The Mechanism Of Death Receptors DR4/DR5 And Intrinsic Resistance To TRAIL In Leukemia Cells

Part one: The mechanism of death receptors DR4/DR5 and intrinsic resistance to TRAIL in leukemia cellsPurpose:To investigate the role of DR4 gene in the occurrence,development and prognosis of acute myeloid leukemia(AML),find a new regulatory gene,namely DR4 gene,and explore the molecular mechanism of AML in the treatment of AML.Methods:The methylation level and the mRNA expression level of DR4 gene promoters of bone marrow mononuclear cells in 122 patients with newly diagnosed AML and24 patients with iron deficiency anemia(IDA)were detected using Methylation specific PCR(MS-PCR)and Q-RT-PCR,respectively,and a correlation analysis of them was conducted.Results:Compared with the control group,the methylation level(P=0.002)of DR4 genes of bone marrow mononuclear cells in patients with newly diagnosed AML was high.The methylation level(P=0.01)of DR4 genes of bone marrow mononuclear cells in patients of the positive group of enlargement of liver,spleen and lymph node was lower than that of the negative group,and the methylation level(P=0.006)of DR4 genes in patients of the high risk group of clinical stage was lower than that of the low risk group,and the methylation level(P=0.03)of DR4 genes in patients of the group where patients did not achieve complete remission(CR1)after a course of induction chemotherapy was lower than that of the group where patients achieved complete remission(CR1)after a course of induction chemotherapy.There was a significant negative correlation(P < 0.01)between the methylation level and the mRNA expression level of DR4 genes of bone marrow mononuclear cells in 122 patients with newly diagnosed AML.Summary:DR4 gene played an important role in the occurrence and development of AML.Decitabine can effectively inhibit the proliferation of AML,which probably partly because it can terminate the methylation effect of DR4 gene promoters and restore the mRNA expression of DR4 genes.Part two: Relationship between DR4 gene promoter methlyation and TRAIL resistant in K562 cellPurpose:To investigate the relationship between DR4 gene promoter methylation and tumor necrosis factor-related apotosis-inducing ligand resistant in K562 cell.Methods:K562 cell untreated by decitabine and treated by decitabine for 3 days were selected.Cell counting Kit-8(CCK-8)assay was used to determine the inhibitory rate of cell proliferation.The apoptosis of cells was detected by flow cytometry.The protein and m RNA expression levels of DR4 gene in K562 cell were decected by Western blotting and RT-PCR methods.The methylation of DR4 gene promoter region was examined by methylation-specific PCR(MSP).Results:K562 cell was highly resistance to TRAIL at the low concentration.After increasing the concentration of TRAIL(15.625?31.25?62.5?125?250?500?g/ml),cell growth was inhibited and existed in a dose and time dependent after 24 and 48 hours.After cells were treated by decitabine,the inhibitory effects of K562 cell treated by TRAIL was significantly higher than before treatment(P<0.05).After cells were treated by 125?g/ml decitabine,the apoptosis rate of K562 cell induced by TRAIL was significantly much more higher than before treatment(P < 0.05).Expressions of DR4 m RNA and protein in K562 cell were low,and DR4 gene promoter was methylation state.After cells were treated by decitabine,the expressions of DR4 m RNA and protein were more increased significantly than before treatment(P<0.05),and DR4 gene promoter was unmethylated.Summary:Decitabine can reverse DR4 gene promoter methylation state,regulating the level of gene expression,increasing TRAIL-induced apoptosis in K562 cell,furthermore,reversing TRAIL resistance.Therefore,decitabine combined with TRAIL may be a new steategy for the treatment of leukemia.Part three: The mechanism of intrinsic resistance to TRAIL in leukemia cellsPurpose:To verify the differences in TRAIL-sensitivity of K562 and U937 leukemia cell lines.To analyze the relationship of the expression level of death receptors and the sensitivity to TRAIL in K562 and U937 cells.To explore the effect on TRAIL sensitivity of DR4 gene expression suppressed K562 cells.To detect the differences in the level of death receptors redistribution onto membrane lipid rafts induced by TRAIL in K562 and U937 cells.To investigate the correlation between TRAIL sensitivity and the death receptors translocation onto cell membrane lipid rafts in leukemia cells.Methord:TRAIL-induced cytotoxicity of K562 and U937 cells were detected by MTT assay.TRAIL-induced cell apoptosis was analysed by the flow cytometry.The expression levels of DR4/DR5 of K562 and U937 cells were evaluated by RT-PCR and westen blot.K562 cells were transfected by si RNA targeted DR4,then confirmed suppressed expression of DR4 gene througn RT-PCR and western blot,and the TRAIL sensitivity of K562 cells with DR4 gene silence were analysed by MTT assay and flow cytometry.TRAIL-induced membrane aggregation levels of death receptors were detected by immunofluorescenee combined flow cytometry.Lipid rafts and non-lipid raft were separated by discontinuous sucrose gradient centrifugation,where the proteins were analysed by western blot,to investigate the differences in location of DR4,DR5 and pro-caspase 8 of K562 and U937 cells.At last,the correlation between TRAIL sensitivity and the death receptors translocation onto cell membrane lipid rafts was reverse verified by TRAIL-sensitive U937 cells with nystatin pretreatment.Results:It was indicated that U937 cells were sensitive to TRAIL and K562 cells were intrinsic resistant to TRAIL by MTT assay and flow cytometric analysis.TRAIL-sensitive U937 cell and TRAIL-resistant K562 cell showed the similar m RNA and protein level of DR5,whereas K562 cells showed a higher expression level of DR4 than U937 cells.Under DR4-si RNA transfection,the expression of DR4 was restrained and DR5 was not changed in K562 cells,but there was almost no change in its apoptosis induced by TRAIL.With TRAIL pretreatment,U937 cells showed a elevated formation of TRAIL contained immunocomplex in a time-dependent manner.Howerer,the uptake of TRAIL in K562 cells is almost no change.After lipid rafts and non-lipid rafts were separated by discontinuous sucrose gradient centrifugation,it was identified fractions 4 and 5 as lipid rafts by the presence of the lipid raft marker caveolin-1,and just U937 cells not K562 cells treated by TRAIL resulted in the redistribution of DR4,DR5 and procaspase-8 from the nonraft to the lipid raft fractions.At last,U937 cells with nystatin pretreatment showed the failure of DR4,DR5 and procaspase-8 migrate into lipid rafts.As a result,the nystatin treatment sigificantly down-regulated the TRAIL-induced apotosis of U937 cells.Conclusion:U937 cells were sensitive to TRAIL,and K562 cells were intrinsic resistant to TRAIL.There is on positive correlation between the exprission levels of death receptors and sensitivity to TRAIL,and DR4 expression levels could not affect the capacity of TRAIL in inducing apoptosis in K562 cells.TRAIL-induced redistribution of DR4 and DR5 in lipid rafts contributed to the sensitivity to TRAIL in TRAIL-sensitive U937 cell line,and the intrinsic risistance to TRAIL of K562 cells is conferred by incapacity of death receptors redistribution in lipid rafts.