Purpose: Poly (ADP-ribose) polymerase (PARP) inhibitors are undergoing clinical evaluation for cancer therapy. Because PARP inhibition has been shown to enhance tumor cell sensitivity to radiation, we investigated the in vitro and in vivo effects of the novel PARP inhibitor E7016. Experimental Design:The effect of E7016 on the in vitro radiosensitivity of tumor cell lines was evaluated using clonogenic survival. DNA damage and repair were measured using gH2AX foci and neutral comet assay. Mitotic catastrophe was determined by immunostaining. Tumor growth delay was evaluated in mice for the effect of E7016 on in vivo (U251) tumor radiosensitivity. Results: Cell lines exposed to E7016 preirradiation yielded an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1 from 1.4 to 1.7. To assess DNA doublestrand breaks repair, gH2AX measured at 24 hours postirradiation had significantly more foci per cell in the E7016/irradiation group versus irradiation alone. Neutral comet assay further suggested unrepaired double-strand breaks with significantly greater DNA damage at 6 hours postirradiation in the combination group versus irradiation alone. Mitotic catastrophe staining revealed a significantly greater number of cells staining positive at 24 hours postirradiation in the combination group. In vivo, mice treated with E7016/irradiation/temozolomide had an additional growth delay of six days compared with the combination of temozolomide and irradiation. Conclusions: These results indicate that E7016 can enhance tumor cell radiosensitivity in vitro and in vivo through the inhibition of DNA repair. Moreover, enhanced growth delay with the addition of E7016 to temozolomide and radiotherapy in a glioma mouse model suggests a potential role for this drug in the treatment of glioblastoma multiforme.

Purpose:Temozolomide, a DNA methylating agent, is currently undergoing clinical evaluation for cancer therapy. Because temozolomide has been shown to increase survival rates of patients with 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 1 h before irradiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1ranging from 1.30 to 1.32. Temozolomide had no effect on radiation-induced apoptosis or on the activation of the G 2 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 combined modality compared with 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 U251 tumor 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 in mitotic 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 O 6 -position of guanine in DNA. The formation of O 6 -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 6 -methlyguanine-DNA-methyltransferase (MGMT; refs. 3,4), which removes the O 6 -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...