Title

Reactivation of mutant p53 by small molecules : development of a reporter gene assay for screening.

Date of Award

5-1-2015

Document Type

Thesis

School

College of Liberal Arts

Degree Name

Bachelor in Arts

Abstract

p53 is a tumor suppressor protein that becomes active when DNA is damaged (Levine, 1997). Once activated, p53 induces the expression of many genes capable of halting the cell cycle or causing apoptosis (Levine, 1997). In some transformed cells that have sustained inactivating mutations in the p53 gene, p53 expression is greatly increased (Sun et al., 1993). Most of these oncogenic mutations destabilize the p53 protein, preventing it from being able to bind to DNA and function as a transcription factor (Levine, 1997). Since its expression is greatly increased in some cancerous cells, mutant p53 is an attractive target for developing cancer therapy. Our lab has previously developed a western blot assay to screen small molecules for their ability to reactivate mutant p53 in human transformed cell lines. We have been successful in identifying compounds with the ability to reactivate mutant p53; however, the potency and selectivity of these molecules towards mutant p53 are very low. In order to screen more compounds efficiently, we are seeking to develop a quick, quantitative reporter gene assay. The assay will be used to screen a large number of random compounds for their ability to reactivate mutant p53 in a transformed cell line with mutant p53 in order to identify possible compounds that reactivate mutant p53 and thus can potentially be used in the development of novel cancer treatments. In developing this reporter assay, we found that the pGL4.38 plasmid produced a strong firefly luciferase signal when transfected into A549 cells, transformed cells with wild type p53, that were subsequently treated with doxorubicin. Transformed cells with mutant p53 (SF295) were transfected with the plasmid pGL4.38; hygromycin B- resistant SF295 cells were selected and studied for luciferase expression following treatment with small molecules. Further research is necessary to optimize the treatment conditions to utilize these cells for screening novel compounds for the ability to reactivate mutant p53.