Optimization of Chemical Inhibition Conditions for NMD Studies in p53‐Positive Cancer Cell Lines (LB234)

Abstract

We report on the treatment of p53‐positive cancer cell lines with chemical inhibitors of transcription, translation and PI‐3 kinase‐like kinases (PIKKs). Our goal was to determine optimal concentrations of, and exposure times for, chemical inhibitor treatments. These experiments were prerequisite for our studies of the role nonsense‐mediated mRNA decay (NMD) plays in p53 expression. Cultured p53‐positive cancer cells were treated with varied concentrations of cycloheximide, caffeine, wortmannin, or actinomycin D to determine optimal concentrations. Cells were also treated with constant inhibitor concentrations in time‐course experiments to determine optimal duration of exposure. Treated cell samples were divided equally for protein and total RNA isolation. Protein samples were subjected to SDS‐PAGE and western analyses for p53 and beta‐actin. RNA samples were used to synthesize cDNA, which was subjected to TaqMan quantitative RT‐PCR (qRT‐PCR) analysis for p53 and beta‐actin. Western and qRT‐PCR data are consistent with optimal chemical inhibitor concentrations being substantially higher for HepG2 cells compared to MCF‐7 and HeLa. However, chemical inhibitor exposure times required to achieve optimal effect was not widely varied between cell lines studied. Through our determination of optimal conditions for chemical inhibition of transcription, translation and PIKK activity in p53‐positive cell lines, we conclude that cell lines studied require widely different concentrations of, but not different exposure times to, chemical inhibitors. Therefore, we determined optimal concentrations and exposure times for chemical inhibition in our studies of the role of NMD in p53 expression.Grant Funding Source: University of Tampa Chemistry, Biochemistry & Physics Departmental Student Research Support Fund