GAS2-Calpain2 Axis Contributes To The Growth Of Leukemic Cells

Objective: GAS2(Growth arrest specific 2) modulates cell cycle, apoptosis and Calpain activity. The GAS2-Calpain axis has dual functions in malignant transformation. However, the GAS2 expression and the role of GAS2-Calpain2 axis in leukemic cells remain unclear. This study aims to investigate the role and the mechanism of GAS2-Calpain2 axis in leukemic cells.Methods:(1) Q-RT-PCR(Quantitative Real-time PCR) and Calpain activity assay were used to measure m RNA expression of GAS2 and Calpain activity in human leukemic cells;(2) Western blot and immunofluorescence were performed to examine GAS2 expression and localization in leukemic cells;(3) Small hairpin RNA(sh RNA) against GAS2 was delivered with lentiviral vector to THP-1 and Jurkat cells, the control and GAS2 silenced cells were assessed for their proliferation, colony–forming cell capacities and Calpain activity;(4) Tumorigenic abilities were compared between control and GAS2 silenced THP-1 cells, when they were injected subcutaneously into nude mice;(5) Co-immunoprecipitation was performed to detect the interaction between endogenous GAS2 and Calpain2 in both THP-1 and Jurkat cells;(6) The effect of GAS2 silence on the colony-foming cell capacities of chronic myeloid leukemia(CML) CD34+ cells was investigated as well.Results:(1) Q-RT-PCR was performed to assess the expression of GAS2 in 119 leukemia patients, and found that all kinds of leukemia patients and various leukemic cell lines had significant higher expression compared with the normal bone marrow cells. It was found that Calpain activity was significantly lower in AML patients(n=22) in comparison to that in normal bone marrow cells(n=7);(2) The Immunofluorescence staining found that GAS2 was higher in CCRF-CEM, SHI-1, THP-1, Jurkat and K562 cells than normal control cells;(3) Two independent sh RNA sequences against GAS2 were validated to suppress the transcript and protein expression of GAS2 as high as 75%;(4) GAS2 silence enhanced the Calpain activity in both THP-1 and Jurkat cells, at the same time, cell proliferation and colony-forming cell capacities were significantly inhibited by GAS2 silence compared to control cells;(5) Tumorigenic capacity of THP-1 cells were strongly inhibited by GAS2 silence as well;(6) Co-immunoprecipitation demonstrated that GAS2 interacted with Calpain2 but not Calpain1 in both THP-1 and Jurkat cells;(7) GAS2 silence significantly inhibited the colony-forming cell ability of chronic myeloid leukemia CD34+ cells(n=3).Conclusion: Our data have demonstrated that GAS2 is aberrantly expressed in various human leukemic cells. GAS2 silence enhances Calpain activity and inhibits the growth of leukemic cells in vitro and in vivo. GAS2 silence also inhibits the in vitro growth of CML stem/progenitor cells. Endogenous GAS2 interacts with Calpain2 rather than Calpain1, and Calpain2 is required for GAS2 silence induced growth inhibiton, which suggests the critical role of GAS2-Calpain2 axis in the growth of leukemic cells.