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. / Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Biological Sciences
| Identifer | oai:union.ndltd.org:WICHITA/oai:soar.wichita.edu:10057/3302 |
| Date | 05 1900 |
| Creators | Devabhakthuni, Rajeswari |
| Contributors | May, Jeffrey V. |
| Publisher | Wichita State University |
| Source Sets | Wichita State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Format | xvi, 64 p., 1403417 bytes, 1843 bytes, application/pdf, text/plain |
| Rights | Copyright Rajeswari Devabhakthuni, 2010. All rights reserved |
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