Differential expression of germ cell nuclear factor (GCNF) in human ovarian cancer cell lines

Abstract

Germ Cell Nuclear Factor (GCNF) is the only existing member of nuclear receptor (NR) super family NR6A1, and an orphan receptor because of an unidentified ligand. Members of NR superfamily act as ligand activated transcription factors which regulate the target gene expression either by activating or repressing transcriptional activity. GCNF is expressed in both embryonic and adult stages in the human. In adults, expression of GCNF is restricted to testis and ovaries. GCNF is also found in other species such as Xenopus leavis, Zebrafish, rats, mice and hamsters. GCNF mRNA and protein were recently found in our lab to be expressed in various human ovarian cancer cell lines; i) ES2 – a clear cell carcinoma, ii) PA1- teratocarcinoma, iii) TOV 112D –an adenocarcinoma and iv) OVCAR–3 another adenocarcinoma. Differences in morphologies and growth rates were observed in the above cell lines. According to the growth rate curve, ES2 cells have faster growth rate than other cancer cells. According to previous studies in our lab, GCNF was also found to be expressed in a nontransformed cell line, MLEC (Mink Lung Epithelial Cells). When MLEC cells were cultured with varying amounts of TGF-β1, a decrease in amount of GCNF mRNA expression was observed. A proportionate decrease in rate of cell proliferation was also observed. Based on these two findings, a direct relationship between growth and GCNF expression was postulated. Based on this proposed relationship experiments were undertaken to establish the potential correlation between cell growth and levels of GCNF mRNA expression. Analysis of GCNF expressed in various cancer cells using Quantitative Real time-PCR proved that PA1 had the highest amount of mRNA expression and ES2 had the least. When growth rates were compared, ES2 had the fastest doubling time when compared to PA1, vii TOV112D and OVCAR cells. This contradicts the hypothesis, in spite of having fastest growth rate, ES2 contained the least amount of GCNF mRNA. The results suggest that there is no direct, linear correlation between cell growth rate and the level GCNF mRNA expression. Expression of GCNF protein in the cancer cell lines was demonstrated via western blot analysis. Newer more efficient polyclonal antibodies have been produced by Santa Cruz Biotechnology. When nuclear and cytoplasmic fractions from cancer cells were subjected to western blotting using the new antibodies, specific bands were exhibited in the range of 50 -75 KD. PA1 cells exhibited the bands with high intensity when compared with other cancer cell lines. To study the GCNF DNA binding properties quantitative analysis of GCNF protein was performed. It is mainly via electrophoretic mobility shift assay, but we could not attain consistency in results. Preliminary studies had demonstrated the homology of the DNA binding domain in TOV112D (epithelial cancer cell) when compared with published human GCNF sequence. The main focus of this objective was to determine if there was any homology in the coding sequence (CDS) of GCNF in other ovarian cancer cells. Cancer cells selected to prove this objective were PA1 (germ cell tumor) and ES2 (epithelial cell tumor). Three different pairs of primers providing overlapping cDNA sequences to amplify the entire GCNF mRNA sequences following reverse transcription were used to illustrate homology. A standard PCR was performed using the different primers followed by gel electrophoresis. DNA was extracted and sent for sequencing. The results indicated that the PA1 cell GCNF RNA was amplified by all 3 primer pairs, whereas ES2 cells did not amplify one of the three overlapping primers (GCNF NH and GCNF DW). These results gave a new dimension to the objective. PA1 cells are of germ cell in origin whereas ES2 is epithelial in origin. When the PA1 sequence was compared to the published human GCNF sequence it showed 98 % homology in the CDS (Coding sequence). The impact of GCNF siRNA on PA1 cells was examined. siRNA was utilized to achieve gene silencing or gene knock down. Previous studies indicated an effect of GCNF siRNA on cancer cells of epithelial origin (TOV 112D and ES2). In terms of growth inhibition, the results showed a proportional decrease in the cell proliferation and GCNF mRNA expression. When cells of (epithelial and germ cell cancers) were compared, similarity of effect was demonstrated. This result demonstrates that GCNF is required for growth. When growth was suppressed sufficiently by siRNA it appeared to directly affect GCNF mRNA expression. Results of the experimentation manifest discrepancies in the expression of GCNF in human ovarian cancer cell lines. Nuclear receptors bind to ligands which can be altered serve as potential pharmacological targets. As GCNF still exists as an orphan nuclear receptor, discovery of the ligand will pave the way for creating new drugs which will be helpful in the future to cure some of the life threatening diseases like Alhzeimers, Diabetes and Cancer etc.