Abstract
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. 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 ((m)) was assessed using the lipophilic cationic membrane potential-sensitive dye JC-1. 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 ((m)) and beta-cell death. Bcl-2 overexpression stabilized (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. 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.
Highlights
Insulin-dependent diabetes mellitus is an autoimmune disorder resulting from the specific and progressive destruction of insulin-producing -cells of the islets of Langherhans
Dysfunction and damage of -cells in the early phase of the disease is thought to result from a direct contact with isletinfiltrating cells and/or exposure to cytotoxic mediators produced by these cells [1,2], leading to -cell death both by apoptosis [3] and necrosis [4]
The statistical differences between the groups are determined by one-way analysis of variance (ANOVA) for repeated measurements and the Bonferroni test
Summary
Insulin-dependent (type 1) diabetes mellitus is an autoimmune disorder resulting from the specific and progressive destruction of insulin-producing -cells of the islets of Langherhans. Bcl-2 overexpression has been reported to prevent cellular death both by apoptosis [5] and necrosis [4] in many cell types including insulin-producing cells, suggesting its potential as a tool for gene therapy in type 1 diabetes. 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 -cell death. The mitochondrial membrane potential (⌬⌿m) was assessed using the lipophilic cationic membrane potential-sensitive dye JC-1
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