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Vascular endothelial growth factor (VEGF) is an important mediator of endothelial cell proliferation and survival. The purpose of the present studies was to investigate the role of VEGF in the tumor response to ionizing radiation.Two ras-transformed murinefibrosarcoma cell lines, VEGF+/+ and VEGF−/− were exposed to ionizing radiation (0, 1, 3, 5, 7 or 9 Gy) in vitro, and clonogenic survival was determined. VEGF+/+ and VEGF-\- xenografts were generated in athymic nude mice and then treated with ionizing radiation (ten 5-Gy fractions = 50 Gy). Mean fractional tumor volume was used to evaluate treatment efficacy. To determine whether VEGF enhances tumor radioresistance by targeting endothelial cells, we performed clonogenic survival assays with human umbilical vein endothelial cells. Surviving fractions were calculated after treatment with ionizing radiation (5 Gy) and recombinant hVEGF165 (0, 1, 10, and 100 ng/mL). To determine whether VEGF neutralization enhances tumor radio-sensitivity, we employed anti-VEGF165 monoclonal antibody to treat human tumor xenografts. Tumors were exposed to ionizing radiation (four 5-Gy fractions = 20 Gy) and treated with anti-VEGF antibody (0, 5, and 25 μg/kg in four intraperitoneal doses). Mean fractional tumor volume was used to evaluate treatment efficacy. To elucidate the molecular mechanism contributing to the observed anti-VEGF/ionizing radiation interaction, we exposed human umbilical vein endothelial cells to ionizing radiation (5 Gy) in the presence of anti-VEGF antibody (1 μg/mL). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of cell lysates was probed for mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK1/MEK2).The in vitro radiosensitivities of the VEGF+/+ and VEGF−/− clones were equivalent (Do = 146 vs 149). However, the VEGF+/+ xenografts were more resistant to the cytotoxic effects of ionizing radiation than the VEGF−/− xenografts. VEGF+/+ xenografts demonstrated a faster doubling time (4.5 vs 6.0 days) and a shorter growth delay (15 vs 23 days) than VEGF−/− xenografts. The surviving fraction of human umbilical vein endothelial cells after exposure to ionizing radiation was significantly enhanced in the presence of VEGF (6.4% vs 12.5%). Western blot analysis demonstrated that stimulation of MAPK and MEK1/MEK2 was abrogated after exposure to anti-VEGF antibody.These findings represent the first genetic evidence that factors other than inherent tumor cell radiosensitivity are important determinants of radiocurability. Antitumor strategies targeting VEGF and other endothelial cell survival mechanisms may be used to enhance the cytotoxic effects of radiotherapy.