[Effect of 5-aza-2'-deoxycytidine on cell of high-risk patients with myelodysplastic syndrome in vitro].
To investigate the effect of 5-aza-2'-deoxycytidine (5-Aza-CdR) on cell of high-risk patients with myelodysplastic syndrome (MDS) in vitro, the growth inhibition of MUTZ-1 cell induced by 5-Aza-CdR was detected by MTT method; apoptosis was detected by morphological observation and translocation of phosphatidylserine (PS) was examined by flow cytometry assay; the expressions of P15INK4B, DNA methyltransferases (DNMT)(1), DNMT(3A) and DNMT(3B) gene on mRNA level were detected by RT-PCR; methylation of p15INK4B gene in MUTZ-1 cells was detected by PCR using methylation specific primer (MSP). The results showed that 5-Aza-CdR inhibited the growth of MUTZ-1 cells. The IC(50) values of 24, 48 and 72 hours were 6.75, 2.82 and 5.45 mmol/L respectively. Characteristic changes of apoptosis emerged in MUTZ-1 cells after being exposed to 5-Aza-CdR in the different concentration from 0.8 mmol/L to 3.2 mmol/L, and the positive cells of annexin V on the membrane of MUTZ-1 cells were analyzed by flow cytometry. 5-Aza-CdR could activate the p15INK4B gene expression in MUTZ-1 cells by demethylation of the p15INK4B gene in a dose-dependent manner after the cells were treated for 48 hours. Furthermore, 5-Aza-CdR could significantly down-regulate the expressions of DNA methyltransferase genes DNMT(3A) at mRNA level in a dose dependent manner. However, it had no effects on DNMT(1) gene and DNMT(3B) gene. It is concluded that 5-Aza-CdR can inhibit the growth of MUTZ-1 cells and induce the apoptosis of these cells within the range of concentration from 0.8 mmol/L to 3.2 mmol/L, which may be one of the mechanisms of antitumor effects of 5-Aza-CdR. The drug can activate the expression of p15INK4B gene in MUTZ-1 cells by demethylation of the p15INK4B gene through inhibiting the expression of DNMT(3A) gene. It may be the mechanism of 5-Aza-CdR in the treatments of MDS.
Tong
Hong-Yan
HY
Department of Hematology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China. tonghy@mail.hz.zj.cn
Lin
Mao-Fang
MF
D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000235:genetics; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D015254:DNA Modification Methylases / Q000378:metabolism; D000077209:Decitabine; D006801:Humans; D009190:Myelodysplastic Syndromes / Q000188:drug therapy* / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis; D025521:Tumor Suppressor Proteins / Q000235:genetics
C493966:CDKN2B protein, human; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; C000717967:DNMT3A protein, human; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D000077209:Decitabine; D015254:DNA Modification Methylases; D000090205:DNA Methyltransferase 3A; D001374:Azacitidine