Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction.

Abstract

Apoptosis has been identified as a mechanism of pancreatic islet beta-cell death in autoimmune diabetes. Proinflammatory cytokines are candidate mediators of beta-cell death in autoimmune diabetes, and these cytokines can induce beta-cell death by apoptosis. In the present study, we examined whether transfection of human islet beta-cells with an anti-apoptotic gene, bcl-2, can prevent cytokine-induced beta-cell destruction. Human islet beta-cells were transfected by a replication-defective herpes simplex virus (HSV) amplicon vector that expressed the bcl-2 gene (HSVbcl-2) and, as a control, the same HSV vector that expressed a beta-galactosidase reporter gene (HSVlac). Two-color immunohistochemical staining revealed that 95+/-3% of beta-cells transfected with HSVbcl-2 expressed Bcl-2 protein compared with 14+/-3% of beta-cells transfected with HSVlac and 19+/-4% of nontransfected beta-cells. The bcl-2-transfected beta-cells were fully protected from impaired insulin secretion and destruction resulting from incubation for 5 days with the cytokine combination of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. In addition, the bcl-2-transfected islet cells were significantly protected from cytokine-induced lipid peroxidation and DNA fragmentation. These results demonstrate that cytokine-induced beta-cell dysfunction and death involve mechanisms subject to regulation by an anti-apoptotic protein, Bcl-2. Therefore, bcl-2 gene therapy has the potential to protect human beta-cells in pancreatic islets, or islet grafts, from immune-mediated damage in type 1 diabetes.

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to β-cell apoptosis

“…3,[5][6][7]13,18,32 Here, we show that cytokines, palmitate and thapsigargin, induce Bax translocation, cytochrome c release and caspase-3 cleavage, independently of changes in Bcl-2, Bcl-XL, Bax and Bak expression levels, but partly as a consequence of Mcl-1 downregulation (Figure 8). Our data are in agreement with previous studies showing that Mcl-1, in spite of its preferential mitochondrial localization, 33 is able to prevent Bax activation and translocation.…”

“…4 The Bax/Bak activation is regulated by a delicate equilibrium between antiapoptotic Bcl-2 proteins and pro-apoptotic BH3-only proteins. Among the antiapoptotic proteins, overexpression of Bcl-2 and Bcl-XL have been shown to prevent b-cell apoptosis in a variety of models, [5][6][7] but they may hamper b-cell function. 7 The molecular role of Mcl-1 in b-cells remains to be characterized.…”

Suppressor of cytokine signalling (SOCS)-3 protects beta cells against IL-1?-mediated toxicity through inhibition of multiple nuclear factor-?B-regulated proapoptotic pathways

“…the free radical scavengers manganese superoxide dismutase (Mn-SOD), glutathione peroxidase and catalase [6,7], and elements with anti-apoptotic properties, e.g. thioredoxin, calbindin, bcl-2 and A20 [8,9,10,11]. In general, however, the ability of the beta cell to activate a sufficient protective response is impaired, which could, in part, explain their sensitivity to the toxic effect of cytokines [12].…”

Gene- and cell-based therapeutics for type I diabetes mellitus

“…In the meantime, antigen nonspecific inflammation progresses within islets because of macrophage and DC secretion of soluble mediators of b-cell dysfunction and apoptosis activation. 233,234 Cationic liposomes 204,205,214 Peptide fusion domains 118,235 Therapeutics for type I diabetes mellitus R Bottino et al intact islet transduction. Equally unknown is the degree to which these vectors can contribute to post-transplantation inflammation.…”