The Non-canonical Growth Activating Functions of Highly Expressed Human Mdm2-C
Item
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Title
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The Non-canonical Growth Activating Functions of Highly Expressed Human Mdm2-C
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Identifier
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d_2009_2013:ed194221ef7c:11850
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identifier
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12475
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Creator
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Okoro, Danielle,
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Contributor
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Jill Bargonettil
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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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Molecular biology | Cellular biology | Biomarker | Cancer | Growth | Mdm2-C | Non-canonical
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Abstract
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Mdm2 is an oncoprotein that regulates the tumor suppressor protein, p53 via the Mdm2 canonical pathway. The pathway involves p53 protein degradation and transcriptional repression. Mdm2 is often found over-expressed in cancers. In the presence of Mdm2 over-expression, the activity of p53 is frequently attenuated and the protein levels remain paradoxically high. Cancers with Mdm2 over-expression also over-express mdm2 splice variant transcripts. There are over forty identified spliced variants of mdm2. Therefore, we hypothesized that in the presence of Mdm2 over-expression, a different form of Mdm2 protein exists that does not function in the Mdm2 canonical pathway. In this study, the functions of an Mdm2 isoform, Mdm2-C, were investigated. We observed that Mdm2 over-expressing cells have high basal levels of mdm2-C transcript. We have cloned and expressed mdm2-C in vitro. We created an Mdm2-C specific antibody, Mdm2 C410, to the splice junction of exons four and ten (Mdm2 C410) and validated the C410 antibody using in vitro translated full-length Mdm2 compared to Mdm2-C. The Mdm2 C410 antibody did not detect Mdm2-FL. We saw that different human cancer cell lines, liposarcoma and breast cancer tissues, over-expressed endogenous Mdm2-C protein. We also observed that there was an estrogen-dependent increase in endogenous Mdm2-C protein in ER+ mdm2 SNP309 breast cancer cells that was p53-independent. In addition, the exogenous expression of Mdm2-C in human p53-null cancer cells showed that Mdm2-C does not function in the Mdm2 canonical pathway. Immunofluorescence utilizing the Mdm2 C410 antibody displayed that Mdm2-C was localized to the cytoplasm and nucleolus in a speckled pattern that might be integral to its cellular functions. We observed that the over-expression of Mdm2-C in the presence or absence of p53 in human and mouse cell lines promoted cell growth. Furthermore, the partial down regulation of mdm2-C via siRNA in mutant p53 G/G mdm2SNP309 breast cancer cells, T47D resulted in increased cell death. Thus suggesting that unlike other Mdm2 isoforms and full-length Mdm2, Mdm2-C has distinct roles in cell survival and p53-independent Mdm2 molecular pathways. Here we report the first identification of an endogenous tumor-associated splice variant Mdm2 protein, and document that Mdm2-C functions through a non-canonical growth activation pathway that is p53-independent.
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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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Biology
