Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes

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Abstract

Cholangiocytes are the target of a group of liver diseases termed the cholangiopathies that include conditions characterized by periductal inflammation and cholangiocyte apoptosis. Because inflammation is associated with oxidative stress, we developed the hypothesis that cholangiocytes exposed to oxidative stress will be depleted of endogenous cytoprotective molecules, leading to cholangiocyte apoptosis. To begin to test this hypothesis, we explored the relationships among glutathione (GSH) depletion, expression of Bcl-2 (a protooncogene that inhibits apoptosis), and apoptosis in a nonmalignant human cholangiocyte cell line. Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using buthionine sulfoximine (BSO). Bcl- 2 expression was assessed by quantitative immunoblot analysis, and apoptosis quantified by fluorescence microscopy using the DNA binding dye 4',6'- diamidino-2-phenylindole. Bcl-2 message was assessed by RNase protection assay, and Bcl-2 protein synthesis and half-life by pulse-chase analysis. Exposure of human cholangiocytes in culture to BSO reduced GSH levels by 93 ± 3% (P < 0.01). In addition, treatment of cholangiocytes with BSO reduced Bcl-2 levels by 87 ± 2% (P < 0.01) and was associated with a time-dependent increase in the number of cells undergoing apoptosis; ~11 ± 1% of cultured cells demonstrated morphological changes of apoptosis by 72 h compared with 1.5 ± 0.1% in untreated cholangiocytes (P < 0.01). Maintenance of GSH levels by addition of glutathione ethyl ester in the presence of BSO blocked the BSO-associated increase in apoptosis in BSO-treated cholangiocytes and also prevented the decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady-state levels of bcl-2 mRNA or Bcl-2 protein synthesis. However, Bcl-2 protein half-life decreased 57% in BSO-treated vs. untreated cells. Our results using a human cholangiocyte cell line demonstrate that reduction in the cellular levels of an antioxidant such as GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis, an observation that may be important in the pathogenesis of the inflammatory cholangiopathies.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume275
Issue number4 38-4
StatePublished - 1998

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Buthionine Sulfoximine
Glutathione
Apoptosis
Oxidative Stress
Proteins
Half-Life
Inflammation
Cell Line
Ribonucleases
Bile Ducts
Fluorescence Microscopy
Liver Diseases
Cultured Cells
Coloring Agents
Cell Count
Antioxidants
Epithelial Cells
Maintenance
Observation
Messenger RNA

Keywords

  • Cholangiopathies
  • Ductopenia
  • Glutathione ethyl ester

ASJC Scopus subject areas

  • Gastroenterology
  • Physiology
  • Physiology (medical)

Cite this

@article{e6619733817e487e9f34d76235698e9d,
title = "Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes",
abstract = "Cholangiocytes are the target of a group of liver diseases termed the cholangiopathies that include conditions characterized by periductal inflammation and cholangiocyte apoptosis. Because inflammation is associated with oxidative stress, we developed the hypothesis that cholangiocytes exposed to oxidative stress will be depleted of endogenous cytoprotective molecules, leading to cholangiocyte apoptosis. To begin to test this hypothesis, we explored the relationships among glutathione (GSH) depletion, expression of Bcl-2 (a protooncogene that inhibits apoptosis), and apoptosis in a nonmalignant human cholangiocyte cell line. Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using buthionine sulfoximine (BSO). Bcl- 2 expression was assessed by quantitative immunoblot analysis, and apoptosis quantified by fluorescence microscopy using the DNA binding dye 4',6'- diamidino-2-phenylindole. Bcl-2 message was assessed by RNase protection assay, and Bcl-2 protein synthesis and half-life by pulse-chase analysis. Exposure of human cholangiocytes in culture to BSO reduced GSH levels by 93 ± 3{\%} (P < 0.01). In addition, treatment of cholangiocytes with BSO reduced Bcl-2 levels by 87 ± 2{\%} (P < 0.01) and was associated with a time-dependent increase in the number of cells undergoing apoptosis; ~11 ± 1{\%} of cultured cells demonstrated morphological changes of apoptosis by 72 h compared with 1.5 ± 0.1{\%} in untreated cholangiocytes (P < 0.01). Maintenance of GSH levels by addition of glutathione ethyl ester in the presence of BSO blocked the BSO-associated increase in apoptosis in BSO-treated cholangiocytes and also prevented the decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady-state levels of bcl-2 mRNA or Bcl-2 protein synthesis. However, Bcl-2 protein half-life decreased 57{\%} in BSO-treated vs. untreated cells. Our results using a human cholangiocyte cell line demonstrate that reduction in the cellular levels of an antioxidant such as GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis, an observation that may be important in the pathogenesis of the inflammatory cholangiopathies.",
keywords = "Cholangiopathies, Ductopenia, Glutathione ethyl ester",
author = "Adriane Celli and Florencia Que and Gores, {Gregory James} and {La Russo}, {Nicholas F}",
year = "1998",
language = "English (US)",
volume = "275",
journal = "American Journal of Physiology - Renal Fluid and Electrolyte Physiology",
issn = "1931-857X",
publisher = "American Physiological Society",
number = "4 38-4",

}

TY - JOUR

T1 - Glutathione depletion is associated with decreased Bcl-2 expression and increased apoptosis in cholangiocytes

AU - Celli, Adriane

AU - Que, Florencia

AU - Gores, Gregory James

AU - La Russo, Nicholas F

PY - 1998

Y1 - 1998

N2 - Cholangiocytes are the target of a group of liver diseases termed the cholangiopathies that include conditions characterized by periductal inflammation and cholangiocyte apoptosis. Because inflammation is associated with oxidative stress, we developed the hypothesis that cholangiocytes exposed to oxidative stress will be depleted of endogenous cytoprotective molecules, leading to cholangiocyte apoptosis. To begin to test this hypothesis, we explored the relationships among glutathione (GSH) depletion, expression of Bcl-2 (a protooncogene that inhibits apoptosis), and apoptosis in a nonmalignant human cholangiocyte cell line. Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using buthionine sulfoximine (BSO). Bcl- 2 expression was assessed by quantitative immunoblot analysis, and apoptosis quantified by fluorescence microscopy using the DNA binding dye 4',6'- diamidino-2-phenylindole. Bcl-2 message was assessed by RNase protection assay, and Bcl-2 protein synthesis and half-life by pulse-chase analysis. Exposure of human cholangiocytes in culture to BSO reduced GSH levels by 93 ± 3% (P < 0.01). In addition, treatment of cholangiocytes with BSO reduced Bcl-2 levels by 87 ± 2% (P < 0.01) and was associated with a time-dependent increase in the number of cells undergoing apoptosis; ~11 ± 1% of cultured cells demonstrated morphological changes of apoptosis by 72 h compared with 1.5 ± 0.1% in untreated cholangiocytes (P < 0.01). Maintenance of GSH levels by addition of glutathione ethyl ester in the presence of BSO blocked the BSO-associated increase in apoptosis in BSO-treated cholangiocytes and also prevented the decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady-state levels of bcl-2 mRNA or Bcl-2 protein synthesis. However, Bcl-2 protein half-life decreased 57% in BSO-treated vs. untreated cells. Our results using a human cholangiocyte cell line demonstrate that reduction in the cellular levels of an antioxidant such as GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis, an observation that may be important in the pathogenesis of the inflammatory cholangiopathies.

AB - Cholangiocytes are the target of a group of liver diseases termed the cholangiopathies that include conditions characterized by periductal inflammation and cholangiocyte apoptosis. Because inflammation is associated with oxidative stress, we developed the hypothesis that cholangiocytes exposed to oxidative stress will be depleted of endogenous cytoprotective molecules, leading to cholangiocyte apoptosis. To begin to test this hypothesis, we explored the relationships among glutathione (GSH) depletion, expression of Bcl-2 (a protooncogene that inhibits apoptosis), and apoptosis in a nonmalignant human cholangiocyte cell line. Monolayers of human bile duct epithelial cells, derived from normal liver and immortalized by SV40 transformation, were depleted of GSH using buthionine sulfoximine (BSO). Bcl- 2 expression was assessed by quantitative immunoblot analysis, and apoptosis quantified by fluorescence microscopy using the DNA binding dye 4',6'- diamidino-2-phenylindole. Bcl-2 message was assessed by RNase protection assay, and Bcl-2 protein synthesis and half-life by pulse-chase analysis. Exposure of human cholangiocytes in culture to BSO reduced GSH levels by 93 ± 3% (P < 0.01). In addition, treatment of cholangiocytes with BSO reduced Bcl-2 levels by 87 ± 2% (P < 0.01) and was associated with a time-dependent increase in the number of cells undergoing apoptosis; ~11 ± 1% of cultured cells demonstrated morphological changes of apoptosis by 72 h compared with 1.5 ± 0.1% in untreated cholangiocytes (P < 0.01). Maintenance of GSH levels by addition of glutathione ethyl ester in the presence of BSO blocked the BSO-associated increase in apoptosis in BSO-treated cholangiocytes and also prevented the decrease in Bcl-2 protein. BSO treatment of cholangiocytes did not change steady-state levels of bcl-2 mRNA or Bcl-2 protein synthesis. However, Bcl-2 protein half-life decreased 57% in BSO-treated vs. untreated cells. Our results using a human cholangiocyte cell line demonstrate that reduction in the cellular levels of an antioxidant such as GSH results in increased degradation of Bcl-2 protein and an increase in apoptosis. These data provide a mechanistic link between the consequences of oxidative stress and cholangiocyte apoptosis, an observation that may be important in the pathogenesis of the inflammatory cholangiopathies.

KW - Cholangiopathies

KW - Ductopenia

KW - Glutathione ethyl ester

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M3 - Article

C2 - 9756506

AN - SCOPUS:0031754499

VL - 275

JO - American Journal of Physiology - Renal Fluid and Electrolyte Physiology

JF - American Journal of Physiology - Renal Fluid and Electrolyte Physiology

SN - 1931-857X

IS - 4 38-4

ER -