MOLECULAR BIOLOGY OF P53 IN PROSTATE CANCER

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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.