Characterization of a prolactin-synthesizing rat pituitary cell line immortalized by the temperature-sensitive mutant SV40.

A prolactin (PRL) synthesizing rat anterior pituitary (AP) cell line named tAP-CLC-2 was established by transformation with mutant temperature-sensitive A (tsA) simian virus 40 (SV40). The transformed cells were temperature-sensitive for morphology, cell propagation, and expression of phenotypic genes. Monolayer primary rat AP cells were transfected with a mutant SV40 (tsA 205) at 10 times the multiplicity of infection (MOI) at 34 degrees C. After cell clones formed, each clone was isolated and transferred to separate flask until sufficient cells were grown for freezing and characterization. At the permissive temperature (34 degrees C), these cells were spindle-shaped and grew rapidly like tumor cells. However, at the nonpermissive temperature (40 degrees C), the cells exhibited a rounded shape and ceased to propagate because the gene for maintenance of transformation was not expressed. Cell extracts from the cell line tAP-CLC-2 showed an inhibition curve parallel to that of normal rat pituitary cell extracts in a PRL radioimmunoassay (RIA). The gel filtration profile of immunoassayable PRL obtained from fast performance liquid chromatography (FPLC) showed that tAP-CLC-2 cell extract exhibited a PRL peak coincidental with primary rat pituitary cell extracts. Western blot showed that cell extracts from the tAP-CLC-2 cell line and from normal rat pituitary glands shared a similar, major immunoreactive PRL band. The tAP-CLC-2 cell line was responsive to estradiol (E 2; 10(-7) M), progesterone (10(-8) M), and gonadotropin-releasing hormone (GnRH; 10(-9) M) treatments. These hormonal treatments increased (p < 0.05) cell PRL content. Interestingly, treatment with a high dose (10(-7) M) of ovine luteinizing hormone (oLH) also increased (p < 0.05) PRL content in the cell line; a low dose (10(-9) M) of oLH did not. The cell line appeared to synthesize growth hormone (GH) as well. These results indicate this cell line has properties shared by primary AP cells and can provide a unique model for the study of the synthesis and gene regulation of PRL in vitro.