Diabetes, Vol 49, Issue 4 562-570, Copyright © 2000 by American Diabetes Association

ARTICLES

Selection of insulinoma cell lines with resistance to interleukin-1beta- and gamma-interferon-induced cytotoxicity

G Chen, HE Hohmeier, R Gasa, VV Tran and CB Newgard
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235, USA.

Engineered insulinoma cell lines may represent an alternative to isolated islets for transplantation therapy of type 1 diabetes. Success of this approach may require development of cell lines that can withstand cytokine-mediated damage. To this end, we have cultured INS-1 insulinoma cells in increasing concentrations of interleukin-1beta (IL-1beta) + gamma-interferon (IFN-gamma), with approximate weekly iterations over an 8-week period. Based on the C,N diphenyl-N'-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium+ ++ bromide (MTT) viability assay, the selected cells, termed INS-1res, were 100% viable after 5 days of treatment with 10 ng/ml of IL-1beta. These cells were also 78 +/- 1.2% viable after 5 days of exposure to the combination of 10 ng/ml IL-1beta and 100 U/ml IFN-gamma, whereas parental INS-1 cells treated in the same manner were only 0.3 +/- 0.03% viable. INS-1res cells were also resistant to treatment with supernatants from activated rat peripheral blood mononuclear cells, whereas only 20% of parental INS-1 cells survived such treatment. The resistance to IL-1beta conferred by this procedure was stable, whereas the partial resistance to IFN-gamma was transient but reinducible by culture in the presence of cytokines. Stable transfection of INS-1res cells with a plasmid containing the human insulin cDNA and expansion of the transfected colonies in the absence of cytokines produced cell lines that were on average more resistant to IL-1beta + IFN-gamma (53 +/- 11%) than similarly transfected clones derived from parental INS-1 cells (15 +/- 7%). Importantly, several INS-1res-derived clones retained the capacity to secrete insulin in response to glucose concentrations over the normal physiological range. With regard to the mechanism by which selection was conferred, we found normal levels of IFN-gamma receptor mRNA, but a 60% reduction in expression of the IL-1 receptor type I (IL-1RI) in INS-1res cells compared with parental INS-1 cells. IL-1beta signaling through p38 MAP kinase was found to be normal in INS-1res cells, suggesting that their expression of IL-1RI is sufficient to maintain cytokine action. However, normal IL-1beta-mediated translocation of NF-kappaB and induction of inducible nitric oxide synthase expression and nitric oxide production was severely impaired in the INS-1res cell lines, suggesting a mechanism for the IL-1beta resistance. In sum, this study defines a strategy for isolation of cytokine-resistant beta-cell lines and provides a new system for studying the mechanisms by which such resistance can be achieved.
CiteULike    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				O. T. Hardy, H. E. Hohmeier, T. C. Becker, E. Manduchi, N. M. Doliba, R. K. Gupta, P. White, C. J. Stoeckert Jr, F. M. Matschinsky, C. B. Newgard, et al. Functional Genomics of the {beta}-Cell: Short-Chain 3-Hydroxyacyl-Coenzyme A Dehydrogenase Regulates Insulin Secretion Independent of K+ Currents Mol. Endocrinol., March 1, 2007; 21(3): 765 - 773. [Abstract] [Full Text] [PDF]

				S. Kim, I. Millet, H. S. Kim, J. Y. Kim, M. S. Han, M.-K. Lee, K.-W. Kim, R. S. Sherwin, M. Karin, and M.-S. Lee NF-{kappa}B prevents beta cell death and autoimmune diabetes in NOD mice PNAS, February 6, 2007; 104(6): 1913 - 1918. [Abstract] [Full Text] [PDF]

				J. J. Collier, P. T. Fueger, H. E. Hohmeier, and C. B. Newgard Pro- and Antiapoptotic Proteins Regulate Apoptosis but Do Not Protect Against Cytokine-Mediated Cytotoxicity in Rat Islets and {beta}-Cell Lines. Diabetes, May 1, 2006; 55(5): 1398 - 1406. [Abstract] [Full Text] [PDF]

				E. Agbottah, C. de La Fuente, S. Nekhai, A. Barnett, A. Gianella-Borradori, A. Pumfery, and F. Kashanchi Antiviral Activity of CYC202 in HIV-1-infected Cells J. Biol. Chem., January 28, 2005; 280(4): 3029 - 3042. [Abstract] [Full Text] [PDF]

				V. V. Tran, G. Chen, C. B. Newgard, and H. E. Hohmeier Discrete and Complementary Mechanisms of Protection of {beta}-Cells Against Cytokine-Induced and Oxidative Damage Achieved by bcl-2 Overexpression and a Cytokine Selection Strategy Diabetes, June 1, 2003; 52(6): 1423 - 1432. [Abstract] [Full Text] [PDF]

				I. Chang, S. Kim, J. Y. Kim, N. Cho, Y.-H. Kim, H. S. Kim, M.-K. Lee, K.-W. Kim, and M.-S. Lee Nuclear Factor {kappa}B Protects Pancreatic {beta}-Cells From Tumor Necrosis Factor-{alpha}-Mediated Apoptosis Diabetes, May 1, 2003; 52(5): 1169 - 1175. [Abstract] [Full Text] [PDF]

				C. B. Newgard While Tinkering With the {beta}-Cell... Metabolic Regulatory Mechanisms and New Therapeutic Strategies: American Diabetes Association Lilly Lecture, 2001 Diabetes, November 1, 2002; 51(11): 3141 - 3150. [Abstract] [Full Text] [PDF]

				D. Lu, H. Mulder, P. Zhao, S. C. Burgess, M. V. Jensen, S. Kamzolova, C. B. Newgard, and A. D. Sherry 13C NMR isotopomer analysis reveals a connection between pyruvate cycling and glucose-stimulated insulin secretion (GSIS) PNAS, March 5, 2002; 99(5): 2708 - 2713. [Abstract] [Full Text] [PDF]

				M. M.W. Chong, H. E. Thomas, and T. W.H. Kay {gamma}-Interferon Signaling in Pancreatic {beta}-Cells Is Persistent but Can Be Terminated by Overexpression of Suppressor of Cytokine Signaling-1 Diabetes, December 1, 2001; 50(12): 2744 - 2751. [Abstract] [Full Text] [PDF]

				J. Xiao, S. Gregersen, M. Kruhoffer, S. B. Pedersen, T. F. Orntoft, and K. Hermansen The Effect of Chronic Exposure to Fatty Acids on Gene Expression in Clonal Insulin-Producing Cells: Studies Using High Density Oligonucleotide Microarray Endocrinology, November 1, 2001; 142(11): 4777 - 4784. [Abstract] [Full Text] [PDF]

				C. Bonny, A. Oberson, S. Negri, C. Sauser, and D. F. Schorderet Cell-Permeable Peptide Inhibitors of JNK: Novel Blockers of {beta}-Cell Death Diabetes, January 1, 2001; 50(1): 77 - 82. [Abstract] [Full Text]

				G. Chen, H. E. Hohmeier, and C. B. Newgard Expression of the Transcription Factor STAT-1alpha in Insulinoma Cells Protects against Cytotoxic Effects of Multiple Cytokines J. Biol. Chem., January 5, 2001; 276(1): 766 - 772. [Abstract] [Full Text] [PDF]