Dissertations, Theses, and Capstone Projects

Date of Degree

9-2015

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Jill Bargonetti

Committee Members

Benjamin Ortiz

Patricia Rockwell

James J. Manfredi

David Cobrinik

Subject Categories

Molecular Biology

Keywords

chromatin; MDM2; mdm2 SNP309; p53; transcription

Abstract

MDM2 overexpression is a common occurrence in many types of cancer. A single nucleotide polymorphism (T to G) near the mdm2 promoter, termed mdm2 SNP309, leads to MDM2 overexpression. This polymorphism is associated with accelerated tumor formation, decreased sensitivity to DNA damage treatment and compromised p53 transcriptional activity. Two G/G SNP309 cancer cell lines MANCA and A875, a Burkitts' lymphoma and melanoma respectively, express a stable wild-type p53 protein. We previously reported these cells have DNA damage resistant MDM2-p53 chromatin complexes and hypothesized that MDM2 is the contributing factor for the compromised p53 transcriptional activity. We created constitutive mdm2 shRNA cell lines to address MDM2 function. MDM2 knockdown in MANCA and A875 cells moderately increased expression of subsets of p53 target genes in a cell-type specific manner; although no additive effect was seen with DNA damage. Additionally, MDM2 knockdown did not affect p53 protein stability. We explored the mechanism for compromised p53 transcriptional activity in G/G SNP309 MANCA and A875 cell lines using chromatin immunoprecipitation analysis. When compared to fully activated T/T SNP309 ML-1 cells (myeloid leukemia with functional wild-type p53) treated with etoposide, MANCA and A875 cell lines displayed comparable recruitment of total and initiated RNA polymerase II at transcription start sites (TSS) for p21 and puma genes. This indicated that G/G SNP309 cells had functional transcription initiation at p53 target genes after DNA damage treatment. Next, we assessed transcriptional elongation using H3K36 trimethylation (H3K36me3) as a mark for active elongation. In ML-1 cells with etoposide treatment, we observed higher H3K36me3 at p21 and puma TSS than in either MANCA or A875 cells at these same regions. This suggested transcriptional elongation is compromised and also suggests that in G/G SNP309 cancer cells reactivation of p53 transcriptional activity is difficult. We sought to explain this phenomenon by examining the other well-known p53 negative regulator, MDMX, which is a homolog of MDM2. Interestingly, in MANCA cells the knockdown of MDM2 caused a substantial increase in MDMX protein levels. However, in A875 cells this was not observed. This suggests that for certain cell types, MDMX may function in some redundant roles for MDM2. Finally, we tested if inducing p53-independent cell death would be more effective than reactivating the wild-type p53 pathway. We treated the cells with 8-amino-adenosine, an inducer of p53-independent cell death. Indeed, treatment of MANCA and A875 cells with 8-amino-adenosine reduced cell viability more effectively than other chemotherapeutics.

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