title: Atg3 Overexpression Enhances Bortezomib-Induced Cell Death in SKM-1 Cell.
abstract: BACKGROUND
  Myelodysplastic syndrome (MDS) is a group of heterogeneous hematopoietic stem cell malignancies with a high risk of transformation into acute myeloid leukemia (AML). Clonal evolutions are significantly associated with transformation to AML. According to a gene expression microarray, atg3 is downregulated in MDS patients progressing to leukemia, but less is known about the function of Atg3 in the survival and death of MSD/AML cells. Moreover, the role of autophagy as a result of bortezomib treatment is controversial. The current study was designed to investigate the function of Atg3 in SKM-1 cells and to study the effect of Atg3 on cell viability and cell death following bortezomib treatment.
  
  
  METHODS
  Four leukemia cell lines (SKM-1, THP-1, NB4 and K562) and two healthy patients' bone marrow cells were analyzed for Atg3 expression via qRT-PCR and Western blotting analysis. The role of Atg3 in SKM-1 cell survival and cell death was analyzed by CCK-8 assay, trypan blue exclusion assay, DAPI staining and Annexin V/PI dual staining with or without bortezomib treatment. Western blotting analysis was used to detect proteins in autophagic and caspase signaling pathways. Electron microscopy was used to observe ultrastructural changes after Atg3 overexpression.
  
  
  RESULTS
  Downregulation of Atg3 expression was detected in four leukemia cell lines compared with healthy bone marrow cells. Atg3 mRNA was significantly decreased in MDS patients' bone marrow cells. Overexpression of Atg3 in SKM-1 cells resulted in AKT-mTOR-dependent autophagy, a significant reduction in cell proliferation and increased cell death, which could be overcome by the autophagy inhibitor 3-MA. SKM-1 cells overexpressing Atg3 were hypersensitive to bortezomib treatment at different concentrations via autophagic cell death and enhanced sensitivity to apoptosis in the SKM-1 cell line. Following treatment with 3-MA, the sensitivity of Atg3-overexpressing cells to bortezomib treatment was reduced. Atg3 knockdown blocked cell growth inhibition and cell death induced by bortezomib.
  
  
  CONCLUSIONS
  Our preliminary study of Atg3 in the high-risk MDS cell line suggests that Atg3 might be possibly a critical regulator of autophagic cell death and a gene target for therapeutic interventions in MDS.
authors.affiliation: Department of Hematology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhuang
authors.affiliation: Department of Hematology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Department of Hematology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Hematology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Hematology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhu
authors.affiliation: Department of Hematology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Hematology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Xu
mesh_terms: D000071183:Autophagy-Related Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D000069286:Bortezomib / Q000494:pharmacology*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D006801:Humans; D009190:Myelodysplastic Syndromes / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*
chemical_list: D000071183:Autophagy-Related Proteins; D034741:RNA, Small Interfering; D000069286:Bortezomib; D044763:Ubiquitin-Conjugating Enzymes; C455621:ATG3 protein, human