Investigating the Interaction and the Effect of p53 on MTBP Levels in Haematological Malignancies

Abstract

MDM2 binding protein (MTBP) functions to facilitate the ability of MDM2 to ubiquitinylate p53. In this way MTBP helps to maintain low levels of p53 protein in cells experiencing “normal” growth conditions. However, p53 is upregulated during times when cells experience genotoxic stress, causing them to exit the cell cycle and, ultimately, to undergo apoptosis should the stress be too great for the cell to effectively handle. p53 performs this function by acting as a transcription factor that binds DNA via a consensus response element (p53-RE). Thus, p53 is a transcriptional activator of many genes including CDKN1A, which encodes the protein p21 that functions to inhibit cyclin-dependent kinases and cell cycle progression. p53 can also inhibit expression of genes involved in cell cycle progression, though the way p53 does this is mainly indirect. Given the function of MTBP in regulating p53 expression in cells experiencing normal growth conditions, it is logical that expression of MTBP needs to be regulated during times of when cells are experiencing stress. The importance of such regulation increases considering that MTBP additionally participates in initiating DNA replication as well as chromosomal segregation during S- and M-phases of the cell cycle. This thesis describes the identification of a p53-RE located within the MTBP gene. In Chapter 3, the effect of p53 activation upon MTBP levels were investigated using nutlin-3, etoposide, and ionising radiation and concluded that active p53 negatively regulates MTBP levels. In Chapter 4, it was concluded using ChIP assay that p53 represses MTBP levels through direct binding to MTBP promoter. Finally, in chapter 5, the expression of MTBP is investigated in relation to the cell cycle using mass cytometry. Here it was shown that MTBP expression was the highest in cells that are in M and S phase, and that such expression is inhibited in cells exposed to nutlin-3.