Biomedical subjects

S Orrenius

Publications and source records attributed to S Orrenius.

At least 37 records · Page 2Linked to original sources

PubMed2001

Hsp27 inhibits cytochrome c-mediated caspase activation by sequestering both pro-caspase-3 and cytochrome c.

Mitochondrial cytochrome c release in response to pro-apoptotic signals leads to the formation of a cytochrome c/Apaf-1/procaspase-9 complex (the apoptosome) and resultant activation of caspase-9 and caspase-3. Here we demonstrate that the molecular chaperone, Hsp27, inhibits this cytochrome c-mediated activation of caspase-3. Immunodepeletion of Hsp27 from cytochrome c-activated cytosols resulted in decreased caspase activity. Furthermore, immunoprecipitation of Hsp27 resulted in the coprecipitation of both cytochrome c and procaspase-3. In reciprocal experiments, immunoprecipitation of both procaspase-3 and cytochrome c resulted in coprecipitation of Hsp27, indicating two independent interactions. These results point to Hsp27 mediating its inhibition of procaspase-3 activation through its ability to sequester both cytochrome c and procaspase-3, and thus prevent the correct formation/function of the apoptosome complex.

Apoptosis↗

PubMed2000

Distinct pathways for stimulation of cytochrome c release by etoposide.

Induction of apoptosis by DNA-damaging agents, such as etoposide, is known to involve the release of mitochondrial cytochrome c, although the mechanism responsible for this event is unclear. In the present study, using Jurkat T-lymphocytes, a reconstituted cell-free system, or isolated liver mitochondria, we demonstrate the ability of etoposide to induce cytochrome c release via two distinct pathways. Caspase inhibition by either benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) or benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethyl ketone (z-VDVAD-fmk) attenuates cytochrome c release triggered by a low dose of etoposide via an apparent inhibition of nuclear events involving the release of protein factor(s) that is (are) able to interact with mitochondria. In contrast, caspase inhibition has no effect on cytochrome c release induced by a higher dose of etoposide. Moreover, the higher dose of etoposide heightens the sensitivity of Ca(2+)-loaded isolated mitochondria to mitochondrial permeability transition, an effect that is completely abolished by cyclosporin A. Interestingly, cyclosporin A is ineffective at preventing similar mitochondrial damage in Jurkat cells treated with etoposide. We propose that lower doses of etoposide predominantly target the nucleus and stimulate the release of caspase-sensitive protein factor(s) that interact with mitochondria to trigger cytochrome c release, whereas higher doses of the drug impart a more direct effect on mitochondria and thus are not mitigated by caspase inhibition.

Amino Acid Chloromethyl Ketones↗

PubMed2000

[Apoptosis required for maintenance of homeostasis: familial hemophagocytic lymphohistiocytosis caused by too little cell death].

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.

Adult↗

PubMed2000

Review: nuclear events in apoptosis.

Initial apoptosis research characterized this form of cell death based on distinct nuclear morphology that was subsequently shown to be associated with the appearance of oligonucleosomal DNA fragments. More recent evidence has indicated that apoptosis depends upon a tightly regulated cellular program for its successful initiation and execution. Molecular participants in this program are present in different subcellular compartments, including the plasma membrane, cytosol, mitochondria, and nucleus. The interplay among these compartments and the exchange of specific signaling molecules are critical for the systematic progression of apoptosis. While numerous reports have described a key role for caspase activity in the signaling and executive steps of apoptotic cell death, there are some instances where well-established nuclear changes, characteristic of this form of cell death, can occur independently of caspase activity. Moreover, evidence indicates that certain nuclear events, including chromatin condensation and DNA fragmentation, are controlled separately and depend upon a persistent supply of energy in vivo. In this review, we discuss our current understanding of the role and regulation of nuclear events in the apoptotic process with an emphasis on protease and endonuclease activities as well as the ability of certain Bcl-2 family proteins to influence this process.

Animals↗

PubMed2000

Dexamethasone pre-treatment interferes with apoptotic death in glioma cells.

Glucocorticoids are known to influence the ability of cells to undergo apoptosis, directly inducing apoptosis in thymocytes while inhibiting it in hepatoma and carcinoma cells. Dexamethasone, a synthetic glucocorticoid, is reported to induce partial resistance to certain anticancer drugs in glioma cell lines. In the present study, the effect of dexamethasone on apoptosis of glioma and astrocytoma cell lines was investigated. Exposure of D384 human astrocytoma and C6 rat glioma cells to staurosporine induced apoptosis as judged by the formation of condensed nuclei and caspase activation. Pre-treatment of cells with dexamethasone caused a reduction in staurosporine-induced apoptosis. In addition, dexamethasone also conferred protection against the induction of apoptosis by anticancer agents including camptothecin and etoposide. The protective effect of dexamethasone was dose and time dependent, with maximal protection obtained with concentrations equal to or greater than 100 nM and a pre-incubation period of at least 24h. The earliest significant inhibition was seen with a pre-incubation period of 8h. Co-treatment with the glucocorticoid receptor antagonist RU38486 abolished the effect of dexamethasone, indicating that the protection due to dexamethasone is mediated via this receptor. Dexamethasone was found to induce a time-dependent up-regulation of Bcl-x(L) protein expression. However, the ability of cytochrome c/dATP to activate the caspase cascade in cytosolic extracts of D384 cells was unaffected by prior exposure of the cells to dexamethasone (1 microM) for 48 h. In conclusion, dexamethasone inhibits the induction of apoptosis in astrocytoma cells, probably via an up-regulation of Bcl-x(L), which could prevent cytochrome c release from mitochondria and subsequent caspase activation. Since glucocorticoids are often used in the treatment of gliomas to relieve cerebral oedema, the inhibition of apoptosis by these compounds could potentially interfere with the efficacy of chemotherapeutic drugs.

Animals↗

PubMed2000

Triggering and modulation of apoptosis by oxidative stress.

Cell survival requires multiple factors, including appropriate proportions of molecular oxygen and various antioxidants. Although most oxidative insults can be overcome by the cell's natural defenses, sustained perturbation of this balance may result in either apoptotic or necrotic cell death. Numerous, recent studies have shown that the mode of cell death that occurs depends on the severity of the insult. Oxidants and antioxidants can not only determine cell fate, but can also modulate the mode of cell death. Effects of oxidative stress on components of the apoptotic machinery may mediate this modulation. This review will address some of the current paradigms for oxidative stress and apoptosis, and discuss the potential mechanisms by which oxidants can modulate the apoptotic pathway.

Animals↗

PubMed2000

The most unkindest cut of all: on the multiple roles of mammalian caspases.

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.

Animals↗

PubMed2000

Molecular mechanisms of apoptosis induced by cytotoxic chemicals.

The purpose of this review article is to discuss established molecular mechanisms of apoptosis and their relevance to cell death induced by environmental toxicants. Apoptosis is a highly regulated form of cell death distinguished by the activation of a family of cysteine-aspartate proteases (caspases) that cleave various proteins resulting in morphological and biochemical changes characteristic of this form of cell death. Abundant evidence supports a role for mitochondria in regulating apoptosis. Specifically, it seems that a number of death stimuli target these organelles and stimulate, by an unknown mechanism, the release of several proteins, including cytochrome c. Once released into the cytosol, cytochrome c binds to its adaptor molecule, Apaf-1, which oligomerizes and then activates pro-caspase-9. Caspase-9 can signal downstream and activate pro-caspase-3 and -7. The release of cytochrome c can be influenced by different Bcl-2 family member proteins, including, but not limited to, Bax, Bid, Bcl-2, and Bcl-X(L). Bax and Bid potentiate cytochrome c release, whereas Bcl-2 and Bcl-X(L) antagonize this event. Although toxicologists have traditionally associated cell death with necrosis, emerging evidence suggests that different types of environmental contaminants exert their toxicity, at least in part, by triggering apoptosis. The mechanism responsible for eliciting the pro-apoptotic effect of a given chemical is often unknown, although in many instances mitochondria appear to be key participants. This review describes our current understanding of the role of apoptosis in environmental toxicant-induced cell death, using dioxin, metals (cadmium and methylmercury), organotin compounds, dithiocarbamates, and benzene as specific examples. Finally, we conclude with a critical discussion of the current knowledge in this area and provide recommendations for future directions.

Animals↗

PubMed1999

Apoptosis in human disease: a new skin for the old ceremony?

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.

Apoptosis↗

PubMed1999

Phosphatidylserine exposure during apoptosis is a cell-type-specific event and does not correlate with plasma membrane phospholipid scramblase expression.

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.

Annexin A5↗

PubMed1999

The role of calcium in pre- and postmitochondrial events in tributyltin-induced T-cell apoptosis.

Using a novel dual-channel FACS methodology, the organotin compound TBT (2 microM) was shown to induce rapid (maximal by 3 min) and sustained elevations in intracellular calcium levels [Ca(2+)](i) in Jurkat T cells. This was preceded by mitochondrial hyperpolarization (maximal at 1 min), with subsequent loss of membrane potential, (Deltapsi(m)) over the next 15 min and was associated with the release of mitochondrial cytochrome c and the activation of type II caspases. The activation of the caspases was blocked by calcium chelation with EGTA and/or BAPTA. Interestingly, changes in Deltapsi(m) caused by TBT were not affected by chelation of intra- and extracellular calcium or by performing the experiments in a Ca(2+)-free medium. TBT also caused rapid elevation of [Ca(2+)](i) in cells lacking glycolytic ATP production. Despite this, the loss of Deltapsi(m) and the activation of type II caspases were delayed (maximal by 2 h) in these cells. Further, there was a failure to activate type II caspases in cells treated with TBT in a Ca(2+)-free medium, despite rapid release of mitochondrial cytochrome c. Consequently, these cells evaded the induction of apoptosis and were diverted to delayed necrotic deletion. Taken together, these data strongly suggest that the rapid rise in [Ca(2+)](i) caused by TBT in Jurkat T cells is not directly coupled to the induction of mitochondrial permeability transition, which rather results from a direct interaction of TBT with mitochondrial component(s) controlling pore transition. However, the rise in [Ca(2+)](i) is a prerequisite for postmitochondrial events involved in caspase activation prior to the induction of apoptosis.

Apoptosis↗

PubMed1999

Thermotolerance and cell death are distinct cellular responses to stress: dependence on heat shock proteins.

We tested the hypothesis that heat shock protein (Hsp) induction and cell death are mutually exclusive responses to stress. Despite activation of heat shock transcription factor 1 at temperatures ranging from 40 to 46 degrees C, Hsp72 and Hsp27 were not induced above 42 degrees C. Moreover, cells underwent apoptosis at 44 degrees C and necrosis at 46 degrees C, with mitochondrial cytochrome c release at both temperatures. However, only apoptosis was associated with caspase activation. Treatment of cells with z-VAD-fmk prior to heat shock at 44 degrees C failed to restore Hsp induction despite inhibition of heat-induced apoptosis. Furthermore, accumulation of Hsps after incubation at 42 degrees C rendered the cells resistant to apoptosis. These results suggest that lack of Hsp induction is the cause rather than the consequence of cell death.

Apoptosis↗

PubMed1999

Application of a fluorometric assay to detect caspase activity in thymus tissue undergoing apoptosis in vivo.

To date, in vivo apoptosis within the thymus has been assessed using morphological criteria and/or detection of a DNA ladder indicative of oligonucleosomal fragmentation of the DNA. Here, we have used a fluorometric method to investigate activation of the caspase protease family in the thymus following in vivo induction of apoptosis by injection of the synthetic glucocorticoid hydrocortisone. Cleavage of DEVD-MCA by caspase-3 and other group II caspases releases free MCA which can be detected fluorimetrically. We demonstrate a time-dependent increase in DEVD-MCA cleavage activity within this tissue indicating the activation of caspase-3 like enzymes. This activity was inhibited by the specific group II caspase inhibitor DEVD-CHO. The interpretation of increased caspase activity was confirmed by immunoblot analysis to reveal cleavage of the caspase-3 substrate, fodrin. In addition, agarose gel electrophoresis of the DNA yielded a ladder pattern, confirming the occurrence of apoptosis. This study demonstrates that DEVD-MCA cleavage activity may be a useful quantitative method for the analysis of apoptosis in thymus tissue. It is a relatively rapid procedure not requiring thymocyte isolation or gel electrophoresis and detects fairly early biochemical changes occurring during apoptosis. In the present study we have used this method to demonstrate the involvement of caspases in thymocyte apoptotic death induced in vivo by glucocorticoids. Thus, measurement of caspase activity in thymus tissue may have applications for studying the in vivo effects of immunotoxicants.

Animals↗

PubMed1999

Protease activation in apoptosis induced by MAL.

The proteolytic caspase cascade plays a central role in the signaling and execution steps of apoptosis. This study investigated the activation of different caspases in apoptosis induced by MAL (a folding variant of human alpha-lactalbumin) isolated from human milk. Our results show that the caspase-3-like enzymes, and to a lesser extent the caspase-6-like enzymes, were activated in Jurkat and A549 cells exposed to MAL. Activated caspases subsequently cleaved several protein substrates, including PARP, lamin B, and alpha-fodrin. A broad-range caspase inhibitor, zVAD-fmk, blocked the caspase activation, the cleavage of proteins, and DNA fragmentation, indicating an important role for caspase activation in MAL-induced apoptosis. Since an antagonistic anti-CD95 receptor antibody, ZB4, did not influence the MAL-induced killing, we conclude that this process does not involve the CD95-mediated pathway. While MAL did not directly activate caspases in the cytosol, it colocalized with mitochondria and induced the release of cytochrome c. Thus, these results demonstrate that caspases are activated and involved in apoptosis induced by MAL and that direct interaction of MAL with mitochondria leads to the release of cytochrome c, suggesting that this release is an important step in the initiation and/or amplification of the caspase cascade in these cells.

Amino Acid Chloromethyl Ketones↗

PubMed1999

Radical scavenging compound J 811 inhibits hydrogen peroxide-induced death of cerebellar granule cells.

Oxidative stress is considered to be an important pathophysiological condition to promote cell death in a broad variety of disorders, such as cardiovascular and neurodegenerative diseases. Scavestrogens, structurally derived from estradiol, are potent radical scavengers and inhibitors of iron-induced cell damage in vitro. In this study the potential cytoprotective effects of the so-called scavestrogen estra-1,3,5(10),8-tetraene-3,17alpha-diol, J 811, was tested using rat cerebellar granule cells (CGCs) exposed to 25 or 50 microM hydrogen peroxide (H2O2). H2O2-induced apoptotic cell death was detected by the appearance of high molecular weight DNA fragments and nuclear condensation. The addition of J 811 before or shortly after the exposure to H2O2 prevented CGC apoptosis in a dose-dependent manner. The estrogen receptor antagonist ICI 182.780 failed to prevent the protective effect of J 811, suggesting that the latter is not dependent on estrogen receptor activation. The lack of protection against apoptosis caused by colchicine suggests that J 811 is neither interfering with the activation of caspase-3, nor acting downstream of caspase-3. Therefore, the protective effect observed against H2O2 seems to be upstream caspases activation, pointing to a scavenging action of J 811. Thus the scavestrogen J 811 is a powerful antioxidant able to interfere with radical-mediated cell death and is potentially useful in diseases where reactive oxygen species are involved.

Animals↗

PubMed1999

Caspase involvement in the induction of apoptosis by the environmental toxicants tributyltin and triphenyltin.

Organotin compounds such as tributyltin (TBT) and triphenyltin (TPT) can kill target cells by triggering apoptosis. The mechanism by which these environmental toxicants activate the apoptotic program is currently unclear. We have studied the effect of TBT and TPT in the human Hut-78 and Jurkat T-lymphocyte cell lines. Within 1 h there was a 30-fold increase in caspase activity, as measured by the cleavage of the fluorescent peptide DEVD-AMC. Morphological changes characteristic of apoptosis, such as membrane blebbing and nuclear fragmentation, were readily detectable. Blocking caspase activity with the peptide inhibitor z-VAD-fmk prevented all subsequent apoptotic changes. The optimal concentration range for induction of apoptosis was 0.5 to 5 microM TBT. TPT was also able to trigger caspase activity in the lymphocyte cell lines, but it took over 2 h to detect and occurred at a lower concentration range of 0.01 to 1 microM. Higher concentrations of TBT and TPT caused cell necrosis, and we showed that these concentrations were able to inhibit caspase activity in apoptotic cells. TBT and TPT were able interact with a vicinal thiol compound, similar to the known caspase inhibitor phenylarsine oxide, providing a potential mechanism for caspase inhibition. We propose that vicinal thiol proteins may be a general biological target of these organotin compounds, leading to the induction of caspase activity and apoptosis at low concentrations, and more extensive cell damage and necrotic cell death at higher concentrations.

Apoptosis↗

PubMed1999

Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of jurkat cells.

Activation of pro-caspase-3 is a central event in the execution phase of apoptosis and appears to serve as the convergence point of different apoptotic signaling pathways. Recently, mitochondria were found to play a central role in apoptosis through release of cytochrome c and activation of caspases. Moreover, a sub-population of pro-caspase-3 has been found to be localized to this organelle. In the present study, we demonstrate that pro-caspase-3 is present in the mitochondrial fraction of Jurkat T cells in a complex with the chaperone proteins Hsp60 and Hsp10. Induction of apoptosis with staurosporine led to the activation of mitochondrial pro-caspase-3 and its dissociation from the Hsps which were released from mitochondria. The release of Hsps occurred simultaneously with the release of other mitochondrial intermembrane space proteins including cytochrome c and adenylate kinase, prior to a loss of mitochondrial transmembrane potential. In in vitro systems, recombinant Hsp60 and Hsp10 accelerated the activation of pro-caspase-3 by cytochrome c and dATP in an ATP-dependent manner, consistent with their function as chaperones. This finding suggests that the release of mitochondrial Hsps may also accelerate caspase activation in the cytoplasm of intact cells.

Apoptosis↗

PubMed1999

Tumor radiosensitivity and apoptosis.

With approximately 50% of all cancer patients receiving radiation therapy at some point in their treatment, increasing the sensitivity of tumor cells to the lethal effects of irradiation has the potential to significantly improve the rate of recovery from many malignancies. The major biological determinant of radiotherapy failure is tumor radioresistance. It is well known that tumors from the same histological group and stage of development are extremely heterogeneous in their sensitivity to radiotherapy. There are many factors which could affect tumor radiosensitivity. One cellular mechanism common to various therapeutic regiments, including radiation, is killing tumor cells via apoptosis. However, this killing is not always efficient. In this review the link between tumor sensitivity to radiation treatment and the capacity of tumor cells to be killed by apoptotic mechanisms will be discussed.

Animals↗