Stability of p53 Isoforms in MCF7 Cells

Two major p53 mRNA isoforms differ in the length of the 5′‐leader sequence. Longer isoforms (p53 mRNA‐L) contain a putative upstream open reading frame (uORF) not present in those with shorter 5′ leaders (p53 mRNA‐S). We hypothesize p53 mRNA‐L is subject to nonsense‐mediated mRNA decay (NMD). Cell treatment with inhibitors of translation (cycloheximide) and PI‐3 kinase‐like kinases (PIKKs) (caffeine and wortmannin) was employed to diminish cellular NMD. Our objective was to block NMD using chemical inhibitors in MCF7 cells concurrently treated with actinomycin D (Act D). Cellular proteins were subjected to SDS‐PAGE and western analyses for p53 and beta‐actin (ActB). Isolated RNA samples were used to synthesize cDNA, which were subjected to quantitative RT‐PCR (qRT‐PCR) analyses of p53 mRNA‐L & ‐S isoforms, using GAPDH and beta‐actin endogenous controls. Our p53 mRNA‐L/p53 mRNA‐S isoform ratios (L/S) were calculated from Relative Quantification (RQ) values using untreated samples as reference. Two independent qRT‐PCR data sets were averaged and reported as mean L/S ratio and standard deviation (dev). Act D treatment, without inhibitors, resulted in a L/S = 1.135 (dev = 0.1105). Act D concurrent treatment with cycloheximide, caffeine or wortmannin resulted in L/S means of 1.260 (dev = 0.1801), 1.241 (dev = 0.1099) and 1.310 (dev = 0.2117), respectively. Western blot analyses were consistent with an absence of p53 protein cycloheximide treated cells, where Act B was unchanged (Figure 1). Samples from caffeine or wortmannin treated cells contained a prominent p53 protein band consistent with hypo‐phosphorylated p53. We conclude that (1) the chemical treatment conditions effectively inhibited translation and kinase activity, and (2) p53 mRNA‐L is partially rescued in cells treated with chemical inhibitors of NMD.