Roles Of Proapoptotic Protein Bim In Apoptosis Of Mouse Thymocytes And Human T-ALL CEM-C7 Cells Induced By Dexamethasone And Effects Of BimS Recombinant Adenovirus On Human Burkitt Lymphoma Raji Cells

Patient response to glucocorticoids(GC) is an important determinant of clinical outcome in childhood acute lymphoblastic leukemia(ALL)/lymphoma. Primary or secondary resistance to GC is one of the major causes of chemotherapy failure. Although GC has been used to ALL/lymphoma treatment for nearly 50 years, the mechanism of GC induced apoptosis of ALL/lymphoma is still unclear. To explore the molecular mechanism of GC induced apoptosis of ALL/lymphoma cells will help us understand GC resistance mechanism and further overcome this obstacle. Bim (Bcl-2 interacting mediator of cell death), a newly discovered member in pro-apoptotic subgroup of Bcl-2 family, is a novel apoptosis inducer and plays an important anti-tumor activity in lots of drug induced apoptosis models. In this study, we explored the effect and mechanisms of Bim in dexamethasone(DEX) induced apoptosis of sensitive cells.Firstly, as apoptosis of thymocyte induced by DEX is a typical model of apoptosis, 1μmol/L DEX was used to BALB/c mouse thymocyte cultured in vitro. Apoptosis of mouse thymocyte after DEX incubation was observed by the methods of transmission electronic microscope, AnnexinV-FITC/PI flow cytometry and DNA agarose gel electrophoresis. Using semi-quantitative RT-PCR, the increasing expression of Bim mRNA in thymocyte induced by 1μmol/L DEX was detected before obvious apoptosis of thymocyte was appeared, and the levels of Bim mRNA at indicated time points(except at 0h and 24h ) were significantly up-regulated when compared with control(p＜0.05). The peak point of mRNA expression in DEX induced thymocyte was also observed to came much earlier than control cells, especially BimL. When thymocyte was incubated with different concentrations of DEX(0, 10^-2, 10^-1, 10⁰, 10μmol/L), Bim mRNA expression increased in a dose-dependent manner and the increased range of BimL was the most significant among three isoforms of Bim. These results show that pro-apoptotic protein Bim plays an important role in mouse thymocyte apoptosis induced by DEX and BimL is the predominant isoform in this process. The increase of Bim mRNA expression appears in very early stage of thymocyte apoptosis and its expression levels are both time-vary and dose-dependent.Secondly, we explored the role of pro-apoptotic protein Bim in the apoptosis of glucocorticoid sensitive human T-ALL cell lines CEM-C7 cells induced by DEX. Apoptosis of CEM-C7 cells induced by 1μmol/L DEX was observed by transmission electronic microscope and PI flow cytometry after DEX incubation. By Quantitative Real Time PCR, the Bim mRNA expression in DEX sensitive CEM-C7 cells appeared as early as 4 hours after incubation with DEX and the Bim mRNA expression at indicated time points significantly increased when compared with control(without DEX) (p＜0.05). The level of Bim protein in DEX sensitive CEM-C7 cells was also obviously up-regulated after incubation with 1μmol/L DEX for 24h while no Bim protein up-regulation in DEX resistant CEM-C1 cells was observed. These results show that pro-apoptotic protein Bim plays a pivotal role in apoptosis of DEX sensitive CEM-C7 cells, and the absence of increased Bim protein expression after DEX treatment appears to be one of causes of CEM-C1 cells resistant to DEX. To further explore if the up-regulation of Bim was mediated by GC-GR signal transduction, CEM-C7 cells were incubated with GR blocker RU486 and DEX. The percentage of apoptosis in CEM-C7 cells significantly decreased after 36h and 48h incubation, respectively(p＜0.05). The expression levels of Bim mRNA and protein were also reduced at indicated time points when compared with control which was treated with 1μmol/L DEX only, indicating conjugation of GC to GR may be one of the key steps for GC induced Bim expression and the apoptosis of CEM-C7 cells. To know whether P38 MAPK signal pathway participates in the process of DEX induced Bim mRNA up-regulation and apoptosis, CEM-C7 cells were preincubated with different concentrations of P38 MAPK blocking pharmacon(SB203580) for 30 minutes followed by incubation with DEX. The results of Real Time PCR showed that Bim mRNA level in CEM-C7 cells was down-regulated by SB203580, indicating P38 MAPK signaling pathway takes part in the process of Bim expression and apoptosis in CEM-C7 cells.Thirdly, to construct recombinant adenovims vector of human BimS for gene therapy of DEX resistant ALL/lymphoma, BimS was specifically amplified from HL-60 cells by RT-PCR and confirmed to be correct by sequencing. Then BimS was cloned into shuttle vector pAdTrack-CMV carrying a green fluorescence protein(GFP) gene to generate a recombinant plasmid pAdTrack-CMV-BimS. This plasmid and aclenovims backbone plasmid pAdEast-1 were linearized and electroporated into E. coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restrict endonuclease digestion. The recombinant pAdEasy-CMV-BimS was transferred into HEK293 cells for packaging and amplification. The virus titre was determined to be arrived to 1.6×10⁸IU/ml, and the insert of Bums gene and the transient expression of BimS protein were verified by the methods of PCR and Western blot. These results indicated that recombinant human BimS adenovirus(Ad-BimS) was successfully constructed.Lastly, to test whether recombinant human BimS adenovirus has the capability of inducing apoptosis of tumor cells, recombinant of Ad-BimS was used to infect GC resistant Burkitt lymphoma Raji cells. After infected for 2～5 days, BimS expression. in Raji cells was detected by RT-PCR and Western blot. The significant growth retardation and increased apoptosis of Raji cells were also observed by growth curve and flow eytometry. These results imply that transfer of proapoptotic BimS into Raji lymphoma cells leads to inhibition of cell growth and induces apoptosis of DEX resistant Raji cells. This protocol provides a sound base for gene therapy of GC-resistance lymphoid tumor. Altogether, proapoptoic protein Bim participates in the process of DEX induced apoptosis of thymocyte and ALL-CEM-7 cells. The absence of up-regulation in Bim expression is one of the most important mechanisms of GC resistance in ALL. The up-regulation of Bim expression needs GC-GR mediated signal transduction, and P38 MAPK signal pathway also involves in this process. The use of recombinant adenovirus of BimS implies a new therapy for GC resistance ALL/lymphoma.