CPT and zeocin induce increased levels of p53 independent of cell cycle stage.
Item
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Title
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CPT and zeocin induce increased levels of p53 independent of cell cycle stage.
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Identifier
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AAI3083672
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identifier
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3083672
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Creator
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Houser, Sandra Dean.
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Contributor
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Adviser: Jill Bargonetti
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Date
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2003
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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, Cell | Biology, Molecular
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Abstract
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We investigated how DNA damage activates the tumor suppressor p53 during the cell cycle. Centrifugal elutriation was utilized to separate exponentially growing ML-1 cells (containing wild-type p53). Distinct fractions were subjected to the DNA damaging agents Camptothecin (Cpt) (topoisomerase I inhibitor) (Hisang, Y., et al., 1989) and Zeocin (bleomycin/phleomycin family of antibiotics) (Burger, R. M., 1998, et al., 1994,) and analyzed for p53 levels, p53 DNA binding properties to p53-targeted genes, phosphorylation at serine 392 (a phosphoserine site located in the C-terminus of the protein) and cleavage states of p53.;By using gel shift and Western blot analysis we observed that both drugs induced DNA binding p53. However only the Cpt-induced p53 was phosphorylated at serine-392, while cells treated with Zeocin-induced a form of p53 phosphorylated at serine-15. Phosphorylation at serine-392 occurred in every cell cycle stage and this phosphorylated form of p53 (approximately 40 kDa) migrated more rapidly on the SDS-PAGE gel. Apoptosis occurred in every cell cycle fraction, suggesting that all the machinery to initiate the apoptotic response as well as the increase in p53 is present throughout the cell cycle (Houser, S., et al., 2001).;By using Western blot analysis with different p53 specific antibodies, we discovered that p53 was not induced when DNA replication was blocked for a short time with aphidicolin (APH) alone but, transcriptionally active p53 levels were stabilized in the presence of Zeocin and Cpt. This suggests that Zeocin and Cpt both activated p53 and Cpt, whose induction of p53 is said to require the replication process (Hisang, Y., et al., 1989), can signal p53 to be activated when DNA synthesis is blocked. Non-cycling cells treated with higher doses of both drugs incorporated bromodeoxyuridine (BrdU). This incorporation of BrdU is the result of cells utilizing deoxynucleotides (dNTPs) in DNA repair synthesis, which is necessary throughout the cell cycle. We and others have found that the stabilization of p53 is not cell cycle-dependent and that different types of DNA damage (induced by Cpt and Zeocin) can signal for increased levels of differentially modified p53.
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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.
