Gene expression in human keratinocytes containing integrated copies of the SV40 early region
Item
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Title
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Gene expression in human keratinocytes containing integrated copies of the SV40 early region
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Identifier
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d_2009_2013:e39df3780dc2:11660
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identifier
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12192
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Creator
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Liu, Suqing,
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Contributor
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Mark L. Steinberg
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Date
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2013
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biochemistry | Gene expression | Human keritinocytes | SV40 early region
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Abstract
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Simian Virus 40 (SV40) large T antigen is one of the simplest and most reliable agents for induction of immortalization of cultured cells. It is known to act by blocking the activities of the cellular tumor suppressors of p53 and pRb proteins. However, other factors that may act downstream from T antigen and the mode of its regulation in SV40 transformed human keratinocytes remain unknown. In the present study we demonstrate that loss of functional T antigen results in a comprehensive gene regulation pattern that is exploited by p53 during the conversion of immortalized cells into primary cells.;We employed line 130, a permanent line of SV40-immortalized human keratinocytes with a unique integrated copy of the virus to study altered gene expression after silencing of the viral early genes using RNA interference methodology. We characterized the viral integration site in 130 cells by sequence analyses of a cloned XbaI fragment isolated from a lambda bacteriophage library as well as by primer walking using various PCR techniques. The viral integrant was found to consist of two tandemly integrated viral DNA copies but with only a single intact copy of the viral early genes. The viral DNA was found to be joined, at one end, to human chromosome 21q within an intronic region of the homeobox gene, PKNOX1 and, at the other end to a site within a noncoding region of chromosome 10. The late region of the second copy adjacent to chromosome 10 was found to contain numerous rearrangements which may have occurred during the integration process that also brought about the chromosomal breakage and joining of chromosomes 21 and 10. Sequence analyses of the viral mRNAs (via cDNAs cloned in a T vector) showed that there was only a single viral transcript encoding a full length, intact early gene mRNA.;SV40 large T antigen (LT) binds to the cell cycle regulators p53 and pRb, and we found that down regulation of LT antigen expression brought about an accumulation of cells at the G1/S interface, associated with cell cycle arrest. We also found increased expression of BTG2, a novel anti-proliferation protein, as well as induction of the cell cycle inhibitor p21WAF1/CIP1 , murine double minute-2 promoter activity, expression of murine double minute-2 gene product and expression of GADD45A. Expression of p53 was down-regulated, but we observed an increase in p53 activity that was correlated with reduced binding to LT, suggesting that LT silencing led to p53-independent and BTG2-dependent cycle control in cell 130 line. No evidence of apoptosis was found. We attempted to establish a stable LT knock-down subline of 130 using an miRNA expression vector, but we found that while the presence of the plasmid vector was stably maintained over long-term culture, long-term LT silencing was not maintained, supporting the idea that even after long-term culture immortalization remains dependent upon SV40 T antigen expression.
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Biochemistry
