There is increasing evidence that prolonged mitotic arrest initiates apoptosis; however, little is known about the signaling pathways involved. Several studies have associated deregulated Cdc2 activity with apoptosis. Herein, we report that the anti-apoptotic protein, Bcl-2, undergoes cell cycle-dependent phosphorylation during mitosis when there is elevated Cdc2 activity. We found that paclitaxel (Taxol ® ) treatment of epithelial tumor cells induced a prolonged mitotic arrest, elevated levels of mitotic kinase activity, hyperphosphorylation of Bcl-2, and subsequent cell death. The Taxol-induced Bcl-2 phosphorylation was dose-dependent. Furthermore, phosphorylated Bcl-2 remained complexed with Bax in Taxol-treated cells undergoing apoptosis. Immunoprecipitation experiments revealed a Bcl-2-associated kinase capable of phosphorylating histone H1 in vitro. However, the kinase was likely not cyclin B1/Cdc2, since cyclin B1/Cdc2 was not detectable in Bcl-2 immunoprecipitates, nor was recombinant Bcl-2 phosphorylated in vitro by cyclin B1/Cdc2. The results of this study further define a link between mitotic kinase activation and the apoptotic machinery in the cell. However, the role, if any, of prolonged Bcl-2 phosphorylation in Taxol-mediated apoptosis awaits further definition of Bcl-2 mechanism of action. Taxol may increase cellular susceptibility to apoptosis by amplifying the normal downstream events associated with mitotic kinase activation.

In the present study, we investigated the role of p53 in G 2 checkpoint function by determining the mechanism by which p53 prevents premature exit from G 2 arrest after genotoxic stress. Using three cell model systems, each isogenic, we showed that either ectopic or endogenous p53 sustained a G 2 arrest activated by ionizing radiation or adriamycin. The mechanism was p21 and retinoblastoma protein (pRB) dependent and involved an initial inhibition of cyclin B1-Cdc2 activity and a secondary decrease in cyclin B1 and Cdc2 levels. Abrogation of p21 or pRB function in cells containing wild-type p53 blocked the down-regulation of cyclin B1 and Cdc2 expression and led to an accelerated exit from G 2 after genotoxic stress. Thus, similar to what occurs in p21 and p53 deficiency, pRB loss can uncouple S phase and mitosis after genotoxic stress in tumor cells. These results indicate that similar molecular mechanisms are required for p53 regulation of G 1 and G 2 checkpoints.

The LNCaP-luc-M6 cell line is a bioluminescent, hormone-sensitive prostate cancer cell line applicable for BLI studies to non-invasively monitor subcutaneous and orthotopic prostate tumor growth and metastasis in vivo.

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Main purposeVoreloxin is a first-in-class anticancer quinolone derivative that intercalates DNA and inhibits topoisomerase II, inducing site-selective DNA damage. Voreloxin is in clinical studies, as a single agent and in combination with cytarabine, for the treatment of acute myeloid leukemia (AML). The preclinical studies reported here were performed to investigate the activity of voreloxin alone and in combination with cytarabine, in support of the clinical program.Research questionsIs single agent voreloxin active in preclinical models of AML? Does the combination of voreloxin and cytarabine enhance the activity of either agent alone?MethodsInhibition of proliferation was studied in three cancer cell lines: HL-60 (acute promyelocytic leukemia), MV4-11 (AML), and CCRF-CEM (Acute lymphoblastic leukemia). Combination index (CI) analysis established the effect of the drugs in combination. A mouse model of bone marrow ablation was used to investigate in vivo efficacy of the drugs alone and in combination. Peripheral white blood cell and platelet counts were followed to assess marrow impact and recovery.ResultsVoreloxin and cytarabine alone and in combination exhibited cytotoxic activity in human leukemia cell lines and in vivo. The two drugs had additive or synergistic activity in vitro and supra-additive activity in vivo. Bone marrow ablation was accompanied by reductions in peripheral white blood cells and platelets that were reversible within 1 week, consistent with the AML treatment paradigm.ConclusionsThese data support ongoing clinical evaluation of voreloxin both alone and in combination with cytarabine for the treatment of AML.