Xiaoqing Li1, Shitao Zou1, Liangsu Zhou2, Aidi Gao1, Jing Xu3, Chao He1, Jundong Zhou2, Shuhua Wu4, Yihong Chen5. 1. Suzhou Cancer Center Core Laboratory, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, Jiangsu, China. 2. Department of Radiation Oncology, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, Jiangsu, China. 3. Department of Neurology, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, China. 4. Department of Geriatrics, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China. 5. Department of Radiation, The First Affiliated Hospital of Wanna Medical College, Wuhu, Anhui, China.
Abstract
BACKGROUND: Radiotherapy has recently become more common for the treatment of esophageal squamous cell carcinoma (ESCC). Radioresistance, on the other hand, continues to be a major issue because it interferes with the effectiveness of ESCC radiation. It has been demonstrated that RAD18, an E3 ubiquitin-protein ligase that regulates translesion DNA synthesis (TLS), is implicated in the regulation of genomic integrity and DNA damage response. METHODS: In the present study, immunohistochemical staining and western blotting were utilized to determine RAD18 expression in ESCC tissues and cells. ESCC cell proliferation was determined using a colony formation assay. Immunofluorescence staining, comet assay, and homologous recombination (HR)/non-homologous end-joining (NHEJ) assays were conducted to examine the effect of RAD18 on the DNA damage response in ESCC cells. RESULTS: We found that high RAD18 expression was positively associated with a poorer prognosis in patients with ESCC who received radiotherapy. Downregulation of RAD18 expression significantly increased the sensitivity of ESCC cells to irradiation. Moreover, RAD18 knockdown prolonged the repair kinetics of γH2AX foci and resulted in longer comet tails. Furthermore, loss of RAD18 expression markedly decreased non-homologous end-joining (NHEJ) activity, but it did not affect homologous recombination (HR)-mediated double-strand break repair in ESCC cells. RAD18 upregulated p-DNA-dependent protein kinase complex (p-DNA-PKc) expression in vivo and in vitro. CONCLUSIONS: These data indicated that RAD18 may regulate radioresistance by facilitating NHEJ via phosphorylation of DNA-PKcs in ESCC cells, providing a novel radiotherapy target for ESCC.
BACKGROUND: Radiotherapy has recently become more common for the treatment of esophageal squamous cell carcinoma (ESCC). Radioresistance, on the other hand, continues to be a major issue because it interferes with the effectiveness of ESCC radiation. It has been demonstrated that RAD18, an E3 ubiquitin-protein ligase that regulates translesion DNA synthesis (TLS), is implicated in the regulation of genomic integrity and DNA damage response. METHODS: In the present study, immunohistochemical staining and western blotting were utilized to determine RAD18 expression in ESCC tissues and cells. ESCC cell proliferation was determined using a colony formation assay. Immunofluorescence staining, comet assay, and homologous recombination (HR)/non-homologous end-joining (NHEJ) assays were conducted to examine the effect of RAD18 on the DNA damage response in ESCC cells. RESULTS: We found that high RAD18 expression was positively associated with a poorer prognosis in patients with ESCC who received radiotherapy. Downregulation of RAD18 expression significantly increased the sensitivity of ESCC cells to irradiation. Moreover, RAD18 knockdown prolonged the repair kinetics of γH2AX foci and resulted in longer comet tails. Furthermore, loss of RAD18 expression markedly decreased non-homologous end-joining (NHEJ) activity, but it did not affect homologous recombination (HR)-mediated double-strand break repair in ESCC cells. RAD18 upregulated p-DNA-dependent protein kinase complex (p-DNA-PKc) expression in vivo and in vitro. CONCLUSIONS: These data indicated that RAD18 may regulate radioresistance by facilitating NHEJ via phosphorylation of DNA-PKcs in ESCC cells, providing a novel radiotherapy target for ESCC.
Authors: Yang Yang; Yanzhe Gao; Anastasia Zlatanou; Satoshi Tateishi; Vyacheslav Yurchenko; Igor B Rogozin; Cyrus Vaziri Journal: Cell Cycle Date: 2018-05-08 Impact factor: 4.534