Expression Of Death-Associated Protein Kinase Gene In Actute Myeloid Leukemia Cell Line HL-60

bjective:To investigate the effect of methylation transferase inhibitor 5-aza-2'-deoxycitydine (5-aza-2dC) at various concentrations on the apoptosis of human acute myeloid leukemia (AML) cell line HL-60 and the expression of DAPK gene in HL-60 cells,to explore the possible anti-AML mechanism of 5-aza-2dC and provide experimental basis for AML pathogenesis research and clinical application of methylation transferase inhibitors in the treatment of leukemia.Methods:1 HL-60 cell line used as a model of leukemia. 2 Groups and interventions:Were divided into 7 groups:drug groups were respectively joined with the final concentration of 5-aza-2dC in 0.1,0.2,0.4,0.8,1.6 or 3.2 umol/L, by control group without any drug as a comparison. Each group set up three repeat holese, to cultivate HL-60 cells for 72h by the tumor cells culture techiques in vitro.3 The effect of 5-aza-2dC on the proliferation of HL-60 cells were detected by MTT assay.4 The effect of 5-aza-2dC on the apoptosis morphology of HL-60 cells were observed by Wright's staining.5 The effect of 5-aza-2dC on the apoptosis of HL-60 cells were detected by flow cytometry.6 Observing the expression of DAPK mRNA in different groups by PCR.6.1 Each group was extracted from total cellular RNA after cultrued for 72h, and total RNA was electrophoresed in 1% formaldehyde denaturalized agarose gel in order to validate integrity of RNA. 6.2 By random primer to reverse transcribe total RNA to cDNA, real-time fluorescent quantitative reverse transcription polymerase chain reaction (fluorogenic quantitive reverse transcription polymerize chain reaction, FQ-RT-PCR) to detect DAPK gene expression in the cell apoptosis process of each group.6.3 Randomly selected reverse transcriptase of the DAPK gene electrophoresis respectively DAPK gene in the third day electrophoresis. To produce respective standard curve according to gradient dilution of the DAPK gene cDNA and the ACTB gene cDNA. according to the starting point of each sample's exponential growth cycle which is also called the cycle threshold (cycle threshold, Ct) and each sample's standard curve slope, to calculate the starting cDNA template multiple values of each sample and standardize it by ACTB gene as internal reference.6.4 The relative expression of DAPK gene: Results relative DNA copy number and RNA expression of the relative amount of (2-ΔΔCt) said that the relative expression of DAPK gene.7 Statistical Methods:using the mean plus or minus standard deviation (X±S) to say the apoptosis rate and DAPK gene changes. Homogeneity of variance test, ONE-WAY AONVA analysis of variance, groups were compared with the number 22 LSD method, completed by the statistical software SPSS 13.0, P <0.05 was statistically significant. Results:1 By MTT assay,5-aza-2dC inhitited the proliferation of HL-60 cells in a concentration dependant manner.2 By Wright's staining,cell morphology identification demonstrated that the cultured cells were granulocytes. The apoptotic cells were obviously increased with the drug concentration.The inverted optical microscope displayed apoptic cells accurred the morphological changes.For example, cell volume became decrescent,cytoplasm reduced, nuclear became pyknotic, nuclear fractured, Chromatin near to karyotheca, some cell membrane had pseudopods sample protuberance or apoptotic body appearred.3 The results of flow cytometry indicated 5-aza-2dC induced HL-60 cell apoptosis in a concentration manner. Compared with the control group, all drug groups had statistically significant differences.4 RT-PCR results showed that the expression of DAPK gene mRNA in HL-60 cells had a gradually increasing trend with the increasing drug concenrtation; Compared with the control group, HL-60 cells began to appear a differential expression of DAPK gene mRNA when 5-aza-2dC reached a certain concentration. Conclusion:1 5-aza-2dC induced HL-60 cell apoptosis in a concentration manner.2 HL-60 cells in the control group had DAPK gene expression,but there was a higher level of gene expression with the increasing concentration of 5-aza-2dC.3 With the increasing concentration of 5-aza-2dC, the apoptosis rate of HL-60 cells and expression level of DAPK gene had a rising trend. Thus,speculated that DAPK gene may participate in the apoptosis process,induced by 5-aza-2dC.