The Role Of Apg-2 Overexpression In BaF3-BCR/ABL Cells

Objective ATP and peptide-binding protein in germ cells-(Apg-2), a mammalian heat shock protein belonging to the heat shock protein 110 (Hsp110) family, was previously found to be overexpressed in mouse primary B cells (BaF3 cells) stably expressing BCR/ABL cells (named BaF3-BCR/ABL) compared to the control BaF3 cells with empty vector (named BaF3-MIGR1) that were treated with hydrogen peroxide (H2O2) through our comparative proteomics study. The role of Apg-2 in BCR/ABL transformed cells have not been investigated. The present study was initiated to overexpress Apg-2 in BaF3-BCR/ABL and BaF3-MIGR1 cells, clarify the mechanisms of Apg-2 in BaF3-BCR/ABL cells, which would investigate the prospects of Apg-2 protein in the development and clinical therapy of CML with BCR/ABL.Methods The gene of Apg-2 was introduced into the pIRES2-EGFP vector. The recombinant plasmid had been examined by endonuclease restriction and sequencing. The plasmid was transfected into BaF3-MIGR1 cells and BaF3-BCR/ABL cells by electroporation. The stably transfected strain after G418 screening was identified with RT-PCR and western blot. Cell cycle and apoptosis were detected by FCM. The proliferation was analyzed by Am-Blue and colony formation assay. The expression of Bcl-2 and Bax were determined. After being exposed to H2O2, the cell viability was detected by Am-Blue, the release of LDH, content of MDA and activity of SOD were analyzed. The expression ofγ-H2AX was determined by western blot and immunofluorescence.Results (1) Succeed constructing the eukaryotic expressing vector pIRES2-EGFP-Apg-2, transfecting and screening cell lines stably expressing Apg-2(BaF3-BCR/ABL-Apg-2 and BaF3-MIGR1-Apg-2). (2) Apg-2 overexpression promoted cell cycle progression, increased cell proliferation and colony formation in BaF3-BCR/ABL cells and had no effect on apoptosis. (3) Apg-2 overexpression increased cell viability, induced cell damage, induced release of LDH, deceased content of MDA and increased activity of SOD, so that protecting H2O2-induced cell damage. Apg-2 overexpression inhibited the foci formation ofγ-H2AX, inducing oxidative stress induced DNA damage in BaF3-BCR/ABL cells.Conclusion This study explains that Apg-2 overexpression in BaF3-BCR/ABL cells increases cell proliferation and protects cells from H2O2 induced DNA damage that may interact with BCR/ABL, which provides evidence for the development and target therapies of CML.