Normal human cells in culture show a limited division potential known as senescence. Cells divide for a defined number of generations and then fail to enter subsequent S phases. Available evidence suggests that senescent cells have intact a pathway that prevents some aspect of activation of the cell cycle regulatory apparatus. Our overall interests are to determine what aspect of activation is absent, and whether introduction of cell cycle regulatory genes and proteins that normally promote entry into S phase can overcome this inactivation. Recent genetic and biochemical evidence suggests that the G1 to S phase transition in the eukaryotic cell cycle is controlled, in part, by the coordinate action of a protein kinase(s), p34cdc2, and its associated regulatory subunits, cyclins. The principal focus of this program will be the analysis of the role that Cdk2 and Cdc2Hs protein kinases and G1 cyclins play in the process of senescence and the regulation of cell cycle transitions. Cdk2 is a Cdc2-related protein kinase that forms a distinct sub-family of Cdc2 kinases. It is currently believed that Cdk2, which is found complexed with cyclin A, is involved in the control of DNA replication and/or the G1-S transition. Depletion of the Xenopus Cdk2 protein, but not the Cdc2 protein, blocks DNA replication in cycling embryonic extracts in vitro. Further evidence suggestive of an early role in the cell cycle is that some CDK2 mRNA persists in G0 cells and, when G0 cells are stimulated to enter the cell cycle, its mRNA levels increase in G1 prior to the increase in CDC2Hs mRNAs.
The specific aims of this research are to use the G1 cyclins, CDK2 and CDC2Hs genes as tools to: 1) explore the expression state of quiescent and senescent cells with respect to key cell cycle regulators, 2) determine the effects of removal of Cdk2 and other relevant proteins on the progression of the cell cycle, 3) to explore regulation of Cdk2 by phosphorylation, and 4) to determine if expression of combinations of cyclins and p34 protein kinases can promote entry of quiescent or senescent cells into S phase. Our analysis of G1 control in senescent cells is aimed at identifying those key regulators whose functions are negatively regulated in aging cells. This information can be used to further elaborate the mechanisms by which negative regulation is established in senescent cells.
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