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Title: Analysis of stress induced retinoblastoma protein activation using an SiRNA screen
Author: Runnacles, Elizabeth
ISNI:       0000 0004 2683 1080
Awarding Body: Institute of Cancer Research (University Of London)
Current Institution: Institute of Cancer Research
Date of Award: 2009
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Activation of the retinoblastoma protein (RB), by stresses such as ionizing radiation (IR) or hypoxia, causes a cell cycle arrest and has been suggested to convey therapeutic resistance to cancer cells. To investigate the cell signaling networks mediating RB activation, with the aim of uncovering potential targets for sensitisation of cancer cells, I developed and carried out a high-throughput RNA interference screen of the Dharmacon kinome-covering small interfering (si)RNA library. This identified seven siRNA gene targets required for RB activation in response to ionizing radiation; PRPK, PRKACG, STK4, DYRK1A, HK1, CDK4 and p21-cip1/waf1. Silencing of these genes effectively reduced the percentage of cells exhibiting G1-like characteristics after IR treatment. Two of these seven targeted genes, PRPK and STK4, appear to be involved in IR-induced p21-cip1/waf1 upregulation and silencing of these two genes sensitised colon carcinoma cells to radiation treatment. Biochemical characterisation of cells with PRPK knockdown revealed that its expression is required for p53 upregulation and activation in response to IR. The work presented here provides first time evidence of a role for PRPK in the radiation response and implicates this gene product and the gene product of STK4 as targets to sensitise cells to radiation. The work further identifies other gene products necessary for RB activation and G1/S checkpoint activation, yet ablation of these did not confer loss of p21-cip1/waf1 induction or sensitisation to irradiation, indicating wiring of these gene products into mechanisms that promote radiation survival in the absence of RB activity and G1/S checkpoint control.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available