P27kip1 as an early effector of pRB function

DESCRIPTION: (Adapted from the investigator's abstract) The tumor suppressor retinoblastoma protein inhibits cell proliferation when re-expressed in pRB-deficient tumor cells. Our objective is to identify the molecular mechanisms underlying this function of pRB. The currently most accepted model for pRB function is that pRB forms complexes with the transcription factor E2F to repress the expression of certain genes whose encoded proteins promote cell proliferation. The lack of these growth promoting proteins will then pose a block to the initiation of DNA replication. This latter aspect of the model however remains to be tested experimentally. We have discovered that, in time course experiments after controlled re-expression of pRB in pRB-deficient tumor cells, protein levels of cyclin EfCdk2 and cyclin A/Cdk2, perhaps the most important repression targets of pRB, declined after the onset of GI cell cycle arrest. The kinase activities of these complexes however were inhibited with the onset of Gi arrest by an early increase in the protein levels of the cyclin-dependent kinase inhibitor p27Kipl. We therefore hypothesize that p27 is an early effector of pRB function. In this application, we will achieve two specific aims to test this hypothesis. In aim 1, we will determine the role of p27 level increase in pRB-mediated 01 arrest. Our second aim is to determine the mechanisms by which pRB induces p27 levels in the cell. Although not found mutated in human tumors, p27 has been implicated in tumor prognosis by the fact that more aggressive tumors contain lower levels of p27 due to active protein degradation. Successful completion of the proposed studies will not only provide new knowledge about the functional mechanism of pRB but also reveal a new mechanism for the involvement of p27 in cancer. A better understanding of the complex regulatory networks linking various negative regulators of cell proliferation has the great potential of revealing vulnerable steps for therapeutic targeting in the treatment of cancer, a long-term objective of our research.