Wanek, K.; (2011) Development of an shRNA screen to identify mediators of cellular senescence in human breast epithelial cells. Doctoral thesis , UCL (University College London).

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Abstract

Cells from organisms with renewable tissues can permanently withdraw from the cell cycle in response to a variety of stimuli, including dysfunctional telomeres, DNA damage, physiological stress, and activation of certain oncogenes. This phenomenon, cellular senescence, is controlled by the p53 and pRb tumour suppressor proteins, and constitutes a potent anti-tumour mechanism. The underlying mechanism controlling cellular senescence and the signal transduction pathways involved are poorly defined and the critical targets of the p53 and pRb pathways in this process are not well characterised. As most breast cancers originate in epithelial cells, Mike O’Hare, Parmjit Jat and colleagues developed a conditionally immortalized human mammary epithelial line, 226L 8/13, derived from human mammary luminal epithelial cells, as a model to study cellular senescence in epithelial cells. 226L 8/13 cells constitutively express hTERT, the catalytic component of human telomerase, and a temperature sensitive non-DNA-binding mutant of Simian Virus 40 large T (SV40 LT) antigen. These cells grow at the permissive temperature 34°C, but undergo a rapid growth arrest at the non-permissive temperature 38°C, upon inactivation of the thermolabile LT antigen which allows activation of the p53 and pRB tumour suppressor pathways. This arrest displays features of cellular senescence, and is dependent mainly on the p53 pathway, but also on the pRb pathway. The Open Biosystems Human pGIPZ lentiviral shRNAmir library and pSM2c retroviral shRNAmir library were applied to the conditional system to identify mediators of cellular senescence, and several genes whose inhibition leads to cells overcoming the conditional growth arrest were identified, including IRF-1, FOXA1, Rab23 and CCNL1. By acting as mediators of cellular senescence these genes may play a role in the prevention of tumourigenesis. There is already significant evidence suggesting that IRF-1, FOXA1, Rab23 and CCNL1 play a role in cell proliferation, senescence and tumourigenesis, supporting the results presented in this thesis. Genes identified in this study may have prognostic and/or diagnostic value in the context of cancer biology.

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