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Dissertation
Molecular analysis of the mechanisms regulating EPC-1 gene expression in WI-38 fibroblasts
Doctor of Philosophy (Ph.D.), Allegheny University of the Health Sciences
Nov 1996
DOI:
https://doi.org/10.17918/00008919
Abstract
Abstract A cDNA, named EPC-1 for, early population doubling level (PDL) cDNA-1, was isolated by randomly selecting clones from an early PDL, subtracted, G0 library derived from WI-38 fibroblasts. EPC-1 is also known as pigment epithelium-derived factor (PEDF) and encodes for a protein belonging to the serine protease inhibitor (serpin) superfamily. This protein has been shown to induce differentiation of Y74 retinoblastoma cells into neuronal-like cells. In WI-38 fibroblasts, EPC-1 mRNA and secreted EPC-1 protein are detectable in early passage cells which are in G0, a growth-arrested state. The relative EPC-1 mRNA levels, determined by northern analysis, are more than 100-fold less in proliferating and senescent cells as compared to quiescent cells. This work focuses on elucidation of the mechanisms regulating the differential EPC-1 gene expression. Results from transcriptional assays suggest: post-transcriptional regulation of the EPC-1 gene. Measurements of EPC-1 mRNA stability using RNA synthesis inhibitors indicate that the half-life of EPC-1 mRNA is altered between quiescent and proliferating cells. The half-life of EPC-1 mRNA in proliferating cells is less than half that of quiescent cells. A combination of growth factors capable of inducing cellular proliferation, or transforming growth factor-[beta]1 (TGF-[beta]1) can decrease the EPC-1 mRNA levels. These studies also demonstrate that reduction of the EPC-1 mRNA levels caused by TGF-[beta]1 occurs in the absence of cellular proliferation. We propose that EPC-1 mRNA stability is decreased in proliferating cells due to a serum inducible protein. We also examined the relative EPC-1 pre-mRNA levels by reverse transcriptase-Polymerase chain reaction (RT-PCR) and observed that the pre-mRNA levels reflect steady-state mRNA levels. These results lead us to conclude that this quiescent specific differentiation factor is subject to two levels of regulation: both pre-mRNA and mRNA stability contribute to its differential expression.
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Details
- Title
- Molecular analysis of the mechanisms regulating EPC-1 gene expression in WI-38 fibroblasts
- Creators
- Vincent W. Coljee
- Awarding Institution
- Allegheny University of the Health Sciences
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Allegheny University of the Health Sciences; Philadelphia, Pennsylvania
- Number of pages
- xv, 152 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Biochemistry [Historical]; Allegheny University of the Health Sciences (1996-1998); School of Medicine (1996-1998)
- Other Identifier
- 991021888900804721