Gregory, Fiona Janet; (2000) Functional analysis of p16INK4a and p21CIP1 in replicative senescence. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Normal diploid cells have a limited lifespan in culture culminating in a process known as replicative senescence. Since tumour cells have an extended or indefinite lifespan, senescence is viewed as a mechanism for tumour suppression. Senescent cells arrest in the late G1 phase of the cell cycle with the retinoblastoma protein in a predominantly unphosphorylated state. They also accumulate high levels of the cyclin-dependent kinase inhibitors, p16INK4a and p21CIP1 both of which are likely to contribute to the arrest. To dissect the different contributions p16INK4a and p21CIP1 make to replicative senescence cyclin-CDK-CKI complexes have been examined in senescent human diploid fibroblasts and fibroblasts whose lifespan has been extended by DNA virus oncoproteins. The latter proteins, such as SV40 T-antigen or the E6 and E7 proteins of human papilloma virus, interfere with the functions of p53 and pRb. Abrogating pRb renders cells insensitive to p16INK4a whereas abrogating p53 diminishes the expression of p21CIP1. One strain of human diploid fibroblasts, SVts8, have been analysed in detail. These cells express a temperature sensitive form of SV40 T-antigen and are immortal when grown at the permissive temperature but upon inactivation of T-antigen the cells arrest immediately with the characteristics of senescence. Analysis of the cyclin-CDK complexes by gel filtration has shown that while cyclin D1-CDK6 complexes remain intact in senescent cells, the cyclin Dl- CDK6 complexes are completely disrupted, indicating a potential difference between CDK4 and CDK6 in fibroblast senescence. R24P a variant of p16INK4a able to interact with CDK6 but not CDK4 was utilised to dissect the effects of CDK4 and CDK6. The data confirm the critical roles of p16INK4a and p21CIP1 in the implementation of senescence and has provided important insights into different cell cycle regulators in this process.

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