Clay Winterford

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.

Tumor Necrosis Factor-α Induces Apoptosis of Human Adipose Cells

Tumor necrosis factor-α (TNF-α) production by adipocytes is elevated in obesity, as shown by increased adipose tissue TNF-α mRNA and protein levels and by increased circulating concentrations of the cytokine. Furthermore, TNF-α has distinct effects on adipose tissue including induction of insulin resistance, induction of leptin production, stimulation of lipolysis, suppression of lipogenesis, induction of adipocyte dedifferentiation, and impairment of preadipocyte differentiation in vitro. Taken together, these effects all tend to decrease adipocyte volume and number and suggest a role for TNF-α in limiting increase in fat mass. The aim of the present study was to determine if TNF-α could induce apoptosis in human adipose cells, hence delineating another mechanism by which the cytokine could act to limit the development of, or extent of, obesity. Cultured human preadipocytes and mature adipocytes in explant cultures were exposed in vitro to human TNF-α at varying concentrations for up to 24 h. Apoptosis was assessed using morphological (histology, nuclear morphology following acridine orange staining, electron microscopy) and biochemical (demonstration of internucleosomal DNA cleavage by gel electrophoresis and of annexin V staining using immunocytochemistry) criteria. In control cultures, apoptotic indexes were between 0 and 2.3% in all experiments. In the experimental systems, TNF-α induced apoptosis in both preadipocytes and adipocytes, with indexes between 5 and 25%. Therefore, TNF-α induces apoptosis of human preadipocytes and adipocytes in vitro. In view of the major metabolic role of TNF-α in human adipose tissue, and the knowledge that adipose tissue is dynamic (with cell acquisition via preadipocyte replication/differentiation and cell loss via apoptosis), these findings describe a further mechanism whereby adipose tissue mass may be modified by TNF-α.

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 portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease

Although nonalcoholic fatty liver disease (NAFLD) is conventionally assessed histologically for lobular features of inflammation, development of portal fibrosis appears to be associated with disease progression. We investigated the composition of the portal inflammatory infiltrate and its relationship to the ductular reaction (DR), a second portal phenomenon implicated in fibrogenesis. The portal inflammatory infiltrate may contribute directly to fibrogenesis as well as influence the fate of the DR hepatic progenitor cells (HPCs), regulating the balance between liver repair and fibrosis. The presence of portal inflammation in NAFLD was strongly correlated with disease severity (fibrosis stage) and the DR. The portal infiltrate was characterized by immunostaining NAFLD liver biopsy sections (n = 33) for broad leukocyte subset markers (CD68, CD3, CD8, CD4, CD20, and neutrophil elastase) and selected inflammatory markers (matrix metalloproteinase 9 and interleukin [IL]‐17). Cells expressing all markers examined were identified throughout the liver lobules and in portal tracts, although portal tracts were more densely populated (P < 0.01), and dominated by CD68+ macrophages and CD8+ lymphocytes, at all stages of disease. An increase in portal macrophages in NAFLD patients with steatosis alone (P < 0.01) was the earliest change detected, even before elevated expression of the proinflammatory cytokines, IL1B and TNF, in patients with early NASH (P < 0.05). Portal and periductal accumulation of all other cell types examined occurred in progressed NASH (all P < 0.05). Conclusion: Knowledge of the complex cellular composition of the portal inflammatory infiltrate and HPC/DR niche in NAFLD will shape future functional studies to elucidate the contribution of portal inflammation to HPC differentiation and NAFLD pathogenesis. (Hepatology 2014;59:1393‐1405)

Streptozotocin at low doses induces apoptosis and at high doses causes necrosis in a murine pancreatic beta cell line, INS-1.

The ability of ß cells to endure assaults by various environmental agents, including toxins and viruses, may be relevant to the development of diabetes. We have examined the mode of cell death caused by streptozotocin (STZ) in a murine pancreatic ß cell line, INS‐1. Apoptosis was identified by detection of initial endonuclease‐mediated DNA strand breaks by DNA gel electrophoresis. Apoptosis and necrosis were distinguished morphologically by light and electron microscopy. Higher rates of apoptosis, as compared to necrosis, were observed when cells were exposed to 15 mM STZ for 1 hr followed by a 24 hrs recovery period. Higher doses of STZ (30 mM) caused the cells to undergo necrosis (22%) as well as apoptosis (17%). These results suggest that the cytotoxic effect of STZ, at low doses, on ß cells involves the activation of the apoptotic pathway, whereas, at high doses, the mode of ß cell death is predominantly necrosis.

Apoptosis in Vascular Endothelial Cells Caused by Serum Deprivation, Oxidative Stress and Transforming Growth Factor-β

Vascular endothelial cell apoptosis has previously been shown to play a role in the pathogenesis of hypertension-induced vessel deletion and damage. In the present in vitro study we analyse several possible relevant causative factors of vascular endothelial cell apoptosis, namely, serum deprivation and nutrient depletion, oxidative stress in the forms of hypoxia, hyperoxia or free radical damage, and altered levels of transforming growth factor-beta1 (TGF-beta1) protein. An established cell line, bovine aortic endothelial cells (BAEC), was maintained in complete growth medium (RPMI-1640 plus 15% fetal calf serum and antibiotics, abbreviated as RPMI) in 25cm2 flasks or in 12-well plates on glass coverslips. Confluent but actively-growing cultures were treated with either hypoxia (PO2 of RPMI = 50mmHg), serum-free media (SFM), SFM plus hypoxia, hyperoxia (PO2 of RPMI = 450mmHg), hydrogen peroxide (H2O2, 1mM) in SFM, or TGF-beta1 protein (10ng/mL) in SFM. Appropriate control cultures were used. BAEC were collected 48h or 72h after all treatments except for TGF-beta1 and H2O2 treatments that were collected at 16-18h. Cell death was assessed using morphological characteristics or in situ end labeling (ISEL), cell proliferation assessed using proliferating cell nuclear antigen (PCNA), and TGF-beta1 expression assessed using transcript levels or immunohistochemistry. All treatments significantly increased levels of apoptosis over control cultures (P<0.05), and decreased levels of cell proliferation. Treatment with TGF-beta1 protein or SFM plus hypoxia induced greatest levels of apoptosis. TGF-beta1 protein and transcript levels were decreased in treated cultures, results suggesting that a paracrine source of TGF-beta1 protein would be needed as a cause of endothelial cell apoptosis in viva. Future therapies against inappropriate vessel deletion in disease states may use the known gene-driven nature of apoptosis to modify this sort of cell death in endothelial cells.

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.

Apoptosis in normal epithelium, premalignant and malignant lesions of the oropharynx and oral cavity: a preliminary study

To explore the involvement of apoptosis in the development of oral and oropharyngeal squamous cell carcinoma (SCC) in vivo, biopsies were taken from patients with macroscopically normal (n = 6), leukoplakic (n = 12) or malignant (n = 8) mucosa. Leukoplakic lesions were divided histologically into dysplasia (n = 5) or carcinoma in situ (CIS: n = 7). Material was prepared for light and electron microscopy. The apoptotic index (AI), vertical cell position of apoptoses (cp), mitotic index (MI) and AI:MI ratio were calculated for each patient. AI increased from 0.12% +/- 0.07 S.E.M. (normal) to 0.58 +/- 0.13 (CIS) but fell to 0.14 +/- 0.14 in SCC. Apoptoses were suprabasal in normals, but generalised in CIS. MI increased from normal (0.20 +/- 0.06) to SCC (0.32 +/- 0.09), and AI:MI was at its maximum in CIS (1.57; SCC: 0.44). The results suggest that a change in apoptosis accompanies the onset of invasion in a premalignant lesion of the human oral cavity and oropharynx.

Ethionine-induced Atrophy of Rat Pancreas Involves Apoptosis of Acinar Cells

The mechanism of acinar cell loss occurring during ethionine-induced atrophy of the pancreas was investigated. Rats were given a standard diet, a protein-depletion diet (PDD), or a PDD with low- (0.04 g/kg body wt; LDE) or high- (0.4 g/kg; HDE) dose ethionine administered intraperitoneally daily for 10 days. Changes were most extensive in the animals given a PDD and HDE: After 10 days, pancreatic weight was reduced by 72%, and most of the acinar cells had disappeared. Prior to their deletion, these cells showed cytoplasmic vacuolation and enhanced autophagy. The main mechanism involved in their deletion was apoptosis, the apoptotic bodies being phagocytosed and degraded by adjacent acinar cells and intraepithelial macrophages. In contrast, necrosis of acinar cells was rare. Interstitial inflammation and apoptosis of capillary endothelial cells were also observed. In animals given a PDD and LDE, enhanced apoptosis occurred later and was more limited in extent, and additional manifestations of cell injury were not evident. As in other circumstances where glandular atrophy is effected by apoptosis, the basic tissue architecture was preserved, thus explaining the known capacity for the pancreas to regenerate after ethionine is discontinued.