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

Publication ,  Journal Article
Chen, G; Hohmeier, HE; Gasa, R; Tran, VV; Newgard, CB
Published in: Diabetes
April 2000

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.

Duke Scholars

Published In

Diabetes

DOI

ISSN

0012-1797

Publication Date

April 2000

Volume

49

Issue

4

Start / End Page

562 / 570

Location

United States

Related Subject Headings

  • Tumor Cells, Cultured
  • Transfection
  • Receptors, Interleukin-1
  • Receptors, Interferon
  • Rats
  • RNA, Messenger
  • Nitric Oxide Synthase Type II
  • Nitric Oxide Synthase
  • Nitric Oxide
  • Leukocytes, Mononuclear
 

Citation

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MLA
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Chen, G., Hohmeier, H. E., Gasa, R., Tran, V. V., & Newgard, C. B. (2000). Selection of insulinoma cell lines with resistance to interleukin-1beta- and gamma-interferon-induced cytotoxicity. Diabetes, 49(4), 562–570. https://doi.org/10.2337/diabetes.49.4.562
Chen, G., H. E. Hohmeier, R. Gasa, V. V. Tran, and C. B. Newgard. “Selection of insulinoma cell lines with resistance to interleukin-1beta- and gamma-interferon-induced cytotoxicity.Diabetes 49, no. 4 (April 2000): 562–70. https://doi.org/10.2337/diabetes.49.4.562.
Chen G, Hohmeier HE, Gasa R, Tran VV, Newgard CB. Selection of insulinoma cell lines with resistance to interleukin-1beta- and gamma-interferon-induced cytotoxicity. Diabetes. 2000 Apr;49(4):562–70.
Chen, G., et al. “Selection of insulinoma cell lines with resistance to interleukin-1beta- and gamma-interferon-induced cytotoxicity.Diabetes, vol. 49, no. 4, Apr. 2000, pp. 562–70. Pubmed, doi:10.2337/diabetes.49.4.562.
Chen G, Hohmeier HE, Gasa R, Tran VV, Newgard CB. Selection of insulinoma cell lines with resistance to interleukin-1beta- and gamma-interferon-induced cytotoxicity. Diabetes. 2000 Apr;49(4):562–570.

Published In

Diabetes

DOI

ISSN

0012-1797

Publication Date

April 2000

Volume

49

Issue

4

Start / End Page

562 / 570

Location

United States

Related Subject Headings

  • Tumor Cells, Cultured
  • Transfection
  • Receptors, Interleukin-1
  • Receptors, Interferon
  • Rats
  • RNA, Messenger
  • Nitric Oxide Synthase Type II
  • Nitric Oxide Synthase
  • Nitric Oxide
  • Leukocytes, Mononuclear