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. 1988 Jun;85(11):3875–3879. doi: 10.1073/pnas.85.11.3875

Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: sequence analysis of an enzymatically amplified mutant HRAS allele.

C W Stevens 1, T H Manoharan 1, W E Fahl 1
PMCID: PMC280322  PMID: 3131765

Abstract

Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies (11 X 10(-4) consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo[a]pyrene (+/-)anti- 7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cell DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed i in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. Direct sequence analysis of the amplified DNA indicated equal presence of a wild-type (GGC) and mutant (GTC) allele of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC----TA codon 12 transversions that have been commonly observed in human tumors. This oncogene as well as yet to be identified oncogene are also shown to stably confer anchorage-independence to human cells.

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