Expression of p53 isoforms in colorectal cancer cell lines

Abstract

Colorectal cancer (CRC), encompassing colon and rectal carcinomas, ranks as the world’s third most prevalent cancer and the second leading cause of cancer-associated deaths. CRC may be classified into four distinct consensus molecular subtypes (CMS), namely CMS1-CMS4, with CMS1 having the best prognosis and CMS4 the worst. Tumour protein 53 (TP53), encoding the p3 protein, is greatly implicated in CRC. Typically, this protein acts as a tumour suppressor by preventing the propagation of damaged deoxyribonucleic acid (DNA). TP53 expresses thirteen different mRNA variants, due to internal promoter usage and alternative splicing, resulting in thirteen p53 protein isoforms. To date, literature about these isoforms is limited, thereby requiring further research. This project aimed to investigate p53 isoform expression patterns in CMS1 and CMS4 CRC cell lines, at mRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and at protein level by western blotting. RT-qPCR data revealed that all three CRC cell lines (LoVo, RKO, and DLD1) express p53α/β/γ and Δ40α/γ/β variants, with DLD1 showing lower expression. DLD1 also had reduced mRNA expression for Δ133/Δ160α/β/γ transcripts. Protein analysis revealed p53β/γ isoform expression in all cell lines. p53α was detected only in RKO and DLD1, and LoVo exhibited a band likely corresponding to a Δ40 protein isoform. Δ133/Δ160β/γ isoforms were also faintly detected in RKO and DLD1. Furthermore, a novel purification protocol was devised for GST-∆160p53, using a GSTrapTM FF column, and achieving up to 87% purity. Circular dichroism (CD) indicated an ordered structure and higher thermal stability compared to GST-FLp53, the wild type counterpart. Interestingly, GST-Δ160p53 and GST-Δ40p53 isoforms showed a higher tendency for fibril formation than the FLp53 protein.