Characterization of endocrine cell lines immortalized by a temperature-sensitive mutant SV40

An ideal in vitro model for the study of endocrine functions would be one in which cells could propagate in culture and express their specialized functions. Most endocrine studies to date have relied on primary cell culture or on the use of tumor cell lines. This report describes the characterization of three endocrine cell lines immortalized by transfecting endocrine cells with a temperature-sensitive mutant SV40 virus. Rabbit endometrium (HRE-H9), human placenta (SPA209-10) and rat pituitary (RP) cells were immortalized by SV40 virus, a temperature-sensitive (ts) mutant in the A gene, which encodes the large tumor antigen that is required for the maintenance of transformation. The transformed phenotype of the SV40 tsA mutant-immortalized cell line can be reversed simply by a shift in temperature. At the permissive temperature (34 degrees C), all three types of cells exhibited a transformed phenotype, which is characterized by high cell density growth and by the overgrowth of nontransformed cell layers. However, at the non-permissive temperature (40 degrees C) these cells reverted to a non-transformed phenotype as demonstrated by a marked decrease in the overgrowth of nontransformed layers and by the expression of differentiated functions. At the non-permissive temperature (40 degrees C), the endometrial cell line was capable of synthesizing beta-endorphin, and it exhibited hormonally regulated expression of the transfected hybrid uteroferrin gene construct. The human placenta cell line was capable of secreting GnRH upon stimulation by cAMP, forskolin, theophyllin, PGE, catecholamine and Ca++ channel stimulators. Moreover, the rat pituitary cell line was capable of synthesizing and secreting growth hormone (GH) which was stimulated by GHRH and cAMP. The advantage of the temperature-sensitive cell lines is that a single cell line is the source of both the normal and transformed states; thus, studies are internally controlled. These results demonstrate that tsA mutants of SV40 virus are the best available agents for immortalizing mammalian endocrine cells that retain differentiated functions.