Inhibition of chromatin-bound wild-type p53 by Mdm2.
Item
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Title
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Inhibition of chromatin-bound wild-type p53 by Mdm2.
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Identifier
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AAI3213171
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identifier
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3213171
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Creator
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Arva, Nicoleta Catalina.
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Contributor
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Adviser: Jill Bargonetti
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Date
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2006
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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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In cancer cells the function of the tumor suppressor protein p53 is usually blocked. In tumor cells homozygous for a Single Nucleotide Polymorphism (SNP) in the mdm2 gene at position 309 Mdm2 is over-expressed, resulting in impairment of the p53 pathway. In such tumor cells, we found that a chromatin associated Mdm2-p53 complex blocked the p53 transcriptional activity and p53 was not subjected to Mdm2-induced degradation. p53 protein accumulated in the nucleus of mdm2 SNP309 cells after camptothecin, etoposide or mitomycin C treatment, with the p53 protein phosphorylated at Ser-15. Although the p53 protein was able to bind to DNA, quantitative PCR showed compromised transcription of endogenous target genes. Additionally, exogenously introduced p53 was incapable of activating transcription from p53 responsive elements in SNP309 cells, confirming the trans -acting nature of the inhibitor. Chromatin immuno-precipitation experiments demonstrated that p53 and Mdm2 bound to p53 responsive elements. Down-regulation of Mdm2 by siRNA or disruption of the p53-Mdm2 complex with the Mdm2 antagonist Nutlin resulted in transcriptional activation of p53 targets.;Although DNA damage did not activate p53 transcriptional activity in cells homozygous for mdm2 SNP309, p53 target genes were up-regulated when Mdm2 over-expression came from gene amplification, suggesting that over-expression from mdm2 SNP309 gene confers a different phenotype than over-expression from amplified wild-type mdm2 gene. Mass-spectroscopy/mass-spectrometry identified, in SNP309 cells, other interacting proteins (nucleolin, heat shock proteins) that might be part of the inhibitory p53-Mdm2 complex found on chromatin. Sequencing of the mdm2 cDNAs from homozygous cell lines also discovered new mdm2 isoforms that might explain this difference.;p53 is inhibited from activating transcription of its target genes in cells homozygous for mdm2 SNP309 through a chromatin-bound inhibitory protein complex.
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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.
