False positive staining in the TUNEL assay to detect apoptosis in liver and intestine is caused by endogenous nucleases and inhibited by diethyl pyrocarbonate

B, Stähelin; Marti, Ulrich; Solioz, Marc; Zimmermann, Heinz; Reichen, Jürg

doi:10.1136/mp.51.4.204

Cited by 103 publications

(67 citation statements)

References 16 publications

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“…False positives that are known to occur in liver tissue were carefully avoided by additional treatment with diethyl pyrocarbonate. 28 Moreover, this assay also may stain necrotic cells, 39 explaining why we observed less cells positive for activated caspase 3. These results suggest that apoptosis is not massive in the hours after transplantation despite prolonged cold ischemia.…”

Section: Discussionmentioning

confidence: 97%

“…Paraffin-embedded sections (4 m) were deparaffinized by soaking twice in xylol 100% for 10 minutes, then treated for 30 minutes with 4% diethylpyrocarbonate in ethanol at 4°C to avoid false positivity with the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay. 28 The slides were successively soaked in ethanol 100% (2 ϫ 10 min), ethanol 94% (5 min), ethanol 70% (5 min), ethanol 50% (5 min), and double-distilled water (5 min). After treatment with Proteinase K (10 g/mL) at 37°C for 60 minutes in 5 mmol/L ethylenediaminetetraacetic acid in Tris 20 mmol/L (pH 8.1), and 0.3% H 2 O 2 in methanol for 30 minutes to block the endogenous peroxidases, the TUNEL assay was performed by using a commercial kit (Roche Diagnostic, Basel, Switzerland).…”

Section: Methodsmentioning

confidence: 99%

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Extended preservation of rat liver graft by induction of heme oxygenase-1

et al. 2002

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Livers can be preserved only for a short period without jeopardizing the transplantation outcome. Heat shock proteins (HSPs) protect against ischemia and reperfusion injury. We studied whether their induction and, in particular, the induction of heme oxygenase 1 (HO-1), improves transplantation survival after an extended time of cold storage. Rats were subjected to heat preconditioning (42°C for 20 minutes). Livers were harvested 24 hours later, preserved in cold University of Wisconsin solution for 44 hours, and transplanted in isogeneic rats (arterialized transplantation). HO-1 was specifically induced and inhibited by cobalt protoporphyrin and tin protoporphyrin, respectively. All animals receiving a graft without preconditioning and subjected to 44 hours of cold preservation died within 3 days, whereas 89% of rats who received a graft exposed to heat survived for 3 weeks (P ‫؍‬ .0004). Preconditioning reduced serum aspartate transaminase (AST) and lactate dehydrogenase activities after reperfusion, improved bile flow, and decreased the histologic lesions of reperfusion injury. These significant effects of heat preconditioning were prevented by administration of tin protoporphyrin and could be reproduced by administration of cobalt protoporphyrin. In grafts without preconditioning, only a small fraction (<5%) of hepatocytes were positive with the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay, and even less expressed activated caspase 3. Preconditioning tended to reduce the number of positive cells and to stimulate the expression of antiapoptotic Bcl-X L . In conclusion, heat preconditioning and, specifically, overexpression of HO-1 improve posttransplantation survival and graft function after prolonged cold ischemia preservation. The mechanism underlying these beneficial effects does not appear to be prevention of apoptosis. (HEPATOLOGY 2002;35:1082-1092

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Section: Discussionmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Extended preservation of rat liver graft by induction of heme oxygenase-1

et al. 2002

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“…Several concerns have been expressed about pitfalls, lack of specificity, and difficulties to standardize the TUNEL technique. [32][33][34][35] Furthermore, it is a common experience that performance of TUNEL staining depends greatly on the tissue pretreatment and labeling procedures. 36 Another reason for the discrepancy in the number of TUNEL-positive cells and cells revealing caspase activation may be explained by the different time course of biochemical events in apoptosis.…”

Section: Discussionmentioning

confidence: 99%

Caspase activation correlates with the degree of inflammatory liver injury in chronic hepatitis C virus infection

et al. 2001

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Hepatitis C virus (HCV) infection is a major cause of liver disease characterized by inflammation, cell damage, and fibrotic reactions of hepatocytes. Apoptosis has been implicated in the pathogenesis, although it is unclear whether proteases of the caspase family as the central executioners of apoptosis are involved and how caspase activation contributes to liver injury. In the present study, we measured the activation of effector caspases in liver biopsy specimens of patients with chronic HCV infection. The activation of caspase-3, caspase-7, and cleavage of poly(ADP-ribose)polymerase (PARP), a specific caspase substrate, were measured by immunohistochemistry and Western blot analysis by using antibodies that selectively detect the active truncated, but not the inactive precursor forms of the caspases and PARP. We found that caspase activation was considerably elevated in liver lobules of HCV patients in comparison to normal controls. Interestingly, the immunoreactive cells did yet not reveal an overt apoptotic morphology. The extent of caspase activation correlated significantly with the disease grade, i.e., necroinflammatory activity. In contrast, no correlation was observed with other surrogate markers such as serum transaminases and viral load. In biopsy specimens with low activity ( Hepatitis C virus (HCV) infection is one of the major causes of liver disease with an increased risk of cirrhosis and hepatocellular carcinoma. The infection has a high propensity to chronicity, and the majority of HCV carriers have histologic evidence for liver inflammation, cell damage, and fibrotic reactions of hepatocytes. The mechanisms responsible for HCVmediated liver cell damage are poorly understood, and both immune-mediated reactions and direct cytopathic effects of HCV may be involved in its pathogenesis. It has been suggested that apoptosis plays an important role in HCV-associated liver injury, 1-4 although it is unclear which cellular and molecular mechanisms participate in the process.One of the best-defined apoptotic pathways is mediated by the death receptor CD95, a member of the tumor necrosis factor superfamily that is constitutively expressed on hepatocytes. 5 Experiments in mice have shown that agonistic CD95 antibodies cause massive liver cell lysis, resulting in increased serum levels of transaminases and death from fulminant hepatic failure. 6 In patients with chronic HCV infection, expression of CD95 is increased and associated with disease activity and the severity of liver inflammation. 7,8 When HCV-specific T cells migrate towards hepatocytes and recognize viral antigens through the T-cell receptor, they become activated and inducibly express the ligand CD95L that can transduce the apoptotic death signal to CD95-bearing hepatocytes. 2 In addition to CD95L and other cytokines, both structural and nonstructural HCV proteins have been shown to modulate the sensitivity of hepatocytes for cell death. [9][10][11][12][13] Cells undergoing apoptosis show a sequence of morphologic features including membrane...

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“…Because TUNEL assay may not distinguish between apoptotic and necrotic cells (Stahelin et al, 1998), we assessed, in parallel experiments, the DNA content of CBL-treated A2780 and A2780(100) cells by propidium iodide staining (Bresnahan et al, 1997). Histograms generated by¯ow cytometry show that more than 80% A2780 cells are sub-G1 at 48 h after treatment, while drug-treated A2780(100) cells had a cell cycle stage distribution similar to that of the nontreated cells (Figure 1).…”

Section: Induction Of Apoptosis By Chlorambucil In A2780 But Not A278mentioning

confidence: 99%

Acquired alkylating drug resistance of a human ovarian carcinoma cell line is unaffected by altered levels of pro- and anti-apoptotic proteins

et al. 2000

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False positive staining in the TUNEL assay to detect apoptosis in liver and intestine is caused by endogenous nucleases and inhibited by diethyl pyrocarbonate

Abstract: False positive staining in the TUNEL assay in the liver is caused by the release of endogenous endonucleases as a result of proteinase treatment. This can be abolished by pretreatment of tissue slides with DEPC.

Cited by 103 publications

References 16 publications

Extended preservation of rat liver graft by induction of heme oxygenase-1

Extended preservation of rat liver graft by induction of heme oxygenase-1

Caspase activation correlates with the degree of inflammatory liver injury in chronic hepatitis C virus infection

Acquired alkylating drug resistance of a human ovarian carcinoma cell line is unaffected by altered levels of pro- and anti-apoptotic proteins

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