Mechanism of CAG Regimen Eliminating Human T Cell Acute Lymphoblastic Leukemia Cell Line, A3

Objective To explore the mechanism of CAG regimen eliminating human T cell acute lymphoblastic leukemia cell line, A3 and evaluate the role played by G-CSF/G-CSFR system in this process.

Methods

1. The expression of G-CSFR on A3 cells was detected by flow cytometric analysis.

2. Cell cycle parameters of A3 cells treated with different concentration of G-CSF(5ng/ml A10ng/ml A15ng/ml A20ng/ml G0ng/ml as control) were examined by propidium iodide staining.

3. The inhibition and apoptosis rates of A3 cells caused by treatment with various combination of G-CSF, cytarabine (Ara-C), and aclarubicin (ACR) after incubation for 48h were analyzed by Cell Counting Kit (CCK-8) and AnnexinV staining, respectively.

4. After incubation for 48 hours with G-CSF and PD98059(the specific inhibitor of MEK in Ras-MAPK signaling pathway), cell cycle and cell dynamic change were examined.

Results

1. The expression frenquency of G-CSFR on A3 cells was 94.2% which was comparable to that of KG-1 cells.

2. The proportion of A3 cells in S-phase was elevated 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, the proliferation of A3 cells was inhibited more significantly than incubation with incubation with Ara-C alone (P<0.05, Ara-C 10⁻⁵M and 10⁻⁶M) by CCK-8 assay.

4. Incubated with Ara-C, ACR, and G-CSF for 48 hours, the apoptosis of A3 cells was increased than that treated with Ara-C and ACR.

5. With the concentration of PD98059 increased gradually, the proportion of A3 cells in S-phase and OD values of A3 cells decreased, which was less than that of control group (p<0.05).

Conclusion

1. G-CSFR was 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. Apoptosis was one of the mechanisms of CAG regimen eliminating A3 cells.

3. The interaction between G-CSF and G-CSFR activates a series of signaling pathways which includes Ras-MAPK. The inhibition of MAPK phosphorylation by PD98059 contributed partially to the effect of G-CSF on A3 cells.