Mechanism Of CAG Regimen Eliminating T-cell Acute Lymphoblastic Leukemia Cells Of A3 Cell Line

Objective To explore the mechanism of CAG regimen eliminating T-cell acute lymphoblastic leukemia cells of A3 cell line and evaluate the role played by G-CSF/G-CSFR system in this process.Methods (1) Levels of G-CSFR expressed on the A3 cells membrane were detected by flow cytometrical assay. (2) Cell cycle parameters of A3 cells made by different concentration of G-CSF(5ng/ml,10ng/ml,15ng/ml,20ng/ml;0ng/ml as control group) were examined by propidium iodide staining method. (3) The inhibition and apoptosis rates of A3 cells caused by treatment with various combination of G-CSF, cytarabine (Ara-C), aclarubicin (ACR) after incubation for 48h were analyzed by Cell Counting Kit(CCK-8) and AnnexinV Kit, respectively. (4) After incubation for 48 hours with G-CSF and PD98059(the specific inhibitor of MEK in Ras-MAPK channel), cell cycles of A3 cells were analyzed by FCM and cell viabilities by CCK-8.Results (1) The expression levels of G-CSFR on A3 cells were 94.2%, no obvious differentiation between which and KG-1 cells of acute myeloid leukemia(AML) , positive control cells. (2) The proportions of A3 cells in S-phase rised concomitantly with the increasing G-CSF concentrations within 0-20ng/ml, highest at 15ng/ml of G-CSF. (3) After incubation with Ara-C and G-CSF for 48 hours, A3 cells were inhibited more significantly compared with incubation with Ara-C alone(P<0.05, Ara-C 10-5M and 10-6M) by CCK-8. (4) After incubation with Ara-C, ACR and G-CSF for 48 hours, A3 cells were induced into apoptosis much more than incubation with Ara-C and ACR by AnnexinV Kit . (5) With the concentration of PD98059 increased gradually, the proportions of A3 cells in S-phase and OD values of A3 cells decreased, which were less than that of control groups(p<0.05).Conclusion (1) G-CSFR were expressed on A3 cells. (2) G-CSF/G-CSFR system had a synergetic effect on eliminating A3 cells when administrated simultaneously with chemical agents by driving G0-phase cells into S-phase. (3) Inducing apoptosis may be one of the mechanisms of CAG regimen eliminating A3 cells. (4) The integration of G-CSF and G-CSFR activated a series of signaling transduction pathways which included Ras-MAPK channel. PD98059 interfered probably MAPK phosphorylation specifically to inhibit part of the effect of G-CSF.