P16 GENE FAMILY IN HEALTH AND DISEASE

这项提案的目的是确定p16家族的作用， 细胞周期蛋白依赖性激酶抑制剂在人类癌症发生中的作用。 是 很明显，伴随肿瘤的细胞生长的变化 转化必须转化为细胞调节的变化 分裂周期 我们最近在细胞周期中发现了一个新的基序 控制，即通过以下抑制剂调节细胞周期进程： 细胞周期蛋白依赖性激酶（CDKs）。 p16家族蛋白是特异性的 调节细胞周期蛋白依赖性激酶（CDK 4和GDK 6）的抑制剂 通过Gi。p16家族目前包含两个成员， p16 INK 4A和p15 INK 4 B。 p15和p16基因串联排列， 人类肿瘤中常见染色体异常位点（9 p21）， 说明它们是肿瘤抑制剂 p15和p16也经常 在广泛的癌症中缺失或突变。 在本申请中， 我们建议研究p15和p16在细胞调控中的作用， 研究这些基因的缺失如何导致 肿瘤性转化 在具体目标#1中，我们建议隔离 人和鼠p16家族成员的完全补体。 这将是 通过多种方法完成，包括低严格PCR、低 严格杂交、双杂交筛选和蛋白质 纯化/反向遗传学。 在具体目标#2中，我们检查了 p15和p16的启动子结构和转录调控 基因. 在p16的情况下，我们的目的是获得其功能的线索 在控制正常细胞增殖方面。 对于p15，一个目标是 有助于了解TGF-β信号转导 级联。 在具体目标#3中，我们将探索三维 使用X射线晶体学、NMR和分子建模对p16的结构进行了研究。 再加上广泛的诱变，p16结构的知识应该 提供了深入了解p16-CDK相互作用。 #4目标明确 研究p15和p16在人类癌症中的作用。 一是 将决定p15和p16缺失和突变的频率， 原发性人类肿瘤 第二，我们将测试p15和/或p16是否可以 逆转转化细胞的致瘤表型。 最后为 直接解决p15和p16在癌症发展中的作用，我们 将产生基因敲除的小鼠，它们特别缺乏这两种基因之一， 基因.

The goal of this proposal is to determine the role of the p16 family Of cyclin dependent kinase inhibitors in the genesis of human cancer. It is clear that the changes in cell growth that accompany neoplastic transformation must translate into changes in-the regulation of the cell division cycle. We have recently discovered a new motif in cell cycle control, namely regulation of cell cycle progression by inhibitors of cyclin dependent kinases (CDKs). p16 family proteins are specific inhibitors of cyclin dependent kinases (CDK4 and GDK6) that regulate passage through Gi. The p16 family currently contains two members, p16INK4A and p15INK4B. The p15 and p16 genes are tandemly arrayed at a site of frequent chromosomal abnormality in human tumors (9p21), implicating them as tumor suppressors. p15 and p16 are also frequently deleted or mutated in a broad spectrum of cancers. In this application, we propose to examine the role of p15 and p16 in the control of cell proliferation and to investigate how loss of these genes contributes to neoplastic transformation. In Specific Aim #1, we propose to isolate the complete complement of human and murine p16 family members. This will be accomplished through multiple approaches including low stringency PCR, low stringency hybridization, two-hybrid screening and protein purification/reverse genetics. In Specific Aim #2, we examine the promoter structure and transcriptional regulation of the p15 and p16 genes. In the case of p16, our purpose is to gain clues to its function in the control of normal cell proliferation. With p15, one goal is to contribute to an understanding of the TGF-beta signal transduction cascade. In Specific Aim #3, we will probe the three dimensional structure of p16 using X-ray crystallography, NMR and molecular modeling. Coupled with extensive mutagenesis, knowledge of the p16 structure should provide insight into the p16-CDK interaction. Specific Aim #4 is targeted toward investigating the role of p15 and p16 in human cancer. First, we will determine the frequency of p15 and p16 deletions and mutations in primary human tumors. Second, we will test whether p15 and/or p16 can revert the tumorigenic phenotype of transformed cells. Finally, to directly address the role of p15 and p16 in the development of cancer, we will generate knockout mice which specifically lack one of both of these genes.