Detection of apoptosis in tissue sections

During the last few years, detection of apoptosis has evolved from a predominantly morphological basis to the use of ever more specific techniques. The methods widely used to visualize DNA fragmentation in tissue sections are now supplemented by a variety of specific antisera against components of the cell death pathways. Essential requirements for apoptosis detection techniques include high sensitivity for apoptotic cells, the ability to differentiate between apoptotic and necrotic cell death and other forms of DNA damage, and the distinction between different stages of the cell death process. In this overview, we will focus on recent technical advances in apoptosis detection covering improvements of in situ DNA fragmentation techniques, as well as pointing out some of the new tools available for the detection of apoptotic cells in tissue.     

Microwave irradiation of paraffin-embedded tissue sensitizes the TUNEL method for in situ detection of apoptotic cells

Apoptosis is a morphologically distinct form of programmed cell death that plays an important role in the growth regulation of a variety of tissues and also in the elimination of self-reacting immunocompetent cells. Several techniques for the qualitative and quantitative detection of this process have been established; recently, an in situ nick end-labelling technique based on the detection of DNA fragmentation, which is a molecular characteristic of apoptotic cell death, was described. Applying this method to paraffin sections of human tissues, sensitivity was observed to be inconsistently low with regard to the expected number of apoptotic cells. In the present study we show that irradiation of the tissue sections in 10 mM citrate buffer, pH 6.0, by microwaves at 750 W considerably enhances the sensitivity of this nick end-labelling technique.     

Identification of apoptotic cell death in distraction osteogenesis

The purpose of this experimental work was to investigate whether apoptosis contributes to tissue remodelling during distraction bone healing. In a rabbit model of mandibular distraction osteogenesis, we quantitatively analysed the extent of apoptotic cell death in relation to differently applied mechanical loadings. Apoptotic cells were identified by means of an in situ detection assay for nuclear DNA fragmentation using a modified TUNEL procedure and by electron microscopical examination for typical morphological features of programmed cell death. TUNEL-positive cells were frequently detected in samples distracted at higher strain magnitudes. Ultrastructurally, these apoptotic cells displayed a condensed chromatin and fragmented nuclei, while the continuity of their plasma membranes remained intact. Our results clearly indicated that the discontinuous traction of osteotomized mandibles induced enhanced apoptosis. In contrast to non-distracted samples and mandibles distracted at low strain magnitudes, in which only minimal evidence of apoptotic cell death was detected, the application of hyperphysiological strain magnitudes resulted in an increased apoptosis rate. Thus, mechanical loading seems to be a triggering factor for apoptotic changes in osteoblastic cells. These findings suggest a pathophysiological role of apoptotic cell death in the control of tissue integrity during distraction osteogenesis.     

Apoptosis in the lung: induction, clearance and detection

Apoptosis and other forms of programmed cell death are important contributors to lung pathophysiology. In this brief review, we discuss some of the implications of finding apoptotic cells in the lung and methods for their detection. The balance between induction of apoptosis and the normally highly efficient clearance of such cells shows that these are highly dynamic processes and suggests that abnormalities of apoptotic cell clearance may be an alternative explanation for their detection. Because recognition of apoptotic cells by other lung cells has additional effects on inflammation, immunity, and tissue repair, local responses to the dying cells may also have important consequences in addition to the cell death itself.     

Tissue transglutaminase in the small intestine of the mouse as a marker for apoptotic cells. Colocalization with DNA fragmentation

Besides the morphological changes in cells undergoing apoptosis, such as chromatin condensation and cell shrinkage, histological demonstration of DNA fragmentation by in situ end labeling (ISEL) has been widely used for the demonstration of apoptotic cells in tissue sections. Although DNA fragmentation can be demonstrated in apoptotic cells and apoptotic bodies in most cases, there is no clear correlation of ISEL staining with apoptosis. It has often been demonstrated that, in many morphologically intact cells, nuclei with fragmented DNA can be found. Thus staining with ISEL for the detection of apoptosis is useful only in connection with other markers for apoptosis as, for example, characteristic morphological changes. Here we show that tissue transglutaminase protein is unequivocally expressed in apoptotic enterocytes as shown by DNA fragmentation and morphology. Tissue transglutaminase is not expressed in enterocytes with healthy morphology, although DNA fragmentation can be demonstrated in these cells. Thus the immunohistochemical demonstration of tissue transglutaminase may serve as a simple marker for apoptotic epithelial cells in tissue sections.     

Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis.

Differentiating apoptosis from necrosis is a challenge in single cells and in parenchymal tissues. The techniques available, including in situ TUNEL (Terminal deoxyribonucleotide transferase-mediated dUTP-X Nick End-Labeling) staining, DNA ladder assay, and flow cytometry, suffer from low sensitivity or from a high false-positive rate. This study, using a Jurkat cell model, initially evaluated the specificity of the neutral comet assay and flow cytometry compared to the gold standard, electron microscopy, for detection of apoptosis and necrosis. Neutral comet assay distinguished apoptosis from necrosis in Jurkat cells, as evidenced by the increased comet score in apoptotic cells and the almost zero comet score in necrotic cells. These findings were consistent with those of electron microscopy and flow cytometry. Furthermore, using rats with burn or ischemia/reperfusion injury, well-established models of skeletal and cardiac muscle tissue apoptosis, respectively, we applied the comet assay to detect apoptosis in these muscles. Neutral comet assay was able to detect apoptotic changes in both models. In the muscle samples from rats with burn or ischemia-reperfusion injury, the comet score was higher than that of muscle samples from their respective controls. These studies confirm the consistency of the comet assay for detection of apoptosis in single cells and provide evidence for its applicability as an additional method to detect apoptosis in parenchymal cells.     

Caspases: A Molecular Switch Node in the Crosstalk between Autophagy and Apoptosis

Autophagy and apoptosis are two important catabolic processes contributing to the maintenance of cellular and tissue homeostasis. Autophagy controls the turnover of protein aggregates and damaged organelles within cells, while apoptosis is the principal mechanism by which unwanted cells are dismantled and eliminated from organisms. Despite marked differences between these two pathways, they are highly interconnected in determining the fate of cells. Intriguingly, caspases, the primary drivers of apoptotic cell death, play a critical role in mediating the complex crosstalk between autophagy and apoptosis. Pro-apoptotic signals can converge to activate caspases to execute apoptotic cell death. In addition, activated caspases can degrade autophagy proteins (i.e., Beclin-1, Atg5, and Atg7) to shut down the autophagic response. Moreover, caspases can convert pro-autophagic proteins into pro-apoptotic proteints to trigger apoptotic cell death instead. It is clear that caspases are important in both apoptosis and autophagy, thus a detailed deciphering of the role of caspases in these two processes is still required to clarify the functional relationship between them. In this article, we provide a current overview of caspases in its interplay between autophagy and apoptosis. We emphasized that defining the role of caspases in autophagy-apoptosis crosstalk will provide a framework for more precise manipulation of these two processes during cell death.     

Staining apoptosis in paraffin sections. Advantages and limits

OBJECTIVE: To describe the advantages and limits of apoptosis detection on paraffin sections by TdT-mediated dUTP nick end labeling (TUNEL). STUDY DESIGN: Two hundred sixty-five paraffin-embedded samples from malignant and benign human tissue were analyzed by TUNEL. Also, biparametric analysis of apoptosis and proliferation index (MIB-1), apoptosis, cytokeratin or leukocyte common antigen was performed. RESULTS: Our preliminary conclusions are as follows. The limits are that this labelling method might detect cells that have not shown DNA fragmentation specific for apoptosis only. The technique is extremely sensitive to the degree of proteolytic digestion. TUNEL identifies nuclei in areas of necrosis. Indeed, the staining of necrotic areas of tissue with the in situ labelling method should not cause confusion since simple morphologic examination of tissues will suffice to identify areas of necrotic cells. The advantages are that TUNEL is a method of simplifying the identification of apoptotic nuclei in routinely processed tissue sections, maintaining topography. It allows retrospective studies and biparametric analysis of cell death and proliferation on the same sample. Furthermore, with biparametric stain, it could better identify the origin (epithelial, mesenchymal, and so on) of apoptotic cells. CONCLUSION: TUNEL is a good method of detecting apoptotic nuclei in fixed, embedded tissue sections, but, because of the limits of the method, the results should be interpreted in conjunction with apoptosis assessment by routine light microscopy.     

Increased sensitivity to cis-diamminedichloroplatinum induced apoptosis with mitochondrial DNA depletion

Malignant cells harbor mechanisms which allow escape from drug-induced apoptosis, and the drug-resistance phenotype can be significantly associated with resistance to programmed cell death. There is accumulating evidence that mitochondria play a role in the tumorigenic phenotype, including the relative resistance to apoptosis. Whether changes at the mitochondrial level per se, would impact on the relative sensitivity of malignant cells to undergo drug-induced apoptosis, is not know. Accordingly, we determined if depleting mitochondrial DNA (mtDNA) would change the susceptibility of U937 cells to undergo apoptosis. With depletion, increases in sensitivity to cis-diamminedichoroplatinum (cisplatin)-induced apoptosis was observed. This sensitivity could be reverted to the parental phenotype by transforming the depleted cells with normal platelet mitochondria. mRNA expression of BAX, BCL2, MDR1, MRP, ERCC1 and ERCC2, putatively associated with cisplatin resistance to apoptotic death was unchanged. Inhibition of mitochondrial ATP production by oligomycin did not result in a change in ATP levels, indicating energetics were not playing a role in the observed phenotype changes. All U937 cells (with/without mtDNA) continued to respond to cisplatin by an apoptotic death. MtDNA-encoded molecules may be playing a role in the relative sensitivity of cells to undergo a cisplatin-induced apoptotic death, but may not be required for cells to undergo apoptosis per se.     

Imaging apoptosis for detecting plaque instability: rendering death a brighter facade

The relatively poor correlation between the risk of atherosclerotic plaque rupture and the degree of luminal obstruction before this event implies a strong imperative for in vivo detection of the processes underlying progressive plaque destabilization. In addition to the morphologic characteristics, apoptosis and inflammation comprise two important indicators of plaque instability. Apoptotic macrophage death results in enlargement of the plaque necrotic core and positive vascular remodelling, whereas apoptosis of the smooth muscle cells leads to attenuation of the fibrous cap. Imaging of apoptotic cells with annexin A5 provides an opportunity for the non-invasive assessment of cell death, and hence plaque vulnerability. The clinical detection of apoptosis could therefore promote the development of novel intervention strategies.     

Apoptotic force: active mechanical function of cell death during morphogenesis

Apoptosis, or programmed cell death, is an essential process for the elimination of unnecessary cells during embryonic development, tissue homeostasis, and certain pathological conditions. Recently, an active mechanical function of apoptosis called apoptotic force has been demonstrated during a tissue fusion process of Drosophila embryogenesis. The mechanical force produced during apoptosis is used not only to force dying cells out from tissues in order to keep tissue integrity, but also to change the morphology of neighboring cells to fill the space originally occupied by the dying cell. Furthermore, the occurrence of apoptosis correlates with tissue movement and tension of the tissue. This finding suggests that apoptotic forces might be harnessed throughout cell death-related morphogenesis; however, this concept remains to be fully investigated. While the investigation of this active mechanical function of apoptosis has just begun, here we summarize the current understandings of this novel function of apoptosis, and discuss some possible developmental processes in which apoptosis may play a mechanical role. The concept of apoptotic force prompts a necessity to rethink the role of programmed cell death during morphogenesis.     

常规组织切片凋亡细胞原位末端标记方法

凋亡是有别于组织坏死的一种细胞死亡方式,多与基因调控的编程性死亡有关。准确地判断细胞凋亡对探讨程序化细胞死亡诱发机制具有重要意义。本文采用Biotin-16-dUTP对凋亡细胞内DNA断裂片段的末端进行标记,可原位检测常规组织切片上的凋亡细胞,方法具有敏感、直观、重复性好等优点。     

Effects of culture media on the susceptibility of cells to apoptotic cell death

Whether responses of cells to extracellular environments affect the induction of apoptotic cell death is poorly understood. The current study aimed to unravel the different effects of culture media employed in vitro as extracellular environments on the susceptibility of cells to apoptosis. We found that apoptosis is stimulated to the higher levels by culturing human HeLa cells in Opti-MEM with unknown components, a medium that is specifically used for transfections, than by culturing cells in Dulbecco’s modified Eagle’s medium, a medium that is generally used for maintenance of cells. We showed that apoptosis is suppressed partially by culturing cells in heat-treated Opti-MEM, implicating a heat-sensitive component(s) in stimulating the apoptotic response of cells. Thus, different extracellular environments may contribute to different responses of cells to apoptosis, and this should be considered to evaluate the incidences of apoptotic cell death and could be applied to develop an efficient treatment for curing diseases such as cancer.     

Cytoplasmic c-Jun N-terminal immunoreactivity: a hallmark of retinal apoptosis

1. We investigated the association of c-Jun with apoptosis within retinal tissue. Explants of the retina of neonatal rats were subject to a variety of procedures that cause apoptosis of specific classes of retinal cells at distinct stages of differentiation. The expression of c-Jun was detected by Western Blot, and immunohistochemistry was done with antibodies made for either N-terminal or C-terminal domains of c-Jun, and correlated with apoptosis detected either by chromatin condensation or by in situ nick end labeling of fragmented DNA.2. c-Jun protein content was increased in retinal tissue subject to induction of both photoreceptor and ganglion cell death.3. c-Jun N-terminal immunoreactivity was found mainly in the cytoplasm of apoptotic cells regardless of cell type, of the stage of differentiation, including proliferating cells, or of the means of induction of apoptosis.4. The data are consistent with the hypothesis that c-Jun is involved in the control of cell death in retinal tissue, but other proteins that cross-react with c-Jun N-terminal antibodies may also be major markers of retinal apoptosis.5. Antibodies directed to c-Jun N-terminal (aa 91-105) are useful tools to follow apoptotic changes in retinal tissue.     

The mechanism whereby heat shock induces apoptosis depends on the innate sensitivity of cells to stress

The cellular response to heat shock (HS) is a paradigm for many human diseases collectively known as “protein conformation diseases” in which the accumulation of misfolded proteins induces cell death. Here, we analyzed how cells having a different apoptotic threshold die subsequent to a treatment with HS. Cells with a low apoptotic threshold mainly induced apoptosis through activation of conventional stress kinase signaling pathways. By contrast, cells with a high apoptotic threshold also died by apoptosis but likely after the accumulation of heat-aggregated proteins as revealed by the formation of aggresomes in these cells, which were associated with the generation of atypical nuclear deformations. Inhibition of the proteasome or expression of an aggregation prone protein produced similar nuclear alterations. Furthermore, elevated levels of chaperones markedly suppressed both HS-induced nuclear deformations and apoptosis induced upon protein aggregation whereas they had little effect on stress kinase-mediated apoptosis. We conclude that the relative contribution of stress signaling pathways and the accumulation of protein aggregates to cell death by apoptosis is related to the innate sensitivity of cells to deadly insults.     

On the use of an appropriate TdT-mediated dUTP–biotin nick end labeling assay to identify apoptotic cells

Apoptosis is an essential cellular mechanism involved in many processes such as embryogenesis, metamorphosis, and tissue homeostasis. DNA fragmentation is one of the key markers of this form of cell death. DNA fragmentation is executed by endogenous endonucleases such as caspase-activated DNase (CAD) in caspase-dependent apoptosis. The TUNEL (TdT-mediated dUTP–biotin nick end labeling) technique is the most widely used method to identify apoptotic cells in a tissue or culture and to assess drug toxicity. It is based on the detection of 3′-OH termini that are labeled with dUTP by the terminal deoxynucleotidyl transferase. Although the test is very reliable and sensitive in caspase-dependent apoptosis, it is completely useless when cell death is mediated by pathways involving DNA degradation that generates 3′-P ends as in the LEI/L-DNase II pathway. Here, we propose a modification in the TUNEL protocol consisting of a dephosphorylation step prior to the TUNEL labeling. This allows the detection of both types of DNA breaks induced during apoptosis caspase-dependent and independent pathways, avoiding underestimating the cell death induced by the treatment of interest.     

Loss of the maintenance methyltransferase, xDnmt1, induces apoptosis in Xenopus embryos

DNA methylation is necessary for normal embryogenesis in animals. Here we show that loss of the maintenance methyltransferase, xDnmt1p, triggers an apoptotic response during Xenopus development, which accounts for the loss of specific cell populations in hypomethylated embryos. Hypomethylation-induced apoptosis is accompanied by a stabilization in xp53 protein levels after the mid-blastula transition. Ectopic expression of HPV-E6, which promotes xp53 degradation, prevents cell death, implying that the apoptotic signal is mediated by xp53. In addition, inhibition of caspase activation by overexpression of Bcl-2 results in the development of cellular masses that resemble embryonic blastomas. Embryonic tissue explant experiments suggest that hypomethylation alters the developmental potential of early embryo cells and that apoptosis is triggered by differentiation. Our results imply that loss of DNA methylation in differentiated somatic cells provides a signal via p53 that activates cell death pathways.     

Ischemic preconditioning attenuates apoptotic cell death associated with ischemia/reperfusion

Apoptosis or programmed cell death is a genetically controlled response for cells to commit suicide and is associated with DNA fragmentation or laddering. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca2+ which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine whether ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15, 30 or 60 min of ischemia as well as 15 min of ischemia followed by 30, 60, 90 or 120 min of reperfusion. At the end of each experiment, the heart was processed for the evaluation of apoptosis and DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG® in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from the hearts to 1.8% agarose gel electrophoresis and photographed under UV illumination. The results of our study revealed apoptotic cells only in the 90 and 120 min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results were corroborated with the findings of DNA fragmentation which showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA length (about 180 bp). The presence of apoptotic cells and DNA fragmentation in the myocardium were completely abolished by subjecting the myocardium to repeated short-term ischemia and reperfusion which also reduced the ischemic reperfusion injury as evidenced by better recovery of left ventricular performance in the preconditioned myocardium. The results of this study indicate that reperfusion of ischemic heart, but not ischemia, induces apoptotic cell death and DNA fragmentation which can be inhibited by myocardial adaptation to ischemia.     

Isolation of cell death-associated cDNAs from involuting mouse mammary epithelium

Although apoptosis is important in determining cell fate and maintaining tissue homeostasis, the initiation and control of apoptotic cell death in epithelium is not well understood. Post-lactationai involution of the mammary gland provides both an important developmental process and a normal physiological setting for studying apoptosis of epithelium. We used a differential screening strategy, based on previous studies correlating morphology with gene expression and nucleic acid integrity during mammary gland involution, to isolate genes involved in the regulation and execution of apoptotic cell death in regressing mammary epithelium. This screening strategy yielded a large number of genes the expression of which is significantly altered during mammary gland involution. These include genes associated with cell death processes, tissue remodelling and mesenchymal differentiation. In addition, a number of novel genes have been isolated. We have used Northern analysis and in situ hybridisation to study the expression of a selection of these putative death-associated genes during post-lactational mouse mammary gland involution.     

Both the Caspase CSP-1 and a Caspase-Independent Pathway Promote Programmed Cell Death in Parallel to the Canonical Pathway for Apoptosis in Caenorhabditis elegans

Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to the canonical apoptosis pathway involving CED-3 activation.