Study Of The Expression And Effect Of Augmenter Of Liver Regeneration On Cell Proliferation And Survival In T-cell Acute Lymphoblastic Leukemia

Part1：Study of the expression and effect of augmenter of liverregeneratin in T-cell acute lymphoblastic leukemiaObjective: Augmenter of liver regeneration (ALR) is expressed invarious tissues and cells, and involves in many pathologic andphysiological processes. However, the expression and biological effects ofALR have not been studied in T-cell acute lymphoblastic leukemia (T-ALL)cells. In the present study, we evaluated the expression of ALR in T-ALLcells and investigated the role of rhALR in T-ALL cell proliferation andsurvival.Experiment1：Study of the expression and effect of augmenter ofliver regeneratin in Jurkat leukemia cellsMethods: Real-time fluorescent quantitative PCR and western blotwere used to determine the expression of ALR. MTS assay was used tocheck cell proliferation and the effect of ALR on vincristine-induced celldeath.Flow cytometry was used to determine the apoptotic cells and cell cycle distribution. Western blot analysis was used to detect the expressionof apoptosis-related and cell cycle relative proteins.Results: ALR expression in Jurkat cells were significantlyup-regulated. Administration of ALR had little effect on Jurkat cellviability and apoptosis, cell cycle distribution and apoptotic and cell cyclerelated proteins (Control group vs rhALR group, P>0.05). Inhibition ratesappeared to obviously increase after treatment with VCR (VCR group vsVCR+rhALR group, P<0.05). With progressively increasingconcentrations of rhALR, the means of VCR-induced apoptotic rates weredecreased and the G2/M phase cell population decreased significantly(VCR group vs VCR+rhALR group, P<0.05). ALR could regulate theexpression of apoptosis-related and cell cycle relative proteins (VCR groupvs VCR+rhALR group, P<0.05).Experiment2：Study of the expression and effect of augmenter ofliver regeneratin in T-cell acute lymphoblastic leukemiaMethods: T-cell acute lymphoblastic leukemia cells were isolatedwith Ficoll from patients’ bone marrow. Real-time fluorescent quantitativePCR and western blot were used to determine the expression of ALR. MTSassay was used to measure the effect of rhALR on vincristine-inducedT-ALL cell death. The amount of apoptotic cells was measured by flowcytometry. Western blot analysis was used to detect the expression ofapoptosis-related and cell cycle relative proteins. Results: ALR in6patients with T-ALL were significantlyup-regulated. Inhibition rates appeared to obviously increase after treatmentwith VCR (VCR group vs VCR+rhALR group, P<0.05). Withprogressively increasing concentrations of rhALR (0,20ug/ml), the meansof apoptotic rates were41.7±5.98%and26.3±3.56%in the presence ofVCR, respectively. Western blot showed ALR could regulate theexpression of apoptotic and cell cycle related proteins.Conclusions:1. Both on mRNA and protein level of ALR were up-regulated inT-cell acute lymphoblastic leukemia.2. Exogenous ALR had little effect on cell proliferation but renderedcells resistant to VCR in T-cell acute lymphoblastic leukemia.3. Exogenous ALR reduced apopotic cells induced by VCR in T-ALLcells.4. ALR could antagonize the effect of VCR on the G2/M-arrest inT-ALL cells. Part2: Decreased expression of augmenter of liver regenerationresults in enhancing the chemotherapeutic responsed of vincristine inJurkat leukemia cells Objective: As ALR were up-regulated in T-ALL and rhALRdecreased apoptotic cells and regulated cell cycle, in this part of study, wedetected the effect of silenced ALR on cell proliferation, apoptosis, cellcycle and chemotherapeutic sensitivity.Methods: After72hours of siRNA/ALR transfection, real-timefluorescent quantitative PCR was used to detect the efficiency of blockingthe ALR gene expression, western bot and immunofluorescence cellstaining were used to detect the expression of ALR protein. MTS was usedto determine the effect of siRNA/ALR interference on Jurkat cellproliferation and the chemotherapeutic sensitivity of vincrsitine. FCM wasused to analysis the changes of Apoptotic cells and cell cycle. Western blotanalysis was used to detect the expressin of apoptosis-related and cyclerelative proteins.Results: After72hours of transfection, the expression of ALR weresignificantly decreased (siRNA/ALR group vs siRNA/control group,P<0.05). Decreased expression of ALR could inhibit the proliferation ofJurkat cells, increase apoptotic cells and decrease the G2/M ratio(siRNA/ALR group vs siRNA/control group, P<0.05). After transfection,vincristine was added into culture medium. The IC50values ofsiRNA/ALR group was1.32±0.17μg/ml, of the siRNA/control group was4.87±0.52μg/ml, apoptotic rate were43.75±5.96%and24.59±3.76%, theG2/M ratio were85.98±6.67%, and62.21±1.83%, respectively (siRNA/ALR+VCR group vs siRNA/control+VCR group, P<0.05). AftersiRNA/ALR interference, the expression of pro-PARP, pro-caspase8,pro-caspase3and Bcl-2decreased, the expression of cdc25c and cdc2increased, cyclin B1and P53expression decreased. Incubation withvincristine the expression of cdc25c and cdc2decreased, cyclin B1and P53expression increased compared to siRNA/control group.Coclusions:1. SiRNA/ALR interference could block the ALR expressionefficiently.2. SiRNA/ALR interference could increase apoptotic rates anddecrease G2/M ratio, thus inhibit cell proliferation of Jurkat leukemia cells.3. SiRNA/ALR interference could increase vincristine-inducedapoptosis and cell cycle arrest to enhance the chemosensitivity ofvincristine in Jurkat cells.4. The mechanism of that siRNA/ALR interference enhancedapoptosis and chemosensitivity might be relevant to the apoptosis and cyclerelative proteins regulation.