Cotransfection of granulosa cells with simian virus 40 and Ha-RAS oncogene generates stable lines capable of induced steroidogenesis.

Cellular and viral oncogenes are usually defined on the basis of their ability to elicit neoplastic transformation. However, oncogene activity has also been implicated in the control of differentiation. We have tested whether transfection of primary cultured granulosa cells with various oncogenes can yield cell lines that maintain their differentiated properties. Primary granulosa cells were prepared from diethylstilbestrol-treated immature female rats and transfected with simian virus 40 (SV40) DNA or with SV40 plus activated human Ha-RAS oncogene. Transfection with SV40 plus Ha-RAS yielded cell lines that lost response to gonadotropins but, after 48 hr of stimulation with isoproterenol, cholera toxin, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), produced progesterone at levels comparable to those of differentiated primary cells. In contrast, cells transformed only by SV40 lost their ability to produce progesterone. Whereas in primary cell cultures progesterone production was already evident after a 3-hr incubation with 1 mM 8-Br-cAMP, in cotransfected cells progesterone production became evident only after 12 hr. All cotransformed cell lines produced SV40 large tumor antigen as well as human RAS p21 protein. The expression of the expected oncogenes in the various cell lines was confirmed by mRNA analysis. These results suggest that the expression of an activated RAS oncogene in granulosa cells can play a role in preserving inducible steroidogenesis.