Regulation and function of the INK4a/ARF tumour suppressor locus
The CDKN2a locus encodes two important tumour suppressors, pl61NK4a and ARF. The two genes share a common exon which is translated in different reading frames. pl6,NK4a binds to CDK4 and CDK6, preventing them from forming active complexes with D cyclins. As a result, pRb does not undergo the phosphorylation necessary for the transition from the G1 to S phase of the cell cycle. ARF inhibits the ubiquitination of p53 by MDM2, thereby causing the accumulation of p53. There is a growing awareness that the CDKN2a locus plays a central role in the cellular defences against transformation, and in the cellular response to stress. For example, pl6INK4a is involved in senescence, a permanent cell cycle arrest triggered in primary human fibroblasts in response to many stresses, including the overexpression of oncogenes. However, little is known about the regulation of pl6INK4a under these circumstances, and work in this thesis investigates this issue using overexpression of Myc as a model. The thesis also describes the characterisation of human diploid fibroblasts (Milan cells) from a patient homozygous for the R24P mutation of pl6INK4a. As this mutation is in exon la, ARF is unaffected. The mutant pl6INK4a cannot bind to CDK4, but retains some capacity to bind to CDK6. Milan cells have also been used in combination with shRNA targeting ARF to investigate the relative roles of pl6INK4a and ARF in the prevention of transformation. A panel of Milan cells were produced expressing telomerase, with combinations of Myc, Ras and shRNA targeting ARF, and the ability of the cells to grow in soft agar was assessed. A similar panel of Milan expressing p53 shRNA was also built up. These cells were used to investigate whether ablation of ARF can substitute for the loss of p53 function often associated with transformation, and to help identify which aspects of the p53 pathway are activated in the defence against transformation.