Dissection of signal transduction pathways mediated by Ras for the induction of anchorage-independent growth.
Item
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Title
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Dissection of signal transduction pathways mediated by Ras for the induction of anchorage-independent growth.
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Identifier
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AAI9808024
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identifier
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9808024
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Creator
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Yang, Jaw-Ji.
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Contributor
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Adviser: Robert S. Krauss
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Date
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1997
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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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Biology, Molecular | Biology, Cell
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Abstract
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Inappropriate control of Ras proteins will lead to cellular transformation, including anchorage-independent growth, which is a strong correlate of tumorigenicity. Ras proteins exert their biological effects by interacting with effector molecules through an effector loop (residues 32 to 40). There is a growing number of Ras-mediated-effector pathways that have been identified. To understand the relationship between Ras, cell-substratum adhesion, and the cell cycle machinery, we have used three Ras effector loop mutants (12V, 35S, 12V, 37G, and 12V, 40C) to dissect the signaling pathways generated by Ras required for cell cycle progression and stimulation of anchorage-independent growth. Anchorage-independent growth of cells depends on abrogation to the normal adhesion requirement of several cell cycle events: (1) activation of G{dollar}\sb1{dollar} cyclin-dependent kinases (as measured by phosphorylation of the retinoblastoma protein and by cyclin E-dependent kinase activity); and (2) cyclin A expression. These effector site mutants of Ras failed to induce anchorage-independent growth and certain cell cycle events of cultured fibroblast cells, indicating that multiple pathways generated by Ras signal to the cell cycle machinery, which together promote cell cycle progression in non-adherent cultures of cells.;A mutant rat fibroblast cell line (ER-1-2) that fails to form colonies in soft agar when infected with a v-H-ras - expressing retrovirus, yet still undergoes transformation-related changes in morphology and gene expression in response to this oncogene was also studied. This cell line is resistant to Ras because it does not support Ras-mediated expression of cyclin A under non-adherent growth condition. We tested whether secreted factors play an important role in controlling anchorage-independent growth of ER-1-2/ras cells. A low molecular weight, hydrophilic, heat- and protease-resistant, UV-sensitive, secreted factor (designated TRF) that specifically rescued anchorage-independent growth of ER-1-2/ras cells was identified. We then tested small molecules that have similar chemical properties to TRF. We found that extracellular ATP, most likely acting via a P{dollar}\sb2{dollar} purinoceptor, also rescues anchorage-independent growth of ER-1-2/ras 2 cells. Furthermore, ATP, but not TRF, induces expression of cyclin A in non-adherent cultures of ER-1-2/ras cells.
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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.
