Brian V. Harmon

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.

Spermatogonial apoptosis has three morphologically recognizable phases and shows no circadian rhythm during normal spermatogenesis in the rat

Abstract. In this study we examined the possibility that regular or circadian fluctuations occur in the frequency of spontaneous spermatogonial apoptosis. Apoptosis of A2, A3 and A4 type spermatogonia occurring spontaneously in the normal rat testis was studied by light and electron microscopy. Normal and apoptotic A3 spermatogonia were quantified in 36 animals killed at two‐hourly intervals over a 24 h period. Three sequential phases of spermatogonial apoptosis were defined and quantified separately: (i) an early phase in which cells showed margination of nuclear chromatin, (ii) an intermediate phase in which phagocytosed apoptotic bodies were partly degraded and (iii) a late phase in which only debris of degraded apoptotic bodies was evident. Groups of spermatogonia linked by intercellular bridges underwent apoptosis synchronously. Normal and apoptotic A3 spermatogonia occurred at a mean frequency of 33.4 and 9.6 per 10 seminiferous tubule profiles respectively; there was a large variation in these frequencies between animals, but no peaks or circadian periodicity were detected. Progressive degradation of apoptotic bodies was evident, the average ratios of intermediate and late bodies to early bodies being 1.5 and 3.5, respectively. Absence of a circadian rhythm in these data does not exclude the possibility that initiation of apoptosis in susceptible spermatogonial clones is synchronous, and that affected clones undergo lag periods of differing duration before expressing morphological apoptosis.

Apoptosis Is the Mode of β-Cell Death Responsible for the Development of IDDM in the Nonobese Diabetic (NOD) Mouse

The NOD/Lt mouse, a widely used model of human autoimmune IDDM, was used to establish the mode of β-cell death responsible for the development of IDDM. Apoptotic cells were present within the islets of Langerhans in hematoxylin and eosin–stained sections of pancreases harvested from 3- to 18-week-old female NOD/Lt mice (a range of 11–50 apoptotic cells per 100 islets). Immunohistochemical localization of insulin to the dying cells confirmed the β-cell origin of the apoptosis. Although some islets from age-matched control female NOD/scid mice contained apoptotic cells, virtually all of these cells were insulin negative as determined by immunohistochemistry. The small number of apoptotic insulin-positive cells identified in islets from NOD/scid mice (a range of 0–1 apoptotic cells per 100 islets) was not statistically significant, compared with the numbers recorded in NOD/Lt mice. All dying cells showed the morphological changes characteristic of cell death by apoptosis and stained positively with the TUNEL method for end-labeling DNA strand breaks. The maximum mean amount of β-cell apoptosis occurring in NOD/Lt mice was at week 15 (50 apoptotic cells per 100 islets), which coincided with the earliest onset of diabetes as determined by blood glucose, urine glucose, and pancreatic immunoreactive insulin measurements. While there was no peak incidence of β-cell apoptosis throughout the time period studied (weeks 3–18), the incidence of apoptosis decreased at week 18, by which time 50% of the animals had overt diabetes. The low levels of β-cell apoptosis observed is indicative of a gradual deletion of the β-cell population throughout the extensive preclinical period seen in this model and would be sufficient to account for the β-cell loss resulting in IDDM. Apoptosis of β-cells preceded the appearance of T-cells (CD3-positive by immunohistochemistry) in islets. Lymphocytic infiltration of islets (insulitis) was not detected until week 6. The results show that β-cell apoptosis is responsible for the development of IDDM in the NOD/Lt mouse and that its onset precedes lymphocytic infiltration of the islets.

Beta cell apoptosis is responsible for the development of IDDM in the multiple low-dose streptozotocin model

Although insulin‐dependent diabetes mellitus (IDDM) results from irreversible loss of beta cells, the mode of cell death responsible for this loss has not previously been categorized. In this study, the multiple low‐dose streptozotocin (stz) model (intraperitoneal injection of stz at a concentration of 40 mg/kg body weight per day for five consecutive days) was used to investigate beta‐cell death during the development of IDDM in male C57B1/6 mice. Apoptotic cells were evident by light microscopy within the islets of Langerhans of treated animals from day 2 (the day of the second stz injection) until day 17. Immunohistochemical localization of insulin to the dying cells confirmed the beta‐cell origin of the apoptosis. Two peaks in the incidence of beta‐cell apoptosis occurred: the first at day 5, which corresponded to an increase in blood glucose concentration, and the second at day 11, when lymphocytic infiltration of the islets (insulitis) was maximal. Insulitis did not begin until day 9, by which time treated animals had developed overt diabetes as revealed by blood glucose and pancreatic immunoreactive insulin (IRI) measurements. Beta‐cell apoptosis preceded the appearance of T‐cells in the islets and continued throughout the period of insulitis. Thus, whether induced by stz or a subsequent immune response, apoptosis is the mode of cell death responsible for beta‐cell loss in the multiple low‐dose stz model of IDDM.

The Role of Apoptosis in the Response of Cells and Tumours to Mild Hyperthermia

There is now abundant evidence that apoptosis, the cell death mechanism responsible for physiological deletion of cells, can be triggered by mild hyperthermia. However, the overall importance of this mode of death in heated tumours has not yet been established. In this light and electron microscopic study, apoptosis induced by 43 degrees C or 44 degrees C water bath heating for 30 min in a range of murine and human tumours growing in vitro and in four murine tumours growing as solid nodules in vivo, was identified on the basis of its characteristic morphology, and the amount present quantified. Apoptosis was found to play a variable role in the response of tumours to heating, with the lowest levels produced in human melanoma lines (less than 1%) and the highest levels in some Burkitts lymphoma lines (up to 97%). In these latter tumours the induction of apoptosis is clearly a major component of the hyperthermic response.

The significance of cell death by apoptosis in hepatobiliary disease

Keywords: acidophilic body; apoptosis; cell death; Councilman body; hepatitis; piecemeal necrosis.