Cloning of genes expressing in the early stage of rat bladder carcinogenesis induced by BBN

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We have been studying inactivation of tumor suppressor genes in human urothelial cancer cells. Along with analyzes using human tumor tissues, experimental models should provide data in the early stage of carcinogenesis or in precancerous lesions. A recently developed technique of differential RT-PCR allows us to detect expression of genes highly expressed in such primary lesions. It would also be possible to identify human homologues from clones isolated by the differential RT-PCR method. Total cytoplasmic RNA was isolated from hyperplastic lesions, papilloma, and carcinoma in situ of the rat bladder treated with BBN for six months. Differential display was performed with several sets of random primers and oligo dTs using a normal bladder mucosa as a control. Forty-two bands were chosen because they were specificly found or their intensity was increased. Confirmation of specific expression in each lesions was performed using Northern blot or quantitative RT-PCR assay. Due to a limited amount of RNA from the primary lesions, quantitative RT-PCR was effective for such evaluation. Further characterization of the isolated clones are ongoing.