Analysis of methylation crosstalk in the c-terminus of p53 following chemoresistance in colorectal cancer

Abstract

The p53 protein acts as a tumour suppressor by interacting with numerous other proteins and controlling transcriptional activity. In particular, the C-terminus has regulatory functions, controlled by post-translational modifications (PTMs). The focus of this dissertation was to analyse the crosstalk between K372 methylation, K373 acetylation, and S376 phosphorylation. Understanding the involvement and timing of PTM modifications in the chemoresistance process would allow for a therapy targeting the oncogenic process through PTMs rather than p53 as a whole, which may prove as an effective strategy against cancer. The CRC cell lines Caco-2 and HCT116 were cultured and chemoresistant cultures were generated by treatment with doxorubicin and 5- fluorouracil. The chemoresistant phenotype was then validated by utilising light microscopy to view morphological changes, in conjunction with the MTS assay to help determine cellular growth and viability. Western blotting was conducted to validate and analyse the expression of total p53 protein and the three selected PTMs in the CRC cells. Both Caco-2 and HCT116 cell lines became resistant to DOX and 5-FU respectively. PTM expression patterns detected in both cell lines showed differential expression of methylation and acetylation, with phosphorylation not being detected throughout this study. This project provides a better understanding of PTM crosstalk upon gaining resistance to DOX and 5-FU.