperature immediately before each irradiation. No toxicity occurred as a consequence of multiple exposures to 5 mM misonidazole (MISO) or SR 2508 (2508) during the deoxygenation procedure. Experimental conditions during the pregassing and irradiation (presence of drug and gas mixture) were appropriately manipulated to test for the different mechanisms of radiosensitization demonstrated by nitroimidazoles. A very low oxygen enhancement ratio (OER) results under these conditions (1.34). Exposure to 5 mM MISO or 2508 during the deoxygenation and irradiation of hypoxic cells resulted in greater radiosensitization than could be accounted for by oxygen-mimetic sensitization alone (MISO and 2508 enhancement ratios were greater than the OER). Pregassing cells with N2 in the presence of 5 mM drug sensitized cells which were subsequently irradiated under aerobic conditions (drug free), indicating the occurrence of the "preincubation effect" (which does not require hypoxia or the drug's presence during the irradiation). Thus, for the hypoxic irradiations, the preincubation effect could account for the greater sensitization by nitroimidazoles than by oxygen. The presence of 5 mM drug only during the irradiation of aerobic cells produced radiosensitization in both multifraction and single-dose experiments with delayed plating. This sensitization has been previously shown to involve reduced PLD repair. Finally, maximum radiosensitization occurred in the multifraction schedule when a transient period of hypoxia with drug preceded an aerobic irradiation with drug present, thus combining the benefits of both the preincubation effect and PLD repair inhibition. This work demonstrates the possibility that effects other than oxygen-mimetic radiosensitization could be largely responsible for the sensitization seen in multifraction schedules, particularly when the OER is already low and only transient periods of hypoxia occur.

    View details for Web of Science ID A1987K476800014

    View details for PubMedID 3659294



    In this report we describe various aspects of tumor and normal tissue radiosensitization by nicotinamide. The LD50 for a single injection of nicotinamide in C3H mice was found to be 2050 mg/kg. When a large nonlethal dose (1000 mg/kg) was injected into tumor-bearing mice, peak plasma and tumor levels were reached 30-60 min after injection and decayed with a half-life of about 3 h. This dose of nicotinamide enhanced radiation-induced cell killing in three different tumor models (EMT6, Lewis Lung, and RIF-1) when injected at least 1 h before irradiation and produced enhancement ratios (ERs) of between 1.2 and 1.7. The ER in the EMT6 tumor was dependent on the dose of nicotinamide injected, but even at doses as low as 25% of the LD50 value an ER greater than 1.5 could still be observed. In two normal tissue assays (jejunum crypt cell survival and mean skin reaction) ERs of less than 1.2 were obtained. These results, and the fact that high levels can be tolerated in humans, suggest that nicotinamide, or a structurally related compound, could be a likely candidate for development in clinical trials.

    View details for Web of Science ID A1987G717800013

    View details for PubMedID 2951765



    Inhibition of repair of X-ray-induced potentially lethal damage (PLD) could enhance the curability of radioresistant tumours. We have studied the effect of inhibitors of the enzyme poly (ADP-ribose) polymerase on X-ray PLD repair. Four classes of inhibitors are known: aromatic amides (e.g., 3-aminobenzamide), thymidine, nicotinamides and methyl xanthines (e.g., caffeine). Plateau-phase Chinese hamster ovary (HA-1) cultures were exposed to 10mM concentrations of thymidine, nicotinamide, 3-aminobenzamide (3-ABA) and caffeine prior to irradiation to 12 Gy in air, and then incubated with drug at 37 degrees C for varying times (0-6 h) prior to subculture. Irradiated cells without drug exhibited a 5-6 fold increase in survival over the 6 h period compared to cultures plated immediately after irradiation. Although none of the compounds proved cytotoxic to unirradiated controls over the 6.5 h exposure, all of the compounds except thymidine reduced the capacity of the cells to repair PLD. The order of the inhibitory effect was caffeine greater than 3-ABA greater than nicotinamide, and the inhibition was concentration dependent for nicotinamide and 3-ABA. We also studied the effect of 3-ABA on the radiation response of exponentially growing cells. 5mM 3-ABA for 2 h post-irradiation resulted in a dose-multiplicative sensitization reducing the D0 from 0.88 Gy to 0.69 Gy, indicating an involvement of poly (ADP-ribose) polymerase in the radiosensitivity of exponentially growing as well as plateau-phase cells.

    View details for Web of Science ID A1984SM64200006

    View details for PubMedCentralID PMC2149166


    View details for Web of Science ID A1982NH16600008

    View details for PubMedID 7199747


    View details for Web of Science ID A1981MD98900001

    View details for PubMedID 6457018



    The halogenated pyrimidine analogue 5-bromo-2-deoxycytidine (BCdR) was infused into BALB/C mice bearing EMT-6 tumors via either the intravenous or intra-arterial route. Hepatic dehalogenation of the drug was blocked by 5-diazouracil (DAZU) in order to ascertain its relative importance in the degradation of intravenously administered analogues. Increased radiosensitization was noted with higher intravenous pyrimidine concentrations, but DAZU blockage of dehalogenation had little effect. These studies show that following intravenous infusion, enough BCdR apparently bypasses the hepatic vessels to permit tumor radiosensitization despite dilution of the drug by the systemic circulation.

    View details for Web of Science ID A1977DS55200047

    View details for PubMedID 887782

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