Publication Date
2007
Document Type
Dissertation
Committee Members
Steven Berberich (Advisor)
Degree Name
Doctor of Philosophy (PhD)
Abstract
Mutations in the p53 tumor suppressor gene are among the most prevalent molecular abnormalities in human cancer. While half of all human tumors possess p53 mutations, inactivation of wild-type p53 can also occur through a variety of mechanisms that do not involve p53 gene mutation or deletion. This dissertation focuses on human tumor cell lines harboring wild-type p53 protein and elevated levels of HdmX and/or Hdm2, two critical negative regulators of p53 function. My hypothesis is that loss of HdmX in tumor cells with wild-type p53 and over-expressed HdmX, will activate p53 and induce p53 target genes leading to growth inhibition. To test this hypothesis, RNA interference (RNAi) was utilized to knockdown HdmX protein from HdmX over-expressing breast, colon, and bone tumor cell lines. Alterations in gene expression and effects on cell proliferation following the removal of HdmX were examined. Two RNAi approaches were assessed in this study; transient small interfering RNA (siRNA) transfection and lentivirus-delivered short-hairpin RNA (shRNA) vectors. Multiple siRNA transfections were selected as the method of choice over the shRNA vectors due to the induction of off-target genes in cells expressing the shRNA vectors. Affymetrix GeneChips and subsequent quantitative real time-PCR validations were used to uncover a subset of p53 target genes encoding proteins associated with cell cycle arrest and growth inhibition that were induced upon HdmX knockdown. In contrast, only one p53 dependent pro-apoptotic gene (i.e. Fas) was increased. The induction of these p53 target genes following loss of HdmX was p53-dependent, as no increase in these p53 target genes were observed after HdmX knockdown in two different p53-null tumor cell lines. Cell cycle analysis and cell proliferation assays confirmed that the loss of HdmX led to a significant G1 cell cycle arrest. Similar findings were observed upon Hdm2 knockdown, and removing both HdmX and Hdm2 resulted in even greater p53 activation and a synergistic or additive induction of p53 target genes associated with cell cycle arrest. The increase in p53 transactivation following loss of HdmX was not due to p53 phosphorylation, suggesting a nongenotoxic or genotoxic stress independent p53 activation. Furthermore, the loss of HdmX did not appear to alter p53 localization or stabilization. Although the removal of over-expressed HdmX appears limited to an anti-proliferative effect in tumor cells harboring wild-type p53, loss of HdmX enhanced the cytotoxicity of several chemotherapeutic agents. Cell viability assays showed an increase in chemosensitivity in tumor cells following knockdown of HdmX and/or Hdm2. Taken together, these results suggest that removal of HdmX may be an important therapeutic target that would complement chemotherapy drugs currently used to treat tumors possessing wild-type p53.
Page Count
150
Department or Program
Biomedical Sciences
Year Degree Awarded
2007
Copyright
Copyright 2007, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.
