Activation of mitogen-activated protein kinase is necessary but not sufficient for proliferation of human thyroid epithelial cells induced by mutant Ras

Given the high frequency of ras oncogene activation in several common human cancers, its signal pathways are an important target for novel therapy, Fo r practical reasons, however, these have been studied mainly in the context of transformation of established fibroblast cell lines, whereas ras acts a t an earlier stage in human tumorigenesis and predominantly on epithelial c ells, Here we have developed a more directly relevant model-human primary t hyroid epithelial cells-which are a major target of naturally-occurring Pas mutation, and in which expression of mutant Pas in culture induces clonal expansion without morphological transformation, closely reproducing the phe notype of the corresponding tumour in vivo, Transient or stable expression of mutant H-ras (by scrapeloading or retroviral infection) at levels which stimulated proliferation induced sustained activation and translocation of MAP kinase (MAPK) in these cells, inhibition of the MAPK pathway at the lev el of MAPKK, by expression of a dominant-negative mutant or by the pharmaco logical inhibitor PD98059, efficiently blocked the proliferative response, Conversely, selective activation of MAPK by a constitutively-active MAPKK1 mutant failed to mimic the action of Pas and, although this was achievable with activated Raf, micro-injection of anti-ras antibodies showed that this still required endogenous wild-type Ras function, In contrast to recent re sults obtained with a rodent thyroid cell line (WRT), therefore, activation of the MAPK pathway is necessary, but not sufficient, for the proliferogen ic action of mutant Pas on primary human thyroid cells, These data emphasiz e the unreliability of extrapolation from cell lines and establish the feas ibility of using a more representative human epithelial model for Pas signa lling studies.