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The heightened interest in the development of novel anti-cancer drugs that trigger apoptotic death in cancer cells stems from the fact that immediately upon execution of the death signal, the corpse is efficiently removed via specific recruitment of phagocytic cells. This prevents spilling of cellular contents and the associated inflammatory response, a likely scenario during necrotic death. Recent evidence has established that phagocytic removal of apoptotic cells is a function of ligand-receptor interaction, whereby the ligand(s) for the scavenger receptor(s) of phagocytic cells is/are specifically expressed on apoptosing cells. Therefore, by implication, enhancing this ligand-receptor interaction could be an alternate means for removing unwanted cells. Here we present a provocative hypothesis that circumvents the need for chemotherapy-induced apoptosis in cancer cells. According to our model, cancer cells need not die in order to be removed by scavenger cells, but could still be effectively phagocytosed provided the cell surface expression of specific molecules that strongly engage phagocytic cells is sufficiently enhanced. In other words, inducing the expression of "eat me" signals on cancer cells could be a novel approach to "bury alive" these unwanted cells without the untoward effects of chemotherapy-induced apoptosis.

Apoptosis, a physiological process of self-annihilation, is essential during development and for the maintenance of tissue homeostasis. Considerable efforts have been made in recent years to elucidate the molecular mechanisms that govern this mode of cellular demise; however, the subsequent recognition and removal of apoptotic corpses by neighboring phagocytes has received less attention. Nevertheless, macrophage engulfment of apoptotic cells is known to be important in the remodeling of tissues, and contributes to the resolution of inflammation through the removal of effete cells prior to the release of noxious cellular constituents. Moreover, apoptotic cells are a potential source of self-antigens, and clearance of cell corpses is thought to preclude the induction of autoimmune responses. The view is thus emerging that tissue homeostasis is dependent not only on the balance between mitosis and apoptosis, but also on the rate of apoptosis versus that of cell clearance. This review aims to discuss the mechanisms and consequences of macrophage recognition and disposal of apoptotic cells, a process which will be referred to as programmed cell clearance.

Programmed cell death (apoptosis) functions as a mechanism to eliminate unwanted or irreparably damaged cells ultimately leading to their orderly phagocytosis in the absence of calamitous inflammatory responses. Recent studies have demonstrated that the generation of free radical intermediates and subsequent oxidative stress are implicated as part of the apoptotic execution process. Oxidative stress may simply be an unavoidable yet trivial byproduct of the apoptotic machinery; alternatively, intermediates or products of oxidative stress may act as essential signals for the execution of the apoptotic program. This review is focused on the specific role of oxidative stress in apoptotic signaling, which is realized via phosphatidylserine-dependent pathways leading to recognition of apoptotic cells and their effective clearance. In particular, the mechanisms involved in selective phosphatidylserine oxidation in the plasma membrane during apoptosis and its association with disturbances of phospholipid asymmetry leading to phosphatidylserine externalization and recognition by macrophage receptors are at the center of our discussion. The putative importance of this oxidative phosphatidylserine signaling in lung physiology and disease are also discussed.

Previous studies have demonstrated that Fas-triggered activation of effector caspases and subsequent nuclear apoptosis either is mitochondria-independent (type I cells) or relies on mitochondrial amplification of the initial stimulus (type II cells). We show herein that Bcl-2 overexpression in a prototypic type I cell line (SKW6.4) promotes mitochondrial generation of ATP and blocks Fas-triggered plasma membrane externalization of phosphatidylserine (PS). Moreover, overexpression of Bcl-2 attenuates macrophage engulfment of Fas-triggered cells. Fas-mediated DNA fragmentation, on the other hand, remains unaffected in SKW6.4-bcl-2 cells. These studies thus demonstrate that PS externalization and clearance of cell corpses are mitochondria-dependent events, and show that these events can be dissociated from other features of the apoptotic program, in Fas type I cells.

Low-risk myelodysplastic syndromes (MDS), including refractory anemia and sideroblastic anemia, are characterized by increased apoptotic death of erythroid progenitors. The signaling pathways that elicit this pathologic cell death in MDS have, however, remained unclear. Treatment with erythropoietin in combination with granulocyte colony-stimulating factor (G-CSF) may synergistically improve the anemia in patients with MDS, with a concomitant decrease in the number of apoptotic bone marrow precursors. Moreover, we have previously reported that G-CSF inhibits Fas-induced caspase activation in sideroblastic anemia (RARS). The present data demonstrate that almost 50% of erythroid progenitor cells derived from patients with MDS exhibit spontaneous release of cytochrome c from mitochondria with ensuing activation of caspase-9, whereas normal erythroid progenitors display neither of these features. G-CSF significantly inhibited cytochrome c release and suppressed apoptosis, most noticeably in cells from patients with sideroblastic anemia. Furthermore, inhibition of caspase-9 suppressed both spontaneous and Fas-mediated apoptosis of erythroid progenitors in all low-risk MDS cases studied. We propose that the increased sensitivity of MDS progenitor cells to death receptor stimulation is due to a constitutive activation of the mitochondrial axis of the apoptotic signaling pathway in these cells. These studies yield a mechanistic explanation for the beneficial clinical effects of growth factor administration in patients with MDS, and provide a model for the study of growth factor-mediated suppression of apoptosis in other bone marrow disorders.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disorder of immune regulation. Mutations in the gene encoding perforin were previously identified in a subset of FHL patients. The present analysis of two novel candidate genes, granzyme B and granulysin, by direct sequencing in a total of 16 FHL families, disclosed several sequence variations. However, none of these sequence variations were associated with the manifestations of FHL. These data do not support the notion that granulysin and granzyme B are candidate genes for FHL.

Inflammation is a beneficial host response to foreign challenge involving numerous soluble factors and cell types, including polymorphonuclear granulocytes or neutrophils. Programmed cell death (apoptosis) of neutrophils has been documented in vitro as well as in vivo, and is thought to be important for the resolution of inflammation, as this process allows for engulfment and removal of senescent cells prior to their necrotic disintegration. Studies in recent years have begun to unravel the mechanism of macrophage clearance of apoptotic cells, and evidence has accrued for a critical role of externalization and oxidation of plasma membrane phosphatidylserine, and its subsequent recognition by macrophage receptors, in this process. Activated neutrophils generate vast amounts of reactive oxygen species for the purpose of killing ingested micro-organisms, and these reactive metabolites may also modulate the life-span, as well as the clearance, of the neutrophil itself. This review aims to address the latter topic, as well as to summarize current knowledge on the molecular mechanisms of neutrophil apoptosis and macrophage clearance of these cells at the site of inflammation.

Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Furthermore, phorbol myristate acetate stimulation of the NADPH oxidase caused recognition and ingestion of dimethylsulfoxide-differentiated HL-60 cells by J774A.1 macrophages. To reveal the apoptosis-related component of oxidative stress in the phorbol myristate acetate-induced response, we pretreated cells with a pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), and found that it caused partial inhibition of hydrogen peroxide formation as well as selective protection of only phosphatidylserine, whereas more abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, were oxidized to the same extent in the absence or presence of z-VAD-fmk. In contrast, inhibitors of NADPH oxidase activity, diphenylene iodonium and staurosporine, as well as antioxidant enzymes, superoxide dismutase/catalase, completely protected all phospholipids against peroxidation, inhibited expression of apoptotic biomarkers and externalization of phosphatidylserine, and reduced phagocytosis of differentiated HL-60 cells by J774A.1 macrophages. Similarly, zymosan-induced activation of the NADPH oxidase resulted in the production of superoxide and oxidation of different classes of phospholipids of which only phosphatidylserine was protected by z-VAD-fmk. Accordingly, zymosan caused apoptosis in differentiated HL-60 cells, as evidenced by caspase-3 activation and phosphatidylserine externalization. Finally, zymosan triggered caspase-3 activation and extensive SOD/catalase-inhibitable phosphatidylserine exposure in human neutrophils. Overall, our results indicate that NADPH oxidase-induced oxidative stress in neutrophil-like cells triggers apoptosis and subsequent recognition and removal of these cells through pathways dependent on oxidation and externalization of phosphatidylserine.

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is required for viral transformation and has been shown to protect lymphocytes from apoptosis. However, the effect of LMP1 on cells of epithelial origin remains poorly understood. Using the epithelial cell line HeLa in which the expression of LMP1 is inducibly regulated by tetracycline, we demonstrate that apoptosis triggered by ligation of the death receptor, Fas, or by the chemotherapeutic agent, etoposide, is potentiated by LMP1. Apoptosis was assessed by nuclear condensation and activation of caspase-3-like enzymes with concomitant proteolysis of the nuclear caspase substrate, poly(ADP-ribose) polymerase. However, the effect of LMP1 in HeLa cells appeared to be stimulus-dependent since apoptosis induced by tumor necrosis factor (TNF) was inhibited. Moreover, we observed an upregulation of the zinc finger protein A20 and a decrease in expression of Bcl-2 upon induction of LMP1 in HeLa cells. Taken together, these data further our understanding of the function of LMP1 in epithelial cells and suggest that LMP1, similar to its mammalian homolog CD40, can exert opposing effects on cell survival depending on the nature of the apoptosis trigger., (Copyright 2002 Elsevier Science (USA))

Exposure of phosphatidylserine (PS) on the surface of apoptotic cells has been suggested to serve as an important recognition signal for macrophages. In this work we show that triggering of the death receptor Fas on Jurkat cells results in the generation of reactive oxygen species with oxidation and externalization of PS but not of the other major aminophospholipid, phosphatidylethanolamine. These cells were readily ingested by several classes of macrophages, whereas Raji cells, which are defective for Fas-induced PS exposure, remained unengulfed. However, when Raji cells were incubated with the thiol-reactive agent N-ethylmaleimide to induce PS exposure in the absence of other features of apoptosis, these cells were also engulfed by macrophages. Phagocytosis of Fas-triggered Jurkat cells was inhibited by superoxide dismutase and catalase, which prevent oxidation of PS while allowing PS to remain externalized on these cells. Moreover, liposomes containing oxidized PS (PS-OX) were more potent inhibitors of phagocytosis than those containing its nonoxidized counterpart. Finally, enrichment of the plasma membrane of Jurkat or Raji cells, or myeloid leukemic HL-60 cells, with exogenous PS resulted in phagocytic cell clearance, and this process was further enhanced when PS was substituted for by PS-OX. Taken together, our data suggest that the presence of PS-OX in conjunction with nonoxidized PS on the cell surface is an important signal for macrophage clearance of apoptotic cells.

Recognition signals are displayed on the cell surface during apoptosis that enable macrophages to engulf and dispose of the dying cell. A common signal is the externalization of phosphatidylserine (PS). Studies in erythrocytes and platelets have suggested that PS exposure requires the concomitant activation of a phospholipid scramblase (PLS) and inhibition of an adenosine triphosphate (ATP)-dependent aminophospholipid translocase. However, the molecular mechanism underlying PS exposure during apoptosis remains poorly understood. In this study, we provide evidence that expression of PLS is neither necessary nor sufficient for PS exposure during Fas-triggered apoptosis. On the other hand, egress of PS is shown to correlate with a decline in intracellular ATP and inhibition of aminophospholipid translocase activity upon Fas stimulation. Moreover, suppression of intracellular ATP levels by the glucose anti-metabolite, 2-deoxyglucose, alone or in combination with glucose-free medium, potentiates Fas-induced PS exposure in the PLS-expressing Jurkat cell line and enables PLS-defective Raji cells to externalize PS in response to Fas ligation. These studies suggest that intracellular ATP levels can modulate the externalization of PS during apoptosis, and implicate the ATP-dependent aminophospholipid translocase in this process.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare and fatal disease of early childhood characterized by a non-malignant accumulation of activated T lymphocytes and histiocytes in the reticuloendothelial system. Moreover, immune system derangement, with prominent hypercytokinemia and low or absent cytotoxic T and natural killer (NK) cell activity, is a consistent feature of this autosomal recessive disorder. Recent work has demonstrated that the degree of spontaneous caspase activation in FHL lymphocytes is attenuated in vitro whereas Fas-mediated caspase activation and apoptosis induction remains unmitigated, and FHL can thus be distinguished from the related chronic disorder of immune regulation termed autoimmune lymphoproliferative syndrome or ALPS. However, subsequent studies have identified mutations in the gene encoding perforin, a cytotoxic granule constituent required for apoptotic killing of target cells, in a number of FHL patients. Hence, the underlying defect in FHL may be conceived of as a lack of apoptosis triggering within the immune system, rather than apoptosis resistance per se. These observations represent an important step in our understanding of the pathogenesis of FHL and also serve to emphasize the pivotal role of cellular (perforin-based) cytotoxicity in the regulation of immune homeostasis.

Treatment with granulocyte colony-stimulating factor (G-CSF) plus erythropoietin may synergistically improve hemoglobin levels and reduce bone marrow apoptosis in patients with refractory anemia with ringed sideroblasts (RARS). Fas-induced caspase activity is increased in RARS bone marrow cells. We showed that G-CSF significantly reduced Fas-mediated caspase-8 and caspase-3-like activity and the degree of nuclear apoptotic changes in bone marrow from nine RARS patients. A decrease in mitochondrial membrane potential and an increase in intracellular reactive oxygen species occurred in Fas-treated cells, but became significant only 24 h after changes in caspase activity and decrease in proliferation. G-CSF also reduced the magnitude of these late apoptotic changes. In CD34-selected normal cells, G-CSF induced myeloid colony growth, and an overall small decrease in the number of erythroid colonies. By contrast, G-CSF induced a 33-263% increase of erythroid colony formation in CD34+ cells from four of five RARS patients with severely reduced erythroid growth, while the normal or slightly reduced erythroid growth of three other patients was not influenced by G-CSF. This study suggests that G-CSF may reduce the pathologically increased caspase activity and concomitant apoptotic changes, and promote erythroid growth and differentiation of stem cells from RARS patients. Our data support the clinical benefit of G-CSF in this subgroup of myelodysplastic syndromes.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disease of early childhood characterized by nonmalignant accumulation and multivisceral infiltration of activated T lymphocytes and histiocytes (macrophages). Cytotoxic T and natural killer (NK) cell activity is markedly reduced or absent in these patients, and mutations in a lytic granule constituent, perforin, were recently identified in a number of FHL individuals. Here, we report a comprehensive survey of 34 additional patients with FHL for mutations in the coding region of the perforin gene and the relative frequency of perforin mutations in FHL. Perforin mutations were identified in 7 of the 34 families investigated. Six children were homozygous for the mutations, and one patient was a compound heterozygote. Four novel mutations were detected: one nonsense, two missense, and one deletion of one amino acid. In four families, a previously reported mutation at codon 374, causing a premature stop codon, was identified, and, therefore, this is the most common perforin mutation identified so far in FHL patients. We found perforin mutations in 20% of all FHL patients investigated (7/34), with a somewhat higher prevalence, approximately 30% (6/20), in children whose parents originated from Turkey. No other correlation between the type of mutation and the phenotype of the patients was evident from the present study. Our combined results from mutational analysis of 34 families and linkage analysis of a subset of consanguineous families indicate that perforin mutations account for 20%-40% of the FHL cases and the FHL 1 locus on chromosome 9 for approximately 10%, whereas the major part of the FHL cases are caused by mutations in not-yet-identified genes.

Treatment with granulocyte colony-stimulating factor plus erythropoietin may improve haemoglobin levels in patients with ringsideroblastic anaemia (RARS) and reduce bone marrow apoptosis. We studied bone marrow from 10 RARS patients, two of whom were also investigated after successful treatment. Mononuclear, erythroid and CD34+ cells were analysed with regard to proliferation, apoptosis, clonogenic capacity and oncoprotein expression, in the presence or absence of Fas-agonist, Fas-blocking antibody 2 and caspase-3 inhibitor. During culture, RARS bone marrow cells showed higher spontaneous apoptosis (P < 0.05) and caspase activity (P < 0.05)) than bone marrow cells from healthy donors. Eight out of nine patients had reduced growth of erythroid colony-forming units (CFU-E) (< 10% of control) and granulocyte-macrophage CFU (CFU-GM) (< 50% of control) from CD34+ cells. Fas ligation increased apoptosis and decreased colony growth equally in RARS and controls, but caused significantly more caspase activation in RARS (P < 0.01). Fas-blocking antibody showed no significant inhibitory effect on spontaneous apoptosis or ineffective haematopoiesis, as measured using phosphatidylserine exposure, the terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling technique, caspase activity or clonogenic growth. Caspase inhibition reduced apoptosis, increased proliferation and enhanced erythroid colony growth from CD34+ cells in RARS, but showed no effect on normal cells. CFU-E improved > 1000% after successful treatment. Thus, erythroid apoptosis in RARS is initiated at the CD34+ level and growth factor treatment may improve stem cell function. Enhanced caspase activation at the stem cell level, albeit not mediated through endogenous activation of the Fas receptor, contributes to the erythroid apoptosis in RARS.

The caspases, first discovered almost a decade ago, are intracellular cysteine proteases which have been shown to play an essential role in the initiation and execution phases of apoptotic cell death. Numerous strategies for the activation and inhibition of these 'killer' proteases have evolved, including the regulation of caspase expression and function at the transcriptional and post-translational level, as well as the expression of viral and cellular inhibitors of caspases. Emerging evidence in recent years has also implicated the caspases in various, nonapoptotic aspects of cellular physiology, such as cytokine processing during inflammation, differentiation of progenitor cells during erythropoiesis and lens fiber development, and proliferation of T lymphocytes, thus attesting to the pleiotropic functions of these proteases. The present review aims to discuss the multiple roles of the mammalian caspases with particular emphasis on their activation and regulation in cells of leukemic origin and the attendant possibilities of therapeutic intervention.

Apoptosis or programmed cell death is essential for the maintenance of tissue homeostasis. Recent studies have implicated the dysregulation of apoptosis in a plethora of human diseases. Hence, perturbation of the death program intrinsic to every cell may result in essentially too little or too much apoptosis, which may in turn lead to proliferative or degenerative diseases respectively. In the present review, we discuss our current molecular understanding of the apoptotic process and its possible role in human disease, with particular emphasis on the rare and invariably fatal disease of early childhood, termed familial hemophagocytic lymphohistiocytosis, in which mutations in the perforin gene were recently identified.

Familial hemophagocytic lymphohistiocytosis (FHL) is an invariably fatal disease typically seen in infancy and early childhood, with a median survival without therapy of two months. It is characterized by prolonged fever, hepatosplenomegaly, cytopenia, and deficient NK-cell activity and T-cell cytotoxic capacity. Severe neurological symptoms as well as coagulation disorders and abnormalities in liver function and lipid status may also develop. Since the mid 1980's there has been a remarkable increase in our understanding of this disease. In a large-scale international collaborative effort mediated through the Histiocyte Society, diagnostic criteria and an international treatment protocol (HLH-94) based on immunochemotherapy and BMT have been developed. A large proportion of affected children can now be cured and, moreover, successful chemotherapy in utero of FHL has been achieved. It has been shown that the symptoms and signs are mediated through a pronounced hypercytokinemia. Previous suggestions that FHL may be caused by a deficiency in apoptosis were recently confirmed when perforin gene defects were described, which may well explain the disastrous lymphohistiocytic accumulation and subsequent T-cell activation.

Naturally occurring cell death or apoptosis is essential for the maintenance of tissue homeostasis and serves to remove extraneous or dangerous cells in a swift and unobtrusive manner. Recent studies have indicated a role for apoptosis in a plethora of human diseases. Hence, dysregulation of apoptosis has been implicated in autoimmune disease, acquired immune deficiency syndrome, and other viral (and bacterial) infections, as well as in neurodegenerative disorders and cancer. Furthermore, dysregulated apoptosis signaling may impinge on other age-related disorders such as osteoporosis and atherosclerosis and perhaps on the process of aging itself. The present review provides an overview of human diseases, which are associated with defective or inadvertent apoptosis, with examples of pathological conditions in which putative apoptosis defects have been elucidated at the molecular level. Novel apoptosis-modulating therapeutic strategies are also discussed., (Copyright 1999 Academic Press.)

Phosphatidylserine (PS) exposure on the surface of cells has been considered a characteristic feature of apoptosis. However, we demonstrate herein that externalization of PS occurs in a cell-type-specific, albeit caspase-dependent, manner. Moreover, we could find no correlation in six different cell lines between the level of expression of the phospholipid (PL) scramblase and the capacity of these cells to externalize PS during apoptosis. Overexpression of PL scramblase in Raji cells, which exhibit low constitutive expression of this enzyme, by retroviral transduction of PL scramblase or treatment of the cells with interferon-alpha, failed to confer the capacity to expose PS in response to apoptotic stimuli. However, the lack of PS exposure in some cell types was not due to their inability to translocate PS molecules to the cell surface, since incubation with thiol reactive agents, such as N-ethylmaleimide, disulfiram and diamide, yielded rapid and pronounced PS exposure in all cell lines. These data suggest that plasma membrane PS exposure is not an obligatory component of the apoptotic phenotype, and that PL scramblase is not the sole determinant of PS externalization in apoptotic cells when this occurs., (Copyright 1999 Academic Press.)

Members of the expanding family of Bcl-2-like proteins have emerged as important regulators of programmed cell death, and recent studies have unearthed numerous mechanisms for regulating the function of these death agonists and antagonists. In addition to the transcriptional control of gene expression, these mechanisms include posttranslational events such as phosphorylation, proteolysis, and the induction of conformational changes, which may either activate or inactivate these molecules. Interaction with homologous and nonhomologous proteins and specific subcellular targeting of Bcl-2-like proteins are other means of fine-tuning the cellular response to noxious stimuli. Recently, considerable attention has turned to the regulation of so-called BH3-only molecules, which appear to act as stress sensors that relay signals to other pro- or antiapoptotic family members. We discuss how the regulation of these apoptosis regulators may control the ultimate fate of the cell.

Familial haemophagocytic lymphohistiocytosis (FHL) is a rare and uniformly fatal disorder of early childhood characterized by fever, hepatosplenomegaly, cytopenia and widespread infiltration of vital organs by activated lymphocytes and macrophages. In order to test whether the massive accumulation of immune cells in these patients is associated with a perturbation of apoptosis, lymphocytes were isolated from eight patients and subjected to the chemotherapeutic agent etoposide or agonistic anti-Fas monoclonal antibodies in vitro. These stimuli elicited a normal apoptotic response in FHL patient cells when compared to healthy controls, as determined by phosphatidylserine exposure, DNA fragmentation, in vitro cleavage of the caspase-3-like substrate aspartate-glutamate-valine-aspartate-7-amino-4-methyl-coumarin (DEVD-AMC) and proteolysis of the anti-apoptotic protein Bcl-2. In addition, the degree of constitutive and Fas-triggered apoptosis in freshly isolated neutrophils was monitored in three children, with similar results. These studies indicate that immune cells derived from FHL patients are not inherently resistant to apoptosis induction. Specifically, etoposide-induced and Fas-triggered activation of intracellular caspases appears to remain intact in these individuals. However, the degree of spontaneous activation of caspase-3-like enzymes in activated lymphocytes was attenuated in three of the four patients tested prior to initiation of therapy, suggesting a possible biological deficiency in these individuals.

P39/Tsugane is a myelomonocytoid cell line derived from a patient with myelodysplastic syndrome (MDS). The cells readily undergo apoptosis in response to various agents, and the cell line has been suggested as a useful model to study apoptosis in MDS. The aims of the present study were to assess differentiation and apoptosis induced with all-trans retinoic acid (ATRA) and etoposide, to characterize the mode of apoptosis in these two model systems, and to assess the influence of granulocyte colony-stimulating factor (G-CSF), which in combination with erythropoietin has been shown to inhibit apoptosis in MDS. ATRA induced differentiation and apoptosis in a concentration- and time-dependent manner. Differentiated cells were partially rescued (by 50%) from apoptosis with G-CSF. Etoposide induced apoptosis in a concentration- and time-dependent manner, but no signs of preceding maturation or G-CSF rescue were detected. ATRA- and etoposide-induced apoptosis were both mediated through the caspase pathway and were partially blocked with the general caspase inhibitor zVAD-fmk. Simultaneous treatment with G-CSF and zVAD-fmk additively blocked ATRA-induced apoptosis. However, the two pathways differed in terms of substrate cleavage during apoptosis. ATRA-induced apoptosis caused actin cleavage, which was not affected by G-CSF, and Bcl-2 downregulation. Etoposide induced a caspase-dependent cleavage of Bcl-2, while actin remained intact. The Fas system did not seem to play a major role in any of these apoptotic pathways. Our results may provide new tools to study the mechanisms of apoptosis in MDS.

The proto-oncogene product Bcl-2 protects a wide variety of cell types from apoptosis via a hitherto unknown mechanism. Bcl-2 has been shown to function upstream of the death proteases (caspases) in some, but not all, occurrences of apoptotic cell death. Using the myeloid leukemic cell line P39 we report the chemotherapy-induced caspase-dependent cleavage of endogenous Bcl-2. Etoposide treatment of these cells triggered a time-dependent activation of type II and type III caspases and cleavage of Bcl-2 yielding a 23 kDa cleavage fragment. The emergence of this cleavage product was blocked by the general caspase inhibitor zVAD-fmk, as well as the type III caspase inhibitor IETD-fmk and the caspase-9-selective inhibitor LEHD-fmk, while the type II caspase inhibitor DEVD-fmk proved considerably less efficient. Bcl-2 cleavage preceded cleavage of the known caspase-3 substrate, poly(ADP-ribose) polymerase (PARP), as well as that of the caspase-6 substrate, lamin B, indicating that Bcl-2 cleavage is a relatively early event in the apoptosis cascade in this experimental model. While evidence for cleavage of Bcl-2 in several subcellular compartments of etoposide-treated cells was obtained, this cleavage was detected predominantly in the mitochondrial fraction, thus providing further support for the central role of mitochondria in apoptosis. Caspase-mediated cleavage following etoposide treatment of these myeloid leukemic cells may represent a means for the attenuation of Bcl-2 function upon apoptosis induction.

Human neutrophils have a short half-life and are believed to die by apoptosis or programmed cell death both in vivo and in vitro. We found that caspases are activated in a time-dependent manner in neutrophils undergoing spontaneous apoptosis, concomitant with other characteristic features of apoptotic cell death such as morphologic changes, phosphatidylserine (PS) exposure, and DNA fragmentation. The treatment of neutrophils with agonistic anti-Fas monoclonal antibodies (MoAbs) significantly accelerated this process. However, in cells treated with the potent neutrophil activator phorbol 12-myristate 13-acetate (PMA), caspase activity was only evident after pharmacologic inhibition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Similarily, inhibition of the NADPH oxidase in constitutive and Fas/APO-1-triggered apoptosis resulted in increased rather than suppressed levels of caspase activity, suggesting that reactive oxygen species may prevent caspases from functioning optimally in these cells. Moreover, oxidants generated via the NADPH oxidase were essential for PS exposure during PMA-induced cell death, but not for neutrophils undergoing spontaneous apoptosis. We conclude that caspases are an important component of constitutive and Fas/APO-1-triggered neutrophil apoptosis. However, these redox sensitive enzymes are suppressed in activated neutrophils, and an alternate oxidant-dependent pathway is used to mediate PS exposure and neutrophil clearance under these conditions.

Apoptosis is now widely recognized as being a distinct process of importance both in normal physiology and pathology. In the current paradigm for apoptotic cell death, the activity of a family of proteases, caspases, related to interleukin-1 beta-converting enzyme (ICE) orchestrates the multiple downstream events, such as cell shrinkage, membrane blebbing, glutathione (GSH) efflux, and chromatin degradation that constitute apoptosis. Recent studies suggest that mitochondria could be the principle sensor and that the release of mitochondrial factors, such as cytochrome c, is the critical event governing the fate of the cell.--One of the most reproducible inducers of apoptosis is mild oxidative stress, although it is unclear how an oxidative stimulus can activate the caspase cascade. Oxidative modification of proteins and lipids has also been observed in cells undergoing apoptosis in response to nonoxidative stimuli, suggesting that intracellular oxidation may be a general feature of the effector phase of apoptosis. The caspases themselves are cysteine-dependent enzymes and, as such, appear to be redox sensitive. Indeed, our recent work on hydrogen peroxide-mediated apoptosis suggests that prolonged or excessive oxidative stress can actually prevent caspase activation. A physiological example of this is the NADPH oxidase-derived oxidants generated by stimulated neutrophils that prevent caspase activation in these cells. Pursuant to these findings, stimulated neutrophils appear to use a specialized caspase-independent pathway to initiate phosphatidylserine (PS) exposure and subsequent phagocytic clearance. The possible implications of these dual roles for reactive oxygen species in apoptosis, that is, induction and inhibition of caspases, are discussed in the present review.

Fas/APO-1 is a member of the tumor necrosis factor (TNF)/nerve growth factor receptor family. This cell surface protein, when associated with the Fas/APO-1 ligand or specific mAb, elicits an apoptotic response in susceptible cells via an oligomerization of its intracellular domains, termed the'death domains'. We have previously mapped the epitopes of a panel of Fas/APO-1-reactive mAb to a series of linear portions of the Fas/APO-1 molecule. In order to gain a greater understanding of the mode of interaction of these antibodies with the Fas/APO-1 antigen, we constructed a homology-based model of the extracellular portion of the molecule, based on the crystallographic coordinates of the TNF type I receptor. The model clearly demonstrates that the antibodies do not identically mimic the endogenous ligand to achieve their effect, but probably act in an analogous manner by recruiting Fas/APO-1 molecules into clusters which may lead to oligomerization of 'death domains'. Moreover, the apparent cross-reactivity observed for the monoclonal anti-Fas antibodies between different linear regions of the Fas/APO-1 molecule was found to be due, most likely, to the structural mimicry of these epitopes. Reduction of the Fas/APO-1-derived cross-reactive peptides by dithiothreitol completely abrogated their antigenic reactivity with the anti-Fas mAb CH-11, thus indicating that the establishment of intrapeptide disulfide bonds is critical for antigenic reactivity.

Objective: To study the apoptosis-inducing capacity of HIV-1 primary isolates in human peripheral blood mononuclear cells (PBMC) in relation to the viral biological phenotype., Design and Methods: Four HIV-1 primary isolates capable of replicating and inducing syncytia in the MT-2 cell line and two primary isolates lacking these properties were used to infect PBMC with the same infectious doses. The kinetics of virus production in the culture supernatants were followed in relation to apoptosis induction in PBMC as determined by intracellular labelling of apoptotic DNA strand breaks and flow cytometry analysis., Results: When low virus dose was used (0.001 m.o.i.), productive virus infection, with peak reverse transcriptase (RT) activity at days 5-7, was followed by high numbers of apoptotic cells at day 10 post infection. Tenfold higher inoculum dose (0.01 m.o.i.) resulted in enhanced virus production with peak RT activity at day 3 followed by high numbers of apoptotic cells at day 5 after infection. The apoptosis-inducing capacity of virus isolates was independent of their capacity to induce syncytia or replicate in the MT-2 cell line. However, upon cocultivation of infected PBMC with MT-2 cells, only virus with the MT-2 tropic phenotype initiated productive infection and induced apoptosis in MT-2 cells., Conclusions: These results show that apoptosis induction in PBMC by primary HIV-1 isolates is closely related to the kinetics of virus replication but is not influenced by other biological properties of the virus such as syncytium-inducing capacity and MT-2 tropism.

Fas/APO-1 is a cell surface glycoprotein that mediates programmed cell death or apoptosis when cross-linked with agonistic anti-Fas or anti-APO-1 mAb or the endogenous Fas/APO-1 ligand. In this report, we examined the in vitro biological properties of a panel of anti-human Fas mAb of IgG1 subclass (ZB4, VB3, WB3 and CBE). We found that anti-Fas clone VB3 induced marked apoptotic cell death in Fas/APO-1-expressing Jurkat cells, although this cell killing was delayed when compared to the cytolytic effect mediated by the prototypic anti-Fas antibody of IgM subclass (clone CH-11). The ZB4 antibody, on the other hand, efficiently blocked apoptosis induced by CH-11. The WB3 and CBE clones neither induced or inhibited apoptosis. These antibodies were all found to recognize one and the same linear site on the Fas/APO-1 molecule, despite their different biological effects. The ability of these anti-Fas mAb to induce or inhibit apoptosis appeared to correlate with their relative affinity for the Fas/APO-1 molecule. These results provide further evidence for the potential of anti-Fas antibodies of the IgG1 subclass to elicit signals via the Fas/APO-1 molecule.

Fas/APO-1 is a member of the nerve growth factor/tumor necrosis factor (TNF) family of receptors and has been shown to mediate apoptotic cell death upon binding of specific mAb. We report here that the prototypic anti-human Fas mAb (clone CH-11) induces apoptosis by binding to a linear epitope present on the extracellular domain of the Fas/APO-1 protein. Synthetic peptides corresponding to this epitope blocked the apoptotic effect of the antibody in a susceptible Jurkat T cell line. Based upon the similarity between the Fas/APO-1 protein and the recently crystallized soluble TNF receptor type I, we generated a molecular model of Fas/APO-1, Our computer modeling indicates that the antibody binding region forms a hairpin loop on the surface of the Fas/ APO-1 protein. These findings further our understanding of the Fas/APO-1-mediated apoptotic signal and may provide a useful tool in future investigations of programmed cell death.

The spread of HIV-1 to the nervous system occurs early during infection in a large number of asymptomatic virus carriers. In order to address the question whether the brain is targeted by a group of HIV-1 variants with increased neurotropic properties we have obtained 20 paired isolates from the blood and cerebrospinal fluid (CSF) of patients with varying severity of HIV-1 infection. We determined the nucleotide sequence of variable region 3 (V3) of the gp 120 envelope protein and studied the growth capacity of the virus isolates in primary monocyte/macrophage cultures. Blood and CSF V3 sequences could be grouped according to the host, whereas particular amino acid sequences related to tissue specificity were not identified. Moreover, the majority of HIV-1 isolates, independently of the tissue source, replicated in macrophage cultures. The isolates derived from the brain and blood compartments thus appeared genetically similar and could not be grouped according to their replicative capacities. The genetic distance between paired CSF and blood sequences was more pronounced in the group of patients with AIDS than in asymptomatic virus carriers. These observations indicate that the viruses present in the blood and CSF in the early stage of infection are genetically similar. Different mechanisms of adaptation or immunomodulation of the virus in CSF and blood may account for the increased intra-patient variability observed in the AIDS group.