Oncogenes in human tumor cell lines: molecular cloning of a transforming gene from human bladder carcinoma cells.

Abstract

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.

Suppression of anchorage-independent growth of human cancer cell lines by the drs gene

“…The simplest explanation for the reduced expression of h-drs mRNA in human cancer cell lines is that expression of h-drs mRNA is downregulated by the activation of endogenous oncogenes. In fact, the activation of endogenous proto-oncogenes such as ras and src has been reported for colon, pancreatic, gastric, and bladder carcinoma cell lines including LoVo, DLD-1, WiDr, and T24 (Pulciani et al, 1982;Shih and Weinberg, 1982;Taparowsky et al, 1982;Der and Cooper, 1983;Bolen et al, 1987;Bos, 1989;Cartwright et al, 1989;Shirasawa et al, 1993;Nagase et al, 1997). Another possible mechanism for the downregulation of the h-drs gene involves regulation by the APC-bcatenin pathway.…”

“…The T24 bladder carcinoma cell line is known to have an activating mutation of the endogenous H-ras gene that seems to be responsible for the malignant phenotype of this cell line (Pulciani et al, 1982;Shih and Weinberg, 1982;Taparowsky et al, 1982). To test whether the expression of drs suppresses the down- stream pathways of activated H-ras, we investigated the activation of Erk2 kinase and the expression of cfos, c-jun and c-myc mRNA in both T24-BP and T24-drs cells under adhesion or suspension culture conditions.…”

Evidence of oncogene-induced senescence in thyroid carcinogenesis

“…Cells were refed 24 h later and subjected to G418 (400 mg/ml) selection. Plasmid vectors carrying TRK-T3 and H-RAS G12V oncogenes have been previously described (Pulciani et al 1982, Greco et al 1995.…”

p53 mutations have no additional prognostic value over stage in bladder cancer

“…Restriction fragment length polymorphism The DNA probes used were as follows: chromosome 9q, EFD 126 (Nakamura et al, 1987); chromosome lip, H-ras (Pulciano et al, 1982); chromosome 16q, pV962 (Mansouri et al, 1988) and79.2.23 (Bufton et al, 1986); chromosome 17p, 144D6 (Schwartz et al, 1988); chromosome 18q, 15.65 (Fearon & Vogelstein, 1990). RFLP analysis was performed on the 24 patients from whom normal DNA was available (Poddighe et al, submitted).…”