Defense Date

2005

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry

First Advisor

Sumitra Deb

Abstract

It was hypothesized that the C-terminal sequences for mutant p53 would be required for oligomerization. and oligomerization may be critical for gain-of-function. An N-terminal deletion mutant of p53 that deletes amino acids 1-293 was used as a tool to perform hetero-oligomerization studies. This mutant retains the entire oligomerization domain but dispenses off the transactivation domain and a large portion of the sequence- specific DNA-binding domain. Co-transfection experiments show that p53 del. 1-293 forms hetero-oligomeric complexes with p53-D281G. Also. co-expression of p53 del. 1- 293 with p53-D281G inhibited p53-D28lG-mediated transactivation of the EGFR and MDRl promoters suggesting that hetero-oligomerization inactivates transcriptional functions of mutant p53. The interaction of p53 deli 1-293 and p53-D281G reduced transactivation potential of p53-D281G in stably transfected 10(3) murine cells. Therefore, the data presented supports the idea that proper oligomeric forms of mutant p53 are required for its transactivation function. Expression of mutant p53-D2810 also resulted in increased growth rate (H1299 cells), decreased chemosensitivity (H1299 and 21PT cells) and increased plating efficiency (Saos-2 cells). Expression of a transactivation deficient mutant p53 did not induce gain-of-function properties (increased growth rate and decreased chemosensitivity). Unlike the other gain-of-function properties tested, soft agar plating efficiency in Saos-2 cells was not significantly affected by the expression of a transactivation deficient mutant p53, suggesting that transactivation may not be the only factor affecting this gain-of-function property In order to identify the genes responsible for the observed phenotypes, global gene expression analyses were carried out using p53-null H1299 cell stably transfected to express mutant p53 (-Rl75H, -R273H and -D281G). A thorough and stringent analysis revealed 150 genes up-regulated by the expression of mutant p53. Up-regulation of a number of these genes was confirmed by QPCR and transient transcriptional promoter analyses; expression of the transactivation deficient mutant p53-D2810 (L22Q/W23S) did not result in up-regulation of the tested genes further supporting the idea that transactivation of genes is directly related to gain-of-function phenotypes. Using the ASNS gene as a model, this transactivation by mutant p53 was concentration dependent and that the increased transcription did indeed result in increased protein levels.

Comments

Scanned, with permission from the author, from the original print version, which resides in University Archives.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

6-18-2018

Included in

Biochemistry Commons

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