Armstrong, Alice and Taylor, Elaine and Lindsay, Howard (2016) Characterisation of CIP29 : a potential target of the eukaryotic DNA damage response. Masters thesis, Lancaster University.
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Abstract
Cytokine Induced Protein 29 (CIP29) was originally identified as protein over-expressed in hepatocellular carcinoma, and expression of the protein has since been found to be upregulated in multiple other cancers (Fukuda et al, 2002; Leaw et al, 2004). Several potential roles have been proposed for CIP29. It has been suggested that the protein plays a role in cellular proliferation, perhaps through cell cycle regulation (Fukuda et al, 2002). Additionally, studies have suggested that CIP29 may be an RNA binding protein (RBP) involved in mRNA export (Dufu et al, 2010). Previous research by this laboratory found that the X.laevis ortholog of CIP29, XCip29, is phosphorylated in response to DNA double strand breaks (DSBs). This phosphorylation was shown to be dependent on the activity of ATM, one of the three PI3KK kinases that orchestrate the DNA damage response (DDR). The purpose of our research was to investigate the function of the human CIP29 protein and to explore a potential role for the protein in the cellular response to DNA damage. DNA-damage dependent phosphorylation of human CIP29 could not be detected in this study. CRISPR genome editing was used to generate two CIP29 mutant cell lines, named C9 and C60. The C60 cells were shown to express a mutant form of the CIP29 protein, in which the SAP domain was deleted, whilst in the C9 cells, expression of CIP29 is effectively abolished. Both cell lines demonstrated reduced proliferation in comparison with wild-type cells, with the defect in the C9 cells more pronounced. FACS analysis revealed a slightly reduced proportion of the C60 cells in the S and G2/M phases of the cell cycle compared to wild-type cells, which could account for the slightly reduced proliferation rate seen in these cells. FACS analysis performed on the C9 cells revealed evidence of a cytokinesis defect, implicating the CIP29 protein in cytokinesis. In order to further investigate the potential involvement of CIP29 in the DDR, sensitivity assays using DSB-inducing agents were performed. These assays did not reveal any increased sensitivity of either the C9 or C60 cells to the drugs, indicating that CIP29 may not play a role in the DDR to DSBs.