A transforming gene isolated from T24 human bladder carcinoma cells is closely related to the BALB murine sarcoma virus (MSV) onc gene (v-bas). This transforming gene is localized to a 4.6 kilobase pair (kbp) region and is expressed as a 1.2-kbp polyadenylated transcript, which contains v-bas related sequences. Moreover, antisera known to detect the immunologically related onc gene products of BALB- and Harvey-MSVs recognized elevated levels of a related protein in T24 cells. The normal human homologue of v-bas was found to be indistinguishable from the T24 oncogene by heteroduplex and restriction enzyme analysis. These results imply that rather subtle genetic alterations have led to the activation of the normal human homologue of v-bas as a human transforming gene.

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The presence of dominant transforming genes in human tumor cell lines has been investigated. High molecular weight DNAs isolated from celllines established from carcinomas and sarcomas ofvarious organs as well as from a glioblastoma and two melanomas were utilized to transfect NIH/3T3 mouse fibroblasts. The DNAs of T24 and A2182, two cell lines derived from a bladder and a lung carcinoma, respectively, and of HT-1080, a cell line established from a fibrosarcoma, were able to transform recipient NIH/3T3 cells. First-cycle transformants exhibited anchorage-independent growth and were tumorigenic in athymic and immunocompetent mice. Moreover, they contained human DNA sequences and were able to transmit their malignant phenotype in additional cycles of transfection. Southern blot analysis of T24-derived. transformants-showed that a single fragment of human DNA specifically cosegregated with the malignant phenotype, suggesting that it contained the T24 oncogene. Therefore, these human sequences were molecularly cloned with A Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated AT24-15A, was shown to contain a 15-kldobasepair EcoRI insert of human cellular DNA. AT24-15A DNA (either intact or EcoRI digested) transformed NIH/3T3 fibroblasts with a specific activity of20,000 focus-forming units per pmol of'cloned DNA. Our results indicate that we have molecularly cloned a biologically active oncogene present in T24 human bladder carcinoma cells.

Morphologic transformation of NIH 3T3 mouse cells occurs upon transfection of these cells with large amounts (greater than or equal to 10 micrograms) of recombinant DNA molecules carrying the normal human H-ras-1 proto-oncogene. We provide experimental evidence indicating that transformation of these NIH 3T3 cells results from the combined effect of multiple copies of the H-ras-1 proto-oncogene rather than from spontaneous mutation of one of the transfected H-ras-1 clones (E. Santos, E.P. Reddy, S. Pulciani, R.J. Feldman, and M. Barbacid, Proc. Natl. Acad. Sci. USA 80:4679-4683, 1983). Levels of H-ras-1 RNA and p21 expression are highly elevated in the NIH 3T3 transformants, and in those cases examined, these levels correlate with the malignant properties of these cells. We have also investigated the presence of amplified ras genes in a variety of human carcinomas. In 75 tumor biopsies, we found amplification of the human K-ras-2 locus in one carcinoma of the lung. These results indicate that ras gene amplification is an alternative pathway by which ras genes may participate in the development of human neoplasia.