Stability of p53 mRNA Isoforms in MCF7 Cells

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Date

2017

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College of Natural and Health Sciences, The University of Tampa

Abstract

Tumor protein 53 (p53) is a tumor suppressor gene that has two key functions. This protein regulates cell cycle and induces apoptosis, or programmed cell death. TP53 mRNA isoforms differ in lengths of the 5’-leader sequence. Longer isoforms (p53 mRNAL) contain a putative upstream open reading frame, not present in shorter 5’ leaders (p53 mRNA-S). We hypothesize p53 mRNAL is subject to nonsense-mediated mRNA decay (NMD). Treatment with cycloheximide, caffeine, and wortmannin diminish NMD. Our objective was to chemically inhibit NMD in MCF7 cells concurrently treated with Actinomycin D. Cellular proteins were subjected to SDSPAGE and western analyses for p53. Isolated RNA samples were synthesized into cDNA, then subjected to qRT-PCR analyses of p53 mRNA isoforms. p53 mRNA-L/ p53 mRNA-S isoform ratios (L/S) were calculated from Relative Quantification (RQ) values obtained from p53 mRNA isoforms, by comparing treated to untreated samples and were reported as mean L/S ratios and standard deviations. Actinomycin D treatment, without inhibitors, resulted in a L/S = 1.070 ( 0.05). Actinomycin D co-treatment with cycloheximide, caffeine or wortmannin resulted in L/S means of 1.159 ( 0.07), 1.181 ( 0.18) and 1.279 ( 0.15), respectively. Western blot analyses were consistent with reduced translation of p53 protein in cycloheximide treated cells. Caffeine and wortmannin treated cells contained a prominent p53 protein band consistent with hypo-phosphorylated p53. In conclusion, chemical treatment effectively inhibited translation and kinase activity. p53 mRNA-L is partially rescued in cells treated with inhibitors of translation and kinase activity.

Description

Recommended Citation: Connelly, Zachary M. . “Stability of p53 MRNA Isoforms in MCF7 Cells.” Acta Spartae, 2017. https://doi.org/10.48497/D9CP-J317.

Keywords

Tumor protein 53, Department of Chemistry, Biochemistry and Physics, Research Subject Categories::NATURAL SCIENCES, Research Subject Categories::NATURAL SCIENCES::Chemistry

Citation

Connelly, Zachary M. . “Stability of p53 MRNA Isoforms in MCF7 Cells.” Acta Spartae, 2017. https://doi.org/10.48497/D9CP-J317.

URI

http://hdl.handle.net/20.500.11868/326

DOI

https://doi.org/10.48497/d9cp-j317

Collections

Volume 3, No. 1, 2017