Allele loss and point mutation in codons 12 and 61 of the c‐Ha‐ras oncogene in carcinogen‐transformed human breast epithelial cells

There is significant evidence that the ras oncogene plays a role in experimental mammary carcinogenesis; the evidence in human breast cancer, however, is more limited. We induced the expression of transformation phenotypes in the human breast epithelial cell line MCF‐10F with the chemical carcinogens 7,12‐dimethylbenz[a]‐anthracene, N‐methyl‐N‐nitrosourea, N‐methyl‐N‐nitro‐N′‐nitrosoguanidine, and benzo[a]pyrene. This work was designed to clarify whether chemically induced neoplastic transformation correlates with alterations in the ras gene. MCF‐10F cells have two c‐Ha‐ras alleles, identified by 1.0‐kb and 1.2‐kb restriction fragments. Treatment with carcinogens resulted in the loss of one of the alleles (1.0 kb). Polymerase chain reaction–amplified DNA from all carcinogen‐treated cells was analyzed for point mutations in c‐Ha‐ras at codons 12 and 61. All of the carcinogens induced a mutation of the remaining allele at the first position of codon 12 (GGCÀC). Another frequent mutation occurred at the first position of codon 61 (CAG←GAG). The changes in c‐Ha‐ras were associated with the emergence of colony formation in agar‐methocel, but no specific changes in this gene correlated with the emergence of invasiveness or tumorigenesis, indicating that other genes may be involved in the process. © 1994 Wiley‐Liss, Inc.