2′,5′-oligoadenylate synthetase gene expression in revertants of ras-transformed NIH3T3 fibroblasts

Abstract

Persistent revertant (PR) cells of Ha- ras-transformed NIH3T3 fibroblasts, isolated after prolonged treatment with interferon (IFN), have been previously described. PR cells remain nontumorigenic even after IFN withdrawal. To investigate the mechanisms responsible for the stable phenotypic reversion, we have now examined the potential involvement of an endogenous IFN and the 2′,5′-oligoadenylate (2–5A) synthetase pathway. Northern blot analysis revealed an increased level of 2–5A synthetase transcripts in PR cells compared to parental Ha- ras-transformed cultures. Although inducible on treatment with exogenous IFN α β , this mRNA was not detectable in untreated NIH3T3 cells. 2–5A synthetase expression following IFN treatment was also significantly higher in PRs than in the normal or ras-transformed NIH3T3. The increased levels of synthetase mRNA correlated with a similarly elevated enzymatic activity in cell extracts from PR cells. This increased expression was biologically functional, since the revertant cells were more resistant to the cytolytic action of mengovirus than normal or ras-transformed NIH3T3 fibroblasts. Another class of IFN-induced genes, H-2 class I antigens, showed enhanced expression in PRs. Antibodies directed against mouse IFN α β did not reduce the constitutive expression of 2–5A synthetase in PR cells. Furthermore, conditioned medium from PR cultures or cocultivation with PRs failed to induce the enzyme message in NIH3T3 cells. Finally, there was no detectable elevation in the mRNA specific for IFN β in the PR cultures, as determined using a sensitive polymerase chain reaction amplification protocol. These results show that the Ha- ras revertants constitutively produce a functional 2–5A synthetase, which appears to be independent of the production of an endogenous interferon α or β.