J. F. R. Kerr

Apoptosis: A Basic Biological Phenomenon with Wide-ranging Implications in Tissue Kinetics

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.

Cell Death: The Significance of Apoptosis

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.

Apoptosis : its significance in cancer and cancer therapy

Apoptosis is a distinct mode of cell death that is responsible for deletion of cells in normal tissues; it also occurs in specific pathologic contexts. Morphologically, it involves rapid condensation and budding of the cell, with the formation of membrane‐enclosed apoptotic bodies containing well‐preserved organelles, which are phagocytosed and digested by nearby resident cells. There is no associated inflammation. A characteristic biochemical feature of the process is double‐strand cleavage of nuclear DNA at the linker regions between nucleosomes leading to the production of oligonucleosomal fragments. In many, although not all of the circumstances in which apoptosis occurs, it is suppressed by inhibitors of messenger RNA and protein synthesis. Apoptosis occurs spontaneously in malignant tumors, often markedly retarding their growth, and it is increased in tumors responding to irradiation, cytotoxic chemotherapy, heating and hormone ablation. However, much of the current interest in the process stems from the discovery that it can be regulated by certain proto‐oncogenes and the p53 tumor suppressor gene. Thus, c‐myc expression has been shown to be involved in the initiation of apoptosis in some situations, and bcl‐2 has emerged as a new type of proto‐oncogene that inhibits apoptosis, rather than stimulating mitosis. In p53‐negative tumor‐derived cell lines transfected with wild‐type p53, induction of the gene has, in rare cases, been found to cause extensive apoptosis, instead of growth arrest. Finally, the demonstration that antibodies against a cell‐surface protein designated APO‐1 or Fas can enhance apoptosis in some human lymphoid cell lines may have therapeutic implications.

Anatomical methods in cell death

Publisher Summary This chapter describes methods for studying the morphology of cell death and the criteria used in identifying apoptosis and necrosis. Electron microscopy provides the most reliable method for recognizing the two processes; in many cases, however, they can be identified confidently using light microscopy alone. The recognition of apoptosis and necrosis is based primarily on the distinctive changes that take place within the affected cells. However, when these two processes occur in vivo, they also differ in their distribution and in the tissue reactions that are associated with them. These latter features may be of subsidiary use in identification. Thus, apoptosis involves scattered individual cells in a tissue, whereas necrosis involves groups of adjoining cells. Necrosis is accompanied by an acute inflammatory response with exudation of neutrophil leukocytes and monocytes; this event is characteristically absent in apoptosis. The light microscopic recognition of apoptosis depends on the detection of discrete well-preserved apoptotic bodies. Although convoluted budding cells are sometimes observed in smears, they are rarely seen in paraffin sections of immersion-fixed tissue.

Internucleosomal DNA Cleavage Should not be the Sole Criterion for Identifying Apoptosis

Evidence is presented that questions the validity of using DNA electrophoresis in isolation for identifying apoptosis.

Cell Death Induced in a Murine Mastocytoma by 42–47°C Heating in Vitro: Evidence that the Form of Death Changes from Apoptosis to Necrosis Above a Critical Heat Load

The pathogenesis of heat-induced cell death is controversial. Categorizing the death occurring after various heat loads as either apoptosis or necrosis might help to elucidate this problem, since it has been shown that these two processes differ in their mode of initiation as well as in their morphological and biochemical features. Log-phase cultures of mastocytoma P-815 x 2.1 were heated at temperatures ranging from 42 to 47 degrees C for 30 min. After 42 degrees C heating a slight increase in apoptosis was observed morphologically. However, after heating at 43, 43.5 and 44 degrees C, there was marked enhancement of apoptosis, and electrophoresis of DNA showed characteristic internucleosomal cleavage. With heating at 45 degrees C both apoptosis and necrosis were enhanced, whereas at 46 and 47 degrees C only necrosis was produced. DNA extracted from the 46 and 47 degrees C cultures showed virtually no degradation, which contrasts with the random DNA breakdown observed in necrosis produced by other types of injury; lysosomal enzymes released during heat-induced necrosis may be inactivated at the higher temperatures. It is suggested that apoptosis following heating may be triggered either by a limited increase in cytosolic calcium levels resulting from mild membrane changes or by DNA damage. Necrosis, on the other hand, is likely to be a consequence of severe membrane disruption.

Spontaneous programmed death (apoptosis) of B-chronic lymphocytic leukaemia cells following their culture in vitro.

Summary When B‐cell lymphocytic leukaemia (B‐CLL) cells derived from peripheral blood were cultured in vitro, a substantial proportion of them spontaneously died by apoptosis. This type of cell death is morphologically and biochemically distinct from necrosis and has previously been found to occur under physiologic and certain pathologic conditions where cell deletion appears controlled and biologically meaningful. By 30 h of culture, approximately 20% of the unfractionated B‐CLL cells were affected. There was no significant difference in the incidence of apoptosis in T‐cell depleted and undepleted cultures or when either autologous or normal human serum was used. Furthermore, seeding densities of 2 × 106 and 5 × 105 cells/ml resulted in a similar incidence of apoptosis, indicating that cell density was unlikely to be a contributing factor in producing the death. The finding that B‐CLL cells spontaneously die in vitro has at least two important implications. Firstly, previous work relating to some of the functions of B‐CLL cells and their interactions with T cells may require re‐evaluation. Secondly, an understanding of the mechanisms involved in the induction of apoptosis in this disease may have therapeutic consequences.

History of the events leading to the formulation of the apoptosis concept

Histological studies of ischaemic liver injury performed between 1962 and 1964 distinguished two types of cell death: classical necrosis, and a process involving conversion of scattered cells into small round masses of cytoplasm that often contained specks of condensed nuclear chromatin. Enzyme histochemistry demonstrated rupture of lysosomes in the former, but preservation of lysosomes in the latter. Similar small round masses were also observed sparsely in normal liver. Electron microscopy showed that the small round bodies resulted from cellular condensation and budding, that they were bounded by membranes and contained intact organelles, and that they were phagocytosed and digested by resident tissue cells, including epithelial cells. In work done in association with Jeffrey Searle, the process was found to occur spontaneously in a variety of malignant tumours and to be enhanced in squamous cell carcinomas of skin responding to radiotherapy. During 1971-1972, I collaborated with Andrew Wyllie and Alastair Currie while on sabbatical leave in Scotland. The newly defined type of cell death was shown to be regulated by hormones in the adrenal cortex and in breast carcinomas. Further, review of published electron micrographs of the cell death known to play an essential role in normal development revealed the same morphological pattern. We proposed that this distinctive phenomenon subserves a general homoeostatic function and suggested it be called apoptosis.

Effects of cycloheximide on B-chronic lymphocytic leukaemic and normal lymphocytes in vitro: induction of apoptosis.

A number of reports indicate that protein synthesis is a requirement for the occurrence of apoptosis. In this study, the effect of the protein synthesis inhibitor cycloheximide (CHM) on spontaneous apoptosis of B-chronic lymphocytic leukaemia (B-CLL) cells, previously shown to occur when they are cultured in RPMI-1640 medium with autologous or heterologous serum, was examined. No definite inhibition of apoptosis was observed. Indeed, CHM-treatment augmented apoptosis in the B-CLL cultures and also induced apoptosis of cultured normal peripheral blood lymphocytes. Augmentation was dose-dependent for B-CLL cells over the concentration range 10(-6) M (0.28 micrograms ml-1) to 10(-2) M (2800 micrograms ml-1), resulting in 9% to 98% apoptosis respectively by 24 h of culture (r = 0.619, P = 0.0008). Normal lymphocytes were affected by CHM over the range 10(-4) M to 10(-2) M, resulting in 7% to 74% apoptosis respectively (r = 0.794, P = 0.0001). Inhibition of protein synthesis in these cells by CHM was virtually complete at a concentration of 10(-3) M. The findings are in accord with some recent reports indicating that suppression of protein synthesis by CHM does not inhibit apoptosis in all circumstances. They also illustrate the marked susceptibility of B-CLL cells, compared with normal lymphocytes, to the induction of apoptosis by this drug. The manner in which CHM triggers apoptosis of some cell types is at present uncertain.

Ultrastructure of the rat pancreas after experimental duct ligation. II. Duct and stromal cell proliferation, differentiation, and deletion.

Ligation of the pancreas in rats was followed by rapid atrophy of the distal part of the gland, where deletion of the acinar cells by apoptosis and simultaneous extensive proliferation of duct cells resulted in the lobules being converted into groups of closely packed small ducts within 5 days. We found no ultrastructural evidence that cells lining these small ducts arose from acinar cells by a process of dedifferentiation, as has been suggested by some investigators. During the succeeding weeks, some of the ductal lining cells developed islet cell or partial acinar cell differentiation. The latter soon died by apoptosis, and some ductlike and islet cells were also deleted by this means. Most of the apoptotic bodies formed in the ducts were phagocytosed by intra-epithelial macrophages. In the longer term, continuing apoptosis eventually resulted in the disappearance of many ducts, only their thickened basal laminae remaining. Differentiation of stromal fibroblasts into contractile rnyofibro-blasts may have contributed to shrinkage of the duct-obstructed glandular tissue, and apoptosis of endothelial cells probably accounted for the associated reduction of the capillary bed.