Proapoptotic bax is hyperexpressed in isolated human islets compared with antiapoptotic BCL-2

Dolca Thomas, Hua Yang, Daniel J. Boffa, Ruchuang Ding, Vijay K. Sharma, Milagros Lagman, Baogui Li, Bernhard Hering, Thalachallour Mohanakumar, Jonathan Lakey, Sandip Kapur, Wayne W. Hancock, Manikkam Suthanthiran

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Abstract

Background. Apoptosis is a well-documented pathway for islet cell death. One potential mechanism is overexpression of death-promoting Bax compared with antiapoptotic Bcl-2 in islets. Methods. We isolated islets from 10 human pancreata and measured the expression of Bax mRNA and Bcl-2 mRNA by real-time quantitative polymerase chain reaction; islet and pancreas expression of Bax, Bcl-2, activated caspase-3, and cleaved poly (ADP-ribose) polymerase were also assessed by immunohistochemistry. Islet cell apoptosis was evaluated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay and by flow cytometry. Results. The mean (±SE) level of Bax mRNA was 336±79 copies per nanogram of total RNA, and the level of Bcl-2 mRNA was 36±10 (P=0.001). A positive correlation existed between islet expression of Bax mRNA and Bcl-2 mRNA (P=0.001). The islet Bax to Bcl-2 ratio was 10.8±1.3 and 1.71±0.3 for the spleens (P=0.0001). Bax mRNA (P=0.04), but not Bcl-2 mRNA, was expressed at a higher level in islets compared with spleens. Human islets contained large numbers of cells expressing Bax protein, whereas only infrequent islet cells expressed Bcl-2 protein, activated caspase-3, and poly (ADP-ribose) polymerase. The apoptotic index was 5% by TUNEL assay, and the percentage of apoptotic islet cells was 9.7±2.5% by flow cytometry. Sections of human pancreas before islet isolation showed islet staining for Bax but not Bcl-2. Conclusions. Our finding that isolated human islets express Bax at a higher level compared with Bcl-2 suggests a molecular mechanism for islet cell death by apoptosis. We hypothesize that reducing islet expression of Bax, or regulating its activation, will help preserve islet cell mass after islet transplantation.

Original languageEnglish
Pages (from-to)1489-1496
Number of pages8
JournalTransplantation
Volume74
Issue number11
DOIs
Publication statusPublished - 15 Dec 2002
Externally publishedYes

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Islets of Langerhans
Messenger RNA
Pancreas
Poly(ADP-ribose) Polymerases
Apoptosis
Transferases
Caspase 3
Flow Cytometry
Cell Death
Spleen
bcl-2-Associated X Protein
Islets of Langerhans Transplantation
Real-Time Polymerase Chain Reaction
Cell Count
Immunohistochemistry
RNA
Staining and Labeling
Proteins

ASJC Scopus subject areas

  • Transplantation

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Proapoptotic bax is hyperexpressed in isolated human islets compared with antiapoptotic BCL-2. / Thomas, Dolca; Yang, Hua; Boffa, Daniel J.; Ding, Ruchuang; Sharma, Vijay K.; Lagman, Milagros; Li, Baogui; Hering, Bernhard; Mohanakumar, Thalachallour; Lakey, Jonathan; Kapur, Sandip; Hancock, Wayne W.; Suthanthiran, Manikkam.

In: Transplantation, Vol. 74, No. 11, 15.12.2002, p. 1489-1496.

Research output: Contribution to journalArticle

Thomas, D, Yang, H, Boffa, DJ, Ding, R, Sharma, VK, Lagman, M, Li, B, Hering, B, Mohanakumar, T, Lakey, J, Kapur, S, Hancock, WW & Suthanthiran, M 2002, 'Proapoptotic bax is hyperexpressed in isolated human islets compared with antiapoptotic BCL-2', Transplantation, vol. 74, no. 11, pp. 1489-1496. https://doi.org/10.1097/00007890-200212150-00003
Thomas, Dolca ; Yang, Hua ; Boffa, Daniel J. ; Ding, Ruchuang ; Sharma, Vijay K. ; Lagman, Milagros ; Li, Baogui ; Hering, Bernhard ; Mohanakumar, Thalachallour ; Lakey, Jonathan ; Kapur, Sandip ; Hancock, Wayne W. ; Suthanthiran, Manikkam. / Proapoptotic bax is hyperexpressed in isolated human islets compared with antiapoptotic BCL-2. In: Transplantation. 2002 ; Vol. 74, No. 11. pp. 1489-1496.
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abstract = "Background. Apoptosis is a well-documented pathway for islet cell death. One potential mechanism is overexpression of death-promoting Bax compared with antiapoptotic Bcl-2 in islets. Methods. We isolated islets from 10 human pancreata and measured the expression of Bax mRNA and Bcl-2 mRNA by real-time quantitative polymerase chain reaction; islet and pancreas expression of Bax, Bcl-2, activated caspase-3, and cleaved poly (ADP-ribose) polymerase were also assessed by immunohistochemistry. Islet cell apoptosis was evaluated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay and by flow cytometry. Results. The mean (±SE) level of Bax mRNA was 336±79 copies per nanogram of total RNA, and the level of Bcl-2 mRNA was 36±10 (P=0.001). A positive correlation existed between islet expression of Bax mRNA and Bcl-2 mRNA (P=0.001). The islet Bax to Bcl-2 ratio was 10.8±1.3 and 1.71±0.3 for the spleens (P=0.0001). Bax mRNA (P=0.04), but not Bcl-2 mRNA, was expressed at a higher level in islets compared with spleens. Human islets contained large numbers of cells expressing Bax protein, whereas only infrequent islet cells expressed Bcl-2 protein, activated caspase-3, and poly (ADP-ribose) polymerase. The apoptotic index was 5{\%} by TUNEL assay, and the percentage of apoptotic islet cells was 9.7±2.5{\%} by flow cytometry. Sections of human pancreas before islet isolation showed islet staining for Bax but not Bcl-2. Conclusions. Our finding that isolated human islets express Bax at a higher level compared with Bcl-2 suggests a molecular mechanism for islet cell death by apoptosis. We hypothesize that reducing islet expression of Bax, or regulating its activation, will help preserve islet cell mass after islet transplantation.",
author = "Dolca Thomas and Hua Yang and Boffa, {Daniel J.} and Ruchuang Ding and Sharma, {Vijay K.} and Milagros Lagman and Baogui Li and Bernhard Hering and Thalachallour Mohanakumar and Jonathan Lakey and Sandip Kapur and Hancock, {Wayne W.} and Manikkam Suthanthiran",
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T1 - Proapoptotic bax is hyperexpressed in isolated human islets compared with antiapoptotic BCL-2

AU - Thomas, Dolca

AU - Yang, Hua

AU - Boffa, Daniel J.

AU - Ding, Ruchuang

AU - Sharma, Vijay K.

AU - Lagman, Milagros

AU - Li, Baogui

AU - Hering, Bernhard

AU - Mohanakumar, Thalachallour

AU - Lakey, Jonathan

AU - Kapur, Sandip

AU - Hancock, Wayne W.

AU - Suthanthiran, Manikkam

PY - 2002/12/15

Y1 - 2002/12/15

N2 - Background. Apoptosis is a well-documented pathway for islet cell death. One potential mechanism is overexpression of death-promoting Bax compared with antiapoptotic Bcl-2 in islets. Methods. We isolated islets from 10 human pancreata and measured the expression of Bax mRNA and Bcl-2 mRNA by real-time quantitative polymerase chain reaction; islet and pancreas expression of Bax, Bcl-2, activated caspase-3, and cleaved poly (ADP-ribose) polymerase were also assessed by immunohistochemistry. Islet cell apoptosis was evaluated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay and by flow cytometry. Results. The mean (±SE) level of Bax mRNA was 336±79 copies per nanogram of total RNA, and the level of Bcl-2 mRNA was 36±10 (P=0.001). A positive correlation existed between islet expression of Bax mRNA and Bcl-2 mRNA (P=0.001). The islet Bax to Bcl-2 ratio was 10.8±1.3 and 1.71±0.3 for the spleens (P=0.0001). Bax mRNA (P=0.04), but not Bcl-2 mRNA, was expressed at a higher level in islets compared with spleens. Human islets contained large numbers of cells expressing Bax protein, whereas only infrequent islet cells expressed Bcl-2 protein, activated caspase-3, and poly (ADP-ribose) polymerase. The apoptotic index was 5% by TUNEL assay, and the percentage of apoptotic islet cells was 9.7±2.5% by flow cytometry. Sections of human pancreas before islet isolation showed islet staining for Bax but not Bcl-2. Conclusions. Our finding that isolated human islets express Bax at a higher level compared with Bcl-2 suggests a molecular mechanism for islet cell death by apoptosis. We hypothesize that reducing islet expression of Bax, or regulating its activation, will help preserve islet cell mass after islet transplantation.

AB - Background. Apoptosis is a well-documented pathway for islet cell death. One potential mechanism is overexpression of death-promoting Bax compared with antiapoptotic Bcl-2 in islets. Methods. We isolated islets from 10 human pancreata and measured the expression of Bax mRNA and Bcl-2 mRNA by real-time quantitative polymerase chain reaction; islet and pancreas expression of Bax, Bcl-2, activated caspase-3, and cleaved poly (ADP-ribose) polymerase were also assessed by immunohistochemistry. Islet cell apoptosis was evaluated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay and by flow cytometry. Results. The mean (±SE) level of Bax mRNA was 336±79 copies per nanogram of total RNA, and the level of Bcl-2 mRNA was 36±10 (P=0.001). A positive correlation existed between islet expression of Bax mRNA and Bcl-2 mRNA (P=0.001). The islet Bax to Bcl-2 ratio was 10.8±1.3 and 1.71±0.3 for the spleens (P=0.0001). Bax mRNA (P=0.04), but not Bcl-2 mRNA, was expressed at a higher level in islets compared with spleens. Human islets contained large numbers of cells expressing Bax protein, whereas only infrequent islet cells expressed Bcl-2 protein, activated caspase-3, and poly (ADP-ribose) polymerase. The apoptotic index was 5% by TUNEL assay, and the percentage of apoptotic islet cells was 9.7±2.5% by flow cytometry. Sections of human pancreas before islet isolation showed islet staining for Bax but not Bcl-2. Conclusions. Our finding that isolated human islets express Bax at a higher level compared with Bcl-2 suggests a molecular mechanism for islet cell death by apoptosis. We hypothesize that reducing islet expression of Bax, or regulating its activation, will help preserve islet cell mass after islet transplantation.

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