Glucocorticoids, oxysterols, and cAMP with glucocorticoids each cause apoptosis of CEM cells and suppress c-myc.

In clones of the CEM human acute lyumphoblastic leukemic cell line, glucocorticoids, oxysterols and activators of the cAMP pathway acting synergistically with glucocorticoids, each can cause apoptotic cell death. Morphologically and kinetically, these deaths resemble one another. The kinetics are striking: in each case, after addition of the lethal compound(s), an interval of approximately 24 h follows, during which cell growth continues unabated. During this “prodromal” period, removal of the apoptotic agent leaves the cells fully viable. We hypothesize that a sequence of biochemical events occurs during the prodrome which eventually results in the triggering of the full apoptotic response as evidenced by the activation of caspases and DNA fragmentation. At some point, the process is irreversible and proceeds relatively rapidly to cell death. Suppression of c-Myc seems a universal early event evoked by each of these lethal compounds or combinations, and we conclude that the negative regulation of this proto-oncogene is an important aspect of the critical pre-apoptotic events in these cells.     

Apoptosis and the conformational change of Bax induced by proteasomal inhibition of PC12 cells are inhibited by bcl-xL and bcl-2

The function of the proteasome has been linked to various pathologies, including cancer and neurodegeneration. Proteasomal inhibition can lead to death in a variety of cell types, however the manner in which this occurs is unclear, and may depend on the particular cell type. In this work we have extended previous findings pertaining to the effects of pharmacological proteasomal inhibitors on PC12 cells, by examining in more detail the induced death pathway. We find that cell death is apoptotic by ultrastructural criteria. Caspase 9 and 3 are processed, cytochrome c is released from the mitochondria and a dominant negative form of caspase 9 prevents death. Furthermore, Bax undergoes a conformational change and is translocated to the mitochondria in a caspase-independent fashion. Total cell levels of Bax however do not change, whereas levels of the BH3-only protein Bim increase with proteasomal inhibition. Transient overexpression of bcl-xL or, to a lesser extent, of bcl-2, significantly decreased apoptotic death and prevented Bax conformational change. We conclude that death elicited by proteasomal inhibition of PC12 cells follows a classical “intrinsic” pathway. Significantly, antiapoptotic bcl-2 family members prevent apoptosis by inhibiting Bax conformational change. Increased levels of Bim may contribute to cell death in this model.     

Fas Induces Cytoplasmic Apoptotic Responses and Activation of the MKK7-JNK/SAPK and MKK6-p38 Pathways Independent of CPP32-like Proteases

IL-1β converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1–like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.     

Clearing the Dead: Apoptotic Cell Sensing,Recognition, Engulfment,and Digestion

Clearance of apoptotic cells is the final stage of programmed cell death. Uncleared corpses can become secondarily necrotic, promoting inflammation and autoimmunity. Remarkably, even in tissues with high cellular turnover, apoptotic cells are rarely seen because of efficient clearance mechanisms in healthy individuals. Recently, significant progress has been made in understanding the steps involved in prompt cell clearance in vivo. These include the sensing of corpses via “find me” signals, the recognition of corpses via “eat me” signals and their cognate receptors, the signaling pathways that regulate cytoskeletal rearrangement necessary for engulfment, and the responses of the phagocyte that keep cell clearance events “immunologically silent.” This study focuses on our understanding of these steps.Multicellular organisms execute the majority of unwanted cell populations in a regulated fashion via the process of apoptosis (Henson and Hume 2006; Nagata et al. 2010). Examples of unwanted cells include excess cells generated during development, cells infected with intracellular bacteria or viruses, transformed or malignant cells capable of tumorigenesis, and cells irreparably damaged by cytotoxic agents. Swift removal of these cells is necessary for maintenance of overall health and homeostasis and prevention of autoimmunity, pathogen burden, or cancer. Quick removal of dying cells is a key final step, if not the ultimate goal of the apoptotic program.The term “phagocytosis” refers to an internalization process by which larger particles, such as bacteria and dead/dying cells, are engulfed and processed within a membrane-bound vesicle called the phagosome (Ravichandran and Lorenz 2007). A phagocyte is any cell that is capable of engulfment, including “professional” phagocytes such as macrophages, immature dendritic cells, and neutrophils. Metazoa have multiple mechanisms for clearing apoptotic cells, often depending on the tissue and apoptotic cell type (Gregory 2009). Macrophages and immature dendritic cells readily engulf dead or dying cells in tissues such as bone marrow (where a large number of new hematopoietic cells are generated), spleen (during or after an immune response), and the thymus (in young animals during T-lymphocyte development). In other tissues, neighboring “nonprofessional” phagocytes can also mediate the clearance of apoptotic targets. For example, in the mammary epithelium, viable mammary epithelial cells engulf apoptotic mammary epithelial cells after cessation of lactation (Monks et al. 2005, 2008). What distinguishes the phagocytosis of apoptotic cells from the phagocytosis of most bacteria or necrotic cells is the lack of a pro-inflammatory immune response (Henson 2005). This article discusses apoptotic cell engulfment, specifically the recruitment of phagocytes, through “find me” signals, the recognition of apoptotic cells by phagocytes via “eat me” signals, the internalization process and signaling pathways used for cytoskeletal rearrangement, and finally the digestion of apoptotic cells and phagocytic response to this process (Fig. 1).Open in a separate windowFigure 1.The steps of efficient apoptotic cell clearance. First, “find me” signals released by apoptotic cells are recognized via their cognate receptors on the surface of phagocytes. This is the sensing stage and stimulates phagocyte migration to the location of apoptotic cells. Second, phagocytes recognize exposed “eat me” signals on the surface of apoptotic cells via their phagocytic receptors, which leads to downstream signaling events culminating in Rac activation. Finally, further signaling events within the phagocyte regulate the digestion and processing of the apoptotic cell meal and the secretion of anti-inflammatory cytokines.     

An Early and Robust Activation of Caspases Heads Cells for a Regulated Form of Necrotic-like Cell Death

Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as “apoptosis-necrosis continuum.” To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death.     

Accumulation of Phosphorylated β-Catenin Enhances ROS-Induced Cell Death in Presenilin-Deficient Cells

Presenilin (PS) is involved in many cellular events under physiological and pathological conditions. Previous reports have revealed that PS deficiency results in hyperproliferation and resistance to apoptotic cell death. In the present study, we investigated the effects of PS on β-catenin and cell mortality during serum deprivation. Under these conditions, PS1/PS2 double-knockout MEFs showed aberrant accumulation of phospho-β-catenin, higher ROS generation, and notable cell death. Inhibition of β-catenin phosphorylation by LiCl reversed ROS generation and cell death in PS deficient cells. In addition, the K19/49R mutant form of β-catenin, which undergoes normal phosphorylation but not ubiquitination, induced cytotoxicity, while the phosphorylation deficient S37A β-catenin mutant failed to induce cytotoxicity. These results indicate that aberrant accumulation of phospho-β-catenin underlies ROS-mediated cell death in the absence of PS. We propose that the regulation of β-catenin is useful for identifying therapeutic targets of hyperproliferative diseases and other degenerative conditions.     

Effective Blockage of Both the Extrinsic and Intrinsic Pathways of Apoptosis in Mice by TAT-crmA

Evidence accumulates that in clinically relevant cell death, both the intrinsic and extrinsic apoptotic pathway synergistically contribute to organ failure. In search for an inhibitor of apoptosis that provides effective blockage of these pathways, we analyzed viral proteins that evolved to protect the infected host cells. In particular, the cowpox virus protein crmA has been demonstrated to be capable of blocking key caspases of both pro-apoptotic pathways. To deliver crmA into eukaryotic cells, we fused the TAT protein transduction domain of HIV to the N terminus of crmA. In vitro, the TAT-crmA fusion protein was efficiently translocated into target cells and inhibited apoptosis mediated through caspase-8, caspase-9, and caspase-3 after stimulation with α-Fas, etoposide, doxorubicin, or staurosporine. The extrinsic apoptotic pathway was investigated following α-Fas stimulation. In vivo 90% of TAT-crmA-treated animals survived an otherwise lethal dose of α-Fas and showed protection from Fas-induced organ failure. To examine the intrinsic apoptotic pathway, we investigated the survival of mice treated with an otherwise lethal dose of doxorubicin. Whereas all control mice died within 31 days, 40% of mice that concomitantly received intraperitoneal injections of TAT-crmA survived. To test the ability to comprehensively block both the intrinsic and extrinsic apoptotic pathway in a clinically relevant setting, we employed a murine cardiac ischemia-reperfusion model. TAT-crmA reduced infarction size by 40% and preserved left ventricular function. In summary, these results provide a proof of principle for the inhibition of apoptosis with TAT-crmA, which might provide a new treatment option for ischemia-reperfusion injuries.     

Adult neurogenesis and neuronal regeneration in the brain of teleost fish

Whereas adult neurogenesis appears to be a universal phenomenon in the vertebrate brain, enormous differences exist in neurogenic potential between “lower” and “higher” vertebrates. Studies in the gymnotiform fish Apteronotus leptorhynchus and in zebrafish have indicated that the relative number of new cells, as well as the number of neurogenic sites, are at least one, if not two, orders of magnitude larger in teleosts than in mammals. In teleosts, these neurogenic sites include brain regions homologous to the mammalian hippocampus and olfactory bulb, both of which have consistently exhibited neurogenesis in all species examined thus far. The source of the new cells in the teleostean brain are intrinsic stem cells that give rise to both glial cells and neurons. In several brain regions, the young cells migrate, guided by radial glial fibers, to specific target areas where they integrate into existing neural networks. Approximately half of the new cells survive for the rest of the fish’s life, whereas the other half are eliminated through apoptotic cell death. A potential mechanism regulating development of the new cells is provided by somatic genomic alterations. The generation of new cells, together with elimination of damaged cells through apoptosis, also enables teleost fish rapid and efficient neuronal regeneration after brain injuries. Proteome analysis has identified a number of proteins potentially involved in the individual regenerative processes. Comparative analysis has suggested that differences between teleosts and mammals in the growth of muscles and sensory organs are key to explain the differences in adult neurogenesis that evolved during phylogenetic development of the two taxa.     

Programmed cell death in Xenopus laevis spinal cord, tail and other tissues, prior to, and during, metamorphosis

Programmed cell death is necessary for the shaping and remodelling of nervous and non-nervous tissues during development. Amphibia, whose body undergoes profound modifications during metamorphosis, are particularly useful models for studying the relationship between cell death in muscles and other non-nervous tissues on the one hand, and in the nervous system connected with these tissues on the other hand. We checked the occurrence of apoptotic cells (identified by TUNEL labelling) in different organs and regions from hatching (stages 35-36) to climax (stages 63-64) in the African Clawed Frog Xenopus laevis. Some organs (e.g., skin and digestive tract) contained apoptotic cells during the entire period studied. In transitory organs (cement gland and gills), a single wave of cell death occurred during the regression of these tissues. In order to compare the timing of cell death in the spinal cord with that of tail regression, we counted the number of TUNEL-positive cells in spinal cord sections taken from animals between stages 54 and 64. Three-dimensional reconstructions using confocal microscopy of vibratome slices immunostained for the detection of c-Jun-like protein accumulated in the cytoplasm of apoptotic cells showed numerous cells at various degrees of degeneration. Many of these cells still presented the morphological characteristics of neurones. The peak of apoptosis was found at stage 58, preceding tail regression. This suggests that neural cell death is not a consequence but rather an element upstream in the chain of events leading to tail degeneration.     

Mechanisms involved in tissue-specific apopotosis regulated by glucocorticoids

Physiological cell turnover is under the control of a sharp and dynamic balance of different homeostatic mechanisms such as the equilibrium between cell proliferation and cell death. These mechanisms play an important role in maintaining normal tissue function and architecture. It is well known that apoptosis is the prevalent mode of physiological cell loss in most tissues. Steroid hormones like glucocorticoids have been identified as key signals controlling cell turnover by modulating programmed cell death in a tissue- and cell-specific manner. In this sense, several reports have demonstrated that glucocorticoids are able to induce apoptosis in cells of the hematopoietic system such as monocytes, macrophages, and T lymphocytes. In contrast, they protect against apoptotic signals evoked by cytokines, cAMP, tumor suppressors, in glandular cells such as the mammary gland epithelia, endometrium, hepatocytes, ovarian follicular cells, and fibroblasts. Although several studies have provided significant information on hormone-dependent apoptosis in an specific tissue, a clearly defined pathway that mediates cell death in response to glucocorticoids in different cell types is still misunderstood. The scope of this review is held to those mechanisms by which glucocorticoids control apoptosis, emphasizing tissue-specific expression of genes that are involved in the apoptotic pathway.     

The morphology of apoptosis

The concept of apoptotic cell death as an essential part of the development and life of complex organisms has been devised in different situations and tested from various angles. This review article discusses the morphological changes during death by apoptosis. In cells undergoing apoptosis, an intracellular signalling pathway operates cell autonomously to implement the death and disposal of the cell. The similarity of the biochemical events during apoptosis in different situations is reflected by a high uniformity of morphological changes in many situations of naturally occurring or experimentally induced cell death. The unifying concept of apoptosis has been derived from the observation of this morphological consistency of dying cells almost 30 years ago. Since then, we have learned much about the intracellular signalling in the apoptotic process and the molecular background has been delineated which guides the initiation of the morphological changes. Here, an attempt is made to present the current knowledge about the molecular events in the development of these morphological alterations and to place these changes in the context of apoptotic cell death.     

Cardiotoxicity of calmidazolium chloride is attributed to calcium aggravation, oxidative and nitrosative stress, and apoptosis

The intracellular calcium concentration ([Ca]_i) regulates cell viability and contractility in myocardial cells. Elevation of the [Ca]_i level occurs by entry of calcium ions (Ca²⁺) through voltage-dependent Ca²⁺ channels in the plasma membrane and release of Ca²⁺ from the sarcoplasmic reticulum. Calmidazolium chloride (CMZ), a subgroup II calmodulin antagonist, blocks L-type calcium channels as well as voltage-dependent Na⁺ and K⁺ channel currents. This study elaborates on the events that contribute to the cytotoxic effects of CMZ on the heart. We hypothesized that apoptotic cell death occurs in the cardiac cells through calcium accumulation, production of reactive oxygen species, and the cytochrome c-mediated PARP activation pathway. CMZ significantly increased the production of superoxide (O₂^•–) and nitric oxide (NO) as detected by FACS and confocal microscopy. CMZ induced mitochondrial damage by increasing the levels of intracellular calcium, lowering the mitochondrial membrane potential, and thereby inducing cytochrome c release. Apoptotic cell death was observed in H9c2 cells exposed to 25 μM CMZ for 24 h. This is the first report that elaborates on the mechanism of CMZ-induced cardiotoxicity. CMZ causes apoptosis by decreasing mitochondrial activity and contractility indices and increasing oxidative and nitrosative stress, ultimately leading to cell death via an intrinsic apoptotic pathway.     

Cyclosporine A Induces Apoptotic and Autophagic Cell Death in Rat Pituitary GH3 Cells

Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the development of chronic nephrotoxicity. In this study, we investigated CsA treatment induced apoptotic and autophagic cell death in pituitary GH3 cells. CsA treatment (0.1 to 10 µM) decreased survival of GH3 cells in a dose-dependent manner. Cell viability decreased significantly with increasing CsA concentrations largely due to an increase in apoptosis, while cell death rates due to autophagy altered only slightly. Several molecular and morphological features correlated with cell death through these distinct pathways. At concentrations ranging from 1.0 to 10 µM, CsA induced a dose-dependent increase in expression of the autophagy markers LC3-I and LC3-II. Immunofluorescence staining revealed markedly increased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating increases in autophagosomes. At the same CsA doses, apoptotic cell death was apparent as indicated by nuclear and DNA fragmentation and increased p53 expression. In apoptotic or autophagic cells, p-ERK levels were highest at 1.0 µM CsA compared to control or other doses. In contrast, Bax levels in both types of cell death were increased in a dose-dependent manner, while Bcl-2 levels showed dose-dependent augmentation in autophagy and were decreased in apoptosis. Manganese superoxide dismutase (Mn-SOD) showed a similar dose-dependent reduction in cells undergoing apoptosis, while levels of the intracellular calcium ion exchange maker calbindin-D9k were decreased in apoptosis (1.0 to 5 µM CsA), but unchanged in autophagy. In conclusion, these results suggest that CsA induction of apoptotic or autophagic cell death in rat pituitary GH3 cells depends on the relative expression of factors and correlates with Bcl-2 and Mn-SOD levels.     

Apoptotic Cell Death Induced by Resveratrol Is Partially Mediated by the Autophagy Pathway in Human Ovarian Cancer Cells

Resveratrol (trans-3,4,5’ –trihydroxystilbene) is an active compound in food, such as red grapes, peanuts, and berries. Resveratrol exhibits an anticancer effect on various human cancer cells. However, the mechanism of resveratrol-induced anti-cancer effect at the molecular level remains to be elucidated. In this study, the mechanism underlying the anti-cancer effect of resveratrol in human ovarian cancer cells (OVCAR-3 and Caov-3) was investigated using various molecular biology techniques, such as flow cytometry, western blotting, and RNA interference, with a major focus on the potential role of autophagy in resveratrol-induced apoptotic cell death. We demonstrated that resveratrol induced reactive oxygen species (ROS) generation, which triggers autophagy and subsequent apoptotic cell death. Resveratrol induced ATG5 expression and promoted LC3 cleavage. The apoptotic cell death induced by resveratrol was attenuated by both pharmacological and genetic inhibition of autophagy. The autophagy inhibitor chloroquine, which functions at the late stage of autophagy, significantly reduced resveratrol-induced cell death and caspase 3 activity in human ovarian cancer cells. We also demonstrated that targeting ATG5 by siRNA also suppressed resveratrol-induced apoptotic cell death. Thus, we concluded that a common pathway between autophagy and apoptosis exists in resveratrol-induced cell death in OVCAR-3 human ovarian cancer cells.     

Fanconi Anemia C Protein Acts at a Switch between Apoptosis and Necrosis in Mitomycin C-Induced Cell Death

Deregulation of apoptosis seems to be a hallmark of the Fanconi anemia (FA) syndrome. In order to further define the role of the FA protein from complementation group C (FAC) in apoptosis, we characterized parameters modified during the mitomycin-C (MMC)-induced apoptotic program. It is shown that despite a higher level of cell death for FA compared to normal lymphoblasts after MMC treatment, FA cells do not display a marked DNA fragmentation. Furthermore, while playing a central role in MMC apoptosis of normal lymphoblasts, the activity of caspase-3-like proteases is altered in FA cells. Interestingly, the disruption of the mitochondrial transmembrane potential (Δψ), an early event that can lead to apoptotic or to necrotic death, is accomplished similarly in FA and in normal cells. Finally, it is shown that the overexpressed FAC protein inhibited the apoptotic steps, with the exception of the decrease of the Δψ. Altogether, our results indicate that the FAC protein acts at a step preceding the activation of the caspases and after the modification of the Δψ, a decision point at which cells can be pushed toward either apoptosis or necrosis and which, consequently, regulates the balance between the two modes of cell death.     

Two Modes of Cell Death Caused by Exposure to Nanosecond Pulsed Electric Field

High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity (“nanoelectroporation”), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1–2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6–24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling.     

Regulation of apoptosis in S49 cells

Apoptosis, or programmed cell death, is a highly regulated physiological process by which individual cells die and are removed from a given population. This process, defined by both morphological and biochemical characteristics, has been extensively studied in the glucocorticoid-induced immature thymocyte model. In the present study we explore the effects of glucocorticoids on variants of the S49.1 thymocyte without (S49-NEO) or with (S49-bcl-2) the bcl-2 proto-oncogene. In S49-NEO cells dexamethasone induced a time- and dose-dependent loss of viability and increase in DNA internucleosomal fragmentation (a biochemical hallmark of apoptosis). Glucocorticoid treatment was also associated with an apoptotic morphology (cell shrinkage, chromatin condensation) and the effects of this steroid could be reversed by the glucocorticoid antagonist RU486. In contrast, S49-bcl-2 cells showed no change in viability, DNA fragmentation or apoptotic morphology. Interestingly, the apoptotic effects of glucocorticoid in S49-NEO cells were mimicked by the translation inhibitor cycloheximide and the zinc chelator 1,10-phenanthroline, suggesting that zinc and translational events are necessary to maintain the nonapoptotic state. Finally, nuclease activity was extracted from glucocorticoid-treated S49-NEO cells but not control cells. Together the results further define the effects of glucocorticoids on these cells and provide insight into the mechanisms controlling apoptosis.