MOLECULAR BIOLOGY OF P53 IN PROSTATE CANCER

P53 在前列腺癌中的分子生物学

• 批准号: 6217435
• 负责人: RUSSELL M LEBOVITZ
• 金额: $ 17.47万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2000-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6217435
• 关键词: artificial chromosomes; biomarker; gene deletion mutation; gene expression; genetic library; genetic mapping; genetic markers; human genetic material tag; human tissue; laboratory mouse; loss of heterozygosity; metastasis; molecular cloning; molecular oncology; neoplasm /cancer classification /staging; neoplasm /cancer genetics; nucleic acid sequence; oncogenes; p53 gene /protein; prognosis; prostate neoplasms; transfection; tumor suppressor genes

The search for genetic and environmental factors responsible for the increasing incidence of prostate cancer in our society has been hampered by a dearth of molecular and genetic markers associated with either initiation or progression of prostate neoplasia. Recent studies, however, have shown that at least two prostate tumor suppressor (PTS) genes are likely to be present on the short arm of chromosome 8 (8p) near the MSR 8p22) and ANK (8p11-21) loci, respectively, and that these chromosomal regions undergo loss of heterozygosity (LOH) in at least 50% of clinical prostate cancers. The long-term goal of this project is to identify and characterize PTS genes on 8p, as well as to study their respective role(s) in the development of prostate neoplasia. We will focus initially on 8p22, since presumptive LOH hotspots in this region have recently been identified. Many of the resources developed for the 8p22 studies will also be useful for subsequently mapping the 8p11-21 region. We propose to further localize PTS genes on 8p by mapping lOH breakpoints in this region from an additional 150 prostate tumors. As an important part of this effort, we will clone and map athe genomic sequences on 8p which are deleted most frequently in prostate cancers using YAC, cosmid, and other large-insert libraries. As a prelude to this proposal, we have already isolated and mapped a series of overlapping yeast artificial chromosome (YAC) clones covering the entire 8p22 region, and we have screened a chromosome-8- specific cosmid library with the goal of identifying and mapping approximately 20 cosmid markers spanning 8p22 at approximately 500 kb intervals. Our ability to rapidly develop a highly successful, large scale effort in this area has been greatly facilitated by direct access to a large number of well-characterized human prostate-cancers through the Baylor College of Medicine SPORE and by access to the core facilities of the Baylor College of Medicine human Genoma Center. We are requesting support for five years to; (1) continue our construction of 8p22 and 8p11-21 chromosomal maps including identification of YAC and cosmid markers spaced at approximately 500 kb intervals throughout both regions; (2) Prepare DNA, touch preps, and cytospins from approximately 150 prostate cancer and non-tumor specimens for mapping LOH hotspots identified at 8p22 and 8p11--21; (4) Identify candidate PTS genes which map within these LOH hotspots by cDNA screening, exon trapping, and biological assays; and (5) Prepare YAC and cosmid contigs from mouse chromosomal regions systemic with human 8p22 and 8p11-21. As part of specific aim 3, we will develop a transfection-based functional assay for identification and confirmation of PTS genes using large-insert contigs from lOH hotspot regions. After stable transfection into metastatic rat and mouse prostate carcinoma cell lines; the inserts carrying functional PTS genes will be identified on the basis of their ability to suppress metastasis or growth rate after transplantation into recipient mice. The sensitivity and specificity of this assay will be evaluated initially using a series of YACs and cosmids from 17p, some of which carry a functional p.53 gene.

对遗传和环境因素的研究 我们社会中前列腺癌发病率的上升受到以下因素的阻碍 缺乏与任何一种启动相关的分子和遗传标记 或前列腺瘤的进展。然而，最近的研究表明， 至少有两个前列腺癌抑制因子(PTS)基因可能是 位于8号染色体(8p)的短臂上，靠近MSR 8p22)和ANK (8p11-21)基因座，这些染色体区域经历 至少50%的临床前列腺癌患者存在杂合性缺失(LOH)。 该项目长期目标是确定和描述临时秘书处 8P上的基因，以及它们各自在植物生长发育中的作用(S) 前列腺瘤的发展。我们将首先关注8p22，因为 最近在这一地区发现了推定的陆恭蕙热点。 为8p22研究开发的许多资源也将是有用的。 用于随后定位8p11-21区域。我们建议进一步 定位8P上PTS基因的LOH断裂点 增加150个前列腺癌。作为这一努力的重要组成部分，我们 将在8P上克隆和定位缺失最多的基因组序列 在前列腺癌中经常使用YAC、COSMID和其他大插入物 图书馆。作为这项提议的前奏，我们已经孤立了 定位了一系列重叠的酵母人工染色体(YAC)克隆 覆盖了整个8p22区域，我们已经筛选出一个8号染色体- 特定的粘粒文库，目标是识别和映射 大约20个COSMID标记，跨越8p22，约500kb 间隔时间。我们有能力迅速发展一个非常成功、大规模的 这一领域的努力得到了极大的促进，因为直接访问 大量特征良好的人类前列腺癌通过 贝勒医学院的孢子和通过进入核心设施 贝勒医学院人类基因组中心。我们要求 支持五年；(1)继续建设8p22和 包括YAC标记和粘粒标记的8p11-21染色体图谱 在两个区域中以大约500kb的间隔间隔；(2) 从大约150个前列腺组织中制备DNA、触觉准备材料和细胞旋体 用于定位位于8p22的LOH热点的癌和非肿瘤标本 和8p11--21；(4)确定在这些LOH中定位的候选PTS基因 通过cDNA筛选、外显子捕获和生物检测获得热点；以及(5) 从小鼠系统染色体区域制备YAC和粘粒重叠群 人类8p22和8p11-21。作为具体目标3的一部分，我们将制定一项 基于转染法的功能分析用于鉴定和确认 使用来自LOH热点区域的大插入重叠群的PTS基因。之后 转移性大鼠和小鼠前列腺癌细胞的稳定表达 品系；携带功能性PTS基因的插入片段将在 根据它们抑制转移或生长速度的能力 移植到受体小鼠体内。其敏感性和特异度 这项测试将使用一系列YAC和COSMID进行初步评估 从17P开始，其中一些携带有功能的p.53基因。