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In vitro Studies of β-cell Death and Survival. Modulation by Adenoviral Vectors and Bcl-2 Overexpression
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Cell Biology.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type 1 diabetes is a multifactorial disease resulting from the selective destruction of insulin-producing β-cells within the pancreatic islets of Langerhans. The mechanisms of β-cell death are not fully understood but cytokines are important mediators of this process. In the present study we found that the combination of IL-1β, TNF-α and IFN-γ induced a nitric oxide-dependent disruption of the mitochondrial membrane potential in rat insulin-producing RINm5F-cells, which seems to be a necessary event for both RINm5F-cell apoptosis and necrosis. The antiapoptotic protein Bcl-2 was able to prevent cellular death in RINm5F cells, most probably by counteracting the mitochondrial permeability transition. These results pointed out the potential of such antiapoptotic genes as gene therapy tools, to allow enhanced resistance against autoimmune destruction of β-cells in type 1 diabetes. For this purpose we used a progesterone-antagonist (RU 486)-inducible gene transfer system to achieve an efficient and controlled Bcl-2 overexpression in primary rat β-cells. However, in our experience, prolonged in vitro culture revealed adenoviral-induced islet cell necrosis, a process that was not prevented by Bcl-2 overexpression. Moreover, we observed that specific adenoviral genotypes correlate with differential induction of necrosis in both human and rat pancreatic islet cells. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell-toxicity, our results showed that they were unable to build up an efficient antiviral response following infection and that their survival was dependent on the exogenous addition of α-interferon.

In conclusion, adenoviral techniques for overexpression of antiapoptotic proteins in insulin-producing cells may provide useful tools against β-cell directed autoimmune destruction. However, understanding the specific interactions of the viral gene products with cellular proteins and how they are involved in β-cell death regulation is fundamental for an efficient and safe application of gene therapy approaches to type 1 diabetes.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2004. , p. 40
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1320
Keyword [en]
Cell biology, β-cell, cytokine, mitochondrial membrane potential, Bcl-2, Adenoviral vector, Adenovirus, IFN-α, Diabetes
Keyword [sv]
Cellbiologi
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-3973ISBN: 91-554-5859-9 (print)OAI: oai:DiVA.org:uu-3973DiVA, id: diva2:163990
Public defence
2004-02-28, B21, Biomedical Centre, BMC Husargatan 3, Uppsala, 09:15
Opponent
Supervisors
Available from: 2004-02-03 Created: 2004-02-03 Last updated: 2018-01-13Bibliographically approved
List of papers
1. Cytokine-induced apoptosis and necrosis are preceded by disruption of the mitochondrial membrane potential in pancreatic RINm5F cells: prevention by Bcl-2
Open this publication in new window or tab >>Cytokine-induced apoptosis and necrosis are preceded by disruption of the mitochondrial membrane potential in pancreatic RINm5F cells: prevention by Bcl-2
2002 In: Molecular and Cellular Endocrinology, Vol. 190, p. 75-82Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91301 (URN)
Available from: 2004-02-03 Created: 2004-02-03Bibliographically approved
2. Adenoviral-induced islet cell cytotoxicity is not counteracted by Bcl-2 overexpression
Open this publication in new window or tab >>Adenoviral-induced islet cell cytotoxicity is not counteracted by Bcl-2 overexpression
2002 (English)In: Molecular medicine (Cambridge, Mass. Print), ISSN 1076-1551, E-ISSN 1528-3658, Vol. 8, no 11, p. 733-741Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The ability to transfer immunoregulatory, cytoprotective, or anti-apoptotic genes into pancreatic islet cells may allow enhanced resistance against the autoimmune destruction of these cells in type 1 diabetes. We describe here an inducible transduction system for expression of the anti-apoptotic bcl-2 gene in insulin-producing cells as a potential tool for protecting against beta-cell death.

MATERIALS AND METHODS: Isolated pancreatic rat islet cells or rat insulinoma (RINm5F) cells were transduced using a progesterone antagonist (RU 486) inducible adenoviral vector system, expressing the bcl-2 gene. Bcl-2 overexpression was measured by Western blot assays and flow cytometry analysis. Following exposure to cytokines or to the mitochondrial uncoupler FCCP, cell survival was determined using fluorescence and electron microscopy, and a colorimetric assay (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]- 2H-tetrazolium-5-carboxanilide [XTT]-based) for cell viability. The mitochondrial membrane potential ((m)) was assessed using the lipophilic cationic membrane potential-sensitive dye JC-1.

RESULTS: The adenoviral gene transfer system induced Bcl-2 expression in more than 70% of beta-cells and the protein expression levels were successfully regulated in response to varying concentrations of progesterone antagonist RU 486. Exposure of islet cells to proinflammatory cytokines IL-1beta, TNF-alpha, and IFN-gamma, or to the mitochondrial uncoupler FCCP resulted in disruption of the mitochondrial membrane potential ((m)) and beta-cell death. Bcl-2 overexpression stabilized (m) and prevented cell death in RINm5F cells but not in islet cells. In addition, prolonged in vitro culture revealed adenoviral-induced islet cell necrosis.

CONCLUSIONS: The RU 486-regulated adenoviral system can achieve an efficient control of gene transfer at relatively low doses of the adenoviral vector. However, Bcl-2 overexpression in islet cells did not prevent adenoviral- or cytokine-induced toxicity, suggesting that the specific death pathway involved in adenoviral toxicity in beta-cells may bypass the mitochondrial permeability transition event.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-91302 (URN)12520090 (PubMedID)
Available from: 2004-02-03 Created: 2004-02-03 Last updated: 2017-12-14Bibliographically approved
3. Adenoviral-mediated transduction of human pancreatic islets: importance of adenoviral genome for cell viability and association with a deficient antiviral response
Open this publication in new window or tab >>Adenoviral-mediated transduction of human pancreatic islets: importance of adenoviral genome for cell viability and association with a deficient antiviral response
2005 (English)In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 146, no 5, p. 2406-2414Article in journal (Refereed) Published
Abstract [en]

As adenoviral vectors are extensively used for genetic manipulation of insulin-producing cells in vitro, there is an increasing need to evaluate their effects on the function, morphology, and viability of transduced pancreatic islets. In the present study we observed that specific adenoviral genotypes, carrying E4 and E1/E3 deletions, correlate with differential induction of necrosis in pancreatic islet cells. In particular, the adenovirus death protein encoded from the E3 region of the adenoviral genome was able to modulate the changes induced in the morphology and viability of the transduced cells. We also propose a putative role for the transcriptional regulator pIX. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell toxicity, our results showed that they were unable to build up an efficient antiviral response after transduction and that their survival was dependent on the exogenous addition of alpha-interferon. An intact and fully functional beta-cell is crucial for the successful application of gene therapy approaches in type 1 diabetes, and therefore, the implications of our findings need to be considered when designing vectors for gene transfer into pancreatic beta-cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-91303 (URN)10.1210/en.2004-1667 (DOI)15705772 (PubMedID)
Available from: 2004-02-03 Created: 2004-02-03 Last updated: 2017-12-14Bibliographically approved

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