The subject of this proposal is the mechanism of mitotic catastrophe, a major form of cell death that develops through abnormal mitosis and is induced by different classes of anticancer agents. Mitotic catastrophe is observed in cells that enter mitosis prior to repairing the damage; this early form of catastrophe is promoted by G2 checkpoint abrogation. The delayed form of mitotic catastrophe develops in cells that reenter cell cycle after prolonged growth arrest induced by drugs or cyclin-dependent kinase (CDK) inhibitors. The early and delayed forms of mitotic catastrophe show a different assortment of mitotic abnormalities.
The first aim of the proposed program is to characterize early mitotic catastrophe in gamma irradiated tumor cells that enter mitosis under the conditions where both G1 and G2 checkpoints have been abrogated. The experiments in this Aim will investigate the mechanism of transient increase in mitotic index induced by radiation under these conditions, through the analysis of changes in mitosis-initiating Cdc2 kinase and in expression and localization of different mitosis-controlling proteins. The morphology of radiation-induced abnormal mitosis will be compared among cell lines with different G1 and G2 checkpoint status.
The second Aim i s to develop a defined cellular model of early catastrophe, based on inducible expression of constitutively active Cdc2 kinase. This model will be used to characterize the effects of Cdc2 activation on the initiation of mitosis, expression of mitotic proteins and mitotic morphology, and to develop cell lines resistant to Cdc2-induced mitotic catastrophe.
The third Aim i nvolves analysis of the mechanism of delayed mitotic catastrophe that develops after release from growth inhibition by CDK inhibitors. These studies will include selection and characterization of cell lines resistant to delayed catastrophe and identification of genes responsible for such resistance, through differential gene expression analysis and functional testing.
The fourth Aim will utilize different catastrophe-resistant cell lines, to investigate the rote of early and delayed forms of mitotic catastrophe in the antiproliferative effect of CDK inhibitors, anticancer drugs and radiation. Our long-term goal is to develop molecular targets and screening systems for agents that would induce or potentiate mitotic catastrophe in tumor cells.
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