Access Type

Open Access Dissertation

Date of Award

January 2012

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Cancer Biology

First Advisor

Ayad M. Al-Katib

Abstract

Lymphomas frequently retain wild-type (wt) p53 function but overexpress HDM2, compromising p53 activity. Therefore, lymphoma is a suitable model for studying therapeutic value of disrupting HDM2-p53 association by small-molecule inhibitors (SMIs). HDM2 SMIs have been developed and are currently under various stages of preclinical and clinical investigation. This study examined various molecular mechanisms associated and biological effects of two different classes of HDM2 SMIs: the spiro-oxindoles (MI-219) and cis-imidazoline (Nutlin-3) in lymphoma cell lines and patient-derived B-lymphoma cells. Surprisingly, results revealed significant quantitative and qualitative differences between these two agents. At the molecular level, effect of Nutlin-3 was generally more delayed (48h) and was notable for inducing cell cycle arrest. These findings indicate a response to a low level cellular stress and are supported by lower levels of p53 expression in Nutlin-3-treated cells. In contrast, MI-219 triggered an earlier response (12-24h), predominantly in the form of cell death associated with higher levels of p53 expression. Neither agent interfered with the E3 ligase function of HDM2, as confirmed in a cell-free autoubiquitination assay. Interestingly, these results report for the first time a novel mechanism of HDM2 antagonism by MI-219 in wt-p53 lymphoma cells that stimulates HDM2 autoubiquitination. Additionally, it corresponds with biological response of anti-lymphoma activity and may provide an explanation for the differences in efficacy between MI-219 and Nutlin-3. This study indicates that p53-HDM2 interaction and methods of its disruption are more complex than is currently realized and suggests that stimulation of HDM2 autoubiquitinating activity may be a novel treatment strategy for lymphoma.

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