Our overall aim is to elucidate the early cytogenetic and molecular genetic events underlining estrogen-induced renal tumorigenesis in the hamster kidney and compare these findings to the hamster renal tumor and to our two """"""""semi-immortal"""""""" estrogen-derived kidney cell lines. The strength of this application resides in the experience of two well established groups working with hamster systems with complementary abilities. The collaborating group will develop a battery of expression tagged sites (ETS) to established a physical map of the hamster genome, and eventually a genetic map which will be used to identify the affected genes being altered during renal tumorigenesis. To pursued these aims, the following studies are proposed: 1. Systematic cytogenetic analyses of the kidney tumor and kidney during estrogen-induced tumorigenesis. This will be done by detail studies of the temporal relationship of various cytogenetic alterations during renal tumorigenesis. Nonrandom chromosome alterations (gains and losses) will be determined at monthly intervals of estrogen treatment, they will be related to nonrandom numerical changes, and the frequency and location of fragile sites will be determined on hamster kidney chromosomes in estrogenized kidneys, renal tumor, and DES-derived kidney cell lines. 2. Screen for cDNA probes for protooncogene and other pertinent receptor protein expression during estrogen-induced tumorigenesis. This will be done by hybridization of newly synthesized RNA from hamster kidneys at different stages of estrogen treatment to an array of cDNA clones. The clones that will exhibit changed expression will be confirmed by Northern hybridization. These studies will complement Specific Aims 1 and 4. The study of cellular expression of the cDNA probes will be a prelude to in-situ hybridization studies both on the chromosome and within estrogenized kidney and tumor cells. 3. Create a battery of expression tagged sites (ETS) to establish a physical map of the Syrian hamster genome. Hamsters will be mapped with probes from a random sample of transcription units. A hamster genetic map will be established and will then be used as a road map to identify genes involved in the carcinogenic process. cDNA clones will be isolated, and 150-300 bp regions sequenced from both ends. Sequence information will be used to search the genome database. 4. Establish a genetic map with ETS and other relevant loci. Develop syntenic groups. This will be done by in situ hybridization of hamster metaphase spreads. Syntenic groups will be generated by comparison analysis of hamster data to the mouse and human genome database.
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