Ditzel, Mark Clifford; (2000) Modulation of the p53 response: Effects of DNA damage and MDM2 overexpression. Doctoral thesis (Ph.D), UCL (University College London).

Text Modulation of the p53 response - Effects of DNA damage and MDM2 overexpression.pdf Download (29MB)

Abstract

p53 is known to play a major role in the cellular response to stress, capable of mediating either a cell cycle arrest or apoptosis. MDM2 has recently been implicated as a crucial regulator of p53 activity, affecting p53's protein level, transcriptional capabilities and subcellular localisation. An intimate relationship also exists between these two proteins and MDM2's negative regulator, ARF. Transcriptional- and protein-protein-relationships between these proteins have been highlighted through a number of observations, although the regulatory mechanisms governing the relationships remain unclear. Examination of the irradiation responses of different human cell lines revealed similar increases in p53 expression, but fundamental differences in the p53-inducible MDM2 and p21 protein responses. UV-irradiation induced apoptosis in U2OS cells and reduced the expression of both MDM2 and p21 protein, while only mediating a reduction in mdm2 mRNA levels. A transient reduction in MDM2 protein, but not mRNA, levels was apparent in a variety of X-ray-irradiated cell lines. Closer examination revealed potential UV- and X-ray- mediated p21 and MDM2 protein degradation events, respectively. These results suggested the temporal importance of MDM2 and p21 protein levels in irradiation-mediated cell cycle arrest and apoptosis. Furthermore, immunofluorescence analysis of irradiated cells also revealed differential p53 and MDM2 subcellular localisation patterns, perhaps reflecting apoptotic-specific spatial processes. Analysis of ARF-induced and X-ray- and UV-irradiated cells, exhibiting elevated p53 levels, surprisingly showed no negative effects on p53:MDM2 association. Hence, negative regulation of MDM2-mediated p53 degradation seemed to occur downstream of p53:MDM2 association, perhaps at the stage of ubiquitin transfer or the actual proteosomal degradation event.

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