To investigate the role of ras mutations in thyroid epithelial tumorigenesis, we introduced wild-type or mutant v- or c-Ha-ras genes into a sub-cloned rat thyroid follicular epithelial cell line using retroviral vectors and a neutral selection method (G418 resistance). Mutant, but not wild-type, ras induced a spectrum of clonal phenotypes. Interestingly, many clones showed an unchanged phenotype despite ras expression at levels greater than that of the endogenous gene. Further increase in ras expression was associated with altered morphology, loss of thyroid-specific differentiation (thyroglobulin synthesis) and growth factor independence, but not. anchorage-independence or tumorigenicity. The mutation site (codon 12 or 61) did not have a significant influence. The results clearly emphasize the critical importance of expression level in determining the phenotypic effect of mutant ras on epithelial cells.

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