Adenoviral-induced islet cell cytotoxicity is not counteracted by bcl-2 overexpression

被引:25
作者
Barbu, AR
Akusjärvi, G
Welsh, N
机构
[1] Univ Uppsala, Ctr Biomed, Dept Med Cell Biol, S-75123 Uppsala, Sweden
[2] Univ Uppsala, Dept Med Biochem & Microbiol, S-75123 Uppsala, Sweden
关键词
D O I
10.1007/BF03402037
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Background: The ability to transfer immunoregulatory, cytoprotective, or anti-apoptotic genes into pancreatic islet cells may allow enhanced resistance against the autoimmune destruction of these cells in type 1 diabetes. We describe here an inducible transduction system for expression of the anti-apoptotic bcl-2 gene in insulin-producing cells as a potential tool for protecting against beta-cell death. Materials and Methods: Isolated pancreatic rat islet cells or rat insulinoma (RINm5F) cells were transduced using a progesterone antagonist (RU 486) inducible adenoviral vector system, expressing the bcl-2 gene. Bcl-2 overexpression was measured by Western blot assays and flow cytometry analysis. Following exposure to cytokines or to the mitochondrial uncoupler FCCP, cell survival was determined using fluorescence and electron microscopy, and a colorimetric assay (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-5-carboxanilide [XTT]-based) for cell viability. The mitochondrial membrane potential (DeltaPsi(m)) was assessed using the lipophilic cationic membrane potential-sensitive dye JC-1. Results: The adenoviral gene transfer system induced Bcl-2 expression in more than 70% of beta-cells and the protein expression levels were successfully regulated in response to varying concentrations of progesterone antagonist RU 486. Exposure of islet cells to proinflammatory cytokines IL-1beta, TNF-alpha, and IFN-gamma, or to the mitochondrial uncoupler FCCP resulted in disruption of the mitochondrial membrane potential (DeltaPsi(m)) and beta-cell death. Bcl-2 overexpression stabilized DeltaPsi(m) and prevented cell death in RINm5F cells but not in islet cells. In addition, prolonged in vitro culture revealed adenoviral-induced islet cell necrosis. Conclusions: The RU 486-regulated adenoviral system can achieve an efficient control of gene transfer at relatively low doses of the adenoviral vector. However, Bcl-2 overexpression in islet cells did not prevent adenoviral- or cytokine-induced toxicity, suggesting that the specific death pathway involved in adenoviral toxicity in beta-cells may bypass the mitochondrial permeability transition event.
引用
收藏
页码:733 / 741
页数:9
相关论文
共 38 条
[1]   Transgenic overexpression of human Bcl-2 in islet β cells inhibits apoptosis but does not prevent autoimmune destruction [J].
Allison, J ;
Thomas, H ;
Beck, D ;
Brady, JL ;
Lew, AM ;
Elefanty, A ;
Kosaka, H ;
Kay, TW ;
Huang, DCS ;
Strasser, A .
INTERNATIONAL IMMUNOLOGY, 2000, 12 (01) :9-17
[3]  
BARBU A, 2002, MOL CELL ENDOCRINOL, V190, P72
[4]   Adenovirus-mediated gene expression in vivo is enhanced by the antiapoptotic Bcl-2 gene [J].
Bilbao, G ;
Contreras, JL ;
Zhang, HG ;
Pike, MJ ;
Overturf, K ;
Mikheeva, G ;
Krasnykh, V ;
Curiel, DT .
JOURNAL OF VIROLOGY, 1999, 73 (08) :6992-7000
[5]   Induction of cell death by adenoviruses [J].
Braithwaite, AW ;
Russell, IA .
APOPTOSIS, 2001, 6 (05) :359-370
[6]   Gene transfer of the Bcl-2 gene confers cytoprotection to isolated adult porcine pancreatic islets exposed to xenoreactive antibodies and complement [J].
Contreras, JL ;
Bilbao, G ;
Smyth, C ;
Eckhoff, DE ;
Xiang, XL ;
Jenkins, S ;
Cartner, S ;
Curiel, DT ;
Thomas, FT ;
Thomas, JM .
SURGERY, 2001, 130 (02) :166-174
[7]   Cytoprotection of pancreatic islets before and soon after transplantation by gene transfer of the anti-apoptotic Bcl-2 gene [J].
Contreras, JL ;
Bilbao, G ;
Smyth, CA ;
Jiang, XL ;
Eckhoff, DE ;
Jenkins, SM ;
Thomas, FT ;
Curiel, DT ;
Thomas, JM .
TRANSPLANTATION, 2001, 71 (08) :1015-1023
[8]   Lentivirus-mediated Bcl-2 expression in βTC-tet cells improves resistance to hypoxia and cytokine-induced apoptosis while preserving in vitro and in vivo control of insulin secretion [J].
Dupraz, P ;
Rinsch, C ;
Pralong, WF ;
Rolland, E ;
Zufferey, R ;
Trono, D ;
Thorens, B .
GENE THERAPY, 1999, 6 (06) :1160-1169
[9]  
DYSPERSIN G, 1999, BIOCHIM BIOPHYS ACTA, V1428, P357
[10]  
Eizirik DL, 1997, DIABETES METAB REV, V13, P293, DOI 10.1002/(SICI)1099-0895(199712)13:4<293::AID-DMR195>3.3.CO