Apoptosis and autoimmune disease

The process of programmed cell death or apoptosis was already noted in 1842 by Vogt [1], but it was not until the more recent studies of Kerr et al. 1972 [2] that an explosion of interest in apoptosis research occurred. Genetic, biochemical and cellular analysis in certain mammals, in the nematode Caenorhabditis elegans and in the fruitfly Drosophila melanogaster have identified several apoptosis regulating genes. This indicates that programmed cell death is an active, genetically controlled process. Many of the known cell death regulators are homologous in mammals, nematodes and insects, indicating that apoptosis is an evolutionarily conserved process. Apoptosis can be induced via multiple independent signalling pathways which converge upon a common final effector machinery. This stimulates activation of latent cysteine proteases (caspases), which cleave vital cellular substrates and thereby lead to the death of cells. The regulatory pathways of apoptosis are becoming clear with the discovery of specific signalling molecules. It has become evident that many disease processes including autoimmunity and cancer can be caused by deregulation of the apoptotic process. With the discovery of novel cell surface-bound death receptors, their ligands and further insight into the apoptotic machinery within the cell, research may ultimately lead to the design of therapies that allow intervention in the apoptotic process. The aims of such strategies would be to turn on apoptosis in neoplastic cells or in lymphocytes that are causing autoimmune disease or to prevent cell death in degenerative disorders. This review describes current understanding of the molecular regulation of apoptosis, and focuses on issues relating to possible roles of defective cell death control in autoimmunity.     

Apoptosis, cancer and cancer therapy

Apoptosis is a specific process that leads to programmed cell death through the activation of an evolutionary conserved intracellular pathway leading to pathognomic cellular changes distinct from cellular necrosis. Apoptosis is essential in the homeostasis of normal tissues of the body, especially those of the gastrointestinal tract, immune system and skin. There is increasing evidence that the processes of neoplastic transformation, progression and metastasis involve alterations in the normal apoptotic pathways. Furthermore, the majority of chemotherapeutic agents as well as radiation utilize the apoptotic pathway to induce cancer cell death. Resistance to standard chemotherapies also seems to be determined by alterations in the apoptotic pathways of cancer cells. Therefore, understanding the signals of apoptosis and the mechanism of apoptosis may allow the development of better chemo- or radiotherapeutic regimens for the treatment of cancer. Finally, components of the apoptotic pathway may represent potential therapeutic targets using gene therapy techniques.     

Signal transduction pathways that regulate cell survival and cell death

Apoptosis or programmed cell death (PCD) is a physiological process critical for organ development, tissue homeostasis and elimination of defective or potentially dangerous cells in complex organisms. Apoptosis permits cell death without a concomitant inflammatory response in the surrounding tissues. The process of apoptosis depends on the reception of multiple extracellular and intracellular signals, integration and amplification of these signals by second messengers and finally, activation of the death effector proteases. Defects in control of apoptotic pathways may contribute to a variety of diseases including cancer, autoimmune and neurodegenerative conditions and AIDS. While many components of the regulatory network controlling apoptosis have been defined, the mechanisms of action and patterns of interaction of these factors remain controversial. This article summarizes some of the known aspects of signaling pathways involved in apoptosis.     

Mechanisms of resistance to therapy and tumor cell survival

The failure to cure patients with cancer continues to be primarily because of the development of treatment resistance. Both normal and malignant cells die by either programmed cell death (apoptosis) or by cytolysis. Malignant cells have developed mechanisms of resistance that prevent them from entering the programmed cell death pathway as well as mechanisms of escaping immune recognition and cytolysis. These mechanisms include specific protective adaptations involving drug transport, metabolism, and target interactions. Malignant cells may also become resistant to therapy through alterations in genes encoding proteins involved in the initiation of apoptotic pathways. Finally, tumor cells may develop mechanisms to escape immune recognition, making them resistant to T-cell destruction. This article provides an overview of these mechanisms, with emphasis on published articles reported within the past year.     

Knowledge and attitudes about AIDS among deaf and hard of hearing persons

Apoptosis in cells of different lineages is restrained by survival signals which depend upon cell-to-cell communication. The aim of this study was to determine whether colonic cells deprived of crypt ambient are doomed to die prior to their normal chronological demise. Apoptosis was studied in rat whole colonic tissue, in isolated intact crypts, and in colonic cell populations collected from the crypt axis at different stages of proliferation and differentiation. In a number of experiments, cell harvest was performed in the presence of either a tetrapeptide (YVAD-CMK) inhibitor of interleukin-1beta-converting enzyme (ICE), or tyrphostin A25, a protein tyrosine kinase inhibitor, or sodium-orthovanadate, a phosphatase inhibitor. DNA fragmentation was assessed by electrophoretic and nonisotopic-labeling procedures. The ultrastructure of colonic tissue specimens and isolated cells was examined by transmission electron microscopy. Apoptosis in whole colonic tissue and in isolated crypts was confined predominantly to cells resident in the upper crypt regions. In contrast, extensive apoptotic death was observed in isolated colonic cells, irrespective of their developmental stage and positional hierarchy within the crypt continuum at harvest time. An apoptotic gradient, however, was evident. Exposure to YVAD-CMK resulted in a marked decrease in the number of apoptotic cells. Treatment with tyrphostin A25 caused a sharp rise in the apoptotic index; conversely, vanadate significantly impeded apoptosis. Cumulatively, these results indicate that disordered intercellular communication provokes unscheduled ICE-mediated apoptosis of colonocytes, and that local signals along the crypt continuum control both the reprieve from death and the timely demise of distinct colonic cell populations. Attenuation of tyrosine phosphorylation may be a contributory event in the acquisition of the apoptotic phenotype.     

Genetic analysis with hierarchical models

Apoptosis is a genetically programmed series of events that results in cell death. As a consequence, it is difficult to identify dominant genes that play a role in this process using genetic selections in conventional cell culture systems. Accordingly, we have established an efficient expression screen to isolate dominant, apoptosis-inducing genes. The assay is based on the apoptotic morphology induced in the human kidney cell line 293 after transient transfection of small plasmid pools from normalized cDNA expression libraries. Using this assay, we isolated a novel isoform of the proto-oncogene Neu differentiation factor (NDF), a ligand for erbB receptor tyrosine kinases. Several lines of experimental evidence indicate that this gene kills in a cell-autonomous fashion and independently of known erbB receptors. This apoptotic property of an NDF isoform is readily contrasted with NDF's transforming potential and might balance the tendency to tumorigenesis in cells that overexpress NDF.     

Facing death in the fly: genetic analysis of apoptosis in Drosophila

Apoptosis, a gene-directed form of cell death, occurs normally during development and plays a major role in many diseases, including cancer and neurodegenerative disorders. Molecular genetic studies in Drosophila have revealed the existence of three novel apoptotic activators, reaper, head involution defective and grim. Additionally, Drosophila homologs of evolutionarily conserved IAPs (inhibitor of apoptosis proteins) and CED-3/ICE-like proteases have been identified and characterized. Through the combined use of genetic, molecular, biochemical and cell biological techniques in Drosophila it should now be possible to elucidate the precise mechanism by which apoptosis occurs, and how the death program is activated in response to many distinct death-inducing signals.     

The time window of apoptosis: a new component in the therapeutic strategy for cardiovascular remodeling

APOPTOSIS AND EXPERIMENTAL HYPERTROPHY: Apoptosis (programmed cell death) is a physiological counterpart of cell replication with shared as well as specific pathways. Our initial studies have demonstrated increased apoptosis in the heart, kidney and brain of spontaneously hypertensive rats (SHR) and mice, persisting in cultured vascular smooth muscle cells. In these target organs of hypertension, programmed cell death paralleled the well known hypertrophy/hyperplasia. We also observed that the two processes can be dissociated in time, as in experimental hypertrophy of the heart induced by pressure overload. In this context, only a short-lived apoptotic window precedes the overt development of cardiac hypertrophy. EFFECTS IN HYPERTENSION: We now propose that a more prolonged apoptotic window is present in hypertension. Apoptosis seems to be significantly reduced during the first weeks of life in SHR, possibly contributing to the early cardiac hyperplasia. However, increased apoptosis is clearly evident thereafter throughout the development of hypertension and fades below the levels observed in normotensive animals after the age of 24 weeks. ANTIHYPERTENSIVE THERAPY AND APOPTOSIS: In addition to the apoptotic windows that suggest a dynamic regulation of this process in disease states, antihypertensive therapy with angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists and calcium channel blockers can also modify the contribution of apoptosis, independently of the blood pressure fall. We propose that the presence of apoptotic windows and the involvement of this biological process as a primary or secondary event in cardiovascular remodeling should be taken into account when designing innovative therapeutic approaches.     

Role of programmed (apoptotic) cell death during the progression and therapy for prostate cancer

Cells possess within their epigenetic repertoire the ability to undergo an active process of cellular suicide termed programmed (or apoptotic) cell death. This programmed cell death process involves an epigenetic reprogramming of the cell that results in an energy-dependent cascade of biochemical and morphologic changes (also termed apoptosis) within the cell, resulting in its death and elimination. Although the final steps (i.e., DNA and cellular fragmentation) are common to cells undergoing programmed cell death, the activation of this death process is initiated either by sufficient injury to the cell induced by various exogenous damaging agents (e.g., radiation, chemicals, viruses) or by changes in the levels of a series of endogenous signals (e.g., hormones and growth/survival factors). Within the prostate, androgens are capable of both stimulating proliferation as well as inhibiting the rate of the glandular epithelial cell death. Androgen withdrawal triggers the programmed cell death pathway in both normal prostate glandular epithelia and androgen-dependent prostate cancer cells. Androgen-independent prostate cancer cells do not initiate the programmed cell death pathway upon androgen ablation; however, they do retain the cellular machinery necessary to activate the programmed cell death cascade when sufficiently damaged by exogenous agents. In the normal prostate epithelium, cell proliferation is balanced by a equal rate of programmed cell death, such that neither involution nor overgrowth normal occurs. In prostatic cancer, however, this balance is lost, such that there is greater proliferation than death producing continuous net growth. Thus, an imbalance in programmed cell death must occur during prostatic cancer progression. The goal of effective therapy for prostatic cancer, therefore, is to correct this imbalance. Unfortunately, this has not been achieved and metastatic prostatic cancer is still a lethal disease for which no curative therapy is currently available. In order to develop such effective therapy, an understanding of the programmed death pathway, and what controls it, is critical. Thus, a review of the present state of knowledge concerning programmed cell death of normal and malignant prostatic cells will be presented.     

Apoptosis--programmed cell death

Apoptosis or programmed cell death is the physiologic, natural form of cell death, exhibiting characteristic morphological and biochemical features. Apoptosis occurs upon developmental processes and represents an important defense mechanism. Defective apoptosis can result in a variety of diseases. Rapidly growing knowledge upon this mode of cell death, especially the identification of genes involved in the regulation of apoptosis raise the hope for new therapies in the treatment of cancer, autoimmune and neurodegenerative disorders, as well as viral infections.     

Use of echocardiography in the management of congestive heart failure in the community

BACKGROUND: Inhibition of apoptosis, or programmed cell death, may be critical both in the development of cancer and in determining response to therapy. The authors examined the expression of two related apoptotic inhibitors, Bcl-2 and Bcl-xL, in pretreatment biopsies from a series of 42 patients with squamous cell carcinoma of the head and neck. The observed pattern of apoptotic inhibitor expression was compared with that of the p53 gene product, another factor implicated in carcinogenesis and therapeutic responsiveness. METHODS: Formalin fixed, paraffin embedded tumor biopsies from 42 patients with locally advanced squamous cell carcinoma of the head and neck were analyzed by immunohistochemistry using antibodies specific for Bcl-xL, Bcl-2, and p53. Measures of clinical outcome, including disease specific survival and overall survival, were compared among the groups. RESULTS: The majority of the tumors demonstrated enhanced expression of either Bcl-2 or Bcl-xL compared with surrounding normal epithelium. Fifty-two percent of the tumors had up-regulated Bcl-xL, and 17% had up-regulated Bcl-2. There was no overlap between these groups. Expression of Bcl-2, but not Bcl-xL, was correlated with improved disease specific survival. Immunohistochemically detectable p53 expression (48% of tumors) was not found to correlate with expression of either Bcl-xL or Bcl-2 and, in this series, was not a predictor of clinical outcome. CONCLUSIONS: These results suggest that disruption of apoptotic control pathways is an important event in the evolution of squamous cell carcinoma of the head and neck. A common mechanism for this disruption involves overexpression of Bcl-xL, Patients whose tumors demonstrate Bcl-2 positivity, even with locoregionally advanced disease, appear to have a high likelihood of cure with aggressive combined modality therapy and may be treated successfully with less toxic therapy.     

Apoptosis and its role in the myelodysplastic syndromes: implications for disease natural history and treatment

Apoptosis (programmed cell death) is an active cellular process which regulates cell population size by decreasing cell survival. In this review the underlying cellular and molecular mechanisms of apoptosis in hemopoietic and non-hemopoietic cells are described, with specific focus on these issues in the myelodysplastic syndrome (MDS), a myeloid clonal hemopathy. Apoptosis-regulating genes exist as families whose protein products are either anti-apoptotic or pro-apoptotic. Numerous stimuli can serve as initiators of the cell death pathway, including essentially all chemotherapeutic drugs, irradiation, certain inhibitory cytokines and deprivation of relevant growth factors. Morphological evidence of increased apoptosis in marrow hemopoietic cells has been demonstrated in patients with MDS. The reviewed data provide support for the hypothesis that early in MDS, increased apoptosis is associated with ineffective progenitor and maturing hemopoietic cell survival, and occurs concomitant with cytopenias/ineffective hemopoiesis; conversely, the progression of MDS toward AML occurs in concert with decreased apoptosis and an increased degree of neoplastic cell survival, leading to subsequent expansion of the abnormal precursor cells. These processes are associated with alterations in the balance between pro- and anti-apoptotic oncoprotein expression within the hemopoietic precursors, which may be modified by cytokine treatment. Investigations evaluating apoptotic events in MDS have improved our understanding of the biology of hemopoietic cell survival as related to pathogenetic features of this disease. By modifying levels of apoptosis, such studies provide a framework for future potentially beneficial therapeutic approaches to treat patients with MDS.     

Measuring food intake in wild animals: primates

Rabbit hemorrhagic disease is a rapidly lethal infection caused by a calicivirus, characterized by acute liver damage and disseminated intravascular coagulation (DIC). Following morphological criteria and using a specific in situ labeling technique, we have found that liver cell death induced upon infection is due to apoptosis, and that programmed cell death is a constant feature in rabbits experimentally infected with RHDV. The process affected mainly hepatocytes, but also macrophages and endothelial cells presented morphologic hallmarks of apoptosis, expressing all these cell types viral antigens as determined by immunohistochemistry. The occurrence of programmed cell death was correlated with the appearance of the RHDV induced pathology in tissues by DNA fragmentation detection in situ. Hepatocyte apoptosis produced extensive parenchymal destruction causing a lethal, acute fulminant hepatitis that is characteristic of RHD. Apoptosis of intravascular monocytes and endothelial cell was observed together with fibrin thrombi in blood vessels. Since apoptotic cells are known sites of enhanced procoagulant activity, apoptosis of these cell populations might constitute a first step in the pathogenesis of DIC and a common pathway to other viral hemorrhagic fevers. In conclusion, apoptosis in RHD may be determinant in the development of the pathogenesis of this disease.     

Programmed cell death and radioresistance

Whereas apoptosis is a critical mode of cell deletion in normal organism development, apoptotic cells are also observed in tumors, especially following cytotoxic treatments, leading to questions about their role in tumor response to therapy. We have conducted a series of studies using murine tumor models and found that the ability of the tumor cells to undergo apoptosis correlates with tumor response to radiation. The best correlation was with the pretreatment apoptotic index, suggesting that apoptosis in some tumors may govern radiocurability by regulating the number of tumor clonogens. However, other roles for apoptosis in tumor response to radiation have not been ruled out. One of the important observations that has come from this work has been the heterogeneity in apoptosis propensity both within the cell population of a given tumor and among different types of tumors. Such findings underscore the fact that apoptosis is under complex genetic control and that some of the same oncogenes and tumor suppressor genes that are responsible for tumor initiation and progression to malignancy also dictate the apoptotic response to treatment. Understanding the biochemical and molecular pathways that govern this process may ultimately allow the development of strategies for modulating apoptosis for therapeutic benefit.     

Regional myocyte hypertrophy parallels regional myocardial dysfunction during post-infarct remodeling

The molecular genetic events involved in the etiology of human granulosa cell (GC) tumors, which represent approximately 7% of all malignant ovarian neoplasms, are unknown. Amplification and/or overexpression of the ERBB genes are a feature of many cancer types, and overexpression of erbB2 correlates with poor prognosis in epithelial ovarian cancer. In the present study, we used immunohistochemistry to determine the level and frequency of expression of different erbB receptors in GC tumors. Ten of 12 tumors expressed erbB4 at moderate to high levels in >50% of cancer cells, whereas erbB2 (6 of 12) and erbB3 (2 of 12) were expressed less frequently. Western blot experiments showed that the only available GC tumor cell line, COV434, also expressed erbB receptors. Heregulin (HRG)-beta2, a ligand for erbB3 and erbB4 receptors, stimulated tyrosine phosphorylation of the erbB receptors, which was accompanied by activation of Erk1 and Erk2, two mitogen-activated protein kinases with a functional role in mitogenesis. Importantly, HRG increased cell proliferation in COV434 cells, and treatment with HRG/PE40, a ligand toxin shown previously to be cytotoxic against human breast cancer cells overexpressing erbB receptors, led to a dramatic and irreversible decrease in cell number. These results indicate that erbB receptor signaling pathways may be critical in the control of GC tumor cell proliferation and that HRG/PE40 is a potential therapeutic agent for the treatment of GC tumors.     

Morphological and biochemical characterization and analysis of apoptosis

Apoptosis is a form of programmed cell death serving physiologic and homeostatic functions. However, recent evidence implicating apoptosis in the etiology and pathophysiology of known human diseases, such as heart diseases, cancer, AIDS, and neurodegenerative and autoimmune diseases are continually surfacing. This has spawned the need for identifying which methods are the most effective and well accepted to decipher its presence in a variety of research settings. We have therefore detailed the morphology and biochemical features of apoptotic cell death, with an emphasis on discriminating it from necrosis. In addition, we describe specific and selective techniques which are optimal to target hallmark apoptotic features, such as microscopy, Annexin V labeling, in situ nick-end labeling (TUNEL), and DNA fragmentation analysis by gel electrophoresis and ELISA for oligonucleosome-sized DNA. The advantages and disadvantages of each technique are discussed, as well as their experimental importance relative to one another. The methods have been described in a stepwise fashion, and can readily be applied in the majority of cell systems. Whether working on the tissue or single cell level, these methods are highly effective in qualifying and quantifying apoptosis. The application of these methods in conjunction with molecular techniques can further delineate the underlying mechanisms of apoptosis.     

Mechanisms and control of programmed cell death in invertebrates

Apoptosis is a morphologically distinct form of programmed cell death that plays important roles in development, tissue homeostasis and a wide variety of diseases, including cancer, AIDS, stroke, myopathies and various neurodegenerative disorders (see Thompson (1995) for review). It is now clear that apoptosis occurs by activating an intrinsic cell suicide program which is constitutively expressed in most animal cells, and that key components of this program have been conserved in evolution from worms to insects to man. Genetic studies of programmed cell death in experimentally highly accessible invertebrate model systems have provided important clues about the molecular nature of the death program, and the intracellular mechanisms that control its activation. This review summarizes some of the key findings in this area, but also touches on some of the many unresolved questions and challenges that remain.     

Apoptosis after traumatic human spinal cord injury

OBJECT: Apoptosis is a form of programmed cell death seen in a variety of developmental and disease states, including traumatic injuries. The main objective of this study was to determine whether apoptosis is observed after human spinal cord injury (SCI). The spatial and temporal expression of apoptotic cells as well as the nature of the cells involved in programmed cell death were also investigated. METHODS: The authors examined the spinal cords of 15 patients who died between 3 hours and 2 months after a traumatic SCI. Apoptotic cells were found at the edges of the lesion epicenter and in the adjacent white matter, particularly in the ascending tracts, by using histological (cresyl violet, hematoxylin and eosin) and nuclear staining (Hoechst 33342). The presence of apoptotic cells was supported by staining with the terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling technique and confirmed by immunostaining for the processed form of caspase-3 (CPP-32), a member of the interleukin-1beta-converting enzyme/Caenorhabditis elegans D 3 (ICE/CED-3) family of proteases that plays an essential role in programmed cell death. Apoptosis in this series of human SCIs was a prominent pathological finding in 14 of the 15 spinal cords examined when compared with five uninjured control spinal cords. To determine the type of cells undergoing apoptosis, the authors immunostained specimens with a variety of antibodies, including glial fibrillary acidic protein, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), and CD45/68. Oligodendrocytes stained with CNPase and a number of apoptotic nuclei colocalized with positive staining for this antibody. CONCLUSIONS: These results support the hypothesis that apoptosis occurs in human SCIs and is accompanied by the activation of caspase-3 of the cysteine protease family. This mechanism of cell death contributes to the secondary injury processes seen after human SCI and may have important clinical implications for the further development of protease inhibitors to prevent programmed cell death.     

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Refinement of the cell number by programmed cell death is a major morphogenetic mechanism of the developing central nervous system (CNS) in vertebrates including mammals, which determines to a significant degree its mature cytoarchitecture. We have examined the topography and the extent of cell death in different regions of the human CNS prenatally (11 fetuses), and in the early post-natal weeks (three newborns). Attention was focused on the wall of the telencephalon during a relatively short time period (12th-23rd week of gestation), corresponding to the time of major proliferation in the ventricular zone and to the peak of neuronal migration; both these mechanisms are crucial for corticogenesis. The TUNEL method was used, allowing the recognition of cell death because of its ability to label blunt ends of double-stranded DNA breaks. Morphological features of nuclei at different stages of apoptosis were identified, providing better evidence of the extent of the process than histological stains. Cell labelling was seen in either post-mitotic elements in the ventricular zone, or along the migratory pathways in the intermediate zone and subplate at all prenatal ages examined. No apoptotic nuclei were seen in the cortical plate. These findings suggest that apoptotic cell death drives the selection of cells which are committed to play a role during the early stages of corticogenesis. Lack of evidence of clonally related apoptotic cells also indicates that cell death occurs randomly. Therefore, molecular signals from the surrounding microenvironment seem to be necessary for the apoptotic pathway to be turned on, thus determining the fate of post-mitotic cells.     

Genes, molecules, and mechanisms regulating programmed cell death

Apoptosis is a morphologically distinct form of programmed cellular death that plays a central role during embryogenesis, tissue homeostasis, and to remove not necessary or potentially dangerous cells. Moreover, disregulation of genes mediating or modulating apoptosis contributes to the pathogenesis of a number of human diseases, including cancer, autoimmune diseases, neurodegenerative disorders, viral infections and acquired immunodeficiency syndrome. A number of genes and molecules promoting or protective against cell death is at present-day known and an important information about the external and internal signals involved in stimulation and suppression of apoptosis is also emerging. In the intracellular pathway of the death deregulation of [Ca2+](i) plays a pivotal role. Increased ionized intracellular calcium stimulates both the activation of enzymes (protein kinases, endonucleases, proteases and phospholipases) and plasma membrane K+ channels. This calcium-mediated activation leads to morphostructural changes, such as cell shrinkage, cytoplasmatic blebbing, nuclear chromatin condensation and DNA degradation into oligonucleosomal fragments. At least some genes of the cell death pathway have been conserved throughout animal evolution; ced-3 e ced-9 that regulate the initiation of cellular suicide in the nematode Caenorhabditis elegans are homologous to genes that in mammalian cells are thought to play a similar role (interleukin-1 beta converting enzyme [ICE] family, Bcl-2). It is possible to suppose that these regulators could constitute a target for treatment of disorders related with disregulation of apoptosis.