Genetic and proteomic analysis of the P33ING1b tumour suppressor in melanoma

The ING1 (In hibitor of G rowth) gene is the founding member of at least five related human genes associated with tumour suppressive properties. ING genes are evolutionarily conserved and express cofactors of histone acetyltransferases (HAT) and historic deacetylases (HDAC). The ING1 locus encodes at least three detectable protein isoforms, including the well-studied p33ING1b protein. Through associated HAT and HDAC activity, p33ING1b is capable of regulating the transcription of various genes, including the p21waf1 and cyclin B1 cell cycle regulators and the pro-apoptotic Bcl-2 family member Bax, leading to inhibition of cell cycle progression and sensitization of cells to apoptosis. P33ING1b also enhances the nucleotide excision repair of ultraviolet-damaged DNA. This work describes (1) the generation and use of rabbit polyclonal antiserum that can specifically recognize the p33ING1b isoform; (2) the status of the ING1 gene in malignant human melanoma; and (3) the regulation of the p33ING1b protein through protein phosphorylation and degradation. Mutational alterations were found within the ING1 gene of nearly a fifth of the melanoma biopsies examined. Two common alterations within the ING1 gene at codons 102 and 260 were found to be detrimental to p33ING1b-mediated enhancement of nucleotide excision repair. Mutations within the ING1 gene may also be indicative of a poorer 5-year patient survival. The ING1 gene was also found to be over-expressed in melanoma cells and biopsies compared to normal melanocytes. There was, however, widespread loss of nuclear expression of p33ING1b in the melanoma tumours. This thesis further describes the regulation of the p33ING1b protein through phosphorylation of serine 126 by the cyclin-dependent kinase 1 in the absence of DNA damage and by the checkpoint kinase 1 upon genotoxic stress. Although serine 126 is near the nuclear-localization sequence, it was not found to affect the sub-cellular localization of the p33ING1b protein. Phosphorylation did however alter the half-life of the protein. Serine to alanine site-directed mutagenesis of codon 126 or the inhibition of the Cdk1 kinase both resulted in a higher turnover rate of the p33ING1b protein. Although p33ING1b did incorporate ubiquitin moieties, it was found not to undergo proteolysis through the classical ubiquitin-proteasome pathway.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat