Up-regulated A20 promotes proliferation, regulates cell cycle progression and induces chemotherapy resistance of acute lymphoblastic leukemia cells
Introduction
Acute lymphoblastic leukemia (ALL) is a hematological malignancy characterized by an accumulation of immature cells in the bone marrow and peripheral blood. It is recognized as the most common type of childhood leukemia [1], [2]. Although great improvements have been made in the treatment of pediatric leukemia, adult ALL remains a high burden [3]. This burden is partly due to uncontrolled cell proliferation and poor response to chemotherapy in adult patients [4]. Leukemic cells abnormally express numerous genes that are associated with proliferation and apoptosis compared to normal hematopoietic cells, and these genes are thought to be the main cause of drug resistance [5]. An examination of abnormally expressed genes in leukemic cells, and of the pathogenic mechanisms associated with these genes, would therefore have great value for the development of targeted therapies.
The zinc-finger protein A20, also known as tumor necrosis factor-α-induced protein 3(TNFAIP3), was first identified as a cytokine-induced gene in human umbilical vein endothelial cells [6]. Many studies have suggested that A20 is an ubiquitin editing protein that regulates inflammation signaling pathways [7] and negatively regulates the nuclear factor kappa B (NF-κB) activation pathway [8]. Some autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis, are closely related with A20. Growing evidence also supports a role for A20 in the pathogenesis of cancer [9]. Dysregulation of A20 expression was observed in many types of solid tumor cells; for example, A20 expression is increased in glioblastoma cells [10], [11], hepatocellular carcinoma cells [12], estrogen receptor(ER)-negative or tamoxifen-resistant ER-positive breast cancer cells [13], [14], undifferentiated nasopharyngeal carcinoma cells, and poorly differentiated head and neck cancer cells [15]. These studies suggest an oncogenic role for A20 in these tumors and also suggest that A20 may be a useful new biomarker. However, little is known about the role of A20 in ALL.
In this study, we investigated the expression and biological function of A20 in several types of ALL cells. The results revealed that there was a significantly higher level of A20 protein in cell lines and in bone marrow mononuclear cells (BMMNC) obtained from ALL patients than in cells obtained from healthy donors. In addition, A20 promoted cell proliferation, induced cell cycle arrest, and reduced the sensitivity of three ALL cell lines to daunorubicin via a mechanism involving the ERK, p53 and p21 signal pathways. However, the role of A20 in different ALL cells varied, and the mechanisms by which A20 acted were not completely consistent. This suggests that the effects of A20 are pleotropic and complex in ALL.
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Patients and cell lines
Bone marrow samples were obtained from 5 newly diagnosed adult T-ALL or B-ALL patients who were enrolled in the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, from January 2014 to January 2015. Control bone marrow samples were obtained from 3 healthy hematopoietic stem cell transplantation donors. Diagnosis and leukemia classification were based on 2008 World Health Organization criteria. All subjects provided informed
A20 is expressed at high levels in ALL patients and cell lines
To investigate the function of A20 in ALL, we first analyzed the expression of A20 in newly diagnosed ALL patients and healthy donors. Western blot analysis revealed a significant increase in A20 protein expression in BMMNCs in ALL patients compared to healthy donors (Fig. 1A). In addition, analysis of protein and mRNA levels showed that A20 was highly expressed in several lymphoid cell lines, and particularly in ALL cell lines, compared to normal PBMCs (Fig. 1B and C).
A20 silencing reduces cell proliferation
Following our finding
Discussion
ALL is the most common primary cause of cancer-related death in children [1], [2]. However, the mechanisms leading to uncontrolled cell proliferation in these patients remain elusive. Multiple molecular dysfunctions associated with ALL leukemogenesis may enhance tumor growth and chemotherapy resistance, and these have been attributed to Notch1, MDM2, p53 and microRNAs [17], [18], [19], [20]. Considerable effort has been made to improve our understanding of the pathogenesis of ALL so that we can
Authors’ contributions
Shuying Chen performed most of the experiments, analyzed the data and drafted the manuscript. Haiyan Xing, Shouyun Li, Jing Yu, Huan Li, Shuang Liu, Zheng Tian, Kejing Tang assisted in molecular studies and plasmid construction. Qing Rao and Min Wang contributed to the design of experiments and data interpretation and helped revise the manuscript. Jianxiang Wang contributed to the approval of the final revision of the manuscript.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 81430004, 81370599, 81300380).
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