BI-TRANSGENIC MICE REVEAL THAT K-RAS(VAL12) AUGMENTS A P53-INDEPENDENT APOPTOSIS WHEN SMALL-INTESTINAL VILLUS ENTEROCYTES REENTER THE CELL-CYCLE

Studies in cell culture systems have indicated that oncogenic forms of Ras can affect apoptosis. Activating mutations of Ras occur in simila r to 30% of all human tumors and 50% of colorectal carcinomas, Since t hese mutations appear at early or intermediate stages in multistep jou rneys to neoplasia, an effect on apoptosis may help determine whether initiated cells progress towards a more neoplastic state, We have test ed the effects of K-ras(Val12) on apoptosis in transgenic mice, A line age-specific promoter was used to direct expression of human K-ras(Val 12), with or without wild-type (wt) or mutant SV-40 T antigens (TAg), in postmitotic villus enterocytes, the principal cell type of the smal l intestinal epithelium, Enterocytes can be induced to reenter the cel l cycle by TAgWt. Reentry is dependent upon the ability of TAg to bind pRB and is associated with a p53-independent apoptosis. Analyses of K -ras(Val12) x TAgWt bi-transgenic animals indicated that K-ras(Val12) can enhance this apoptosis threefold but only in cycling cells; increa sed apoptosis does not occur when K-ras(Val12) is expressed alone or w ith a TAg containing Glu(107,108) --> Lys(107,108) substitutions that block its ability to bind pRB. Analysis of bi-transgenic K-ras(Val12) x TAgWt mice homozygous for wild-type or null p53 alleles established that the enhancement of apoptosis occurs through a p53-independent mec hanism, is not attributable to augmented proliferation or to an increa se in abortive cell cycle reentry (compared to TAgWt mice), and is not associated with detectable changes in the crypt-villus patterns of ex pression of apoptotic regulators (Bcl-2, Bcl-x(L), Bak, and Bar) or me diators of epithelial cell-matrix interactions and survival (e.g., alp ha(5) beta(1) integrin and its ligand, fibronectin), Coexpression of K -ras(Val12) and TAgWt produces dysplasia. The K-ras(Val12)-augmented a poptosis is unrelated to this dysplasia; enhanced apoptosis is also ob served in cycling nondysplastic enterocytes that produce K-ras(Val12) and a TAg with a COOH-terminal truncation The dysplastic epithelium of K-ras(Val12) x TAgWt mice does not develop neoplasms. Our results are consistent with this finding: (a) When expressed in initiated enteroc ytes with a proliferative abnormality, K-ras(Val12) facilitates progre ssion to a dysplastic phenotype; (b) by diminishing cell survival on t he villus, the oncoprotein may impede further progression; and (c) add itional mutations may be needed to suppress this proapoptotic response to K-ras(Val12).