Benvenuti, Silvia; (2002) Differential gene expression in rodent embryo fibroblasts upon replicative senescence. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cancer arises as a consequence of the accumulation of multiple independent mutations in genes that regulate cell proliferation and survival. The acquisition of an unlimited proliferative potential is probably a critical step in this process since normal somatic cells can only undergo a finite number of divisions. One approach that has been extensively used to study this phenomenon has been the serial cultivation of normal somatic cells. When these cells are cultured in vitro, they undergo a finite number of divisions and then cease dividing and enter a state of replicative senescence, in which they are viable and remain metabolically active, but cannot be induced to undergo mitosis, re-enter the cell cycle and divide. To identify the underlying mechanisms that determine the finite mitotic potential of normal somatic cells, and how they may be abrogated in cancer cells, we first undertook a high-resolution differential proteome analysis aimed at identifying proteins that are differentially expressed upon replicative senescence. Since replicative senescence in primary fibroblast cultures is asynchronous, two complimentary systems were used. We analysed a group of conditionally immortalised rat embryo fibroblast cell lines that have previously been shown to undergo synchronous senescence upon inactivation of SV40 tsA58 large T antigen. Moreover since it has been shown that rodent embryo fibroblasts only become dependent upon the immortalising gene when their finite life span has elapsed, we complemented these studies by analysing replicative senescence in the asynchronous serially passaged rat embryo fibroblast cultures. In these two proteome analyses a number of features have been identified as differentially expressed upon replicative senescence; 49 features were found to be up or down-regulated upon serial passaging of the primary rat embryo fibroblasts, and 44 were found to be differentially expressed in the conditionally immortalised cell lines. Some of the identified changes were also shown to occur in two other models of senescence: oncogene induced premature senescence of REF52 cells and replicative senescence of mouse embryo fibroblasts. Comparison of the identity of the features found to be differentially expressed in the two complimentary studies identified 9 differential features that were in common. Although the majority of the identified proteins have previously not been recognised to be involved with replicative senescence, they are likely to represent novel starting points for elucidating the underlying mechanisms that regulate the finite mitotic life span of somatic cells and how it can be overcome in cancer cells. We then extended our studies by comparing the mRNA level of growing versus senescent cells using mouse cDNA microarrays to determine if there was any correlation between differential protein expression and RNA expression, and also to achieve higher sensitivity for identifying changes in gene expression.

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