Cell cycle effects and induction of premitotic apoptosis by irofulven in synchronized cancer cells

Unlike postmitotic cell death, direct premitotic apoptosis diminishes the risk of clonal selection and allows for the elimination of slowly growing cancer cells. This study characterized the ability to induce premitotic apoptosis by irofulven (hydroxymethylacylfulvene), a novel alkylating drug which targets cellular DNA and proteins. Irofulven effects were examined in HeLa-derived BH2 cancer cells with conditional overexpression of antiapoptotic Bcl-2. Cells were synchronized in either early S or in G₁. Following 12 h exposure to irofulven, cells that were originally in early S accumulated in late S or remained in early S phase (at 0.5 and 2.5 μM drug, respectively). Drug treatment of cells in the G₁ cohort prevented their entry into the S phase. Significant apoptosis was detected based on the appearance of sub-G ₁ particles and cells with DNA strand breaks in both G₁ and S cohorts. Apoptotic cells were mostly recruited from the G₁/S border ("G₁" cohort) and from the S phase ("early S" cohort). All the cell cycle and apoptotic effects were only marginally affected by Bcl-2 overexpression. Similar results were obtained with irofulven-treated synchronized cultures of leukemic CEM cells. Collectively, these observations indicate that irofulven-treated cells become committed to death early. Neither active DNA replication nor traverse through mitosis are necessary for irofulven-induced cell death. The ability to promote direct premitotic apoptosis is likely to play a role in the consistently potent apoptotic effects of irofulven and its ability to cause tumor regression in vivo.