P16 GENE FAMILY IN HEALTH AND DISEASE

这项提案的目标是确定p16家族在 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。它是 很明显，伴随肿瘤而来的细胞生长变化 转化必须转化为细胞调节的变化 组织周期。我们最近在细胞周期中发现了一个新的基序 控制，即通过抑制因子对细胞周期进程的调节 细胞周期蛋白依赖性蛋白激酶(CDK)。P16家族蛋白具有特异性 调控细胞周期蛋白依赖性激酶的抑制物(CDK4和GDK6) 通过美国退伍军人。P16家族目前包含两个成员， P16INK4a和p15INK4b。P15和p16基因排列在一个 人类肿瘤中常见的染色体异常部位(9p21) 暗示它们是肿瘤抑制因子。P15和p16也经常出现 在广泛的癌症中缺失或突变。在此应用程序中， 我们建议研究p15和p16在细胞调控中的作用。 并研究这些基因的丢失是如何导致 肿瘤性转化。在特定的目标#1中，我们建议将 人类和小鼠p16家族成员的完整补充。这将是 通过多种方法完成，包括低严格的PCR、低 严格杂交、双杂交筛选和蛋白质 纯化/反向遗传学。在特定的目标#2中，我们检查 P15和p16基因启动子结构及其转录调控 基因。以p16为例，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 有助于理解转化生长因子-β信号转导 卡斯卡德。在具体目标#3中，我们将探索三维 用X射线结晶学、核磁共振和分子模拟研究了p16的结构。 再加上广泛的突变，对p16结构的了解应该 深入了解p16-CDK的相互作用。具体目标#4有针对性 研究p15和p16在人类癌症中的作用。首先，我们 将决定p15和p16基因缺失和突变的频率 人类原发肿瘤。其次，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，为了 直接解决p15和p16在癌症发展中的作用，我们 会产生特别缺乏这两种基因的基因敲除小鼠 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.