The Role Of Interference Apg-2Gene In BaF3-IGR1, BaF3-p210and BaF3-p210-the T315I Cells

Objective Heat shock protein Apg-2widely expresses in a variety ofeukaryotic and prokaryotic cells with an important role in cell proliferationand apoptosis. However, its role in chronic myelogenous leukemia (CML)cells hasn’t been fully investigated. In our previous proteomics studies, wefound a high expression of Apg-2in mice pre-B lymphocytes(BaF3-MIGR1) and BaF3-p210cells with stable expression of Bcr-Ablfusion gene (referred as Bp210) after exposed to H2O2. Further more,wefounded that overexpression of Apg-2gene can promote the proliferation ofBp210cells and protect them from H2O2induced danmages.In this studywe inhibited expression of Apg-2by RNA interference in BaF3-MIGR1、Bp210and Bp210-T315I cells for exploring the effects on the cells, aimingto find a new target for CML treatment.Methods (1) The shRNA (Hsp41-43) plasmid which specificallytargeted Apg-2gene and the disorder of shRNA (HspHK) plasmid havebeen constructed and transfected into BaF3-MIGR1、Bp210andBp210-T315I cells by electroporation, then selested by G418pressureculturing for obtaining cells with best inhibition of Apg-2.(2). Cell proliferation was analyzed by colony formation and Am-blue test. Cellcycles was measured by FCM. Apoptosis related protein of Bax、Bcl-2andBCR/ABL was detected by western-blot.（3）After STI571treatment, cellapoptosis was detected by flow cytometry and Wright staining. Cellviability and proliferation were analyzed by Am-blue and colony formationtest.Results (1) Cell lines BaF3-MIGR1-Hspa42, Bp210-Hspa42andBp210-T315I-Hspa42with stable suppression of Apg-2gene and stabletransfected disorder shRNA BaF3-MIGR1-HspaHK, Bp210-HspaHK andBp210-T315I-HspaHK has been established.(2) Interference Apg-2expression can inhibit Bcr-Abl positive Bp210，Bp210-T315I cell cycleprogression，cell proliferation and colony formation. However，BaF3-MIGR1cell cycle progression and cell proliferation and clone formationhave been promoted. Apoptosis can be induced in all these3cell lines.(3)Inhibition of Apg-2enhanced the sensitivity of the Bp210on STI571,reduced cell viability and increased apoptosis rate.Conclusion This study demonstrated that interference of Apg-2genecan inhibit the proliferation and colony formation in Bcr-Abl positive cellsBp210and Bp210-the T315I, however, counterproductive in the Bcr-Ablnegative cells BaF3-MIGR1. Suppression Apg-2can promote the apoptosisby decreasing Bcl-2expression with the result of reducing Bp210-T315I onSTI571resistance and thus provide an experimental basis for further researches.