Cell cycle arrest and CHK2 activation induced by the anticancer agent, Irofulven

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Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114) is one of a new class of anticancer agents that are semi-synthetic derivatives of the mushroom toxin illudin S. Preclinical studies and clinical trials have demonstrated that irofulven is effective against multiple tumor types. Mechanisms of action studies suggest that irofulven induces DNA damage, ATM-dependent CHK2 activation and apoptosis. In this study, the cell cycle effects and role of p53 and CHK2 activation in cell cycle control after irofulven treatment were investigated in ovarian cancer and isogenic colon cancer cell lines. By using propidium iodide staining and FACS analysis, it was shown that cell cycle arrest induced by irofulven was dependent on both drug concentration and p53 status. At low (one hour 1xIC50 exposure) irofulven concentrations, p53-wild type cells (A2780) arrest mainly in G1 phase and to a lesser extent in S phase. In contrast, p53-mutated cells (CAOV3) arrested primarily in S phase at 12 hours and accumulated in G2/M phases at 24 hours after irofulven treatment. However, when cells were treated at higher irofulven concentrations (one hour, 3xIC50) or had prolonged exposure to low drug concentrations (24 hour, 1xIC50), cells arrested at the G1/S border independent of p53 status. These results were further corroborated by demonstrating p53 accumulation and disparate p21 induction patterns as well as modulation of cyclin E, A and B expression using western blot assays and variant DNA synthesis rates using a BrdU incorporation assays. Furthermore, differential cell cycle effects were observed when paired isogenic human colon cancer cell lines, HCT116 p53 (+/+) or (−/−), were treated with irofulven. Previous studies in our laboratory have demonstrated irofulven-induced ATM-dependent CHK2 activation. CHK2 is a checkpoint kinase that is activated in response to DNA damage and transduces cell cycle regulation signals. In order to understand the influence of CHK2 on irofulven-induced cell cycle arrest, isogenic human colon cancer cell lines, HCT116 CHK2 (+/+) or (−/−), were treated with irofulven and assayed using BrdU incorporation and FACS. These analyses demonstrated that CHK2 activation induced by irofulven contributed to S phase arrest. In summary, the anticancer agent, irofulven, induced G1 and S phase arrest in p53 wild-type cells and S phase arrest and G2/M accumulation in p53 mutant cells. Therefore, the data support the conclusion that p53-mediated p21 induction controls irofulven-induced G1 arrest; whereas, CHK2 activation contributes to irofulven-induced S phase arrest. These observations suggest that irofulven may have enhanced antitumor activity in combination with radiation, S phase abrogators, or inhibitors of the ATM-CHK2 signaling pathway.