Bone Marrow Mesenchymal Stem Cells Affect The Cell Cycle Arrest Effect Of Genotoxic Agents On Acute Lymphocytic Leukemia Cells Via p21 Down-Regulation
Authors: Zhang Yiran , Hu Kaimin , Hu Yongxian , Wang Binsheng , Huang He 
Published: October 2019

Abstract
The recurrence rate of acute lymphocytic leukemia (ALL) in adult is high, and the five-year survival rate of recurrent patients is as low as 7%. Increasing evidence indicates that bone marrow mesenchymal stem cell (BMSC), which is a pivotal component of the bone marrow microenvironment, can enhance the survival and drug resistance of ALL cells, which is a reason for the high recurrence rate of ALL in adult. However, whether BMSCs promote or block the cell-cycle and proliferation of leukemia is indefinite, and whether BMSCs affects the the cell cycle arrest effect of genotoxic agents on ALL cells is unclear.
In this study, human BMSCs was obtained from normal bone marrow donors, and ALL cell line Reh was cultured with or without BMSCs in the presence or absence of etoposide (VP16) or idarubicin (IDA). MLN4924, the inhibitor of SCF complex, was used to induce the high expression of cell-cycle inhibitor p21. The proliferation of ALL cells was detected by CCK-8 kit and cell cycle was analyzed by flow cytometry with PI staining. The protein expression of ALL cells was determined by western blot and the mRNA expression was detected by qRT-PCR.
The results indicated that BMSCs affected the cell cycle arrest effect of genotoxic agents on ALL cells. In the absence of drugs, BMSCs had no significant effect on the cell-cycle of Reh cells. Under the treatment of VP16, the S phase ratio of Reh cultured with BMSCs was higher than Reh cultured alone (10.893±1.077% and 6.842±0.652% respectively, p<0.05), the G2/M phase ratio of Reh cultured with BMSCs was lower than Reh cultured alone (25.812±1.720% and 38.818±3.508% respectively, p<0.05). Under the treatment of IDA, the S phase ratio of Reh cultured with BMSCs was higher than Reh cultured alone (58.720±5.765% and 45.519±7.886% respectively, p<0.05), the G2/M phase ratio of Reh cultured with BMSCs was lower than Reh cultured alone (1.888±0.809% and 6.449±0.325% respectively, p<0.05).
At both protein and mRNA level, treatment of VP16 or IDA induced the expression of p21 in Reh cells, while BMSCs down-regulated its expression. MLN4924-induced p21 high expression in Reh cells eliminated the cell-cycle promotion effect of BMSCs on Reh cells under the treatment of genotoxic agents, which confirmed that BMSCs affect the cell cycle arrest effect of genotoxic agents on ALL cells via p21 down-regulation.
Moreover, in the presence of VP16 or IDA, BMSCs reduced the p53 mRNA expression and increase the c-myc mRNA expression in Reh cells, and BMSCs also up-regulated the protein expression of p-Erk and β-catenin in Reh cells. Since p53 is the major transcription factor of p21 and c-myc is the transcription inhibitor of p21, our results suggested that the BMSCs induced down-regulation of p21 in ALL cells might be through down-regulation of p53 and activation of Wnt/β-catenin/c-myc and erk/c-myc pathways.
In conclusion, the present study indicates that BMSCs affect the cell cycle arrest effect of genotoxic agents on acute lymphocytic leukemia cells via p21 down-regulation. This finding suggests that targeting the effect of microenvironment on leukemia might be a novel approach for ALL therapy.

Disclosures:
No relevant conflicts of interest to declare.