Investigating the molecular basis for p53 haploinsufficiency

A haploid genotype may be insufficient to support nonnal wild-type function. Such haplo-insufficiency has recently been documented for sevefill tumour suppressor genes. p53 is a crucial tumour suppressor orchestrating DNA repair, cell cycle arrest and apoptosis via its role as a stress-responsive transcription factor. p53 haploinsufficiency is observed in vivo with Li-Fraumeni Syndrome (LFS), a human familial cancer predisposition arising from inheritance of a gennline mutation in one p53 allele.· p53 haploinsufficiency has been replicated wi~ p53-knockout cell- and mouse-models of LFS, where loss ofone p53 allele has been'associated with reduced p53-dependent stress ,- responses. However, the underlying biochemical basis for such attenuated responses in p53+/- cells remains unclear. Here, basal p53 mRNA and protein levels were detennined, and the p53 stress response was compared in human HCTl16 p53+/+, p53+/- and p53-/- isogenic clones. Basal p53 expression in p53+/- cells was 25% relative to p53+/+ cells, and this 4-fold differential was maintained following oncogenic stress. This deficiency was manifest at both p53 mRNA and protein levels and resulted in reduced p53 stress responses, in particular for P21 WAF! up-regulation and SURVIVIN down-regulation, and aberrant cell cycle control and apoptosis. That a 2-fold reduction in p53 gene dosage results in a 4-fold reduction in p53 mRNA levels was unexpected and ,' was explored further since p53 expression levels are thought to be under strict control. Splicing efficiency,' the nature of p53 gene knockout, and the expression potential of wild-type and null p53 alleles were characterised here in the widely used HCTl16 cell model. The expression ofall ten p53 splice variant mRNA transcripts was assessed. The 4-fold differential in p53 mRNA levels between p53+/+ and p53+/- cells was persistant after several fonns of stress, was not caused by a change in mRNA stability and was evident in multiple p53 splice variant mRNAs. Epigenetic features and transcriptional regulation of the wild-type and null p53 alleles were also compared in HCTl16 p53 knockout isogenic clones. In summary, the observations here suggest a limiting mRNA model for setting p53 expression levels and identify a molecular basis for wild-type p53 haploinsufficiency which may explain the attenuated tumour-suppressive phenotype observed in cells and mice with a single wild-type p53 allele and in humans with LFS.EThOS - Electronic Theses Online ServiceGBUnited Kingdo