The Role of p53 in Human Epidermal Keratinocyte Terminal Maturation

[摘要] Two cell lines, SCC12F and SCC12B were derived from the same squamous cell carcinoma of the facial epidermis and shown to display different phenotypes. SCC12B is more resistant to the induction of terminal differentiation and also more tumorigenic than SCC12F. The aim of this thesis was to investigate any genetic differences between SCC12B and SCC12F that could be responsible for their different phenotypes and therefore possibly be an important genetic event in the progression of squamous cell carcinomas of the head and neck (SCC-HN). Microsatellite analysis of SCC12B and SCC12F revealed no differences in loss of heterozygosity (LOH) at the loci investigated on chromosome 3p and 9 which have previously been reported to show frequent LOH in SCC-HN. No overexpression of cyclin D1 was observed in the two cell lines and others have reported the absence of any H-ras mutations or abnormalities in Rb-1. Analysis of the tumour suppressor gene p53 however revealed different levels of the protein between the two cell lines. SCC12B was shown to express much higher levels of p53 protein than SCC12F. Sequencing of p53 revealed a novel heterozygous mutation at codon 216, a T→G transversion substituting a valine for a glycine. Interestingly, whilst the mutant allele was visible in both cell lines SCC12B appeared to express much more mutant p53 than SCC12F which mostly retained wild-type p53 expression. Dot blot analysis suggested that mutant p53 expression in SCC12B was double that of SCC12F. Investigations were then undertaken to investigate whether the variations in this mutant to wild-type gene dosage could explain the different abilities of SCC12F and SCC12B to undergo suspension induced terminal differentiation. A clone of SCC12F, clone 19, expressing low levels of p53 and therefore presumably expressing a more normal phenotype was used as a target for alterations in mutant p53 expression. The use of this clone had the advantage that it retained a related genetic background to SCC12F and SCC12B and therefore is a more relevant target cell for investigating the effects of increased mutant p53216 on terminal differentiation. The results in this thesis show that increasing the mutant p53216 dosage in clone 19 decreased the cells ability to express involucrin and form cornified envelopes and increased cell survival in response to suspension induced terminal maturation. An increase in tumorigenicity was however not observed. Taken together these results therefore suggest that the acquisition of a p53 mutation is an early event in this SCC and its accumulation leads to a dramatic progression of this cancer. Inactivation of p53 appears to inhibit the cells ability to terminally differentiate and the possible roles of p53 in tumour progression are discussed.

[发布机构] University:University of Glasgow

[关键词] Molecular biology, Oncology