Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.
TL;DR: The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.
Abstract: Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy-transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.
Citations
More filters
TL;DR: This work has identified endostatin, an angiogenesis inhibitor produced by hemangioendothelioma, a 20 kDa C-terminal fragment of collagen XVIII that specifically inhibits endothelial proliferation and potently inhibitsAngiogenesis and tumor growth.
4,613 citations
TL;DR: It is shown that the inhibition of metastases by a primary mouse tumor is mediated, at least in part, by angiostatin, and a corresponding fragment of human plasminogen has similar activity.
3,516 citations
Journal Article•
TL;DR: Some of the typical features of apoptosis are discussed, such as budding (as opposed to blebbing and zeiosis) and the inflammatory response, and stands in contrast to apoptosis, which leads to necrosis with karyorhexis and cell shrinkage.
Abstract: The historical development of the cell death concept is reviewed, with special attention to the origin of the terms necrosis, coagulation necrosis, autolysis, physiological cell death, programmed cell death, chromatolysis (the first name of apoptosis in 1914), karyorhexis, karyolysis, and cell suicide, of which there are three forms: by lysosomes, by free radicals, and by a genetic mechanism (apoptosis). Some of the typical features of apoptosis are discussed, such as budding (as opposed to blebbing and zeiosis) and the inflammatory response. For cell death not by apoptosis the most satisfactory term is accidental cell death. Necrosis is commonly used but it is not appropriate, because it does not indicate a form of cell death but refers to changes secondary to cell death by any mechanism, including apoptosis. Abundant data are available on one form of accidental cell death, namely ischemic cell death, which can be considered an entity of its own, caused by failure of the ionic pumps of the plasma membrane. Because ischemic cell death (in known models) is accompanied by swelling, the name oncosis is proposed for this condition. The term oncosis (derived from onkos, meaning swelling) was proposed in 1910 by von Reckling-hausen precisely to mean cell death with swelling. Oncosis leads to necrosis with karyolysis and stands in contrast to apoptosis, which leads to necrosis with karyorhexis and cell shrinkage.
3,462 citations
TL;DR: Findings suggest that in the developing nervous system, Akt is a critical mediator of growth factor-induced neuronal survival.
Abstract: A signaling pathway was delineated by which insulin-like growth factor 1 (IGF-1) promotes the survival of cerebellar neurons. IGF-1 activation of phosphoinositide 3-kinase (PI3-K) triggered the activation of two protein kinases, the serine-threonine kinase Akt and the p70 ribosomal protein S6 kinase (p70S6K). Experiments with pharmacological inhibitors, as well as expression of wild-type and dominant-inhibitory forms of Akt, demonstrated that Akt but not p70S6K mediates PI3-K-dependent survival. These findings suggest that in the developing nervous system, Akt is a critical mediator of growth factor-induced neuronal survival.
2,514 citations
TL;DR: The results show that PTEN may exert its role as a tumor suppressor by negatively regulating the PI3'K/PKB/Akt signaling pathway.
2,482 citations
Cites methods from "Identification of programmed cell d..."
...Apoptosis, as measured by TUNEL assay (Gavrieli et al., 1992) or DAPI staining, showed no obvi- embryos analyzed gave an index of 0.8 6 0.1....
[...]
References
More filters
TL;DR: Apoptosis seems to be involved in cell turnover in many healthy adult tissues and is responsible for focal elimination of cells during normal embryonic development, and participates in at least some types of therapeutically induced tumour regression.
Abstract: The term apoptosis is proposed for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations. Its morphological features suggest that it is an active, inherently programmed phenomenon, and it has been shown that it can be initiated or inhibited by a variety of environmental stimuli, both physiological and pathological.The structural changes take place in two discrete stages. The first comprises nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultrastructurally well-preserved fragments. In the second stage these apoptotic bodies are shed from epithelial-lined surfaces or are taken up by other cells, where they undergo a series of changes resembling in vitro autolysis within phagosomes, and are rapidly degraded by lysosomal enzymes derived from the ingesting cells.Apoptosis seems to be involved in cell turnover in many healthy adult tissues and is responsible for focal elimination of cells during normal embryonic development. It occurs spontaneously in untreated malignant neoplasms, and participates in at least some types of therapeutically induced tumour regression. It is implicated in both physiological involution and atrophy of various tissues and organs. It can also be triggered by noxious agents, both in the embryo and adult animal.
15,416 citations
TL;DR: It has proved feasible to categorize most if not all dying cells into one or the other of two discrete and distinctive patterns of morphological change, which have, generally, been found to occur under disparate but individually characteristic circumstances.
Abstract: Publisher Summary The classification of cell death can be based on morphological or biochemical criteria or on the circumstances of its occurrence. Currently, irreversible structural alteration provides the only unequivocal evidence of death; biochemical indicators of cell death that are universally applicable have to be precisely defined and studies of cell function or of reproductive capacity do not necessarily differentiate between death and dormant states from which recovery may be possible. It has also proved feasible to categorize most if not all dying cells into one or the other of two discrete and distinctive patterns of morphological change, which have, generally, been found to occur under disparate but individually characteristic circumstances. One of these patterns is the swelling proceeding to rupture of plasma and organelle membranes and dissolution of organized structure—termed “coagulative necrosis.” It results from injury by agents, such as toxins and ischemia, affects cells in groups rather than singly, and evokes exudative inflammation when it develops in vivo. The other morphological pattern is characterized by condensation of the cell with maintenance of organelle integrity and the formation of surface protuberances that separate as membrane-bounded globules; in tissues, these are phagocytosed and digested by resident cells, there being no associated inflammation.
7,417 citations
"Identification of programmed cell d..." refers background in this paper
..., 1987), and apoptotic bodies in diverse forms are seen for only a few hours before they are phagocytized (Wyllie et al., 1980; Bursch et al., 1990)....
[...]
...APOptotic cell appearance is limited to only a few minutes (Russell et al., 1972; Sanderson, 1976; Matter, 1979; Kerr et al., 1987), and apoptotic bodies in diverse forms are seen for only a few hours before they are phagocytized (Wyllie et al., 1980; Bursch et al., 1990)....
[...]
TL;DR: A flow cytometric method for measuring the percentage of apoptotic nuclei after propidium iodide staining in hypotonic buffer is developed and shown an excellent correlation with the results obtained with both electrophoretic and colorimetric methods.
4,660 citations
TL;DR: It is shown here that this morphological change is closely associated with excision of nucleosome chains from nuclear chromatin, apparently through activation of an intracellular, but non-lysosomal, endonuclease.
Abstract: In near-physiological concentrations, glucocorticoid hormones cause the death of several types of normal and neoplastic lymphoid cell, but the mechanisms involved are unknown. One of the earliest structural changes in the dying cell is widespread chromatin condensation, of the type characteristic of apoptosis, the mode of death frequently observed where cell deletion seems to be 'programmed'. It is shown here that this morphological change is closely associated with excision of nucleosome chains from nuclear chromatin, apparently through activation of an intracellular, but non-lysosomal, endonuclease.
4,605 citations
"Identification of programmed cell d..." refers background in this paper
...These experiments showed that PCD is associated with endogenous endonuclease activity (Wyllie, 1980; Wyllie et al., 1984; Cohen and Duke, 1983, 1984; McConkey et al., 1989, 1990; Shi et al., 1990; Brune et al., 1991)....
[...]
...These experiments showed that PCD is associated with endogenous endonuclease activity ( Wyllie, 1980; Wyllie et al., 1984; Cohen and Duke, 1983, 1984; McConkey et al., 1989, 1990; Shi et al., 1990; Brune et al., 1991)....
[...]
TL;DR: The data confirm that the condensed chromatin which characterizes apoptosis morphologically consists of endogenously digested chromatin fragments, and provide support for the view that at least some cells enter apoptosis by a process dependent upon macromolecular synthesis.
Abstract: In glucocorticoid-treated rat thymocytes and the murine lymphoid cell lines L5178 and S49 the morphology of apoptosis is associated with chromatin cleavage. The cleavage is at internucleosomal sites, apparently through activation of an endogenous endonuclease. In variants of the cell lines selected for resistance to glucocorticoid, neither apoptosis nor chromatin cleavage were observed after steroid treatment, and steroid receptors were undetectable. In thymocytes, both the morphological changes of apoptosis and chromatin cleavage were inhibited by cycloheximide and actinomycin D. The calcium-magnesium ionophore A23187 induced apoptosis and chromatin cleavage in thymocytes, and these effects were also inhibited by cycloheximide. The data confirm that the condensed chromatin which characterizes apoptosis morphologically consists of endogenously digested chromatin fragments. They also provide support for the view that at least some cells enter apoptosis by a process dependent upon macromolecular synthesis.
1,588 citations
"Identification of programmed cell d..." refers background in this paper
...Its gross features, identified by EM, include nuclear chromatin condensation, compactness of cytoplasmatic organelles, and the appearance of pedunculated protuberances on the cell surface (Wyllie et al., 1984; Kerr et al., 1987)....
[...]
...It seems that chromatin cleavage is the most characteristic biochemical feature of the process (Wyllie et al., 1984; Kerr et al., 1987; Martz and t. Abbreviations used in th/s paper."...
[...]
...These experiments showed that PCD is associated with endogenous endonuclease activity (Wyllie, 1980; Wyllie et al., 1984; Cohen and Duke, 1983, 1984; McConkey et al., 1989, 1990; Shi et al., 1990; Brune et al., 1991)....
[...]
...Its gross features, identified by EM, include nuclear chromatin condensation, compactness of cytoplasmatic organelles , and the appearance of pedunculated protuberances on the cell surface (Wyllie et al., 1984; Kerr et al., 1987)....
[...]