Mechanism Of Senescence Regulation In Acute B Cell Lymphoblastic Leukemia

PART ? THE CELL SENESCENCE OF ACUTE B-CELL LYMPHOBLASTIC LEUKEMIA IS POSTPONED BY ?-ARRESTIN1Objective:Cell senescence refers to the irreversible cell cycle arrest of cells in active growth state under various stress conditions,which is a part of the normal physiological process of cells.Abnormal senescence has been demonstrated to be critical for tumorigenesis and development.Although the prognosis of acute B cell lymphoblastic leukemia(B-ALL)has been significantly improved,the survival rate of relapsed B-ALL is still low,and the quiescent leukemia initiating cells(LICs)have been found pivotal in relapse for B-ALL.In recent years,inducing senescence of tumor cells has become one of the therapeutic targets of cancer.Therefore,the mechanism of senescence regulation in B-ALL LICs is worthy of further study.Cell signaling protein?-arrestin1 is a scaffold protein which involved in many signaling pathways about cancer occurence and development.In the previous study of our group,we found that?-arrestin1 postoned the senescence and lengthened telomere of bone marrow cells of B-ALL LICs derived NSG mice,suggesting a worse prognosis.In this study,we further studied the effect of?-arrestin1 on cell senescence in B-ALL and its possible mechanism.Method:The?-galactosidase staining assay was used to detect the senescent cell ratio of clinical samples in B-ALL.The expression of?-arrestin1,p53,p27,p21 mRNA,and?-arrestin1protein were detected by RT-PCR and Western Blot respectively.The correlation analysis was used to explore the relationship between?-arrestin1 and cell senescence.Lentivirus was used to construct Reh cell lines with impaired?-arrestin1.The effect of?-arrestin1 knockdown on cell senescence and double population time in Reh cell lines was studied by?-galactosidase staining assay.The expression of p53,p27,p21 mRNA,CBX and HIRA protein were detected by RT-PCR and Western Blot respectively.RT-PCR,PCR-ELISA and Southern blot were used to detect hTERT mRNA expression,telomerase activity and telomere length.Results:Clinical data showed that compared with the control group,the?-arrestin1 expression of bone marrow mononuclear cells in B-ALL increased 1.02 times(P<0.01),the expression of p53 and p21 increased(P<0.01),and the ratio of senescent cells decreased 31%(P<0.01).The expression of?-arrestin1 was negatively correlated with the proportion of senescent cells(R~2=0.4,P=0.003).Using Reh as B-ALL LICs model cells,the cell line Reh-Si?1 was successfully constructed with impaired?-arrestin1.Compared with the control group,the proportion of?-galactosidase positive cells and the expression of p53,hTERT,CBX and HIRA increased(P<0.05),the doubling time and the length of telomere shortened(P<0.05,P<0.01),and the activity of telomerase decreased(P<0.05).Conclusion:The expression of?-arrestin1 in B-ALL was increased,which was negatively correlated with senescent cells ratio.The stable knockdown Reh cells of?-arrestin1 was successfully constructed,which was the basis of further study in mechanism about cell senescence in B-ALL LICs.PART ? ?-ARRESTIN1 INHIBITS THE SENESCENCE OF REH CELLS BY PROMOTEING THE BINDING OF SP1 AND HTERT PROMOTERObjective : Replicative senescence is mainly mediated by human telomerase reverse transcriptase(hTERT)-telomerase activity-telomere length axis,in which hTERT m RNA expression affects telomerase activity and telomere length.The transcription activity of hTERT is regulated by many transcription factors,among which Sp1 is one of the most important.This part focuses on the molecular mechanism of ?-arrestin1 regulating hTERT transcription in B-ALL.Method:The binding of Sp1 protein to hTERT promoter was verified by electrophoretic mobility shift assay(EMSA),and the expression of Sp1 protein was detected by Western Blot.The vector containing different Sp1 binding sites of hTERT promoter(Sp11,Sp11-2,Sp15 + E-box,Sp11-5,NSp1 + E-box)was constructed on p GL3-basic plasmid.The transcriptional activity was detected by double luciferase reporter gene assay to explore the most important Sp1 binding site of hTERT promoter,and the specificity was verified by antisense nucleotide fragment.Results : The results of EMSA showed that there were macromolecular substances specifically binding to SP1 probe of nucleoprotein in Reh cells.The band with delayed migration was Sp1 hTERT promoter complex.After knocking down ?-arrestin1,the content of complex was significantly reduced,and there was no change in the expression of Sp1 protein in Reh-scram and Reh-Si?1 cells(P>0.05),indicating that ?-arrestin1 affected the binding of Sp1 to hTERT gene.Based on p GL3 basic plasmid,the vectors containing different binding site fragments of Sp1 in hTERT promoter region were successfully constructed: SP11,Sp11-2,Sp15 + E-box,Sp11-5,NSp1 + E-box.The results of double luciferase reporter gene assay showed that the luciferase activity of Reh-Si?1 cells was lower than that of Reh-scram group,and the fluorescence intensity of Sp11-2 was the highest(P<0.05),which was significantly higher than that of other fragments(P<0.05).The antisense nucleotide fragment of Sp12 was designed.After adding the antisense nucleotide fragment of Sp12,compared with the control group,the luciferase activity and hTERT expression decreased(P<0.05),telomere length shortened and the ratio of senescent cells increased.Conclusion:?-arrestin1 promoted Sp1 binding to hTERT promoter region,thus enhancing hTERT gene transcription activity.The binding of Sp1 to-28 to-36 bp in hTERT promoter region maximizes the transcription activity of hTERT.PART ? EFFECT AND MECHANISM OF SMALL MOLECULAR COMPOUNDS ON B-ALL REH CELL SENESCENCEObjective : The results of first two parts and previous animal experiments have proved the importance of B-ALL LICs senescence in the occurrence and development of B-ALL,and explored the mechanism of replicative senescence.The studies and clinical trials on the application of drugs targeting senescence in cancer have been continuously carried out.This part mainly studied the effects of the above two small molecular compounds(telomerase inhibitor,BIBR1532 and cyclin dependent kinase inhibitor,SNS-032)on B-ALL Reh cells.Method:Different concentration of BIBR1532 and SNS-032 were performed on Reh cells,and CCK-8 was used to detect cell proliferation,RT-PCR to detect hTERT expression,Southern blot and ?-galactosidase staining to detect telomere length and senescent cells ratio.Reh cells was treated with IC50 concentration of SNS-032,?-galactosidase staining were performed to detect senescent cells ratio,RT-PCR to detect the expression of p53,p21,p27,CDK1,CDK2,CDK9,XIAP,MCL1,Hoechst staining to detect cell cycle and Annexin V-APC/7-AAD staining to detect apoptosis.Results:BIBR1532 3?mol/l decreased the expression of hTERT in Reh cells by about 40%,shortened the telomere length(4.9% in Reh-Scram,6.1% in Reh-Si?1)and increased the proportion of ?-galactosidase positive senescent cells,but there was no difference in cell growth between the two groups(P>0.05).SNS-032 inhibited the proliferation of Reh cells in a dose-dependent manner,IC50 = 0.119344?mol/l.Reh cells was treated with SNS-032 0.12?mol/l,compared with the control group,the proportion of senescent cells increased 46.8%(P<0.01),the expression of hTERT decreased 65%(P<0.05),the expression of p53 increased 2.52 folds (P<0.05).There was no significant difference in the expression of p27 and p21(P>0.05).The cell cycle was blocked in G1 phase and the expression of CDK2 decreased(P>0.05),but there was no significant difference in the expression of CDK1 and CDK9(P>0.05).The ratio of apoptosis cells increased 89%(P<0.001),the expression of Mcl1 decreased(P<0.05),and the expression of XIAP had no significant difference(P>0.05).Conclusion:Telomerase inhibitor BIBR1532 can down regulate the expression of hTERT,but can't effectively inhibit the proliferation of B-ALL LICs.SNS-032 can effectively inhibit the proliferation of B-ALL LICs,and its mechanism may involve senescence,cell cycle arrest and apoptosis.