Apoptosis and expression of bcl-2 protein in invasive ductal adenocarcinoma of the pancreas

Apoptosis plays a central role in the development and/or progression of cancer. There are several methods for detection of apoptotic cells in tissue sections including light and electron microscopy, in situ nick end-labeling (ISEL), TdT-mediated dUTP nick-end labeling (TUNEL) and immunohistochemical detection of proteins associated with apoptosis. Apoptosis was assessed by the monoclonal antibody M30 CytoDEATH (M30), which is specific for neo-epitope in cytokeratin 18 that becomes available at an early caspase cleavage during apoptosis. Expression of bcl-2 protein was evaluated, because bcl-2 protein plays an important role in the regulation of apoptosis. Twenty-six invasive ductal adenocarcinomas of the pancreas were studied immunohistochemically with antibodies M30 and bcl-2. The mean apoptotic index (AI, the percentage of apoptotic cells of the total tumor cells number) was 2.75%. High AI (> 10%) was observed in 4 cases of the 26 pancreatic carcinomas (15%). Protein bcl-2 was expressed in 3 cases (11.5%). The AI did not correlate with the expression of protein bcl-2. In conclusion, the detection of neo-epitope in cytokeratin 18 by monoclonal antibody M30 can be used for quantification of apoptotic cells with immunohistochemical techniques in tissue sections. It is a new approach to evaluate apoptosis in pancreatic carcinomas. The low positivity of bcl-2 expression in pancreatic adenocarcinomas suggests that bcl-2 protein does not play a central role in pancreatic tumorigenesis and cancer progression.     

Trophoblast apoptosis in human placenta at term as detected by expression of a cytokeratin 18 degradation product of caspase

CONTEXT: Apoptosis occurs in the normal placenta. The monoclonal antibody M30 is directed against a novel epitope of cytokeratin 18 (CK18) that is formed by caspase cleavage early in the apoptotic cascade, and this antibody may therefore be useful for evaluating trophoblast apoptosis. OBJECTIVE: We undertook the present study to evaluate the use of monoclonal antibody M30 to assess trophoblast apoptosis in placenta at term. METHODS: We stained paraffin-embedded placental tissues from 15 deliveries at term with M30. We compared positive M30 staining and CK18 staining (as detected by a monoclonal antibody directed against CK18) of trophoblasts in serial slides. We also compared apoptotic rates as detected by M30 and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) in 7 of the placentas. RESULTS: In fields of villous tissue, most M30-positive cells were CK18-positive syncytiotrophoblasts. Approximately half of M30-positive cells occurred as focal positive staining in the syncytial layer, and half occurred as abundant staining of syncytiotrophoblasts in areas with increased intervillous or perivillous fibrinoid. We found very few M30-positive cells in villous stroma. In decidual/basal plate tissues, most (two thirds) of the M30-positive cells were CK18-positive extravillous trophoblasts, whereas one third were syncytiotrophoblasts of anchoring villi. Since TUNEL detects apoptosis in both epithelial and nonepithelial cells, more cells were positively stained with TUNEL than with M30 in some tissue fields. However, our observations suggest that M30 was more sensitive than TUNEL in recognizing apoptotic trophoblasts and had less nonspecific staining than TUNEL. CONCLUSION: We recommend the use of monoclonal antibody M30 for apoptosis studies in placental tissues. This antibody is easy to handle, the staining obtained seems specific, and the nonspecific staining seems negligible.     

Simultaneous detection of a cell surface antigen and apoptosis by microwave-sensitized TUNEL assay on paraffin sections

The TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique has been described as a sensitive method for detection of apoptotic nuclei in tissues and preferential staining of apoptotic strand breaks. Short-term microwave pre-treatment, a non-enzymatic pre-treatment technique of antigen retrieval, has been demonstrated to optimize the TUNEL method for in situ detection of apoptotic cells in formalin-fixed paraffin-embedded tissue sections. In the present study, we sensitized internal mammary artery sections by short-term microwave pre-treatment and used a two-step indirect enzymatic method to gain as an end product differentially stained cells, namely TUNEL-positive cells and these positive for the surface marker von Willebrand factor (vWF). This technique enables to clearly distinguish between apoptotic, non-apoptotic and vWF-positive cells that are phenotypic for endothelial cells. Phenotypic identification of cells is simplified by double staining with cell surface markers. This rapid, sensitive and reproducible technique allows simultaneous detection of DNA fragmentation and phenotypic markers in the same paraffin-embedded human tissue section.     

General suitability of techniques for in situ detection of apoptosis in small intestinal epithelium

The present study was designed to evaluate different techniques for the in situ detection of apoptosis in human and rat small intestinal epithelium. The techniques included light microscopy (LM) and transmission electron microscopy (TEM) observation of epoxy resin-embedded tissue, scanning electron microscopy (SEM), TUNEL assay, and antibodies directed against caspase cleavage products of caspase 3, cytokeratin 18 (CK 18), and apoptotic single-strand DNA (ssDNA). All techniques, if the labeling was positive, showed apoptotic cells exclusively at the villus tip. LM and TEM were the most reliable and revealed morphological signs typical of cells that have died via apoptosis. SEM indicated the extension of the process. The antibody recognizing cleaved caspase 3 could be considered an appropriate marker for apoptotic epithelial cells in human and rat small intestine. However, the majority of epithelial cells lining the proximal small intestinal villus contained only low levels of intact CK 18. Therefore, sufficient amounts of cleaved CK 18 for immunohistochemical detection were not generated during apoptosis, rendering the application of the antibody inappropriate. The antibody detecting formamide-denatured ssDNA in apoptotic cells was both suitable and reliable; however, the particular staining procedure used compromised the tissue preservation. In comparison to this, the TUNEL assay was less reliable. Although it was performed with a commercially available ready-to-use kit, its application conditions had to be adjusted for each specimen on the basis of the findings produced by other techniques.     

Annexin V联合PI染色法定量检测凋亡细胞

为评价ＡｎｎｅｘｉｎＶ／ＰＩ双参数法在凋亡检测中的价值,用ＰＩ染色法、ＴＵＮＥＬ法及双参数ＡｎｎｅｘｉｎＶ／ＰＩ法定量检测地塞米松处理小鼠胸腺细胞凋亡发生率。结果发现其凋亡检出率分别为ＰＩ法２７１９％,ＡｎｎｅｘｉｎＶ／ＰＩ法３２２８％,ＴＵＮＥＬ法５０１７％。三者之间均有显著差异（Ｐ＜００１）。但ＴＵＮＥＬ法不能将死亡细胞、凋亡细胞区分开来。ＡｎｎｅｘｉｎＶ／ＰＩ法可将正常细胞（ＡｎｎｅｘｉｎＶ ＰＩ ）、凋亡细胞（ＡｎｎｅｘｉｎＶ＋ＰＩ ）、死亡细胞（ＡｎｎｅｘｉｎＶ＋ＰＩ＋）分开来。ＡｎｎｅｘｉｎＶ／ＰＩ法能检出早期凋亡细胞且更为简单、灵敏、特异,是较为理想的凋亡定量检测方法。     

Detection of VP-16-treated HL-60 cell apoptosis by TUNEL electron microscopy

Apoptosis is essential to many physiological processes, including maturation of cells and the immune system. Deficient regulation of apoptosis may play an important role in many pathological conditions, such as autoimmunity, AIDS, and myelodysplastic syndrome. Several methods have been described to identify apoptotic cells. DNA strand breaks can be identified by labeling free 3'-OH termini with modified nucleotides by enzymatic reaction (TUNEL method). In this study, apoptosis was introduced in cultured HL-60 cells treated with VP-16, and the TUNEL method was adapted for electromicroscopy, called the EM TUNEL method. The results of the EM TUNEL method were compared with those of light microscopy and flow cytometry. Apoptotic cells following VP-16 (10 micrograms/mL) treatment were detected after 3 h by all methods (light microscopy, Erythrocin B, electron microscopy, flow cytometry, TUNEL, and EM TUNEL). EM TUNEL was the most sensitive method of detection, with a detection rate of 32%. Furthermore, EM TUNEL was more suitable for distinguishing cell lineage than light microscopy TUNEL. The results indicate that EM TUNEL can be used to detect apoptosis in bone marrow species in vivo.     

流式细胞术定量检测细胞凋亡3种方法的比较研究

目的：探讨流式细胞术定量检测细胞凋亡３种方法的价值。方法：同时使用ＰＩ染色，ＴＵＮＥＬ及Ａｎｎｅｘｉｎ Ｖ／ＰＩ３壹量检测地塞米松处理小鼠的胸腺细胞凋亡发生率。结果：ＰＩ染色，Ａｎｎｅｘｉｎ Ｖ／ＰＩ，ＴＵＮＥＬ３种方法凋亡检出率分别为２７．１９％，３２．２８％，５０．１７％，两者之间均有显著差异。     

Annexin V labelling and terminal transferase-mediated DNA end labelling (TUNEL) assay in human arrested embryos

Confocal laser scanning microscopy was used to observe human arrested and fragmented preimplantation embryos obtained by in-vitro fertilization. Observation of the cellular actin cortex and chromatin showed a high frequency of embryos with blastomeres exhibiting two or more nuclei, while others had nuclei displaying chromatin condensation and fragmentation patterns. Many of the abnormal chromatin images could be due to the process of programmed cell death (apoptosis). The possible link between abnormalities of the blastomeres and apoptosis was investigated using two detection methods for cells undergoing apoptosis. Detection of phosphatidylserine exposure was performed using annexin V; the chromosomal breakdown preceding the nuclear collapse of apoptotic nuclei was tested using the terminal transferase-mediated DNA end labelling (TUNEL) assay. Annexin V staining was observed in all arrested and/or fragmented human embryos, but not in cryopreserved embryos which continued to develop normally after thawing. The TUNEL assay was positive in 30% (15/50) of arrested embryos, all of which had cytoplasmic fragments. In contrast, embryos showing regular size blastomeres without fragments were TUNEL negative.      

Antibody to caspase-cleaved actin detects apoptosis in differentiated neuroblastoma and plaque-associated neurons and microglia in Alzheimer's disease.

During apoptosis, activation of a family of cysteine proteases related to interleukin-1beta-converting enzyme (ICE)-related proteases or "caspases" results in endoproteolytic cleavage of multiple substrates at specific aspartate residues. We have sought to develop new antibody probes for the neoepitopes in protein fragments produced by ICE-related proteolytic cleavage as specific markers of events tightly linked to apoptotic mechanisms. Here, we demonstrate that an antibody probe specific for the C terminus of a 32-kd actin fragment produced by ICE-like activity specifically labels apoptotic but not necrotic, differentiated human neuroblastoma cells in culture. Unlike probes for nonspecific DNA strand breaks confined to the nucleus or cell body, this method allows the detection of cytoskeletal fragments in cell processes as well as the perikaryon long before DNA fragmentation and cell death and therefore serves as a novel marker of apoptosis-related events in distal parts of cells such as axons and dendrites. To illustrate this new tool, we show that the antibody detects the processes and cell bodies of degenerating neurons and plaque-associated microglia in Alzheimer's disease. In situ detection of caspase-cleaved actin provides a new means to evaluate the role of caspase activation in pathological and physiological processes.     

Ex vivo detection of apoptotic trophoblast cells applying flow cytofluorometry and immunocytochemistry using M30 antibody directed to the cytokeratin 18 neo-epitope

Apoptosis of placental trophoblast cells has become the subject of intensive research. Recently, a monoclonal antibody (M30) directed against a neo-epitope of cytokeratin 18, that is formed after cleavage of this cytoskeletal protein by caspases, was shown to be of advantage over other tests for the detection of trophoblast cell apoptosis. In the present study, we describe a method for the enrichment of highly pure villous trophoblast cells based on the proteolytic digestion of placental tissue, density gradient separation of dissected cells, and immunoelimination of contaminating, non-trophoblast cells employing an antibody to the HLA class I antigen. The high purity (94-99%) of the trophoblast cell preparation was shown by antibody staining for cytokeratin 7 and absence of vimentin. Furthermore, we demonstrate that after a simple permeabilization and fixation step with 90% methanol and using the M30 CytoDeath, FITC-conjugated antibody, apoptotic trophoblast cells could be distinguished from non-apoptotic cells by flow cytofluorometry in a highly quantitative and sensitive fashion. Our protocol is an improvement over previously used methods such as immunocytochemistry as it allows to differentiate rapidly between competent and apoptotic trophoblast cells by the quantitative method of flow cytofluorometry.     

Perinatal development of the rat kidney: Apoptosis and epidermal growth factor

ABSTRACT  Localization of apoptotic cells in the kidney of perinatal rats was examined by the terminal deoxynucleotidyl transferase-mediated d-UTP-biotin nick end labeling (TUNEL) method and electron microscopy. Perinatal changes in the percentage of kidney cells with DNA fragmentation were determined by flow cytometric analysis. Through observation of two successive sections, the relationship between the localization of the epidermal growth factor receptor (EGFR) positive cells and TUNEL positive cells in the kidney was determined. From fetal day 18 to neonatal day 5, TUNEL positive cells were noted in immature glom-eruli, collecting ducts and interstitium. Electron microscopically, chromatin condensed nuclei and apoptotic bodies were seen in the same tissue component as the TUNEL positive cells. The percentage of DNA fragmented cells significantly increased from fetal days 18 to 20 and significantly decreased from fetal days 20 to 22, while they still remained low in the neonatal period. The TUNEL positive cells in immature glom-eruli and collecting ducts were not reactive to the EGFR antibody. The TUNEL positive cells were not observed in the proximal tubular cells, which were positive to EGFR antibody. These results indicate that apoptotic cells are present in the kidney throughout the perinatal period in the rat and that EGF plays an important role in perinatal development of the rat kidney.     

抗ssDNA单克隆抗体的研制和在检测凋亡细胞中的应用

为了制备抗单链DNA单克隆抗体和建立检测细胞凋亡的新方法 ,采用杂交瘤细胞融合技术用于筛选分泌抗单链DNA抗体的单克隆杂交瘤细胞株 ,并用斑点印迹和竞争ELISA法鉴定单抗的特异性 ,进而结合免疫组织化学和免疫荧光方法用于检测凋亡细胞。通过筛选得到单克隆杂交瘤细胞株B17,其分泌的抗体与单链DNA结合而与双链DNA无交叉反应 ,用此单抗建立的凋亡细胞检测方法能够检测出凋亡细胞 ,区分非凋亡细胞和坏死细胞。实验结果表明 ,成功地获得一株分泌特异性抗单链DNA抗体的单克隆杂交瘤细胞株 ,以此建立的凋亡细胞检测方法特异性高、敏感性强     

挖空细胞的本质及尖锐湿疣病理诊断

目的 从分子生物学角度，寻找尖锐湿疣比较确切可行的病理诊断标准。方法 采用HE、HPV-CAg免疫组化、HPV-DNA原位杂交组化染色和超薄切片等方法检测50例尖锐湿疣；用原位末端标记TUNEL染色，对20例尖锐湿疣进行观察。结果 鳞状上皮呈尖细的乳头状增生，角化不全和乳头纤维轴索中毛细血管丛状增生，棘层中上部和颗粒层有多少不等的挖空细胞，单个散在、成群或广泛分布，胞质空亮，核皱缩成星形或毛虫样，无炎细胞反应。原位杂交组化HPV-DNA的阳性细胞比免疫组化HPV-CAg阳性细胞明显增多。原位末端标记TUNEL染色及光镜、电镜观察，显示所有的挖空细胞均呈现细胞凋亡变化。结论 尖锐湿疣挖空细胞是由HPV所引起的凋亡细胞。从形态学上确认其凋亡特征即可确定尖锐湿疣的诊断，角化不全和乳头间质血管丛状增生可作佐证。难以确认挖空细胞的疑难病例，可采用原位杂交组化技术。     

Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis

A neo-epitope in cytokeratin 18 (CK18) that becomes available at an early caspase cleavage event during apoptosis and is not detectable in vital epithelial cells is characterized. The monoclonal antibody M30, specific for this site, can be utilized specifically to recognize apoptotic cells, which show cytoplasmic cytokeratin filaments and aggregates after immunohistochemistry with M30, while viable and necrotic cells are negative. The number of cells recognized by the antibody increases after induction of apoptosis in exponentially growing epithelial cell lines and immunoreactivity is independent of the phosphorylation state of the cytokeratins. The generation of the M30 neo-epitope occurs early in the apoptotic cascade, before annexin V reactivity or positive DNA nick end labelling. In a flow cytometric assay, the majority of the M30-positive cells appear in the ‘apoptotic’ subG1 peak. Tests with synthetic peptides define positions 387–396 of CK18, with a liberated C-terminus at the caspase cleavage site DALD-S, as the ten-residue epitope of M30. This epitope starts at the end of coil 2 of the predicted CK18 structure, at a probable hinge region, compatible with the sensitivity to proteolytic cleavage. The definition of a specific caspase cleavage site in CK18 as a neo-epitope can be used for quantification of apoptotic epithelial cells with immunocytochemical techniques and is applicable to both fresh and formalin-fixed material. Copyright © 1999 John Wiley & Sons, Ltd.     

Evaluation of caspase activity in apoptotic cells

A family of cysteine proteases, the caspases, plays a central role in the initiation and execution phases of apoptosis. Upon activation, these enzymes cleave specific substrates and thereby mediate many of the typical biochemical and morphological changes in apoptotic cells, such as cell shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing. Hence, the detection of activated caspases can be used as a biochemical marker for apoptosis. Here we review a set of methods available for characterizing and quantifying the activation of caspases, including immunoblotting, cleavage of synthetic substrates, affinity labeling and confocal microscopy. Each method is described in general terms and the advantages and disadvantages of each technique are discussed.     

The fate of monocytes during 24 h of culture as revealed by flow cytometry and electron microscopy

Culturing elutriation-purified and cryopreserved human monocytes gives a cell loss of about 60% after a week. The main loss is during the first 24 h when one cell population dies by apoptosis and secondary necrosis, while another survives with minimal signs of apoptosis and necrosis. We have studied this initial cell loss using flow cytometry (FCM) and electron microscopy (EM) in parallel. Thawed cells were cultured in ultra low attachment wells and studied by FCM using Annexin V, Propidium iodide (PI), JC-1, APO2.7 and APO-BrDU. The EM studies comprised both transmission EM (TEM) and scanning EM (SEM), the latter employing cells labelled with antiCD14/gold and Annexin V/gold. Cells were counted by light microscopy to provide cell recoveries. DNA ladder patterns were investigated by electrophoresis. Camptothecin (CAM) was used as an apoptosis inducer. In the first 6 h of culture, there was an apoptotic phase with Annexin V(+)/PI(-) positive cells in FCM, chromatin condensation in TEM, a rapid and short phase with Annexin V/gold positively labelled cells in SEM and the cells disappeared by 6 h. All of these effects were enhanced by CAM. The necrotic phase (6-24 h) was associated with Annexin V(+)/PI(+) in FCM, and the data at 24 h was in agreement with the semiquantitatvive results from TEM. Discrepancies in the results for CD14 and Annexin V between FCM and SEM indicated phagocytosis. APO2.7 and APO-BrDU increases also indicated an accumulation of ingested material in vital cells. Centrifugation of supernatants, labelling pellets with Annexin V/FITC and examination by flow cytometry revealed no Annexin V positive cell fragments.We found evidence of rapid and efficient phagocytosis. CAM not only induced apoptosis, but also appeared to stabilise the cell membrane and increase both cell recovery and phagocytosis.     

Mechanisms of cell death associated with death-inducing factors from genomically unstable cell lines

We recently described a unique non-targeted effect of ionizing radiation whereby growth medium from two clones of GM10115 cells exhibiting radiation-induced chromosomal instability was cytotoxic to parental GM10115 cells. We termed this the death-inducing effect (DIE). The goal of the present study was to determine how DIE killed cells. Our hypothesis was that DIE medium contained either a secreted factor(s) from unstable clones or products from dead/dying cells that were cytotoxic to parental cells. First, we investigated the apoptotic characteristics of our unstable clones by Annexin V binding and TUNEL assays. Both the parental GM10115 cells and cells from the unstable clone LS12 had a low background (approximately 2%) level of apoptosis. The unstable Fe-10-3 clone showed a high spontaneous level of apoptosis, indicating major differences in the spontaneously occurring levels of apoptosis. We then analyzed how medium from these unstable clones killed cells by investigating the induction of DNA breaks, micronucleus formation and apoptosis induction in cells exposed to medium from unstable clones. Medium from unstable clones was capable of eliciting DNA double-strand breaks and increased apoptosis. Increased micronucleus frequencies were also observed in cells exposed to medium from either unstable clone, indicating a role of mitotis-linked cell death in DIE. These data suggest that DIE most likely results from cytotoxic factors secreted into the culture medium that can cause DNA double-strand breaks in recipient cells. These breaks can then lead to mitotis-linked cell death, as measured by micronuclei, or apoptosis, which accounts for the DIE.     

Caspase activity and apoptotic markers in ejaculated human sperm

The objectives of this study were to determine if human ejaculated sperm exhibit active caspases and if caspase-dependent apoptosis markers are identifiable. Sperm from fertile donors and infertile patients were examined after gradient separation into leukocyte-free fractions of high and low motility. Sperm were evaluated for motion parameters, morphology, caspase activation, and apoptosis markers including phosphatidylserine (PS) translocation (annexin V binding) and DNA fragmentation (TUNEL). Active caspase-3 was detected by immunofluorescent microscopy in a small proportion of sperm in situ, in fractions of high and low motility sperm of patients and donors, but low motility fractions had significantly higher numbers of positive sperm. Immunoblot analysis detected inactive procaspase-3 (32 kDa) in all fractions of low sperm motility from patients and donors, while active caspase-3 (17 kDa) was only detected by immunoblotting in a limited number of low motility fractions from patients and in even fewer fractions from donors. Caspase enzymatic activity, as measured using the fluorogenic substrate DEVD-afc, was higher in patients than in donors in both low and high motility fractions. Annexin V staining and DNA fragmentation were detected in a proportion of sperm, with a higher frequency in the low motility fractions. A significant positive correlation between in-situ active caspase-3 in the sperm midpiece and DNA fragmentation was observed in the low motility fractions of patients, suggesting that caspase-dependent apoptotic mechanisms could originate in the cytoplasmic droplet or within mitochondria and function in the nucleus. These data suggest that in some ejaculated sperm populations, caspases are present and may function to increase PS translocation and DNA fragmentation.     

Rapid apoptosis induced by Shiga toxin in HeLa cells

Apoptosis was induced rapidly in HeLa cells after exposure to bacterial Shiga toxin (Stx1 and Stx2; 10 ng/ml). Approximately 60% of HeLa cells became apoptotic within 4 h as detected by DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and electron microscopy. Stx1-induced apoptosis required enzymatic activity of the Stx1A subunit, and apoptosis was not induced by the Stx2B subunit alone or by the anti-globotriaosylceramide antibody. This activity was also inhibited by brefeldin A, indicating the need for toxin processing through the Golgi apparatus. The intracellular pathway leading to apoptosis was further defined. Exposure of HeLa cells to Stx1 activated caspases 3, 6, 8, and 9, as measured both by an enzymatic assay with synthetic substrates and by detection of proteolytically activated forms of these caspases by Western immunoblotting. Preincubation of HeLa cells with substrate inhibitors of caspases 3, 6, and 8 protected the cells against Stx1-dependent apoptosis. These results led to a more detailed examination of the mitochondrial pathway of apoptosis. Apoptosis induced by Stx1 was accompanied by damage to mitochondrial membranes, measured as a reduced mitochondrial membrane potential, and increased release of cytochrome c from mitochondria at 3 to 4 h. Bid, an endogenous protein known to permeabilize mitochondrial membranes, was activated in a Stx1-dependent manner. Caspase-8 is known to activate Bid, and a specific inhibitor of caspase-8 prevented the mitochondrial damage. Although these data suggested that caspase-8-mediated cleavage of Bid with release of cytochrome c from mitochondria and activation of caspase-9 were responsible for the apoptosis, preincubation of HeLa cells with a specific inhibitor of caspase-9 did not protect against apoptosis. These results were explained by the discovery of a simultaneous Stx1-dependent increase in endogenous XIAP, a direct inhibitor of caspase-9. We conclude that the primary pathway of Stx1-induced apoptosis and DNA fragmentation in HeLa cells is unique and includes caspases 8, 6, and 3 but is independent of events in the mitochondrial pathway.     

Detection of apoptosis using in situ markers for DNA strand breaks in the failing human heart. Fact or epiphenomenon?

The role of apoptosis and the methods used for its detection in failing human hearts are controversial. Recent data have challenged the hypothesis that in situ markers for DNA strand breaks mirror apoptotic (TUNEL and Taq in situ ligation assay) and/or necrotic (Pfu in situ ligation assay) cell death, and thus provide evidence that apoptotic cell loss contributes to the progression of heart failure. Experimental data cast doubt not only upon the specificity of the TUNEL technique but also the Taq in situ ligation assay as a reliable method for the detection of apoptotic cell death and provide compelling new evidence that the occurrence of cardiomyocyte cell death as defined by the detection of DNA strand breaks using either TUNEL or Taq and Pfu in situ ligation assays is an epiphenomena that is not related to the evolution of heart failure. Cardiomyocyte positivity for in situ markers of DNA strand breaks is a feature of hypertrophic cardiomyopathic hearts, irrespective of the underlying pathology or the presence or absence of heart failure. These data raise concerns regarding the extent of apoptosis in cardiomyopathy and the contribution of this process to the progression of heart failure.