Molecular mechanisms regulating the G2 checkpoint induced after DNA damage.

Carrassa, Laura (2006). Molecular mechanisms regulating the G2 checkpoint induced after DNA damage. PhD thesis The Open University.



The checkpoint kinase (chk) 1 is an essential component of the DNA damage checkpoint acting as the effector of the DNA damage to block the cell cycle at the G2-M transition. The studies reported here were aimed at elucidating the molecular mechanisms regulating the G2 checkpoint after DNA damage and at investigating Chk1 as a potential therapeutic target for new cancer treatments. The promoter region of the human Chk1 gene was at first isolated and characterized. It was possible to show a p53 dependent downregulation of the Chk1 gene. The transcriptional factor E2F1 was also found to have a role in modulating Chk1 promoter activity. To further understand the interplay between p53 and Chk1 in the G2 checkpoint, the cellular response to anticancer treatments in isogenic cellular systems differing in the expression of Chk1 and p53 was studied. The expression of Chk1 was downregulated by siRNA, in the HCT-116 colon carcinoma cell line and in its isogenic systems lacking either p53 or p21. The inhibition of Chk1 in p21-/- and p53-/- cell lines caused a greater abrogation of the G2 block and a greater sensitization to anticancer treatments than in the parental cells with an intact G1 checkpoint. Tetracycline-inducible expressing Chk1 siRNA clones have been obtained in HCT-116 wt and p53 deficient cells and have been transplanted in nude mice. Experiments are undergoing to verify the downregulation of Chk1 upon addition of tetracycline in mice drinking water. The last part of my thesis focused on the investigation of the physiological role of Chk1 in cancer somatic cell lines. The understanding of this aspect appears particularly important as Chk1 has been proposed to be a potential drug target for anticancer therapy. The effects of the lack of Chk1 by siRNA transfection were studied in different human somatic cell lines. The downregulation of Chk1 by siRNA in the HCT-116 isogenic cellular systems did not alter cell growth or compromise survival of these cells independently from the status of p53 and p21. On the other hand, in different cell lines, like U20S and HeLa, an effect in cell growth, and in the cell phenotype was observed. These data suggest that the lack of Chk1 may have different consequences depending on the intrinsic genetic background of the cell line studied.

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