Tumorigenic methylcholanthrene transformants of C3H/10T1/2 cells have a common nucleotide alteration in the c-Ki-ras gene.

Abstract

The polymerase chain reaction was used to amplify DNA surrounding the codon 12 region of the c-Ki-ras gene from C3H/10T1/2 cells and from a number of 3-methylcholanthrene (MCA)-transformed derivatives of these cells. Sequence analysis demonstrated that tumorigenic MCACl16/39 cells, known by DNA-mediated transfection to contain an activated c-Ki-ras oncogene, had a G --T transversion in the first position of codon 12 of this gene, resulting in a Gly' -*2 Cys mutation. A combination of polymerase chain-reaction amplification and oligonucleotide hybridization demonstrated that three additional tumorigenic MCA transformants of C3H/1OT/2 cells had an identical mutation in the c-Ki-ras gene. In contrast, this mutation was not present in an MCA-induced C3H/1OT/2 transformant that was not tumorigenic. The molecular specificity of this MCA-induced mutation resulting in C3H/1OT/2 tumorigenic transformants should provide an excellent system in which to study the roles of transcription, replication, repair, and exogenous factors in the establishment and expression of transformation and tumorigenicity.It has been argued that transformation in cell culture in response to chemical carcinogens should be a valuable model to elucidate mechanisms of carcinogenesis, because of the opportunity to closely control variables such as time of exposure to carcinogens, concentration of chemicals, cell cycle position, exposure to secondary effectors such as tumor promoters, etc. The murine C3H/10T1/2 cell transformation system, first described by Heidelberger and his colleagues (1), has been used to identify a wide variety of complete carcinogens, initiators, and promoters. DNA samples isolated from tumors derived from 3-methylcholanthrene (MCA)-transformed C3H/10T1/2 cell lines were among the first tumor DNA samples to be used to demonstrate the presence of dominant transforming oncogenes by DNAmediated transfection (2). The activated oncogene in three tumors derived from MCA-transformed C3H/10T1/2 cells was identified as a c-Ki-ras gene (3).Despite the early description of dominant oncogenes in MCA-transformed tumorigenic C3H/10T1/2 cells, the specific mutations associated with activation of oncogenes as a consequence of chemical carcinogenesis have been described in experimental animals, not in cultured cells. Exposure of female rats to the direct-acting carcinogen N-methyl-Nnitrosourea caused mammary tumors with an identical (G35 -+ A) transition in the second position of codon 12 of the c-Ha-ras gene in all 48 tumors analyzed (4). The extraordinary specificity ofthis mutation is surprising, since (i) studies with a shuttle vector (5) suggest that mutations in the 5' G residue of GG pairs also occur in response to N-methyl-N-nitrosourea in mammalian cells and (ii) mutation at the first G residue of codon 12 of the c-Ha-ras gene is also transforming (6). In contrast, Reynolds et al. (7) have shown that a single carcinogen, furfural, can lead both to distinct mutations within the c-Ha-ras gene and to mutations in different ras gen...

Ki‐ras oncogene mutations in tumors and DNA adducts formed by benz[j]aceanthrylene and benzo[a]pyrene in the lungs of strain A/J mice

“…This hypothesis is based upon published data regarding the demethylated, saturated analogue of BUIA, 3-methylcholanthrene (3-MCA). The major adduct of 3-MCA with DNA is also formed with dGuo; however, in Ki-ras codon 12, 3-MCA produces mostly the GGT-TGT mutation at a frequency that mimics that of B[a]P [17,18]. When 3-MCA produces a Ha-ras mutation in mouse tissues, the mutation is usually a first-position mutation in codon 13 (GGC+TGC) [I 91, presumably because a Ha-ras codon 12 GGAjTGA mutation codes for termination (i.e., TGA is a stop codon).…”

Activation of ras oncogenes and expression of tumor‐specific transplantation antigens in methylcholanthrene‐induced murine fibrosarcomas

“…In fact, mutations at 2 different rus genes coexisted in 1 sarcoma, whereas another tumor displayed 2 different mutations (at positions 12 and 13) in the K-rus gene. It is entirely possible that multiplicity of mutations may reflect the different transformation system andlor the higher number of tumors examined in our study, as compared with those of others (Chen and Herschmann, 1989;Eva and Aaronson, 1983). Even the dose of 3-MCA may have played a role, since the sarcomas expressing multiple mutations all belong to the highest dose-treatment group.…”

“…In the present work we extend our previous report (Borrello et ul., 1988) by showing that 3-MCA can activate not only K-rus, as suggested by others (Chen and Herschmann, 1989;Eva and Aaronson, 1983;Eva and Trimmer, 1986) but also N-rus genes of the induced murine sarcomas. No evidence of H-rus activation was obtained in 5 transfection experiments.…”

Effect of myogenic determination on tumorigenicity of chemically transformed 10T1/2 cells

“…Treatment of 1 OT1/2 cells with the aromatic hydrocarbon 3-methylcholanthrene (MCA) results in a highly transformed phenotype, giving rise to type 111 foci in vitro and forming fibrosarcomas with short latency periods in nude or irradiated mice [2,18]. Further analysis of MCA-treated 1 OT1/2 cells has shown that their transformed character is due to the activation of the Ki-rds gene, specificallybya G jTtransversion incodon 12 [19,20].…”