Influence of Cell Cycle Checkpoints and p53 Function on the Toxicity of Temozolomide in Human Pancreatic Cancer Cells

Background: Though an increased efficacy of carmustine and temozolomide (TMZ) has been demonstrated by inactivation of O6-methylguanine-DNAmethyltransferase(MGMT) with O6-benzyl-guanine (BG) in human pancreatic tumors refractive to alkylating agents, the regulatory mechanisms have not been explored. Methods: The effects of TMZ and BG on apoptosis, cell growth, the mitotic index, cell cycle distribution, and protein expression were studied byTUNEL, cell counting, flow cytometry, and Western blot analysis, respectively. Results: The wt-p53 human pancreatic tumor cell line Capan-2 and p53-efficient mouse embryonic fibroblasts (MEFs) were more responsive to treatment with TMZ + BG than mutant p53 Capan-1 and p53-null MEFs. S phase delay with a subsequent G2/M arrest was observed in Capans in response to BG + TMZ. The G1-to-S transition delay in Capan-2 was associated with p53-dependent apoptosis and was distinctly different from the presumed mismatch repair (MMR) killing operative during the G2/M arrest. The effect of p53on BG +TMZ toxicity was supported by a marked change in apoptosis when p53 function was restored/inactivated. There was an early induction of MMR proteins in p53-efficient lines. Conclusion: p53 provokes a classic proapoptotic response by delaying G1-to-S progression, but it may also facilitate cell killing by enhancing MMR-related cell cycle arrest and cell death.