Etoposide-induced DNA damage affects multiple cellular pathways in addition to DNA damage response

// Fengxiang Wei 1 , Peng Hao 2 , Xiangzhong Zhang 3 , Haiyan Hu 4 , Dan Jiang 5 , Aihua Yin 6 , Lijuan Wen 1, 7 , Lihong Zheng 8 , Jeffrey Zheru He 9 , Wenjuan Mei 10, 12 , Hui Zeng 11, 12 and Damu Tang 12 1 The Genetics Laboratory, Shenzhen Longgang District Maternity and Child Healthcare Hospital, Shenzhen, Guangdong, PR China 2 Division of Nephrology, The First Affiliated Hospital of Jiamusi University, Jiamusi, Heilongjiang, PR China 3 Department of Hematology, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, PR China 4 Department of Obstetrics and Gynecology, Shenzhen Maternal and Child Healthcare Hospital, Southern Medical University, Shenzhen, Guangdong, PR China 5 Shenzhen Hua Da Clinical Laboratory Center Co., Ltd., Shenzhen, Guangdong, PR China 6 Maternal and Children Metabolic-Genetic Key Laboratory, Guangdong Women and Children Hospital, Guangzhou, PR China 7 Zunyi Medical University, Zunyi, Guizhou, PR China 8 Department of Biogenetics, Qiqihar Medical University, Qiqihar, Heilongjiang, PR China 9 Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA 10 Department of Nephrology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, PR China 11 Department of Thoracic Surgery, Fourth Hospital of Hebei Medical University, Shijiazhuang City, Hebei, PR China 12 Division of Nephrology, Department of Medicine, McMaster University, Hamilton, ON, Canada Correspondence to: Fengxiang Wei, email: haowei727499@163.com Damu Tang, email: damut@mcmaster.ca Keywords: etoposide; DNA damage response; gene expression; RNA sequencing Received: October 14, 2017      Accepted: February 10, 2018      Epub: February 16, 2018      Published: May 08, 2018 ABSTRACT DNA damage response (DDR) coordinates lesion repair and checkpoint activation. DDR is intimately connected with transcription. However, the relationship between DDR and transcription has not been clearly established. We report here RNA-sequencing analyses of MCF7 cells containing double-strand breaks induced by etoposide. While etoposide does not apparently cause global changes in mRNA abundance, it altered some gene expression. At the setting of fold alteration ≥ 2 and false discovery rate (FDR) ≤ 0.001, FDR < 0.05, or p < 0.05, etoposide upregulated 96, 268, or 860 genes and downregulated 41, 133, or 503 genes in MCF7 cells. Among these differentially expressed genes (DEGs), the processes of biogenesis, metabolism, cell motility, signal transduction, and others were affected; the pathways of Ras GTPase activity, RNA binding, cytokine-mediated signaling, kinase regulatory activity, protein binding, and translation were upregulated, and those pathways related to coated vesicle, calmodulin binding, and microtubule-based movement were downregulated. We further identified RABL6, RFTN2, FAS-AS1, and TCEB3CL as new DDR-affected genes in MCF7 and T47D cells. By metabolic labelling using 4-thiouridine, we observed dynamic alterations in the transcription of these genes in etoposide-treated MCF7 and T47D cells. During 0-2 hour etoposide treatment, RABL6 transcription was robustly increased at 0.5 and 1 hour in MCF7 cells and at 2 hours in T47D cells, while FAS-AS1 transcription was dramatically and steadily elevated in both cell lines. Taken together, we demonstrate dynamic alterations in transcription and that these changes affect multiple cellular processes in etoposide-induced DDR.