Tumor-associated mutations in O6 -methylguanine DNA-methyltransferase (MGMT) reduce DNA repair functionality.

MGMT is the primary vehicle for cellular removal of alkyl lesions from the O-6 position of guanine and the O-4 position of thymine. While key to the maintenance of genomic integrity, MGMT also removes damage induced by alkylating chemotherapies, inhibiting the efficacy of cancer treatment. Germline variants of human MGMT are well-characterized, but somatic variants found in tumors were, prior to this work, uncharacterized. We found that MGMT G132R, from a human esophageal tumor, and MGMT G156C, from a human colorectal cancer cell line, are unable to rescue methyltransferase-deficient Escherichia coli as well as wild type (WT) human MGMT after treatment with a methylating agent. Using pre-steady state kinetics, we biochemically characterized these variants as having a reduced rate constant. G132R binds DNA containing an O6 -methylguanine lesion half as tightly as WT MGMT, while G156C has a 40-fold decrease in binding affinity for the same damaged DNA versus WT. Mammalian cells expressing either G132R or G156C are more sensitive to methylating agents than mammalian cells expressing WT MGMT. G132R is slightly resistant to O6 -benzylguanine, an inhibitor of MGMT in clinical trials, while G156C is almost completely resistant to this inhibitor. The impared functionality of expressed variants G132R and G156C suggests that the presence of somatic variants of MGMT in a tumor could impact chemotherapeutic outcomes.

Hypoxia and radiation therapy: past history, ongoing research, and future promise.

Tumor hypoxia influences the outcome of treatment with radiotherapy, chemotherapy and even surgery, not only for the treatment of large bulky tumors with extensive necrosis, but also in the treatment of very small primary tumors and recurrences, micrometastases, and surgical margins with microscopic tumor involvement. Because hypoxic tumor cells are resistant to radiation and to many anticancer drugs, many approaches to circumventing the therapeutic resistance induced by hypoxia have been examined in laboratory studies and clinical trials. In this review, these approaches and the results of past laboratory and clinical studies are described and the limitations of the past agents and their testing are discussed. We describe the importance of new technologies for measuring hypoxia in human tumors, which allow assessment of pretreatment tumor oxygen levels and changes in hypoxia over the course of prolonged treatment regimens. These offer the possibility of improving the design of clinical trials and the selection of patients who will benefit from hypoxia-directed therapies, as well as the possibility of facilitating the development of better agents and regimens for use in hypoxia-directed therapy. We also discuss how the improved understanding of the abnormal vascular beds in solid tumors and of the effects of hypoxia and related microenvironmental insults, resulting from recent and ongoing research, offers the potential for finding new therapeutic targets, that may lead to the development of new agents and novel therapeutic approaches for selectively targeting cells in the adverse microenvironments within solid tumors.

Decreased expression of the DNA mismatch repair gene Mlh1 under hypoxic stress in mammalian cells.

The hypoxic tumor microenvironment has been shown to contribute to genetic instability. As one possible mechanism for this effect, we report that expression of the DNA mismatch repair (MMR) gene Mlh1 is specifically reduced in mammalian cells under hypoxia, whereas expression of other MMR genes, including Msh2, Msh6, and Pms2, is not altered at the mRNA level. However, levels of the PMS2 protein are reduced, consistent with destabilization of PMS2 in the absence of its heterodimer partner, MLH1. The hypoxia-induced reduction in Mlh1 mRNA was prevented by the histone deacetylase inhibitor trichostatin A, suggesting that hypoxia causes decreased Mlh1 transcription via histone deacetylation. In addition, treatment of cells with the iron chelator desferrioxamine also reduced MLH1 and PMS2 levels, in keeping with low oxygen tension being the stress signal that provokes the altered MMR gene expression. Functional MMR deficiency under hypoxia was detected as induced instability of a (CA)(29) dinucleotide repeat and by increased mutagenesis in a chromosomal reporter gene. These results identify a potential new pathway of genetic instability in cancer: hypoxia-induced reduction in the expression of key MMR proteins. In addition, this stress-induced genetic instability may represent a conceptual parallel to the pathway of stationary-phase mutagenesis seen in bacteria.

Quantification of Tumor Hypoxic Fractions Using Positron Emission Tomography with [18F]Fluoromisonidazole ([18F]FMISO) Kinetic Analysis and Invasive Oxygen Measurements.

PURPOSE: The purpose of this study is to use dynamic [18F]fluoromisonidazole ([18F]FMISO) positron emission tomography (PET) to compare estimates of tumor hypoxic fractions (HFs) derived by tracer kinetic modeling, tissue-to-blood ratios (TBR), and independent oxygen (pO2) measurements. PROCEDURES: BALB/c mice with EMT6 subcutaneous tumors were selected for PET imaging and invasive pO2 measurements. Data from 120-min dynamic [18F]FMISO scans were fit to two-compartment irreversible three rate constant (K 1, k 2, k 3) and Patlak models (K i). Tumor HFs were calculated and compared using K i, k 3, TBR, and pO2 values. The clinical impact of each method was evaluated on [18F]FMISO scans for three non-small cell lung cancer (NSCLC) radiotherapy patients. RESULTS: HFs defined by TBR (≥1.2, ≥1.3, and ≥1.4) ranged from 2 to 85 % of absolute tumor volume. HFs defined by K i (>0.004 ml min cm-3) and k 3 (>0.008 min-1) varied from 9 to 85 %. HF quantification was highly dependent on metric (TBR, k 3, or K i) and threshold. HFs quantified on human [18F]FMISO scans varied from 38 to 67, 0 to 14, and 0.1 to 27 %, for each patient, respectively, using TBR, k 3, and K i metrics. CONCLUSIONS: [18F]FMISO PET imaging metric choice and threshold impacts hypoxia quantification reliability. Our results suggest that tracer kinetic modeling has the potential to improve hypoxia quantification clinically as it may provide a stronger correlation with direct pO2 measurements.

Development of a statewide hospital plan for radiologic emergencies.

Although general guidelines have been developed for triage of victims in the field and for hospitals to plan for a radiologic event, specific information for clinicians and administrators is not available for guidance in efficient management of radiation victims during their early encounter in the hospital. A consensus document was developed by staff members of four Connecticut hospitals, two institutions of higher learning, and the State of Connecticut Department of Environmental Protection and Office of Emergency Preparedness, with assistance of the American Society for Therapeutic Radiology and Oncology. The objective was to write a practical manual for clinicians (including radiation oncologists, emergency room physicians, and nursing staff), hospital administrators, radiation safety officers, and other individuals knowledgeable in radiation monitoring that would be useful for evaluation and management of radiation injury. The rationale for and process by which the radiation response plan was developed and implemented in the State of Connecticut are reviewed. Hospital admission pathways are described, based on classification of victims as exposed, contaminated, and/or physically injured. This manual will be of value to those involved in planning the health care response to a radiologic event.

Efaproxiral (RSR13) plus oxygen breathing increases the therapeutic ratio of carboplatin in EMT6 mouse mammary tumors.

Carboplatin, a member of the platinum family of alkylating agents, is often used in combination with radiotherapy. Some studies, including a recent publication from our laboratory, have suggested that the cytotoxic effects of platinum compounds may be altered by changes in the post-treatment oxygenation. The study reported here assessed whether post-treatment changes in tumor oxygenation caused by oxygen breathing alone or in combination with efaproxiral (RSR13) altered the effects of carboplatin. Efaproxiral, which allosterically modifies hemoglobin-oxygen binding to increase tumor pO(2), has been shown to increase the effects of radiation in animal tumor models and is in a second, confirmatory phase III clinical trial as an adjuvant to radiotherapy. These studies with EMT6 tumors in BALB/c Rw mice used clonogenic assays to assess tumor cell survival and tumor growth studies to assess antineoplastic activity and treatment-related toxicity. Efaproxiral plus oxygen breathing for 5 hrs after carboplatin treatment significantly increased the antineoplastic effects of carboplatin. The increased antineoplastic effects of carboplatin produced by efaproxiral plus oxygen breathing occurred without a concomitant increase in host toxicity. These findings suggest that the increases in tumor oxygenation produced by Efaproxiral plus oxygen breathing increased the therapeutic ratio of carboplatin.

Effects of motexafin gadolinium on DNA damage and X-ray-induced DNA damage repair, as assessed by the Comet assay.

PURPOSE: To investigate the effects of motexafin gadolinium (MGd) on the levels of reactive oxygen species (ROS), glutathione (GSH), and DNA damage in EMT6 mouse mammary carcinoma cells. The ability of MGd to alter radiosensitivity and to inhibit DNA damage repair after X-ray irradiation was also evaluated. METHODS AND MATERIALS: Reactive oxygen species and GSH levels were assessed by 2,7-dichlorofluorescein fluorescence flow cytometry and the Tietze method, respectively. Cellular radiosensitivity was assessed by clonogenic assays. Deoxyribonucleic acid damage and DNA damage repair were assessed in plateau-phase EMT6 cells by the Comet assay and clonogenic assays. RESULTS: Cells treated with 100 mumol/L MGd plus equimolar ascorbic acid (AA) had significantly increased levels of ROS and a 58.9% +/- 3.4% decrease in GSH levels, relative to controls. Motexafin gadolinium plus AA treatment increased the hypoxic, but not the aerobic, radiosensitivity of EMT6 cells. There were increased levels of single-strand breaks in cells treated with 100 mumol/L MGd plus equimolar AA, as evidenced by changes in the alkaline tail moment (MGd + AA, 6 h: 14.7 +/- 1.8; control: 2.8 +/- 0.9). The level of single-strand breaks was dependent on the length of treatment. Motexafin gadolinium plus AA did not increase double-strand breaks. The repair of single-strand breaks at 2 h, but not at 4 h and 6 h, after irradiation was altered significantly in cells treated with MGd plus AA (MGd + AA, 2 h: 15.8 +/- 3.4; control: 5.8 +/- 0.6). Motexafin gadolinium did not alter the repair of double-strand breaks at any time after irradiation with 10 Gy. CONCLUSIONS: Motexafin gadolinium plus AA generated ROS, which in turn altered GSH homeostasis and induced DNA strand breaks. The MGd plus AA-mediated alteration of GSH levels increased the hypoxic, but not aerobic, radiosensitivity of EMT6 cells. Motexafin gadolinium altered the kinetics of single-strand break repair soon after irradiation but did not inhibit potentially lethal damage repair in EMT6 cells.

Concurrent chemo-radiotherapy with mitomycin C compared with porfiromycin in squamous cell cancer of the head and neck: final results of a randomized clinical trial.

PURPOSE: Previous randomized trials have shown a benefit with concurrent use of the hypoxic cell cytotoxin mitomycin C (MC) and radiation (RT) in the management of squamous cell cancer of the head and neck (SCCHN). We conducted a randomized trial comparing MC with porfiromycin (POR) in combination with RT in the management of SCCHN. METHODS AND MATERIALS: Between 1992 and 1999, 128 patients with SCCHN were enrolled in this prospective randomized trial. Patients were stratified by management intent, and balanced with respect to stage and site of disease. They were randomized to receive MC (15 mg/M(2)) or POR (40 mg/M(2)) on Days 5 and 47 (or last day) of RT. Of 121 evaluable patients, 61 were randomized to MC and 60 to POR. Patients were treated with standard daily RT to a total median dose of 64 Gy over 47 days. Patients were well balanced with respect to management intent, stage, site, age, sex, hemoglobin levels, tumor grade, radiation dose, and days on treatment. RESULTS: There were no significant differences between the two arms with respect to acute hematologic or nonhematologic toxicities. As of January 2003 with a median follow-up of 6.3 years, there have been 19 local relapses (4 MC vs. 15 POR), 21 regional relapses (7 MC vs. 14 POR), 24 distant metastases (11 MC vs. 13 POR), and 66 deaths (33 MC vs. 33 POR). MC was superior to POR with respect to 5-year local relapse-free survival (91.6% vs. 72.7%, p = 0.01), local-regional relapse-free survival (82% vs. 65.3%, p = 0.05), and disease-free survival (72.8% vs. 52.9%, p = 0.026). There were no significant differences between the two arms with respect to overall survival (49.2% vs. 54.4%) or distant metastasis-free rate (79.9% vs. 75.9%). CONCLUSIONS: Despite promising preclinical data, and an acceptable toxicity profile, POR was inferior to MC as an adjunct to RT in the management of SCCHN. This randomized trial emphasizes the need for randomized studies to evaluate new agents in the management of SCCHN.

Priority list of research areas for radiological nuclear threat countermeasures.

To help the nation prepare for the possibility of a terrorist attack using radiological and nuclear devices, the Office of Science and Technology Policy and the Homeland Security Council established an interagency working group. The working group deliberated on the research needs for radiological/ nuclear threat countermeasures and identified and prioritized 18 areas for further attention. The highest priorities were given to research on (1) radioprotectors for use prior to exposure; (2) therapeutic agents for postexposure treatment; (3) antimicrobial therapy for infections associated with radiation exposure; (4) cytokines and growth factors; (5) mechanisms of radiation injury at the molecular, cellular, tissue and organism levels; and (6) automation of biodosimetric assays. High priority was given to (1) developing biomarkers for biodosimetry; (2) enhancing training in the radiation sciences; (3) exploring the consequences of combined injury; (4) establishing a repository of information regarding investigational countermeasures; and (5) following the health of an exposed population to better prepare for subsequent events. The research areas that the committee felt required the attention of the radiation research community are described in this report in an effort to inform this community about the needs of the nation and to encourage researchers to address these critical issues.

Relative biological effectiveness of 103Pd and 125I photons for continuous low-dose-rate irradiation of Chinese hamster cells.

Monolayers of Chinese hamster lung cells (CCL-16) in a polystyrene phantom were irradiated in vitro by 103Pd and 125I sources at dose rates of 6 to 72 cGy/h. Cell survival curves for acute high-dose-rate irradiation (over 30 Gy/h) were also measured using nearly monoenergetic X-ray beams which were designed to simulate the mean energies of photons emitted by 125I and 103Pd and also using a clinical 250 kVp X-ray beam. A profound dose-rate effect is observed over the dose-rate range of 6 to 20 cGy/h. An inverse dose-rate effect was observed for both radionuclides, with its onset occurring at a dose rate of about 20-30 cGy/h. The average RBE of 103Pd relative to 125I was determined to be 1.45 +/- 0.07, 1.41 +/- 0.07, 0.70 +/- 0.07 and 1.49 +/- 0.07 at dose rates of 6.9, 12.6, 19.0 and 26.7 cGy/h, respectively. Because 103Pd implants are generally prescribed at a higher initial dose rate (21 cGy/h) than the corresponding 125I implants (7 cGy/h), the effects of both dose rate and photon energy on biological response must be considered together. For the CCL-16 cells, the RBE of 103Pd at 19.0 cGy/h relative to that of 125I at 6.9 cGy/h was estimated to be 2.3 +/- 0.5.

Animal models for radiation injury, protection and therapy.

Current events throughout the world underscore the growing threat of different forms of terrorism, including radiological or nuclear attack. Pharmaceutical products and other approaches are needed to protect the civilian population from radiation and to treat those with radiation-induced injuries. In the event of an attack, radiation exposures will be heterogeneous in terms of both dose and quality, depending on the type of device used and each victim's location relative to the radiation source. Therefore, methods are needed to protect against and treat a wide range of early and slowly developing radiation-induced injuries. Equally important is the development of rapid and accurate biodosimetry methods for estimating radiation doses to individuals and guiding clinical treatment decisions. Acute effects of high-dose radiation include hematopoietic cell loss, immune suppression, mucosal damage (gastrointestinal and oral), and potential injury to other sites such as the lung, kidney and central nervous system (CNS). Long-term effects, as a result of both high- and low-dose radiation, include dysfunction or fibrosis in a wide range of organs and tissues and cancer. The availability of appropriate types of animal models, as well as adequate numbers of animals, is likely to be a major bottleneck in the development of new or improved radioprotectors, mitigators and therapeutic agents to prevent or treat radiation injuries and of biodosimetry methods to measure radiation doses to individuals.

Dose rate dependence of the relative biological effectiveness of 103Pd for continuous low dose rate irradiation of BA1112 rhabdomyosarcoma cells in vitro relative to acute exposures.

PURPOSE: To measure the relative biological effectiveness (RBE) of continuous low dose rate irradiation (CLDRI) using 103Pd sources relative to acute high dose rate irradiations (AHDRI) from a 250 kVp x-ray beam and an x-ray beam having an equivalent mono-energetic photon energy equal to the average energy of the 103Pd source for BA1112 rhabdomyosarcoma cells. MATERIALS AND METHODS: A customized 103Pd irradiator was built to provide CLDRI using 103Pd at different dose rates relevant to clinical interstitial brachytherapy to BA1112 rhabdomyosarcoma cells growing in exponential phase in culture. A special x-ray beam that simulates the photon energies emitted by the 103Pd source was also developed to provide acute high dose rate irradiation at those energies. Cell survival curves from different irradiation conditions were measured. The RBE with respect to AHDRI using standard 250 kVp x-rays was determined from the doses required to achieve a cell surviving faction of 0.01. RESULTS: For acute irradiation, the RBE of the x-rays simulating (103)Pd was 1.24 relative to 250 kVp x-rays. A profound dose rate effect was observed at low dose rates in the range of 6.8 - 14.4 cGy/h that are typical of permanent interstitial brachytherapy. At cell-surviving fraction of 0.01, the RBE of CLDRI at 6.8 and 14.4 cGy/h using 103Pd sources was reduced by a factor of 3 and 2, respectively, relative to the acute exposure. This observation is in good agreement with recent in vivo tumor cure studies performed on BA1112 tumor. CONCLUSION: The relative biological effectiveness of the photons emitted by 103Pd depends on both the linear energy transfer (LET) of the low energy photons and the dose rate of the irradiation. The higher LET of 103Pd photons is biologically more effective in killing BA1112 tumor cells compared to conventional 250 kVp x-rays when both are delivered at the same dose rate. But the gain in RBE that results from the higher LET can be quickly negated by the reduced dose rate of the irradiation.

Role of chitinase 3-like-1 and semaphorin 7a in pulmonary melanoma metastasis.

The prototypic chitinase-like protein Chi3l1 is induced in cancers and portends a poor prognosis, but whether it contributes to cancer progression is unknown. To address this gap in knowledge, we investigated the production of Chi3l1 in melanoma lung metastases. We found that Chi3l1 was induced during pulmonary melanoma metastasis and that this induction was regulated by the semaphorin Sema7a, interacting in stimulatory or inhibitory ways with its ß1 integrin or Plexin C1 receptors, respectively. In mouse strains with genetic deletions of Chi3l1 or Sema7a, there was a significant reduction in pulmonary metastasis. Notably, antiserum raised against Chi3l1 or Sema7a phenocopied the reduction produced by genetic deletions. Melanoma lung metastasis was also decreased in the absence of IL13Rα2, a recently identified receptor for Chi3l1, consistent with a key role for Chi3l1 in melanoma spread. We confirmed roles for Sema7a and Chi3l1 in pulmonary metastasis of EMT6 breast cancer cells. Taken together, our studies establish a novel pathway through which Sem7a and its receptors regulate Chi3l1, revealing a host axis involving IL13Rα2 that plays a critical role in generating a pulmonary microenvironment that is critical to license metastasis.

Preliminary studies of the effects of gadolinium texaphyrin on the growth and radiosensitivity of EMT6 cells in vitro.

PURPOSE: To investigate the effects of gadolinium texaphyrin (GdTx) on the growth and radiation response of cells in vitro, in a limited set of experiments designed to examine some areas of controversy concerning the effects of this compound. METHODS AND MATERIALS: Exponentially growing cultures of EMT6 mouse mammary tumor cells, grown in Dulbecco's Modified Eagle's Medium with 10% dialyzed fetal bovine serum, were treated with GdTx either prepared from powder or obtained as a solution similar to that used clinically, in either the presence or absence of equimolar ascorbic acid. Cell viability was measured using a clonogenic assay. RESULTS: Treatment with GdTx in the presence of ascorbic acid dramatically altered the growth, appearance, and behavior of the cells; treatment with GdTx in the absence of ascorbic acid had only minimal effects. The effects of the powdered drug and the solution were similar. GdTx used with equimolar ascorbic acid altered the radiation dose-response curves of cells irradiated under aerobic and hypoxic conditions; no significant changes were observed without ascorbic acid. CONCLUSIONS: The details of the protocols used in experiments examining the effects of GdTx have major effects on the outcomes. Our results suggest that differences in the protocols used by different groups in past studies with GdTx probably were important in producing the disparate results reported previously.

Effects of texaphyrins on the oxygenation of EMT6 mouse mammary tumors.

PURPOSE: To investigate the effects of texaphyrins on the oxygenation of EMT6 mouse mammary tumors in Balb/c Rw mice. Texaphyrins are synthetic, porphyrin-like molecules capable of stably coordinating lanthanide and nonlanthanide metals. Metallotexaphyrin compounds containing gadolinium (MGd), lutetium (MLu), europium (Eu-Tex), dysprosium (Dy-Tex), and manganese (Mn-Tex) were evaluated. METHODS: Tumor oxygenation was measured using an Eppendorf pO2 histograph when tumors, implanted intradermally in the rear dorsum, reached 150-200 mm3. Oxygen measurements were also made in the leg muscle of tumor-bearing mice, to determine whether changes in oxygenation occurred in nontumor tissue. RESULTS: Motexafin gadolinium (Xcytrin, MGd) seems to be an effective modulator of tumor oxygen tension. The mean of the median tumor pO2 6 hours after injection of MGd was 8.0 +/- 2.4 mm Hg. The control value was 1.5 +/- 0.4 mm Hg. The oxygen levels within EMT6 tumors were shifted significantly toward higher oxygen tensions 6-8 hours after i.v. injection of 40 micromol/kg MGd, thereby reducing the percentage of severely hypoxic readings (MGd, 6 hours: 44.6 +/- 4.3% <2.5 mm Hg; CONTROL: 69.4 +/- 3.0% <2.5 mm Hg). There was no significant change in the oxygenation of the leg muscle after MGd treatment. Eu-Tex and Mn-Tex increased the tumor oxygenation to a much lesser degree than MGd. MLu, Dy-Tex, and the vehicle (a 5% mannitol solution) did not modulate tumor oxygenation. CONCLUSIONS: MGd is an effective modulator of tumor oxygenation. The central metal composition of texaphyrin compounds is an important determinant of the effect of the texaphyrins on tumor oxygenation.

IGF1 receptor expression protects against microenvironmental stress found in the solid tumor.

The insulin-like growth factor 1 receptor (IGF1R) is a tyrosine kinase, transmembrane receptor expressed in most body tissues and required for normal growth of cells. In cell culture, overexpression of the receptor has been shown to promote transformation and enhance cell survival in response to selected cytotoxic agents. As tumors develop, abnormalities in vascularization lead to a heterogeneous environment that includes areas of hypoxia, low pH and low glucose. Here we report that the overexpression of the IGF1R promotes increased survival in cells exposed to hypoxia, low pH and low glucose. Furthermore, cells lacking the receptor due to targeted disruption of the IGF1R gene do not survive as well as normal cells in such conditions. In addition, we find that cells can activate the IGF1R gene promoter in response to these conditions, and immunoblot analyses show increased receptor protein levels in cell exposed to hypoxia. Our results suggest a pathway of cancer cell adaptation to the tumor microenvironment in which conditions of the environment may induce expression of IGF1R, and this subsequent overexpression of the receptor may increase cell survival in such conditions.

Education and training for radiation scientists: radiation research program and American Society of Therapeutic Radiology and Oncology Workshop, Bethesda, Maryland, May 12-14, 2003.

Current and potential shortfalls in the number of radiation scientists stand in sharp contrast to the emerging scientific opportunities and the need for new knowledge to address issues of cancer survivorship and radiological and nuclear terrorism. In response to these challenges, workshops organized by the Radiation Research Program (RRP), National Cancer Institute (NCI) (Radiat. Res. 157, 204-223, 2002; Radiat. Res. 159, 812-834, 2003), and National Institute of Allergy and Infectious Diseases (NIAID) (Nature, 421, 787, 2003) have engaged experts from a range of federal agencies, academia and industry. This workshop, Education and Training for Radiation Scientists, addressed the need to establish a sustainable pool of expertise and talent for a wide range of activities and careers related to radiation biology, oncology and epidemiology. Although fundamental radiation chemistry and physics are also critical to radiation sciences, this workshop did not address workforce needs in these areas. The recommendations include: (1) Establish a National Council of Radiation Sciences to develop a strategy for increasing the number of radiation scientists. The strategy includes NIH training grants, interagency cooperation, interinstitutional collaboration among universities, and active involvement of all stakeholders. (2) Create new and expanded training programs with sustained funding. These may take the form of regional Centers of Excellence for Radiation Sciences. (3) Continue and broaden educational efforts of the American Society for Therapeutic Radiology and Oncology (ASTRO), the American Association for Cancer Research (AACR), the Radiological Society of North America (RSNA), and the Radiation Research Society (RRS). (4) Foster education and training in the radiation sciences for the range of career opportunities including radiation oncology, radiation biology, radiation epidemiology, radiation safety, health/government policy, and industrial research. (5) Educate other scientists and the general public on the quantitative, basic, molecular, translational and applied aspects of radiation sciences.

Effects of RSR13 and oxygen on the cytotoxicity of cisplatin and carboplatin to EMT6 mouse mammary tumor cells in vitro and in vivo.

PURPOSE: RSR13, 2-[4-[2-[(3,5-dimethylphenyl)amino]-2-oxoethyl]phenoxy]-2-methylpropanoic acid monosodium salt, allosterically modifies hemoglobin to increase tumor pO(2), increases the effect of radiation in animal tumor models, and is in phase III clinical trials as an adjuvant to radiotherapy. Cisplatin and carboplatin, two commonly used anticancer drugs have been used in combination with radiotherapy. Some studies have suggested that the cytotoxic effects of these drugs are altered in hypoxia. This study tested whether RSR13 plus oxygen breathing increased the cytotoxicity of cisplatin and carboplatin in vivo. METHODS: Solid EMT6 tumors in BALB/c Rw mice were treated with cisplatin (5-30 microg/g) or carboplatin (5-200 microg/g) along with 150 microg/g RSR13 in combination with oxygen breathing. Tumor cell survival was assayed using clonogenic assays. The effects of pre- and posttreatments with RSR13 and oxygen breathing on the cytotoxicity of cisplatin or carboplatin were also examined. To assess whether RSR13 had direct effects on the cytotoxicity of the drugs, exponentially growing monolayers of EMT6 mouse mammary carcinoma cells were treated with graded concentrations of cisplatin or carboplatin for 2 h along with simultaneous (2 h) RSR13 treatments or with prolonged (22 h) pre- or posttreatment incubations with 100 microM RSR13. RESULTS: Single or multiple treatments with 150 microg/g RSR13 plus oxygen breathing had no effect on the viability of cells in EMT6 tumors in mice. After treatment with cisplatin or carboplatin, the tumor cell survival tended to be lower in oxygen-breathing mice especially at higher doses of cisplatin. Treatment with RSR13 plus oxygen breathing beginning 15 min before administration of the alkylating agents did not alter the cytotoxicity of cisplatin or carboplatin from that seen with oxygen breathing alone. Pretreatment with RSR13 plus oxygen at 22 and 14 h prior to administration of either cisplatin or carboplatin did not alter the effect of either alkylating agent. Treatment with RSR13 plus oxygen breathing beginning 15 min before administration of the alkylating agents and lasting for 2 or 5 h did not alter the cytotoxicity of either drug from that seen with oxygen breathing alone. The cytotoxicity of cisplatin was not altered by treatment with oxygen alone or with RSR13 plus oxygen for 5 h after cisplatin injection. For carboplatin, treatment with oxygen alone and with RSR13 plus oxygen for 5 h after injection increased to similar extents the response of the tumor cells compared to that seen with assays at 2 h. Neither short simultaneous treatments, prolonged pretreatment incubations, nor prolonged posttreatment incubations with RSR13 altered the survival of EMT6 cells in cultures treated with cisplatin or carboplatin. CONCLUSIONS: These findings indicate that RSR13 in combination with oxygen breathing does not alter the cytotoxicity of cisplatin or carboplatin when used simultaneously, as a pretreatment or as a posttreatment in vitro or in vivo. Our in vivo findings indicate trends that support previous findings that cisplatin is more cytotoxic to well-oxygenated tumor cells than to hypoxic tumor cells, and that this effect can be improved by improving tumor oxygenation, but the differences seen in our studies did not achieve statistical significance.

Development of a rat solid tumor model for continuous low-dose-rate irradiation studies using 125I and 103Pd sources.

PURPOSE: To develop an experimental technique for studying the radiobiology of continuous low-dose-rate irradiation (CLDRI) using clinical brachytherapy sources emitting low energy photons for a rat solid tumor model. METHODS AND MATERIALS: BA1112 tumors were grown between the ears of 14-week-old male WAG/Rij rats by interdermal inoculation. A radioactive source afterloading system, which consists of a lightweight helmet sutured to the rat and a nine-source polystyrene applicator, was fabricated for in vivo tumor irradiation by (125)I and (103)Pd brachytherapy sources. This system has a 12 x 12 mm opening in the center to accommodate the tumor and its growth during irradiation (the diameter of a typical BA1112 tumor was about 6 mm when radiation was applied). The spatial locations of the nine sources were optimized to produce an as uniform as possible three-dimensional dose distribution to the central portion of the applicator for both the (125)I and (103)Pd sources. Absolute dose delivered by the applicator was verified by point dose measurements using calibrated TLD in a polystyrene phantom that mimics the scattering environment of the tumor on the rat. RESULTS: The feasibility of tumor cure experiments using the experimental technique presented in this work was demonstrated. The technique was used to study the influence of initial dose rate on the in vivo tumor cure probability of BA1112 tumors irradiated by (125)I and (103)Pd sources at dose rates varying from 8-20 cGy/h. The technique was also used for studying the in vitro tumor cell survival following in vivo CLDRI irradiation of the tumor. CONCLUSION: An experimental technique using an in vivo tumor model has been developed for studying the radiobiological effects of continuous low-dose-rate irradiations using (125)I sources alone, (103)Pd sources alone, or a mixture of (125)I and (103)Pd sources.

Importance of hypoxia in the biology and treatment of brain tumors.

The resistance of gliomas to treatment with radiation and antineoplastic drugs may result in part from the effects of the extensive, severe hypoxia that is present in these tumors. It is clear that brain tumors contain extensive regions in which the tumor cells are subjected to unphysiological levels of hypoxia. Hypoxic cells are resistant to radiation. Hypoxia and the perfusion deficits and metabolic changes that accompany hypoxia in vivo also produce resistance to many commonly used anticancer drugs. The resistance of cells that are hypoxic at the time of therapy may influence the efficacy of the treatment of these tumors with radiation, chemotherapy, and combined modality regimens. Moreover, it is becoming increasingly evident from laboratory studies that exposure of cells to adverse microenvironments produces transient changes in gene expression, induces mutations, and selects for cells with altered genotypes, thus driving the evolution of the cell population toward increasing malignancy and increasingly aggressive phenotypes. Hypoxia may therefore be involved in the evolution of cells in low-grade malignancies to the resistant, aggressive phenotype characteristic of glioblastomas. During the past 50 years, many attempts have been made to circumvent the therapeutic resistance induced by hypoxia, by improving tumor oxygenation, by using oxygen-mimetic radiosensitizers, by adjuvant therapy with drugs that are preferentially toxic to hypoxic cells, by using hyperthermia, or by devising radiation sources and regimens that are less affected by hypoxia. Past clinical trials have provided tantalizing suggestions that the outcome of therapy can be improved by many of these approaches, but none has yet produced a significant, reproducible improvement in the therapeutic ratio, which would be needed for any of these approaches to become the standard therapy for these diseases. Several ongoing clinical trials are addressing other, hopefully better regimens; it will be interesting to see the results of these studies.