MOLECULAR BIOLOGY OF P53 IN PROSTATE CANCER

寻找遗传和环境因素， 在我们的社会中，前列腺癌发病率的增加受到以下因素的阻碍： 缺乏与这两种起源相关的分子和遗传标记， 或前列腺肿瘤的进展。 然而，最近的研究表明， 至少有两个前列腺肿瘤抑制基因（PTS）可能是 存在于8号染色体短臂（8p），靠近MSR 8p22）和ANK （8p11 - 21）基因座，这些染色体区域， 在至少50%的临床前列腺癌中存在杂合性缺失（洛）。 该项目的长期目标是确定和描述临时秘书处的特点 8p上的基因，以及研究它们各自在 前列腺肿瘤的发展。 我们将首先关注8p22，因为 最近已经确定了该区域中推定的洛缺失热点。 为8p22研究开发的许多资源也将是有用的 用于随后绘制8p11 - 21区域。 我们建议进一步 PTS基因定位在8p上，通过从一个基因组中定位该区域中的10个断点， 另外150个前列腺肿瘤。 作为这项工作的重要组成部分，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、粘粒和其他大插入片段 图书馆. 作为这一建议的前奏，我们已经分离和 绘制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，我们筛选了一条染色体-8- 特异性粘粒文库，目的是鉴定和定位 跨越8p22约500 kb的约20个粘粒标记 的间隔 我们能够迅速发展一个非常成功的，大规模的 这方面的努力得到了很大的促进，因为可以直接进入一个 大量特征良好的人类前列腺癌通过 贝勒医学院孢子和访问的核心设施， 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， 8p11 - 21染色体图谱，包括YAC和粘粒标记的鉴定 在整个两个区域中以大约500 kb的间隔隔开;（2） 从大约150个前列腺样本中制备DNA、接触制备物和细胞离心涂片 用于定位在8p22鉴定的洛缺失热点的癌症和非肿瘤标本 （4）鉴定定位在这些洛性缺失中的候选PTS基因 通过cDNA筛选、外显子捕获和生物测定的热点;和（5） 从小鼠染色体区域系统性制备YAC和粘粒重叠群， 人8p22和8p11 - 21。 作为具体目标3的一部分，我们将制定一项 基于转染的功能测定，用于鉴定和确认 使用来自10H热点区域的大插入重叠群的PTS基因。 后 稳定转染转移性大鼠和小鼠前列腺癌细胞 携带功能性PTS基因的插入片段将在 它们抑制转移或生长速度的能力的基础， 移植到受体小鼠中。 的敏感性和特异性 最初将使用一系列YAC和Cosmetic对该测定法进行评价 其中一些携带功能性p.53基因。

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.