Role of checkpoint kinase 1 in malignant melanoma

[摘要] Chk1 is a conserved protein kinase which is activated in response to multiple exogenous and endogenous genotoxic stresses. In response Chk1 mediates several cell cycle checkpoint responses which are important components of the cellular DNA damage response (DDR) pathway and as such are important in maintaining genome integrity throughout the cell cycle. The aim of this study was to investigate the potential role that Chk1 may play in the maintenance and progression of malignant melanoma using both in vitro and in vivo models; and to determine if Chk1 inhibition could be a viable therapeutic approach to malignant melanoma treatment.Initially I investigated the importance of Chk1 in the cells of origin for melanoma; melanocytes. In this thesis I have examined the importance of Chk1 on melanoblast proliferation and survival during development. I demonstrated that targeted deletion of Chk1 in the melanoblasts of developing mice causes complete loss of these cells from the developing embryo. This manifests itself as complete lack of a pigmented coat in adult mice. Chk1 deleted melanoblasts exhibit DNA damage as marked by γH2AX.To examine the role of Chk1 in melanoma maintenance and progression I utilized cell lines generated from a murine model of metastatic melanoma in allograft nude mouse experiments and investigated how the conditional genetic deletion of Chk1 in this model affects disease progression. Using this model I showed that complete loss of Chk1 during tumour development caused a profound reduction in the proliferation potential of melanoma tumour formation with a concurrent significant increase in survival time in these mice. In addition I also showed that hemizygous deletion of Chk1 during tumour development exerts a more modest but nevertheless measurable effect on melanoma tumour formation, however with no demonstrable effect on survival time.To further understand how the loss of Chk1 leads to cell death and how this may be beneficial for the treatment of melanoma I utilised a specific allosteric inhibitor of Chk1 in human melanoma cell lines. I found that Chk1 inhibition led to collapse of replication forks in S-phase cells resulting in the generation of DNA damage specifically in S-phase cells, leading to a cell death signal characterisedby cleavage of PARP and Annexin V positivity. I also showed that Chk1 inhibition has significant toxicity in vitro in all metastatic melanoma cell lines; however there is a broad range of relative toxicities to Chk1 inhibition between cell lines. Further in vivo analysis also showed that Chk1 inhibition caused a measurable reduction in the tumour growth rate of subcutaneously injected metastatic melanoma cells in CD1 nude mice.Overall the work presented here provides evidence that Chk1 is essential for both melanocyte and melanoma cell survival with the essential function of Chk1 being in S-phase of the cell cycle, and that Chk1 inhibition may be a viable therapeutic option for melanoma therapy.

[发布机构] University:University of Glasgow;Department:Institute of Cancer Sciences

[关键词] Melanoma, DNA damage signaling, DNA damage checkpoints, Chk1, cell cycle checkpoints