Activation of Non-Homologous End Joining (NHEJ) DNA Repair by Nuclear Translocation of RON in Cancer Cells under Hypoxia

• Main Authors: Ting-ChiaChang, 張庭嘉
• Other Authors: Nan-Haw Chow
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/26801041836301283739

碩士 === 國立成功大學 === 分子醫學研究所 === 101 === Activation of Non-Homologous End Joining (NHEJ) DNA Repair by Nuclear Translocation of RON in Cancer Cells under Hypoxia Student: Ting-Chia Chang Advisors: Nan-Haw Chow Background： Cancer progression is modulated by alterations of microenvironment, such as nutrient starvation, hypoxia and pH change. To survive under these harsh conditions cancer cells must undergo a series of modifications to overcome the O2- and/or nutrient-deprived stresses. Receptuer d'Origine Nantatise (RON) is a member of c-Met RTK family and requires its cognate ligand in activating the downstream pathways. We have reported that membranous RON receptor, in association with epidermal growth factor receptor, could be transported to the nucleus of cancer cells and acts as a transcriptional regulator in response to serum starvation. Recently, we also demonstrated the nuclear translocation of RON when TSGH8301 bladder cancer cells are exposed to hypoxia. To identify its nuclear targets, nuclear fraction was co-immunoprecipitated (Co-IP) with RON antibody, fractionated by high performance liquid chromatography, and submitted for tandem mass analysis (Co-IP-HPLC- MS/MS). Among 80 candidate proteins, both Ku70 and DNA-PKcs were chosen for further investigation. Hypothesis：Modulation of NHEJ repair by nuclear RON may be one of machineries for cancer cells in response to hypoxic stress. Specific aims： (1) To examine the expression of double strand break (DSB) sensor and repair proteins under hypoxia (2) To investigate the relationship between RON and Ku70/DNA-PKcs complex under hypoxia (3) To investigate the role of nuclear RON in NHEJ repair under hypoxia (4) To examine the chemosensitivity of RON-knockdown stable cells in vitro under hypoxia Results: We found an up-regulation of DNA sensor proteins, i.e. p-ATM, γ-H2AX, under hypoxia, while expression of Ku70 and DNA-PKcs remained unchanged. However, there is time-dependent up-regulation of phospho-DNA-PKcs under hypoxia. On the other hand, expression of γ-H2AX was inhibited by DNA-PK inhibitor (NU7026) under hypoxia for 6 and 24 hr, respectively. The interaction of RON with Ku70 and DNA-PKcs in the nuclear fraction was confirmed by Co-IP of total or nuclear fraction of TSGH8301 cells and confocal microscopy. Domain mapping experiments using HEK293 cells co-transfection suggest that kinase domain of RON is essential for its interaction with Ku70 under hypoxia. The end-joining assay showed an activation of NHEJ repair by nuclear RON under hypoxia. Besides, we also demonstrated interaction of nuclear RON with Ku70/DNA-PKcs complex after treatment with DSB-inducing anticancer drugs. The RON-knockdown stable cells are more sensitive to hypoxic stress or chemotherapy treatment in vitro. Conclusions: 1. When bladder cancer cells are exposed to hypoxic stress, nuclear translocation of RON interacts with Ku70 and DNA-PKs to activate the NHEJ repair. 2. The DSB inducing anti-cancer drugs also initiates nuclear translocation of RON and its interaction with Ku70/DNA-PK complex.