Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 97 (2), 117-22

Expression of a Dominant Negative Inhibitor of NF-kappaB Protects MIN6 Beta-Cells From Cytokine-Induced Apoptosis

Affiliations

Expression of a Dominant Negative Inhibitor of NF-kappaB Protects MIN6 Beta-Cells From Cytokine-Induced Apoptosis

M S Baker et al. J Surg Res.

Abstract

Background: Islet graft injury by cytokines released from inflammatory cells (macrophages) that infiltrate the transplant site is an important mechanism of early islet transplant dysfunction. This detrimental "cytokine effect" is thought to be mediated by NF-kappaB-dependent up-regulation of iNOS gene expression and increased nitric oxide (NO) production by the islet. We attempted to make a beta-cell resistant to cytokine-induced apoptosis by transfecting the parent line with a dominant negative inhibitor of NF-kappaB.

Methods: A flag-tagged IkappaBalphaM cDNA subcloned into an SFFV-neo vector was used to transfect parent beta-Cell line MIN6. MIN6 and the resultant mutant (2Bm) were cultured for 24 h in a cytokine mixture including IL-1beta (50 units/mL), TNF-alpha (1000 units/mL), and IFN-gamma (750 units/mL) and cotreated with either the iNOS inhibitor L-NMMA (1 mM) or the caspase inhibitor Z-VAD (0.1 mM). NF-kappaB translocation was determined by gel shift. Nitrite production was determined by the Griess reaction. Apoptosis was determined by flow cytometry.

Results: When treated with cytokine 2Bm demonstrated significantly less NF-kappaB nuclear translocation, nitrite production, and apoptosis than parent MIN6. The rate of apoptosis in cytokine-treated 2Bm was a third less than that for cytokine-treated MIN6 and was similar to MIN6 cotreated with L-NMMA. Z-VAD cotreatment completely eliminated apoptosis in both MIN6 and 2Bm.

Conclusions: Cytokine-induced cell death in the MIN6 beta-cell line involves mechanisms that are, in part, NF-kappaB and NO dependent. Inhibition of NF-kappaB and NO production by the dominant negative inhibitor of NF-kappaB is cytoprotective. This type of genetic modification may prove to be one avenue for improving efficacy of islet transplantation.

Similar articles

See all similar articles

Cited by 17 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback