A number of different oncogenes have been isolated from different human and animal tumors and tumor cell lines by virtue of their ability to transform NIH/3T3 mouse fibroblasts in DNA transfection assays. However, it is not clear whether and how these oncogenes are related to carcinogenesis in vivo. An oncogene has been isolated by the DNA transfection protocol from 2 different human bladder carcinoma cell lines, EJ and T24, and was shown to be homologous to Ha-ras, the oncogene of Harvey murine sarcoma virus. We propose to study the structure, expression and function of this gene and other related ras genes in bladder tumors either isolated from human patients or induced in rats and mice by the carcinogens FANFT (N-4-(5-nitro-2-furyl)-2-thizolyl) formamide and BBN (butyl-4-hydroxybutylnitrosamine). Specifically, we propose the following: 1) The levels of ras mRNA in FANFT and BBN induced bladder tumors and normal bladders will be quantitated to see whether changes in the level of ras gene expression correlate with carcinogenesis. The level of ras mRNA in bladders will be quantitated by RNA dot blot hybridization during the course of tumor development in mice and rats fed with FANFT or BBN. In situ hybridization to tissue sections will be used to ask, at the single cell level, whether enhanced oncogene expression is correlated with morphological transformation. 2) The ras genes as well as DNAs complimentary to ras mRNAs (ras cDNAs) from several FANFT induced bladder tumors and from normal bladders will be isolated by molecular cloning. These sequences will be compared in terms of primary structure and ability to transform NIH/3T3 cells and other normal fibroblast cells in transfection assays. 3) Human bladder tumor biopsies will be screened for the level of oncogene RNA by dot blot and in situ hybridization. Mutations in the ras protein coding sequences will be analyzed by cloning and sequencing ras cDNAs. Oncogene translocation will be analyzed by cytological hybridization to chromosomes. We hope that these studies will provide some insight into the role of oncogenes and protooncogenes in cancer. Also, these studies might be useful in the development of assays for the diagnosis and prognosis of bladder cancer in humans.
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