| Summary: | Replicative senescence describes the irreversible growth arrest that primary human fibroblasts undergo when cultivated in vitro and represents one example of a biological process known as cellular senescence. Cellular senescence is associated with organismal ageing and functions an important tumour suppressive mechanism. A conditionally immortalised human mammary fibroblast cell line, HMF3A, has been developed with the aim of determining the precise molecular basis of telomere-independent cellular senescence. HMF3A cells constitutively express hTERT, the catalytic component of human telomerase, and a temperature sensitive non-DNA-binding mutant of Simian Virus 40 large T (LT) antigen. At the permissive temperature, 33.5C, HMF3A cells grow normally but at the non- permissive temperature, 39.5C, LT antigen is inactivated and the cells undergo a rapid and synchronous irreversible growth arrest. The stringency of HMF3 A growth complementation has enabled me to functionally analyse the pathways implicated in the induction of senescence in these cells. It was concluded that inactivation of the p53 pathway was sufficient to overcome the conditional HMF3A growth arrest. However, expression of a pl6iNK4a insensitive cvciin D1-CDK4R24C fusion construct indicated that the pRb pathway was not critical for the induction of this process. I subsequently identified three novel markers of telomere-independent cellular senescence by cDNA microarray analysis (namely, AKR1B1, CDH13 and UBE2C), and three potential regulators of senescence by an RNA interference screen (namely, NEUROD2, TARBP1 and RRM2). Finally, I detected a novel functional activity of the Adenovirus El A 13S splice variant that is not shared with the El A 12S splice variant. This activity enabled 13S El A, but not 12S El A, to bypass the conditional HMF3A growth arrest. The genes identified using these independent experimental approaches constitute novel markers of senescence and, upon validation of these findings, may provide prognostic and/or diagnostic value in the context of tumorigenesis.
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