Immortalization of equine trophoblast cell lines of chorionic girdle cell lineage by simian virus-40 large T antigen

Immortalized cell lines have many potential experimental applications inclu ding the analysis of molecular mechanisms underlying cell-specific gene exp ression, We have utilized a recombinant retrovirus encoding the simian viru s-40 (SV-40) large T antigen to construct several immortalized cell lines o f equine chorionic girdle cell lineage - the progenitor cells that differen tiate into the equine chorionic gonadotropin (eCG) producing endometrial cu ps. Morphologically, the immortalized cell lines appear similar to normal c horionic girdle cells. Derivation of the immortalized cell lines front a ch orionic girdle cell lineage was verified by immunological detection of cell -surface antigens specific to equine invasive trophoblasts. The cell lines differed, however, from mature chorionic girdle cells or endometrial cup ce lls in that they did not produce eCG and did express MHC class I molecules. Thus, these cell lines appear to have been arrested at a stage of developm ent prior to final differentiation into endometrial cup cells. It,vas also determined that some of these cell lines as well as endometrial cups expres s the estrogen receptor-related receptor beta gene, but not the glial cell missing gene (GCMa) both of which are expressed in the murine and human pla centa. Among these cell lines, three (eCG 50.5, 100.6 and 500.1) express eC G alpha mRNA. Since regulation of eCG alpha subunit gene is largely unknown , we investigated the signal transduction pathways regulating the eCG alpha subunit gene. Both activators of protein kinase A (PKA) and protein kinase C (PKC) induced the expression of eCG alpha subunit expression 3-2 (P<0.05 )- and 1.9 (P<0.05)-fold respectively, in the eCG 500.1 cell line. However, activation of these pathways failed to induce eCG beta subunit expression. In conclusion, lines of equine trophoblast cells have been immortalized th at display markers characteristic of those with the equine chorionic girdle and endometrial cup cell lineage. A subset of these cells expresses the eC G alpha subunit gene which is responsive to activators of the PKA and PKC s ignal transduction pathways,