Vitamin D Promotes G1 Arrest via Cdk2 Mislocalization

描述（由申请人提供） 维生素D最活跃的代谢物[1，25-（OH）2D 3]抑制几种类型的癌症的生长，最明显的是前列腺癌。 人类前列腺癌细胞系和原代前列腺上皮细胞的范围从强烈生长抑制（例如LNCaP）到对1，25-（OH）2D 3的抗增殖作用不敏感。虽然维生素D受体（VDR）是生长抑制所必需的，但VDR是不够的，如人前列腺癌细胞系阿尔瓦31所例示的，其受到最低程度的抑制，但表达相对高水平的功能性VDR。1，25-（OH）2D 3的抗增殖作用主要通过抑制细胞周期的G1至S进程介导，这在很大程度上是由于细胞周期蛋白依赖性激酶2（Cdk 2）活性降低和诱导细胞周期蛋白依赖性激酶抑制剂（CKIs）p21 Cip 1和p27（ip 1.我们最近进行了新的观察，1，25-（OH）2D 3治疗敏感的人前列腺癌细胞导致Cdk 2错误定位于细胞质。由于Cdk 2被细胞周期蛋白激活激酶（CAK）和Cdc 25 A磷酸酶激活需要Cdk 2的核转位，Cdk 2的胞质螯合将有效地阻止细胞周期蛋白E-Cdk 2活性。1，25-（OH）2D 3介导的Cdk 2核定位的减少和随后的细胞周期蛋白E-Cdk 2结合和激活的减少也可能起到抑制p27降解的作用，这需要细胞周期蛋白E-Cdk 2的磷酸化。我们推测，维生素D介导的Cdk 2的细胞质错误定位是这种营养素抑制前列腺癌生长的关键事件。因此，我们建议研究前列腺癌细胞中Cdk 2核质转运的机制，并确定1，25-（OH）2D 3如何调节这些过程。提出了以下具体目标：确定前列腺癌细胞中Cdk 2核输出的机制和维生素D调节。二.分析Cdk 2核输入的机制及维生素D的调控。三.确定Cdk 2激活和细胞质错误定位对维生素D介导的生长抑制的作用。这些研究将利用当代生物化学，分子生物学和成像技术，包括间接免疫荧光和可视化的高分辨率共聚焦显微镜，以及直接免疫荧光研究后，活细胞光漂白（FRAP和FLIP）。 由于Cdk 2失调与几种人类肿瘤有关，维生素D的这种作用代表了一种独特的机制，用于控制Cdk 2活性，可能用于前列腺癌的化学预防。

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.