Roles Of Sirtuins Expression In The Development Of Leukemia And Its Molecular Mechanism

Part?The study of sirtuins expression in acute leukemiaOBJECTIVE To preliminarily explore the relationship between sirtuins and leukemia,by detecting the expression of sirtuins m RNA and protein in peripheral blood mononuclear cells(PBMCs)and bone marrow hematopoietic stem cells from children with leukemia.METHODS The cohort consisted of forty-eight children with acute lymphoblastic leukemia(ALL),twenty children with acute myeloid leukemia(AML),together with a group of sixteen children with ALL and six children with AML who have achieved complete remission(CR).The proportion of immature cells in peripheral blood was more than 80%in all newly diagnosed cases of acute leukemia.The control was made up of eleven healthy bone marrow donors.Peripheral blood and(or)bone marrow specimens were collected,whereby the bone marrow and PBMCs were isolated by ficoll density gradient centrifugation method.Bone marrow CD34~+and CD33~+cells were separated by immunomagnetic microbeads.SIRT1?SIRT7 m RNA expressions and SIRT1?SIRT3 protein expressions were detected by real-time fluorescent quantitative polymerase chain reaction(RT-PCR)and western blot(WB)respectively.RESULTS 1.Compared with healthy controls,SIRT2 and SIRT6 m RNA expression were markedly elevated(P<0.05)in PBMCs from children with ALL,whereas SIRT3 m RNA expression was low(P<0.05).Their SIRT1,SIRT3 and SIRT5 m RNA expressions were significantly decreased after CR(P<0.05).2.SIRT3 m RNA expression in CD34~+and CD33~+cells from children with ALL was significantly less(P<0.05),whereas SIRT2 m RNA expression was high(P<0.05).3.The SIRT1 and SIRT2 protein levels were markedly elevated in children with ALL(P<0.05),and their SIRT1 and SIRT2 protein expression were decreased after CR(P<0.05).When compared with healthy controls,the SIRT3protein expression was much lower in ALL cases(P<0.001),and further declined SIRT3 protein expression was observed after CR(P<0.001).4.In children with AML,SIRT1,SIRT6 and SIRT7 m RNA expressions in PBMCs were much lower than healthy controls(P<0.05),while the SIRT3m RNA expression was significantly decreased after CR(P<0.05).5.SIRT3 m RNA expression in CD34~+and CD33~+cells from children with AML was much higher than healthy controls(P<0.05).6.The SIRT1 protein expression was markedly elevated in children with AML(P<0.05),and its expression did not changed after CR(P>0.05).The SIRT2 protein expression in AML was slightly higher than that of healthychildren but without statistical significance(P>0.05),and were not different from patients who achieved CR(P>0.05).When compared with healthy controls,the SIRT3 protein expression was much higher in AML cases(P<0.05).After CR,the SIRT3 protein expression in AML was declined(P<0.05).Conclusions Higher expression of SIRT2 and lower expression of SIRT3may be related to the pathogenesis of ALL.Higher expression of SIRT3 may be related to the pathogenesis of AML.SIRT1,SIRT2 and SIRT3 are expected to be the target for the therpy of leukemia.Part ? Effects of altered SIRT2 activity on the proliferation and apoptosis of leukemia cellsOBJECTIVE To investigate the effect of SIRT2 gene on the proliferation,and apoptosis of leukemia cells,and the possible molecular mechanism.METHODS Human myeloid leukemia cell line(HL60 cells)and human Tlymphocyte leukemia cell line(Jurkat cells)cultured in vitro were randomlydivided into experimental and control groups,in which the experimental groupwas further divided into 4 subgroups,which were treated with specific inhibitorof SIRT2–BML266,using concentrations 3UM,4UM,5UM and 6UMrespectively.BML266 was prepared by DMSO,and the final concentration ofDMSO was 0.1%.Cells in the control group were treated with 0.1% DMSO.CCK-8 method was applied to draw the growth curve of the cells.Cellproliferation,cell apoptosis and cell cycle were detected by flow cytometry.Wethen detected the m RNA expressions of apoptosis-related genes including B cellleukemia/ lymphoma 2(bcl2),Bcl-associated X protein(bax),cysteinylaspartate specific proteinase(caspase)3,caspase7 and caspase9,cellproliferation related genes Granulocyte Colony-Stimulating Factor(G-CSF)andGranulocyte Colony Stimulating Factor Receptor(GCSFR),and oncogenesknown to be associate with leukemia,including cyclin D1,Nuclear factor ?B(NF-?B)and c-Myc,tumor suppressor gene p21 and p53 by RT-PCR.Theexpression of activated caspase3 protein was detected by WB.(NF-?B)and c-Myc,tumor suppressor gene p21 and p53 by RT-PCR.Theexpression of activated caspase3 protein was detected by WB.RESULTS 1.When the concentration of BML266 was lower than 5UM,the growth curve of BML266-treated HL60 cells was not different from that of the control group.Once the concentration of BML266 reached to 5UM,growthinhibiion of HL60 cells was obvious.When the concentration of BML266 was lower than 4UM,the growth curve of Jurkat cells was not affected,but if the concentration exceeded 4UM,growth inhibition could be observed.2.The proliferation indexs of HL60 cells which were treated with 5UM or 6UM BML266,and Jurkat cells which were treated with 4UM,5UM or 6UM BML266 were significantly low(P<0.05).We therefore conclude that the growth inhibition effect is dosage dependent.3.The specific inhibitor of SIRT2–BML266 could induce G1/G0 cell cycle arrest in HL60 and Jurkat cells.The proportion of G1/G0 phase cells among the HL60 cells which were treated with 5UM or 6UM BML266,and the Jurkat cells which were treated with 4UM,5UM or 6UM BML266 were markedly increased(P<0.05).4.The apoptosis rate of HL60 and Jurkat cells which were treated with 3UM,4UM,5UM or 6UM BML266 markedly elevated(P<0.05).The pro-apoptotic effect is of dosage-dependence.5.After BML266 treatment,the m RNA expression of apoptosisrelated genes bax,caspase3 and caspase9 were increased(P<0.05)in Jurkat cells but not in HL60 cells(P>0.05).However,cleaved-caspase 3 could be detected both in HL60 and Jurkat cells.6.G-CSF and G-CSFR m RNA expressions were downregulated by BML266(P<0.05).7.The expression of oncogene c-Myc was downregulated in HL60 cells(P<0.05),whereas the expression of tumor suppressor gene p21 was upregulated both in HL60 and Jurkat cells(P<0.05).8.5UM BML266 was sufficient to inhibit the growth and induce apoptosis of HL60 cells and Jurkat cells,but didn't effect on the growth of 293 T cells.CONCLUSIONS BML266,a specific inhibitor of SIRT2,could significantly inhibit cell proliferation of HL60 and Jurkat cells by inducing the G1/G0 cell cycle arrest via the regulation of cell cycle controlled genes,and by downregulating the proliferation associated factors.At the same time,BML266 may induce the apoptosis of leukemia cells by activating the apoptotic signal pathway via the regulation of apoptosis related factors.BML266 had no cytotoxicity on benign cell 293 T cells.Part ? Roles of Ras/Raf/MEK/ERK signal pathway in SIRT2 mediated biological behavior changes in leukemia cellsOBJECTIVE To explore the roles of Ras/Raf/MEK/ERK signal transduction pathway in SIRT2 mediated biological behavior changes in leukemia cells.METHODS Before and after the Ras/Raf/MEK/ERK pathway was blocked by U0126,a specific inhibitor of MEK,changes in SIRT2 m RNA and protein expression were detected by RT-PCR and WB respectively.The expression of phosphorylated extracellular signal-regulated kinases(ERK)1/2 protein was detected by WB and compared in each group before and after BML266 treatment.Cells were treated with BML266,or BML266 combined with U0126.CCK-8 method was applied to draw the growth curve of the cells.Apoptosis rate of each group was detected by flow cytometry.The m RNA expression of downstream factors of ERK was detected by RT-PCR.RESULTS 1.1UM U0126 was the maximum tolerated concentration that does not affect the proliferation of HL60 and Jurkat cells.2.After being treated with 1UM U0126,the SIRT2 protein expression of HL60 and Jurkat cells decreased(P<0.05),whereas the levels of acetylated-a-tubulin increased(P<0.05).3.The phosphorylated ERK1/2 protein expression of HL60 and Jurkat cells declined after BML266 treatment(P<0.05).4.The growth inhibition effect of BML266 on HL60 and Jurkat cells was weakened by U0126,with declined apoptosis rate(P<0.05).5.After the Ras/Raf/MEK/ERK pathway was blocked,c-Myc and c-junm RNA expression were increased in HL60 cells(P<0.05).6.After the Ras/Raf/MEK/ERK pathway was blocked,c-fos,c-Myc and c-jun m RNA expression of Jurkat cells were decreased(P<0.05).CONCLUSIONS Ras/Raf/MEK/ERK signaling pathway has a regulating role for the expression of SIRT2 and its deacetylase activity.SIRT2-mediated biological behavior changes in leukemia cells may partly depend on this pathway.