Vitamin D Promotes G1 Arrest via Cdk2 Mislocalization

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DESCRIPTION (provided by applicant) The most active metabolite of vitamin D [1,25 dihydroxyvitamin D3, (1,25-(OH)2D3)] inhibits the growth of several types of cancer, most notably prostate. Human prostate cancer cell lines and primary prostate epithelial cells range from strongly growth inhibited (e.g. LNCaP) to insensitive to the antiproliferative effects of 1,25-(OH)2D3. While the vitamin D receptor (VDR) is required for growth inhibition, VDR is not sufficient as exemplified by the human prostate cancer cell line, ALVA 31, which is minimally inhibited yet expresses relatively high levels of functional VDR. The antiproliferative effects of 1,25-(OH)2D3 are mediated primarily by inhibition of G1 to S progression of the cell cycle due, in large part, to decreased cyelin dependent kinase 2 (Cdk2) activity and induction of cyclin dependent kinase inhibitors (CKIs), p21Cip1 and p27(ip1. We recently made the novel observation that 1,25-(OH)2D3 treatment of sensitive human prostate cancer cells results in the mislocalization of Cdk2 to the cytoplasm. Since Cdk2 activation by cyclin-activating kinase (CAK) and the Cdc25A phosphatase requires nuclear translocation of Cdk2, cytoplasmic sequestration of Cdk2 would effectively prevent cyclin E-Cdk2 activity. The 1,25-(OH)2D3- mediated decrease in Cdk2 nuclear localization and subsequent decreased cyclin E-Cdk2 association and activation may also act to inhibit p27 degradation, which requires phosphorylation by cyclin E-Cdk2. We speculate that vitamin D-mediated cytoplasmic mislocalization of Cdk2 is the critical event in prostate cancer growth inhibition by this nutrient. We therefore propose to investigate the mechanisms underlying Cdk2 nueleo-cytoplasmic transport in prostate cancer cells and to define how 1,25-(OH)2D3 regulates these processes. The following specific aims are proposed: I. Identify the mechanism(s) and vitamin D regulation of nuclear export of Cdk2 in prostate cancer cells. II. Analyze the mechanisms and vitamin D regulation of Cdk2 nuclear import. III. Determine the role of Cdk2 activation and cytoplasmic mislocalization on vitamin D-mediated growth inhibition. These studies will utilize contemporary biochemical, molecular biological and imaging techniques including indirect immunofluoresence and visualization by high resolution confocal microscopy as well as direct immunofluoreseence studies in live cells after photobleaching (FRAP and FLIP). Since Cdk2 deregulation is associated with several human tumors, this effect of vitamin D represents a unique mechanism for control of Cdk2 activity that might be exploited in chemoprevention of prostate cancer.