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The intermediate lobe (IL) of the pituitary produces a PRL-regulating factor (PRF). Targeted tumorigenesis, using the POMC promoter ligated to SV40 large T antigen (Tag), generated transgenic mice that develop IL tumors with PRF activity. Our goal was to establish and characterize a PRF-producing cell line. Two cell lines, which differ markedly in size and morphology, were independently developed from IL tumors and designated mIL5 and mIL39. These cells are transformed, as judged by rapid proliferation, low serum requirements, and generation of secondary tumors in nude mice. RT-PCR revealed that mIL39, but not mIL5 cells, express POMC and dopamine D2 receptors, typical of a melanotroph phenotype. Although mIL5 cells originated from an IL tumor, they do not express messenger RNA for SV40 Tag. The bioassay for PRF used GH3 cells stably transfected with the PRL promoter ligated to a luciferase reporter gene (GH3/luc). Coculture of mIL5 with GH3/luc cells induced cell-density dependent increases in PRL gene expression and release, whereas mIL39 cells showed negligible PRF activity. Incubation of GH3/luc cells with conditioned media from mIL5, but not mIL39 cells, stimulated PRL gene expression and release up to 10-fold. Coculture of mIL5 cells with primary rat anterior pituitary cells stimulated PRL, but not GH, release. Fractionation of mIL5 cell extracts by reverse phase HPLC resolved PRF activity into one major and one minor peak. In conclusion, we have developed two novel and distinct cell lines from mouse intermediate lobe tumors. The first reported melanotroph cell line, mIL39, could provide a valuable model for studying dopaminergic regulation of POMC gene expression and release. In contrast, the mIL5 cells do not express POMC, D2 receptors, or SV40 Tag and appear to have been immortalized by a spontaneous mutation(s). These cells produce and secrete a potent PRF and could be used for the purification and biochemical characterization of PRF.

Two Distinct Pituitary Cell Lines from Mouse Intermediate Lobe Tumors: A Cell that Produces Prolactin-Regulating Factor and a Melanotroph*