Active Cell Death Process Research Articles

Cadmium-Induced Apoptosis in Mouse Liver

Apoptosis is a process of active cell death, distinct from necrosis and characterized by specific morphological and biochemical features. Although the acute hepatotoxic effects of cadmium (Cd) are well described, little is known about the occurrence of apoptosis in Cd toxicity. Therefore, mice were injected with 5–60 μmol/kg ip of Cd and their livers were removed 1.5–48 h later and examined by light microscopy. Cd induced both a time- and dose-dependent increase in apoptotic index, severity of necrosis, and mitotic index. Apoptotic index peaked at 9–14 h after Cd administration and then decreased. The time course of apoptotic DNA fragmentation index, monitored by quantification of oligonucleosomal DNA fragments, correlated with the results obtained by histopathological analysis and a commercialin situapoptotic DNA detection kit. Liver necrosis, as demonstrated by histology and serum alanine aminotransferase and sorbitol dehydrogenase assays, was most severe 14–48 h after Cd injection. Apoptosis was decreasing by 24 h while necrosis persisted. Replacement of liver tissue by blood lakes (peliosis hepatis) was observed after 14 h. The mitotic index increased gradually with time, indicating compensatory liver cell regeneration. There was a progressive increase in the severity of necrosis, apoptotic index, and mitotic index with increasing dose of Cd. These data demonstrate that apoptosis is a major mode of elimination of critically damaged cells in acute Cd hepatotoxicity in the mouse, and it precedes necrosis.

Apoptosis in Trypanosomatids: Evolutionary and phylogenetic considerations

Programmed cell death (PCD) or apoptosis, an active process of cell death, plays a central role in normal tissue development and organogenesis, as well as in the pathogenesis of different diseases. Although it occurs in diverse cells and tissues under the influence of a remarkable variety of inducing agents, the resultant ultrastructural and biochemical changes are extremely monotonous, indicating the existence of a common biological mechanism underlying its occurrence. It is generally accepted that a developmental program leading to cell death cannot be advantageous to unicellular organisms and that PCD appeared in evolution to fulfill the organizational needs of multicellular life. However, the recent description of apoptotic death occurring in three different species of pathogenic kinetoplastids suggests that the evolutionary origin of PCD precedes the appearence of multicellular organisms. The present study proposes that a population of pathogenic Trypanosomatids is socially organized and that PCD is a prerequisite for this organization and for the fulfillment of the demands of a heteroxenic lifestyle. This proposal includes possible roles for PCD in the development of the parasite in the insect vector and/or in its mammalian host and suggests experimental strategies to localize the evolutionary origin of PCD within the kinetoplastids.

On the Role of DNA Double-Strand Breaks in Toxicity and Carcinogenesis

DNA double-strand breaks are associated with various endogenous processes, such as transcription, recombination, replication, and with the process of active cell death, which aims to eliminate cells. In addition, DNA double-strand breaks can be induced by irradiation, exposure to chemicals, increased formation of reactive oxygen species, and, indirectly, during repair of other types of DNA damage or as a consequence of extranuclear lesions. In addition to the neutral filter elution of DNA, the recently introduced pulsed-field gel electrophoresis is capable of determining DNA double-strand breaks with higher accuracy and sensitivity and is expected to increase our knowledge on the frequency and the role of DNA breakage. Parallel determination of parameters for cytotoxicity is necessary to elucidate the causal primary lesion. Although the repair of DNA double-strand breaks is a complex task, cells are capable of repairing — with or without errors and up to a certain extent — and surviving this DNA lesion. Gene translocations, rearrangements, amplifications, and deletions arising during repair and misrepair of double-strand breaks may contribute to cell transformation and tumor development.

Apoptosis, neurotrophic factors and neurodegeneration.

Apoptosis is an active process of cell death characterized by distinct morphological features, and is often the end result of a genetic programme of events, i.e. programmed cell death (PCD). There is growing evidence supporting a role for apoptosis in some neurodegenerative diseases. This conclusion is based on DNA fragmentation studies and findings of increased levels of pro-apoptotic genes in human brain and in in vivo and in vitro model systems. Additionally, there is some evidence for a loss of neurotrophin support in neurodegenerative diseases. In Alzheimer's disease, in particular, there is strong evidence from human brain studies, transgenic models and in vitro models to suggest that the mode of nerve cell death is apoptotic. In this review we describe the evidence implicating apoptosis in neurodegenerative diseases with a particular emphasis on Alzheimer's disease.

Noradrenaline inhibits the programmed cell death induced by 1,25-dihydroxyvitamin D 3 in glioma

The rat glioma cell line C6.9 has been recently reported to respond to 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) by the induction of a programmed cell death. Since, in vivo, glial cells are thought to be exposed to several neurotransmitters, we investigated the possibility of a neurotransmitter-mediated inhibition of this active cell death process. Noradrenaline and the β-adrenoceptor agonist isoproterenol showed significant inhibition of the 1,25(OH) 2D 3-induced programmed cell death. The β-adrenoceptor antagonist propanolol reversed this inhibition, while the α-adrenoceptor antagonist yohimbin was devoid of any effect. This suggests that the efficiency of antiproliferative vitamin D-related therapies could be influenced by endogenous levels of noradrenaline.

The Mechanism of Interferon-gamma Induced Cytotoxicity on the Lung Cancer Cell Line, A549

Background: Interferon- has various biologic effects, including antiviral effect, antitumor proliferative effect, activation of macrophage and B lymphocyte, and increased expression of major histocompatibility complex. Especially, antitumor proliferative effect of interferon- has already been proved to be important in vivo as well as in vitro. And, clinical studies of interferon- have been tried in lung cancer patients. However, the mechanism of antitumor effect of interferon- has not yet been established despite of many hypotheses. Necrosis is a type of cell death which is well known to occur in the circumstances of severe stresses. In contrast, is another type of cell death which occurs in such biological circumstances as embryonic development, regression of organs, and self-tolerance of lymphocytes. And, apoptosis is an active process of cell death in which cells are dying with fragmentations of their cytoplasms and nuclei. And, in the process of apoptosis the DNAs of cells are cleaved between nucleosomes by unidentified endonuclease and therefore DNAs of apoptotic cells result in a typical electrophoresis pattern known as DNA ladder pattern. Recently it has been suggested that cytotoxic effect of interferon- occurs via apoptosis. To elucidate the mechanism of antitumor cytotoxic effect of interferon-, we microscopically observed a lung cancer cell line, A549 which was treated with interferon-. We observed A545 treated with interferon- was dying fragmented. And so, we performed this study to find out that the mechanism of antitumor cytotoxic effect of interferon- be apoptosis. Method: We treated A549, human lung cancer cell line with various concentration of interferon- and quantified its cytotoxic effect of various periods, 24 hours, 72 hours and, 120 hours by MTT(dimethylthiazolyl diphenyltetrazolium bromide) bioassay. Also, after we treated A549 with 100 units/mi of interferon- for 120 hours, we observed the pattern of cell death with inverted microscope and we extracted DNAs from the dead A549 cells and observed the pattern of 1.5% agarose gel electrophoresis with ethidium bromide staining. Result: 1) Cytotoxic effect of interferon- on A549: For the first 24 hours, threre was little cytotoxic effect and for between 24 hours and 72 hours, there was the beginning of cytotoxic effect and for 120 hours there was increased cytotoxic effect. 2) Pattern of A549 cell death by interferon-: We observed with inverted microscope that A549 cells were dying fragmented. 3) DNA ladder pattern of gel electrophoresis: We observed DNA ladder pattern of gel electrophoresis of extracted DNAs from dead A549 cells. Conclusion: We concluded that the mechanism of interferon-induced cytotoxicity on lung cancer cell line, A549 be via apoptosis.

遺伝子発現 : 総論(<特集>遺伝子発現)

Genetic information is stored in all cells as genomic DNA. Differences among cells of a single organism reflect differences in gene expression. Gene expression is controlled mainly at transcriptional step. Once RNA transcripts are synthesized, they are spliced to become mature mRNA, which are transported from the nucleus to the cytoplasm and used as templates for protein synthesis. Each step is regulated so as to accommodate to the needs of each cell. Gene expression can be greatly altered by environmental stimuli, which are transferred by istracellular signal transduction systems. Gene expression is also regulated during various disease processes. Neoplastic transformation is believed to originate via mutation and anomalous gene expression. Protooncogenes, which are responsible for transformation and progression of neoplasms, are involved in the signal transudation system to the level of gene expression. Brain tumor research, therefore, is closely linked to the study of altered gene expression. The process of cell death in brain tumors is, at least in part, regulated via changes in gene expression. The process of such active suicidal cell death is known as apoptosis. In acute neurosurgical disorders such as head trauma and cerebrovascular diseases, the central nervous system (CNS) is damaged so quickly that there seems to be no possibility of involvement of gene expression. However, a secondary process, which could exacerbate a patient's condition, takes place following the initial injury. In this secondary process, cells in the CNS seem to alter their gene expression in order to survive. One such cellular reaction is the stress response. Expression of genes encoding various growth factors is also important for the survival of CNS cells under disease.

Tumor Cell Death Induced through the Receptor for Interleukin-2

We performed experiments to analyze the possible apoptotic effect of recombinant IL-2 on neoplastic cells expressing the receptor for IL-2. YAC-1 or EL/4 tumor cells, incubated in vitro with IL-2 or with monoclonal antibodies against the alfa-chain of the IL-2 receptor, underwent to apoptosis as analyzed by reduction in nuclear size, derangement in chromatin structure and DNA fragmentation in oligonucleosomal subunits. The apoptotic phenomenon was time- and dose-dependent. Inhibition of mRNA synthesis D-act or addition of IL-4 counteracted the effect of both IL-2 and anti-IL-2/r antibodies. Both rIL-2 or anti-IL-2/r mAb determined an augmentation of phosphorilation pattern as measured by western immunoblotting, and a transient augmentation of Ca++ as evaluated by a spectrophotometrical assay. These data suggest that stimulation of IL-2 receptor may induce an active process of cell death on neoplastic cells in vitro.

Intracellular Alkalinization Suppresses Lovastatin-induced Apoptosis in HL-60 Cells through the Inactivation of a pH-dependent Endonuclease

Protein isoprenylation is a post-translational modification essential for the biological activity of G-proteins. Inhibition of protein isoprenylation by lovastatin (LOV) induces apoptosis in HL-60 cells, a process of active cell death characterized by the internucleosomal degradation of genomic DNA. In this article we show that LOV-induced apoptosis is associated with intracellular acidification and that activation of the Na+/H+ antiporter induces a raise in pHi which is sufficient to prevent or arrest DNA digestion. First, LOV induced a decrease in pHi which was dose-dependent and correlated with the extent of DNA degradation. Flow cytometry analysis revealed that this acidification was due to the appearance of a subpopulation of cells whose pHi was 0.9 pH units below control values. Cell sorting experiments demonstrated that DNA degradation had occurred only in those cells which had suffered intracellular acidification. LOV-induced apoptosis could be suppressed by mevalonate supplementation, inhibition of protein synthesis, and protein kinase C activation by phorbol myristate acetate. In all three cases, intracellular acidification was abolished. Inhibition of the Na+/H+ antiporter by 5-N-ethyl-N-isopropyl amiloride induced DNA degradation in HL-60 cells per se and suppressed the protective effect of phorbol myristate acetate. LOV-induced intracellular acidification was not due to a complete inhibition of the Na+/H+ antiporter. In fact, LOV-treated cells were able to respond to phorbol myristate acetate stimulation of the Na+/H+ antiporter with a marked increase in pHi. This effect was accompanied by a rapid arrest of DNA digestion. These observations illustrate the strong pH dependence of LOV-induced DNA degradation, thus providing a connection between the activation of the Na+/H+ antiporter and the suppression of apoptosis.

Viruses and apoptosis

Apoptosis is an active process of cell death that serves diverse functions in multicellular organisms, and under physiological conditions, it is tightly controlled. Many virus genomes encode gene products that modulate apoptosis, either positively or negatively, and induction of apoptosis often contributes directly to the cytopathogenic effects of the viruses. Inhibition of apoptosis by viruses, on the other hand, may prevent premature death of infected cells, thereby facilitating viral replication, spread, or persistence.

Mechanisms for nitric oxide-induced cell death: Involvement of apoptosis

Recent observations suggest that apoptosis, the natural, active cell death process is also triggered in several pathological conditions including ischemic brain insult, and neurodegenerative and autoimmune diseases. We have investigated the mechanisms involved in the development of apoptosis in neuronal and pancreatic cells and in macrophages, which were exposed to either chemical donors of nitric oxide or to inducers of the nitric oxide synthase. In this overview, we summarize current evidence for the involvement of apoptosis in the cytotoxicity of nitric oxide and discuss possible mechanisms that may lead to cell death.

Expression of clusterin in cell differentiation and cell death.

Clusterin, originally isolated as testosterone-repressed prostate message-2 from regressing rat ventral prostate, has been identified with the process of active cell death (ACD). The clusterin gene product is a glycosylated dimer consisting of alpha and beta subunits, resulting from the 70-kilodalton preprotein. To determine its relationship with ACD, we have examined clusterin expression via in situ hybridization and immunohistochemistry. Clusterin message is detected in the supporting cells in both testes and ovaries and the protein surrounds the mature germ cells. The highest level of expression was found in the head region of the epididymis. Clusterin message is also detected in selected cells of uterine glands and ducts both in the normal and pregnant uterus. The expression of clusterin in the developing embryo is most abundant in the choroid plexus, inner ear, and epithelium of the eye. None of the cells in the testes, epididymis, or embryo that express clusterin are undergoing ACD. The expression of clusterin appears to correlate with cell remodelling or differentiation that occurs during these periods of development. However, in the female reproductive system, we found clusterin to be expressed in both differentiating as well as dying cells. These results suggest that clusterin may provide support for cells undergoing specific biochemical and (or) physical changes. Our results are consistent with the hypothesis that clusterin is an antiinflammatory agent.

Differential Priming to Programmed Cell Death of Superantigen-Reactive Lymphocytes of HIV Patients

Programmed cell death or apoptosis has been shown to play a central role in CD4+ T cell depletion following HIV infection. Because most apoptotic signals are delivered through T cell receptor stimulation, we investigated whether T cell depletion in AIDS is a stochastic phenomenon or if it preferentially affects T cell subsets defined by their interaction with superantigens. To address this problem we have taken advantage of the exclusive property of superantigens to trigger T cells expressing selective sets of T cell receptor V beta elements. Here we report that CD4+ T cells from HIV-infected patients can proliferate in vitro to T cell receptor mobilization by some superantigens, but not others. Furthermore, the failure of T cells to respond to some superantigens was shown to be due to an active cell death process that differentially affected T cells capable of interacting with different superantigens. The selective programmed cell death priming of T cells responsive to particular superantigens, observed in this study, suggests that T cell depletion in HIV infection is not simply due to the cytopathic effect of the virus. The possible link between programmed cell death and T cell receptor variable regions suggested by the present experiments may help to better define current models of AIDS pathogenesis.

Skeletal muscle-derived trophic factors prevent motoneurons from entering an active cell death program in vitro

The purpose of the experiments reported here is to provide evidence that motoneurons (MTNs) isolated from chick embryo spinal cords go through an active process of cell death when deprived of trophic support in vitro. In order to analyze and characterize this process, MTNs were isolated with a metrizamide gradient technique and cultured in the presence of saturating concentrations of soluble muscle extract. When muscle extract was washed off from the cultures, MTNs entered a process of cell death that could be blocked with inhibitors of mRNA and protein synthesis. Two other additional criteria were used to define this process as an active one. First, ultrastructural analysis of MTNs dying as a consequence of muscle extract deprivation showed that some, but not all, of the MTNs displayed clear signs of apoptotic cell death. Those included cytoplasm condensation, fragmentation of chromatin, and preservation of cytoplasmic organelles. Second, internucleosomal degradation of DNA was detected in MTNs deprived of muscle extract. When DNA was analyzed by Southern hybridization techniques using digoxigenin-labeled genomic probes, a clear ladder pattern could be identified on muscle extract-deprived MTNs. The degradation of DNA upon trophic deprivation could be prevented by cycloheximide (CHX). In an attempt to characterize further the process of active cell death in MTNs, we found a time point of commitment to cell death of approximately 10 hr by using three different approaches: muscle extract deprivation plus readdition of muscle extract, muscle extract deprivation plus addition of CHX, and muscle extract deprivation plus addition of actinomycin D. Moreover, we show that MTNs deprived of trophic support from muscle extract but maintained alive with CHX could not be rescued from cell death by reading muscle extract if CHX was washed off the cultures within the first 15 hr of muscle extract deprivation. However, muscle extract alone was able to rescue MTNs that had been kept alive with CHX for periods of time longer than 24 hr after muscle extract deprivation. From these results we postulate that the activation of the cell death program after trophic deprivation is transient.

Inhibition of Apoptosis by BCR-ABL in Chronic Myeloid Leukemia

BCR-ABL expression is presumed to effect clonal expansion in chronic myeloid leukemia (CML) by deregulation of cell proliferation. However, most studies have found that relative rates of cell proliferation are not increased in CML. Moreover, we found that CML progenitors display a normal proliferative response to growth factors and do not manifest greater proliferative potential than normal progenitors. Growth of malignancies depends on an imbalance between the rate of cell production and the rate of cell death. We found that BCR-ABL expression inappropriately prolongs the growth factor-independent survival of CML myeloid progenitors and granulocytes by inhibiting apoptosis, a genetically programmed process of active cell death; inhibition of BCR-ABL expression by antisense oligonucleotides reversed the suppression of apoptosis as well as the enhancement of survival. The decreased rate of programmed cell death appears to be the primary mechanism by which BCR-ABL effects expansion of the leukemic clone in CML.

Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia

BCR-ABL expression is presumed to effect clonal expansion in chronic myeloid leukemia (CML) by deregulation of cell proliferation. However, most studies have found that relative rates of cell proliferation are not increased in CML. Moreover, we found that CML progenitors display a normal proliferative response to growth factors and do not manifest greater proliferative potential than normal progenitors. Growth of malignancies depends on an imbalance between the rate of cell production and the rate of cell death. We found that BCR-ABL expression inappropriately prolongs the growth factor-independent survival of CML myeloid progenitors and granulocytes by inhibiting apoptosis, a genetically programmed process of active cell death; inhibition of BCR- ABL expression by antisense oligonucleotides reversed the suppression of apoptosis as well as the enhancement of survival. The decreased rate of programmed cell death appears to be the primary mechanism by which BCR-ABL effects expansion of the leukemic clone in CML.

Induction of apoptosis in mouse thymocytes by cyclosporin A: in vivo study.

We investigated the in vivo effect of cyclosporin A (CsA) on mouse thymus and thymocytes. Administration of CsA (10 mg/kg of body weight) was found to induce a marked reduction in the size, weight and consistency of the thymus. These modifications were associated with thymic reticulo-epithelial cells (TREC) and thymocyte damage. Some of the damaged thymocytes displayed characteristic of cells undergoing apoptosis. Ultrastructural study of thymocytes and thymic tissue, as well as DNA electrophoresis of thymocytes, showed chromatin condensation, cellular shrinkage, and nuclear fragmentation in oligonucleosomal fragments. DNA labeling with propidium iodide (PI) of thymocytes from CsA treated mice cultured for 24 hrs showed an increased number of apoptotic nuclei. Furthermore, flow cytometric analysis using monoclonal antibodies (mAbs) specific for thymocyte subsets confirmed that CsA induces a large decrease in the relative number of mature single positive (SP) CD4+CD8- and CD8+CD4- thymocytes expressing high densities of CD3 and T cell receptor ab (TCR alpha beta) surface molecules, but also a decrease in the absolute number of the other thymocyte subsets. These results suggest that CsA causes macroscopic and ultrastructural modifications of the thymus, associated with an active process of cell death in mouse thymocytes in vivo. In line with these results we formulate a hypothesis concerning the stage of T-cell development at which CsA induces apoptosis.

Interleukin-4 Protects Double-Negative and CD4 Single-Positive Thymocytes From Dexamethasone-Induced Apoptosis

Glucocorticoid hormones (GCH) and anti-CD3 monoclonal antibodies (MoAbs) induce in mouse thymocytes and T-cell tumor lines an active process of cell death called apoptosis. Interleukins (IL), including IL-1 and IL-2, have been reported to inhibit such apoptosis. In this study we show that IL-4 also reduced the DNA fragmentation characteristic of dexamethasone (DEX)-induced apoptosis in thymocytes. This effect, studied in both time-course and dose-response experiments, was also detected at low IL-4 concentrations (1 U/mL) and against high DEX levels (10(-7) mol/L). The effect of IL-4 was blocked by an anti-IL-4 but not by an anti-IL-1 alpha MoAb, and was thus both specific and direct. Phenotypic analysis showed that IL-4 protects predominantly CD4-CD8- and CD4+CD8- cells. Our findings suggest that intrathymic T-cell development may be influenced by IL-4.

Interleukin-4 protects double-negative and CD4 single-positive thymocytes from dexamethasone-induced apoptosis

Glucocorticoid hormones (GCH) and anti-CD3 monoclonal antibodies (MoAbs) induce in mouse thymocytes and T-cell tumor lines an active process of cell death called apoptosis. Interleukins (IL), including IL- 1 and IL-2, have been reported to inhibit such apoptosis. In this study we show that IL-4 also reduced the DNA fragmentation characteristic of dexamethasone (DEX)-induced apoptosis in thymocytes. This effect, studied in both time-course and dose-response experiments, was also detected at low IL-4 concentrations (1 U/mL) and against high DEX levels (10(-7) mol/L). The effect of IL-4 was blocked by an anti-IL-4 but not by an anti-IL-1 alpha MoAb, and was thus both specific and direct. Phenotypic analysis showed that IL-4 protects predominantly CD4- CD8- and CD4+CD8- cells. Our findings suggest that intrathymic T-cell development may be influenced by IL-4.

Interleukin-2 Induces Apoptosis in Mouse Thymocytes

Interleukins play a role in the process of T-cell development and, like other cytokines, seem able to modulate apoptosis. Interleukin-2 has been reported to inhibit apoptotic cell death of thymocytes induced in vitro by either activation of CD3/TCR complex or treatmet with glu-cocorticoid hormone. We demonstrate here that IL-2 can provoke DNA fragmentation and cell death of CD4 +CD8 + mouse thymocytes by activating an endogenous apoptotic pathway. Thymocytes, incubated with high IL-2 concentrations in vitro, showed the morphological characteristics of apoptotic cells, including, reduction in nuclear size, derangement in chromatin structure, and DNA fragmentation in oligonucleosomal subunits. Inhibition of mRNA and protein synthesis and addition of the PKC-inhibitor H-7, Zn 2+ ions, and IL-4 counteracted the IL-2 effect. These data suggest that high IL-2 concentrations may induce an active process of cell death on mouse thymocytes in vitro.