title: Azacytidine inhibits the proliferation of human promyelocytic leukemia cells (HL60) by demethylation of MGMT, DAPK and p16 genes.
abstract: BACKGROUND
  Azacytidine (Aza) was the first demethylation agent identified that may inhibit DNA methyltransferases and reverse DNA hypermethylation, restoring the expression of silenced tumor suppressor genes in patients with myelodysplastic syndromes (MDS). It is unclear whether  azacytidine can alter the proliferative and apoptotic changes in myeloid leukemia cells, and methylation changes induced by this drug have remained poorly characterized in therapy-related models.
  
  
  METHODS
  The proliferation rate of azacytidine on HL60 cells was determined by the MTT  protocol. Methylation-specific PCR (MSP) and RT-PCR were used respectively to detect gene methylation status changes and expression levels of p16, Death associated protein kinase (DAPK) and O(6)-methylguanine-DNA methyltransferase (MGMT) before and after treatment with azacytidine.
  
  
  RESULTS
  Azacytidine inhibited HL60 cell proliferation and showed a time- and dose-dependent effect. MSP showed hypermethylated p16, DAPK, and MGMT genes before azacytidine treatment. Complete demethylation was seen in p16 and DAPK genes and partial demethylation  in the MGMT gene after co-culture with azacytidine. The expression level of p16, DAPK and MGMT genes in HL60 cells was up-regulated after treatment with azacytidine.
  
  
  CONCLUSIONS
  The CpG islands of p16, DAPK and MGMT genes are hypermethylated  in HL60 cells. Azacytidine inhibits proliferation of leukemic cells by hypomethylation of p16, DAPK and MGMT genes.
authors.affiliation: Children's Hospital of Chongqing Medical University, Chongqing 400014, China.
authors.forename: Yongchun
authors.initials: Y
authors.lastname: Su
authors.forename: Hongzhen
authors.initials: H
authors.lastname: Xu
authors.forename: Youhua
authors.initials: Y
authors.lastname: Xu
authors.forename: Jie
authors.initials: J
authors.lastname: Yu
authors.forename: Ying
authors.initials: Y
authors.lastname: Xian
authors.forename: Qing
authors.initials: Q
authors.lastname: Luo
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D001374:Azacitidine / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D048708:Cell Growth Processes / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000235:genetics* / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000235:genetics* / Q000378:metabolism; D064550:Death-Associated Protein Kinases; D005809:Genes, Regulator / Q000187:drug effects; D019942:Genes, p16 / Q000187:drug effects*; D018922:HL-60 Cells; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D011401:Promoter Regions, Genetic; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D045643:DNA Repair Enzymes; D001374:Azacitidine