In vitro and In vivo Radiosensitization Induced by the DNA

  • Methylating AgentTemozolomide, WhoonJong Kil, +5 authors Kevin Camphausen
  • Published 2008


Purpose:Temozolomide, a DNAmethylating agent, is currently undergoing clinical evaluation for cancer therapy. Because temozolomidehas been shown to increase survival rates of patientswith malignant gliomas when given combined with radiation, and there is conflicting preclinical data concerning the radiosensitizing effects of temozolomide, we further investigated the possible temozolomide-induced enhancement of radiosensitivity. Experimental Design:The effects of temozolomide on the in vitro radiosensitivity of U251 (a human glioma) and MDA-MB231BR (a brain-seeking variant of a human breast tumor) cell lines was evaluated using clonogenic assay. DNA damage and repair were evaluated using phosphorylated histone H2AX (gH2AX), and mitotic catastrophe was measured using nuclear fragmentation. Growth delay was used to evaluate the effects of temozolomide on in vivo (U251) tumor radiosensitivity. Results: Exposure of each cell line to temozolomide for 1h before irradiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fractionof 0.1ranging from 1.30 to 1.32. Temozolomide had no effect on radiation-induced apoptosis or on the activation of the G2 cell cycle checkpoint. As a measure of DNA double strand breaks, gH2AX foci were determined as a function of time after the temozolomide + irradiation combination. The number of gH2AX foci per cell was significantly greater at 24 h after the combinedmodality comparedwith the individual treatments. Mitotic catastrophe, measured at 72 h, was also significantly increased in cells receiving the temozolomide + irradiation combination compared with the single treatments. In vivo studies revealed that temozolomide administration to mice bearing U251tumor xenografts resulted in a greater than additive increase in radiation-induced tumor growth delay with a dose enhancement factor of 2.8. Conclusions:These results indicate that temozolomide can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair leading to an increase inmitotic catastrophe. Temozolomide has known anticancer effects against a broad range of tumor histologies including gliomas (1, 2). Temozolomide is a lipophilic molecule that can be given p.o. and crosses the blood-brain barrier. At physiologic pH, temozolomide is converted to the active metabolite methyltriazenoimidazole-carboxamide, which forms methyl adducts at Oposition of guanine in DNA. The formation of O-methylguanine then results in mismatch pairing with thymine during subsequent cycles of DNA replication, followed by DNA strand-break formation and eventually cell death (2). Critical to its effectiveness as an antitumor agent is the cellular expression of O-methlyguanine-DNA-methyltransferase (MGMT; refs. 3, 4), which removes the O-methyl adducts and, thus, acts to repair temozolomide-induced DNA damage (2, 3). In a recently published study by Stupp et al. (5), patients with primary glioblastoma multiforme (GBM) treated with temozolomide plus radiotherapy followed by 6 months of adjuvant temozolomide were found to have a significant survival advantage compared with those patients treated with radiotherapy alone. Further analyses indicated that the greatest survival benefit was obtained in patients whose tumors contained methylated MGMT, which presumably resulted in a suppression of MGMT expression and activity (6). These results have led to temozolomide plus radiotherapy being the newly accepted standard of care for GBMs. However, whereas this combined modality increased survival times, the 5-year survival for GBM remains dismal; thus, understanding the processes CancerTherapy: Prelinical Authors’ Affiliations: Radiation Oncology Branch, Molecular Radiation Therapeutics Branch, and Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, Maryland; Science Applications International Corporation-Frederick, National Cancer Institute-Frederick, Frederick, Maryland; and Drug Discovery Program, H. Lee Moffitt Cancer Center,Tampa, Florida Received 7/27/07; revised10/8/07; accepted10/29/07. Grant support: Intramural Research Programof theNIH, National Cancer Institute. This research was also supported in part by grant W81XWH-062-0033 from the US Department of Defense Breast Cancer Research Program (P.S. Steeg and K. Camphausen). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Kevin Camphausen, Radiation Oncology Branch, National Cancer Institute, 10 Center Drive 3B42, Bethesda, MD 20892. Phone: 301-4964146; Fax: 301-480-1434; E-mail: F2008 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-1856 Clin Cancer Res 2008;14(3) February1, 2008 931 Research. on August 29, 2017. © 2008 American Association for Cancer Downloaded from responsible for the temozolomide + irradiation effectiveness may lead to further improvements in GBM therapy. In the GBM treatment protocol, temozolomide was delivered during radiotherapy (5, 7), which suggests that survival benefits provided by the combined modality may be the consequence of a temozolomide-mediated radiosensitization. However, initial studies using established GBM cell lines (8), which do not express MGMT, indicated that exposure to temozolomide for 24 to 96 h before irradiation has no effect on their in vitro (intrinsic) radiosensitivity. Temozolomide delivered after irradiation was also reported to have no effect on the radiosensitivity of glioma cell lines that do or do not express MGMT (9). In contrast, Chakravarti et al. (10) recently reported that temozolomide delivered 2 h before radiation significantly enhanced the radiosensitivity of GBM cell lines established from primary culture that do not express MGMT but not of GBM cell lines that do express MGMT. Whether the variations in treatment protocols or the different cell lines used account for the inconsistent conclusions regarding the status of temozolomide as a radiosensitizing agent is unclear. Therefore, to further investigate the effects of temozolomide on tumor cell radiosensitivity, we have extended the studies of Chakravarti et al. (10) to the established GBM cell line U251. Moreover, because of the potential of combining temozolomide with radiation in the treatment of breast tumor metastases to the brain, we also evaluated a human breast tumor cell line that was selected for its proclivity to form brain metastases (MDAMB-231BR). Both of these cell lines are negative for MGMT expression (9, 12) and would apparently represent those tumors that benefit the most from the temozolomide plus radiotherapy combination. The data presented indicate that temozolomide enhances tumor radiosensitivity in vitro and in vivo. Moreover, this sensitization correlates with the delayed dispersion of phosphorylated histone H2AX (gH2AX) foci, suggesting an inhibition of the repair of the DNA double-strand breaks (DSB). Materials andMethods Cell lines and treatment. The U251 human GBM cell line was obtained from National Cancer Institute Frederick Tumor Repository. The breast tumor brain metastatic cell line MDA-MB-231BR (11) was supplied by the laboratory of Patricia Steeg (National Cancer Institute, Bethesda, MD). Cells were grown in DMEM (Invitrogen) with glutamate (5 mmol/L) and 10% fetal bovine serum and maintained at 37jC, 5% CO2. Temozolomide, provided by the Developmental Therapeutics Program of the National Cancer Institute, was reconstituted in DMSO (100 mmol/L) and stored at -20jC. Cultures were irradiated using a Pantak X-ray source at a dose rate of 2.28 Gy/min. Clonogenic assay. Cultures were trypsinized to generate a single cell suspension and a specified number of cells were seeded into each well of a six-well tissue culture plate. After allowing cells time to attach (4 h), cultures received temozolomide (50 Amol/L for U251 and 25 Amol/L for MDA-MB-231BR) or DMSO (vehicle control) for 1 h before irradiation: medium was then removed and replaced with drug-free medium. Ten to fourteen days after seeding, colonies were stained with crystal violet, the number of colonies containing at least 50 cells was determined, and surviving fractions were calculated. Survival curves were then generated after normalizing for the amount of temozolomide-induced cell death. Data presented are the mean F SE from at least three independent experiments. Cell cycle analysis. Evaluation of cell cycle phase distribution was performed using flow cytometry. The treatment protocols were essentially the same as in the clonogenic survival experiments, except that the cells were initially seeded into 10-cm dishes. Samples were fixed, stained with propidium iodide, and analyzed using flow cytometry (Guava Technologies). To evaluate the activation of G2 cell cycle checkpoint, mitotic cells were distinguished from G2 cells, and the mitotic index was determined according to the expression of gH2AX (Upstate Biotechnology) as detected in the 4N DNA content population by the flow cytometric method of Xu et al. (13). In this assay, loss of mitotic cells (reduced mitotic index) reflects the onset of G2 arrest. Apoptotic cell death. The Guava Nexin assay (Part Number 45000161) was performed following the manufacturer’s instructions. Briefly, 3.0 10 cells (50 AL) were added to a 150 AL staining solution containing 135 AL 1 apoptosis buffer, 10 AL Annexin V-PE, and 5 AL of 7-AAD. The cells were incubated in the dark at room temperature for 20 min. Samples (2,000 cells per well) were then acquired on the Guava EasyCyte system. Immunofluorescent staining for gH2AX. Immunofluorescent staining and counting of gH2AX nuclear foci was performed as previously described (14). Slides were examined on a Leica DMRXA fluorescent microscope. Images were captured by a Photometrics Sensys CCD camera (Roper Scientific) and imported into IP Labs image analysis software package (Scanalytics, Inc.). For each treatment condition, gH2AX foci were determined in at least 150 cells. Cells were classified as positive (i.e., containing radiation-induced gH2AX foci) when more than five foci were detected. Mitotic catastrophe. The presence of fragmented nuclei was used as the criteria for defining cells undergoing mitotic catastrophe. To visualize nuclear fragmentation, cells were fixed with methanol for 15 min at -20jC, stained with rabbit anti–a-tubulin antibody (SigmaAldrich) followed by staining with Texas red–conjugated secondary antibody (Jackson ImmunuoResearch laboratories, Inc.). Nuclei were counterstained with 4¶,6-diamidino-2-phenylindole. A single field containing 300 cells was selected at random for each treatment and photographed with epi-fluorescence. Nuclear fragmentation was defined as the presence of two or more distinct nuclear lobes within

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@inproceedings{AgentTemozolomide2008InVA, title={In vitro and In vivo Radiosensitization Induced by the DNA}, author={Methylating AgentTemozolomide and WhoonJong Kil and David and William E. Burgan and Katie Beam and Donna J Carter and Patricia S. Steeg and Kevin Camphausen}, year={2008} }