Delivery of Bcl-XL or its BH4 domain by protein transduction inhibits apoptosis in human islets

doi:10.1016/j.bbrc.2004.08.116

Biochemical and Biophysical Research Communications

Volume 323, Issue 2, 15 October 2004, Pages 473-478

https://doi.org/10.1016/j.bbrc.2004.08.116 Get rights and content

Abstract

Viability of isolated islets is one of the main obstacles limiting islet transplantation success. It has been reported that overexpression of Bcl-2/Bcl-XL proteins enhances islet viability. To avoid potential complications associated with long-term expression of anti-apoptotic proteins, we investigated the possibility of delivering Bcl-XL or its anti-apoptotic domain BH4 to islets by protein transduction. Bcl-XL and BH4 molecules were fused to TAT/PTD, the 11-aa cell penetrating peptide from HIV-1 transactivating protein, generating TAT-Bcl-XL and TAT-BH4, respectively. Transduction efficiency was assessed by laser scanning confocal microscopy of live islets. Biological activity was tested as the ability to protect NIT-1 insulinoma cell line from death induced by staurosporine or serum deprivation. Spontaneous caspase activation in human islets and cytotoxicity caused by IL-1β were significantly reduced in the presence of TAT-Bcl-XL and TAT-BH4. We conclude that both TAT proteins are biologically active after transduction and could be an asset in the improvement of islet viability.

Section snippets

Materials and methods

Cell lines and islet isolation. NIT-1 cells were obtained from American Type Culture Collection (ATCC—Manassas, VA) and cultured according to manufacturer’s instructions. Human and non-human primate islets were isolated using collagenase (Roche Molecular Biochemicals, Indianapolis, IN) and the automated method, as described previously [18], [19]. After isolation, islets were cultured in CMRL medium (Mediatech; Herndon, VA) supplemented with human serum albumin for 24 h at 37 °C followed by 22 °C

Analysis of TAT-Bcl-XL transduction efficiency in live islet cells

We investigated the proficiency of TAT-Bcl-XL to translocate into human islets. Human pancreatic islets were transduced with TAT-Bcl-XL fusion protein labeled with fluorescein isothiocyanate (FITC). To avoid potential artifacts due to fixation [20], transduction was assessed by confocal microscopy on live non-fixed, islets (Fig. 1). Propidium iodide staining (red) excluded dead cells and ruled out false positive. Images were collected at 13 different focal planes, demonstrating the ability of

Acknowledgments

This work was supported by grants (DK-59993, awarded to R.L.P.) and Islet Cell Resources (5U42RR016603), from the National Institute of Health and by the Diabetes Research Institute Foundation and the Foundation for Diabetes Research. We thank Brigitte Shaw (Imaging Core Facility) for her technical assistance.

References (32)

F.T. Thomas et al.
Anoikis, extracellular matrix, and apoptosis factors in isolated cell transplantation
Surgery
(1999)
S.Y. Proskuryakov et al.
Necrosis: a specific form of programmed cell death?
Exp. Cell Res.
(2003)
J.S. Wadia et al.
Protein transduction technology
Curr. Opin. Biotechnol.
(2002)
Z. Mi et al.
Characterization of a class of cationic peptides able to facilitate efficient protein transduction in vitro and in vivo
Mol. Ther.
(2000)
M. Chen et al.
Calpain and mitochondria in ischemia/reperfusion injury
J. Biol. Chem.
(2002)
M. Lundberg et al.
Positively charged DNA-binding proteins cause apparent cell membrane translocation
Biochem. Biophys. Res. Commun.
(2002)
T. Takehara et al.
Expression and role of Bcl-xL in human hepatocellular carcinomas
Hepatology
(2001)
T. Satoh et al.
Survival factor-insensitive generation of reactive oxygen species induced by serum deprivation in neuronal cells
Brain Res.
(1996)
D.J. Kawahara et al.
Species differences in human and rat islet sensitivity to human cytokines. Monoclonal anti-interleukin-1 (IL-1) influences on direct and indirect IL-1-mediated islet effects
Cytokine
(1991)
F. Thomas et al.
A tripartite anoikis-like mechanism causes early isolated islet apoptosis
Surgery
(2001)

A.M. Shapiro et al.

Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen

N. Engl. J. Med.

(2000)

C. Ricordi et al.

Clinical islet transplantation: advances and immunological challenges

Nat. Rev. Immunol.

(2004)

S. Paraskevas et al.

Cell loss in isolated human islets occurs by apoptosis

Pancreas

(2000)

D. Thomas, H. Yang, D.J. Boffa, R. Ding, V.K. Sharma, M. Lagman, B. Li, B. Hering, T. Mohanakumar, J. Lakey, S. Kapur,...

J. Hanke

Apoptosis in cultured rat islets of Langerhans and occurrence of Bcl-2, Bak, Bax, Fas and Fas ligand

Cells Tissues Organs

(2001)

J.L. Contreras et al.

Cytoprotection of pancreatic islets before and early after transplantation using gene therapy

Kidney Int.

(2002)

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Delivery of Bcl-XL or its BH4 domain by protein transduction inhibits apoptosis in human islets

Abstract

Section snippets

Materials and methods

Analysis of TAT-Bcl-XL transduction efficiency in live islet cells

Acknowledgments

Surgery

Exp. Cell Res.

Curr. Opin. Biotechnol.

Mol. Ther.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Hepatology

Brain Res.

Cytokine