Transformation of human diploid fibroblasts by simian virus 40.
Item
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Title
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Transformation of human diploid fibroblasts by simian virus 40.
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Identifier
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AAI9521290
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identifier
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9521290
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Creator
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Li, Gang.
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Contributor
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Co-Advisers: Harvey L. Ozer | Timothy J. Yen
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Date
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1995
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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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Chemistry, Biochemistry | Biology, Cell
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Abstract
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Simian virus 40 (SV40) has been used as a model system to study tumorigenesis. It is known that SV40 large T antigen plays an important role in cell transformation. To study the transformation mechanism in human cells, transformants (HAL and AR5) have been generated by introducing origin-defective SV40 encoding heat-labile large T antigen (SVtsA58) into human diploid fibroblasts. The transformants are temperature-sensitive (ts); they proliferate at 35{dollar}\sp\circ{dollar}C and cease to grow at 39{dollar}\sp\circ{dollar}C.;The temperature-dependent growth was studied by focusing on the interaction between large T antigen and antioncogenes pRb and p53. At 35{dollar}\sp\circ{dollar}C, complexes were observed between large T and pRb or p53 while at 39{dollar}\sp\circ{dollar}C the respective complexes were greatly reduced. Mutations of pRb and p53 have been isolated in a number of tumors and overexpression of the wild-type protein can reduce the rate of cell growth in tumor cells. Thus, inactivation of pRb and p53 by complex formation may mimic mutations in tumor cells.;Cell cycle analysis of ts transformants which were immortal and those which had an extended but limited lifespan (i.e "preimmortal" transformants) was performed. Rapid appearance of a tetraploid DNA content was observed in preimmortal but not in immortal transformants at 39{dollar}\sp\circ{dollar}C. To elucidate the tetraploid phenomenon, SV40-transformants (pRNSVtsA58dl) containing only heat-labile large T and no small t were generated. Flow cytometry showed that tetraploid DNA content was not observed in pRNSVtsA58dl. This result demonstrated that small t may contribute to the tetraploid DNA content in preimmortals.;To further address the basis for tetraploidization, I investigated the role of CENP-E, a protein localized at the centromere of chromosomes at prometaphase and shown to be involved in mitosis. CENP-E was found to accumulate to peak level at late G2 and became phosphorylated. Mutagenesis study demonstrated four consensus sites of phosphorylation for p34{dollar}\sp{lcub}\rm cdc2{rcub}{dollar} at C-terminal CENP-E. I propose that the function of CENP-E is regulated by p34{dollar}\sp{lcub}\rm cdc2{rcub}{dollar} and that interference with the expression or phosphorylation of CENP-E may deregulate its function leading to cessation of mitosis. With cells blocked in G2/M, synthesis of cellular DNA may be continued resulting in tetraploidy in preimmortal transformants.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
