GCNF as a growth regulator in normal and neoplastic cells
Germ cell nuclear factor (GCNF/NCNF/RTR, NR6A1) is an orphan member of the nuclear receptor super family that exhibits repressive transcriptional activity and is expressed in the embryonic stem cells, embryonic carcinoma cells and reportedly relegated to the germ line in adults. We have demonstrated that GCNF mRNA and protein are expressed in four of the four ovarian cancer cell lines tested: two adenocarcinomas, OVCAR-3 and TOV-112; one clear-cell carcinoma, ES-2; and one, teratocarcinoma, PA-1. The four cell lines exhibited varied morphologies in culture and exhibited markedly different growth rates in vitro. Immuno blot analysis of GCNF protein expression by the cell lines revealed several GCNF antibody-specific bands, the sizes of which were consistent with the reported homodimeric and multimeric associations of GCNF. In addition, GCNF mRNA was highly expressed in mink lung epithelial cells (MLEC), a rapidly proliferating, non-transformed epithelial cell line. Taken together, these findings suggest a relationship between GCNF and cell proliferation. GCNF gene silencing was employed to investigate this relationship in ovarian cancer cells maintained in vitro with the help of siRNA synthesized by Santa Cruz Biotechnology. The results showed that GCNF siRNA treatment decreased the monolayer cell content by 45.6% and 36.2% of ES-2 and TOV-112 cells, relative to untreated and control siRNA-treated cells. Consistent with the observed decline in cell proliferation, GCNF siRNA treatment decreased the cellular content of GCNF mRNA by 37.6% in TOV-112 cells. These preliminary results implicate GCNF as a here-to-fore unrecognized and potentially important growth regulator in ovarian cancer cells. Another experiment was also undertaken to determine the effects of growth suppression upon GCNF expression using a non-transformed cell line, the mink lung epithelial cells (MLEC). These non-transformed cells (MLEC) express GCNF and are both contact inhibited and exquisitely sensitive to TGF-β1, which markedly inhibits proliferation. MLEC were cultured in the presence and absence of TGF-β1 (1 and 5ng) which showed a dose dependent decrease in both GCNF mRNA expression and cell proliferation which suggests a relationship between growth and GCNF. To demonstrate potential regulation of GCNF, several of the cell lines were treated with all trans-retinoic acid during moderate-term, monolayer culture. Retinoic acid increased the expression of GCNF mRNA within 48 hours of exposure in ES-2, OVCAR and TOV-112 cells. While retinoic acid increased GCNF mRNA expression it did not decrease Oct-4 mRNA expression. GCNF has been reported to repress several key genes including Oct-4, involved in the maintenance of stem cells and GDF-9, a TGF-β family member. In the ovarian cancer cell lines expressing GCNF mRNA, both Oct-4 and GDF-9 mRNAs were detected despite GCNF expression, suggesting an impaired function of GCNF. A conserved domain search was also performed for both the domains for GCNF (the DNA binding domain and the ligand binding domain) and also a multiple alignment for the highly conserved regions which displayed 3D-structures for both the domains. The DNA binding domain of GCNF shares a 3D homology with 1HLZ, which is the Homo sapiens chain A crystal structure of orphan nuclear receptor Rev-Erb (Alpha), named because it is encoded on the opposite strand of the T3R. The ligand binding domain shares a high degree of structural homology (3D) with 1HG4, chain A ultra spiracle ligand binding domain from Drosophila. As USP has RXR as its mammalian homologue, GCNF (the ligand binding domain) shares a higher degree of homology with RXR than with any other nuclear receptor. Our results altogether demonstrate the novel expression of GCNF in ovarian cancer cells and suggest that at least some of the normal regulatory actions of GCNF may be impaired or altered. To what extent this contributes to the neo plastic basis of the ovarian cancer cells is unclear, but the finding of GCNF expression opens new avenues for future investigation and perhaps a new therapeutical target.