ABSTRACT
Objective To investigate the relation between demethylation effect and apoptosis of valproate (VPA) in primary acute lymphoblastic leukemia (ALL) cells in vitro.Methods 10 cases of ALL patients was choosed to acquire leukemia cells.Cell growth curve were assessed by the MTT assay,the apoptosist of primary ALL was analyzed with DNA Ladder and Annexin-V-FITC/PI by flow cytometry.The expression methylation level of p15 was detected by hn-MSPCR,and p15mRNA were detected by RT-PCR.All dates was analyzed by SPSS16.0.Results The 50 ％ inhibition rate of VPA were 1.898 mmol/L to primary ALL cells assayed by MTT respectively.DNA ladder showed the apoptosis of primary ALL cells increased by adding VPA dose.Annexin-V-FITC/PI tests showed that the apoptosis percentage of primary ALL cells were (0.44±0.04) ％ in control group,(5.80±0.65) ％ in 1.0 mmol/L VPA group,(48.46±2.49) ％ in 2.0 mmol/L VPA group,(76.45±2.98) ％ in 4.0 mmol/L VPA group,the apoptosis percentage increased significantly (P ＜ 0.05).The demethylation of p15 INK4B gene decreased by adding VPA dose,the expression of p15 mRNA expression increased significantly compared with control group by RT-PCR (P ＜ 0.05).Conclusion It is found that VPA could induce demethylation of p15 INK4B gene,which could upregulate the p15 mRNA expression,due to the apoptosis of primary ALL cells.
ABSTRACT
Objective To explore the characteristics of hemi-nested methylation specific polymerase chain reaction (hn-MSP) and to find out the possible relationship between patterns of methylation or deletion and the developmet of adult acute lymphoblastic leukemia(ALL).Methods hn-MSP and bisulfit-sequencing PCR (BSP) were designed and adopted to analyze p15 gene methylation or deletion patterns in 25 adult ALL patients,malignant hematopathy cell lines and normal lymphocytes. hn-MSP and BSP products were cloned and sequenced.The sensitivity and specificity of hn-MSP were also analized.Results The sequencing results of hn-MSP and BSP products were consistent, and the sensitivity of detection of p15 methylation was up to 1.0×10-5.17 adult ALL patients (68 ％) were p15 gene hypermethylation and 3 patients were with deletion of p15 gene exon 1.There were no hypermethylation or deletion in the 10 controls.Conclusions The detection rate of p15 methylation in many tumors,especially in adult ALL,is frequent high.hn-MSP is highly sensitive and specific in analyzing p15 methylation.
ABSTRACT
Objective To investigate the inhibition of proliferation and the regulation of histone acetylation modification in Jurkat cells treated by sodium valproate(VPA). Methods Jurkat cells were treated with VPA.Cell proliferation was determined by CCK-8 assay, and cell cycle were analyzed by flow cytometry (FCM). mRNA of HDAC1 was detected by semi-quantitative RT-PCR, and protein expression of HDAC1 and acetylation of histone H3, H4 was examined by Western blotting. Results VPA inhibited the proliferation of Jurkat cells in concentration-and time-dependent manners. After exposure to VPA in different concentrations for 48h,cell cycle was arrested obviously at G0/G1 phase (P <0.05), and with increasing concentration, the percentage of G0/G1 phase cells was increased and that of S phase were decreased. HDAC1 mRNA expression were inhibited with the increasing concentration of VPA. The protein level of HDAC1 was down-regulated, while acetylation of histone H3、H4 was up-regulated in Jurkat cells by VPA. Conclusion VPA can inhibit proliferation of Jurkat cells and induce G0/G1 phase arrest. The mechanism may be that VPA increase acetylation of histone H3/H4 by inhibiting expressions of HDAC1 gene.