We have been investigating the potential utility of engineered cell lines as surrogates for primary islet cells in treatment of type 1 diabetes. To this end, two strategies that have emerged for procuring cell lines with resistance to immune-mediated damage are 1) selection of cytokine-resistant cell lines by growth of INS-1 insulinoma cells in iteratively increasing concentrations of interleukin (IL)-1β + γ-interferon (IFN-γ), and 2) stable overexpression of the anti-apoptotic gene bcl-2 in INS-1 cells. Herein, we show that bcl-2−overexpressing cells are resistant to the cytotoxic effects of reactive oxygen and nitrogen species (ROS/RNS), but are only modestly protected against high concentrations of IL-1β + INF-γ, whereas the converse is true in cytokine selected cells. We also found that the combination of bcl-2 expression and cytokine selection confers a broader spectrum of resistance than either procedure alone, such that the resultant cells are highly resistant to cytokines and ROS/RNS, with no impairment in glucose-stimulated insulin secretion. INS-1−derived cells with combined bcl-2 expression and cytokine selection are also more resistant to damage induced by coculture with mitogen-activated peripheral blood mononuclear cells. Surprisingly, application of the cytokine selection procedure to bcl-2−overexpressing cells does not result in impairment of nuclear factor-κB translocation, iNOS expression, and NO production, as clearly occurs upon application of the selection procedure to cells without bcl-2 overexpression. Further investigation of the diverse pathways involved in the development of cytokine and ROS/RNS resistance may define simplified and specific strategies for preservation of β-cell mass.
Address correspondence and reprint requests to Christopher B. Newgard, PhD, Department of Pharmacology and Cancer Biology, DUMC 3813, LSRC 351, Duke University Medical Center, Durham, NC 27710. E-mail:.
Received for publication 1 November 2002 and accepted in revised form 28 February 2003.
C.B.N. and H.E.H. are supported by a grant from Takeda Chemicals, Osaka, Japan.
GSIS, glucose-stimulated insulin secretion; IFN-γ, γ-interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MnSOD, manganese superoxide dismutase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; l-NMMA, NG-monomethyl-l-arginine; PBMC, peripheral blood mononuclear cell; PMA, phorbol-12-myristate-13-acetate; PMA/I, PMA + 1 μg/ml ionomycin; PMA/I + LPS, 10 ng/ml PMA + 1 μg/ml ionomycin + 10 μg/ml LPS; RNS, reactive nitrogen species; ROS, reactive oxygen species; SIN-1, 3-morpholinosydnonimine; SNAP, S-nitroso-N-acetylpenicillamine; STAT, signal transducer and activator of transcription; STZ, streptozotocin; TNF-α, tumor necrosis factor-α.