ABSTRACT
Rat nasal squamous cell carcinomas induced by inhalation of three direct-acting alkylating agents yielded DNA containing activated oncogenes with no homology to any member of the ras family. The novel NIH 3T3 transforming oncogenes from tumors induced by beta-propiolactone and methylmethane sulfonate are distinct from each other based on restriction analysis. The gene isolated from beta-propiolactone-induced tumors is between 6 and 9 kb in size. None of the tumors induced by dimethylcarbamyl chloride contained positive DNA in the NIH 3T3 focus assay or in the nude mouse cotransfection assay. The rat nasal tumor model is apparently ideally suited for analysis of the roles of carcinogen and tissue specificity in oncogene activation, especially related to novel (non-ras) transforming oncogenes.
Subject(s)
Carcinoma, Squamous Cell/genetics , Nasopharyngeal Neoplasms/genetics , Oncogenes , Proto-Oncogene Proteins/genetics , Animals , Blotting, Southern , Carbamates/toxicity , Carcinoma, Squamous Cell/chemically induced , DNA Restriction Enzymes , DNA, Neoplasm/metabolism , Disease Models, Animal , Gene Expression Regulation/drug effects , Methyl Methanesulfonate/toxicity , Nasopharyngeal Neoplasms/chemically induced , Oncogenes/drug effects , Propiolactone/toxicity , Proto-Oncogene Proteins p21(ras) , Rats , TransfectionABSTRACT
The protease inhibitors antipain, leupeptin, alpha 1-antitrypsin, and epsilon-aminocaproic acid were found to inhibit transformation of NIH3T3 cells after transfection with an activated H-ras oncogene. Inhibition of focus formation by protease inhibitors was concentration dependent and maximal at 50% of control values. Transfection of a gene for neomycin resistance was not affected by protease inhibitors. Antipain was inactive if present only during the first 2 days of the gene transfer protocol or only during the final 10 days of the experiment. However, the full effect was observed when antipain was added at the subculture step on day 3 and during the subsequent cell proliferation. If cells were not subcultured, the yield of the foci per microgram of DNA was sharply reduced and addition of antipain did not further suppress the transformation rate. Subculture of NIH3T3 cells 3 days after transfection at lower cell densities resulted in higher transformation efficiency. The results suggest that transformation of NIH3T3 cells by a single mutated oncogene may involve multiple stages including cell proliferation and that part of this process is susceptible to inhibition by protease inhibitors.
