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Biomedical subjects

S Orrenius

Publications and source records attributed to S Orrenius.

At least 109 records · Page 6Linked to original sources
PubMed1996

Lamin and beta-tubulin fragmentation precede chromatin degradation in glutamate-induced neuronal apoptosis.

During ischaemic brain injury, glutamate accumulation with overstimulation of postsynaptic glutamate receptors and intracellular Ca2+ overload lead to neuronal death. We have shown previously that delayed neuronal death in cultures of cerebellar granule cells (CGCs) exposed to glutamate occurs by apoptosis. Here, we report that lamin cleavage and dissolution of the microtubule network precede chromatin fragmentation in glutamate-induced CGC apoptosis. Like other events that characterize excitotoxic cell death, degradation of lamins, beta-tubulin and disruption of microtubule architecture is inhibited by the NMDA-receptor antagonist MK-801. Our findings suggest that cleavage of key cytoskeletal elements is an important step in glutamate-induced neuronal apoptosis.

Animals↗
PubMed1996

Proteases in apoptosis.

The interleukin-1 beta-converting enzyme (ICE)-like family proteases have recently been identified as key enzymes in apoptotic cell death. Among these proteases one can identify specific activities which may be involved in cytokine production or in resident protein cleavage. Several factors influence the constitutive apoptotic mechanism and may provide insight into the role of protease(s) in apoptosis. Although it appears that ICE family members play a most important role in promoting apoptotic cell death, evidence has been advanced that other proteases are also involved in sequential or parallel steps of apoptosis. Activation of a particular protease can lead to processing molecules either of the same or different proteases, leading to an activation of a protease cascade. Here we attempt to summarize the current thinking concerning these proteases and their involvement in apoptosis.

Animals↗
PubMed1996

Calcineurin and mitochondrial function in glutamate-induced neuronal cell death.

We have previously reported that glutamate can trigger a succession of necrosis and apoptosis in cerebellar granule cells (CGC). Since specific blockers of the N-methyl-D-aspartate (NMDA) receptor channel prevented both types of cell death, the role of Ca2+-dependent processes in the initiation of glutamate toxicity was further investigated. We examined the possible involvement of mitochondria and the role of the Ca2+/calmodulin-regulated protein phosphatase, calcineurin, in the development of either type of cell death. Cyclosporin A and the more selective calcineurin inhibitor, FK-506, prevented the development of both early necrosis and delayed apoptosis. In addition, cyclosporin A prevented the collapse of mitochondrial membrane potential observed during the exposure to glutamate and the concomitant necrotic phase. When CsA was added immediately after glutamate removal, it also prevented delayed apoptosis of neurons that had survived the necrotic phase. Altogether, these results suggest the involvement of calcineurin and a role for mitochondrial deenergization as early signals in neuronal apoptosis induced by glutamate.

Animals↗
PubMed1996

Chromatin condensation during apoptosis requires ATP.

The processes leading to morphological changes of the chromatin in cells that undergo apoptosis are presently unclear. We have recently shown that chromatin fragmentation and the nuclear morphological changes typically seen in apoptosis were reproduced in an in vitro system comprised of isolated rat thymocyte nuclei incubated in the presence of a lysate from Fas/APO-1-stimulated JURKAT cells [Chow, Weis, Kass, Holmström, Eriksson and Orrenius (1995) FEBS Lett. 364, 134-138]. Using this in vitro system, we now report that the presence of ATP is necessary for chromatin condensation, its movement to the nuclear periphery and apoptotic body formation. In clear contrast, chromatin cleavage into high-molecular-mass and oligonucleosomal-length DNA fragments induced by lysates derived from Fas/APO-1-activated JURKAT cells did not require the presence of ATP. The induction of these morphological changes by ATP could not be substituted by the analogues, adenosine 5'-[beta, gamma-methylene]triphosphate and adenosine 5'-[alpha, beta-methylene]-triphosphate, AMP, cAMP and UTP. However, adenosine 5'-[gamma-thio]triphosphate, and to a lesser degree GTP and ADP, could partially replace ATP in inducing nuclear apoptotic morphological changes. It is concluded that ATP is essential for the morphological changes occurring in nuclei during apoptosis, but not for DNA fragmentation.

Adenosine Triphosphate↗
PubMed1996

Rapid and specific efflux of reduced glutathione during apoptosis induced by anti-Fas/APO-1 antibody.

Although human JURKAT T lymphocytes induced to undergo apoptosis with anti-Fas/APO-1 antibody were observed to rapidly lose reduced glutathione (GSH), increased concentrations of oxidized products were not detectable. Unexpectedly, the reduced tripeptide was instead quantitatively recovered in the incubation medium of the cells. As GSH loss was blocked by bromosulfophthalein and dibromosulfophthalein, known inhibitors of hepatocyte GSH transport, a specific export rather than nonspecific leakiness through plasma membranes is proposed to be responsible. Apoptosis was delayed when GSH-diethylesters were used to elevate intracellular GSH, although the high capacity of the activated efflux system quickly negated the benefit of this treatment. Stimulation of GSH efflux provides a novel mechanism whereby Fas/APO-1 ligation can deplete GSH. We speculate that it enhances the oxidative tonus of a responding cell without requiring an increase in the production of reactive oxygen species.

Apoptosis↗
PubMed1996

CPP32/apopain is a key interleukin 1 beta converting enzyme-like protease involved in Fas-mediated apoptosis.

Cysteine proteases of the interleukin 1 beta Converting Enzyme (ICE)/CED-3 family have been implicated in the effector process of apoptosis in several systems, including Fas-mediated apoptosis. We have recently isolated and partially characterized a protease present in extracts from anti-Fas antibody treated Jurkat T cells that promotes apoptotic changes in isolated nuclei (Schlegel, J., Peters, I., and Orrenius, S. (1995) FEBS Lett. 364, 139-142). We now show that this protease cleaves poly-(ADP-ribose) polymerase (PARP) with high efficiency and specificity. Both PARP proteolysis and the proapoptotic effects of the protease are inhibited by nanomolar concentrations of a selective inhibitor of apopain (CPP32), while an inhibitor of IL-1 beta converting enzyme is much less effective, requiring micromolar concentrations for the inhibition of the isolated protease. Kinetic analysis of the isolated protease reveals kinetic constants similar to those reported for apopain. The isolated protease is recognized by antibodies specific for CPP32/apopain but not by an anti-ICE antibody. Furthermore, a selective inhibitor of apopain prevents Fas-induced apoptosis in intact Jurkat T cells. We therefore conclude that CPP32/apopain is activated in Fas-induced apoptosis.

Amino Acid Sequence↗
PubMed1996

1,10-Phenanthroline stimulates internucleosomal DNA fragmentation in isolated rat-liver nuclei by promoting the redox activity of endogenous copper ions.

Isolated rat-liver nuclei were incubated with a series of membrane-permeable metal-ion-complexing agents and examined for DNA damage. Of the reagents tested, only 1,10-phenanthroline (OP) and neocuproine (NC) were found to induce DNA fragmentation. Agarose-gel electrophoresis of the DNA fragments generated in the presence of OP revealed internucleosomal cleavage, which is widely considered to be a hallmark for the enzymic DNA digestion that occurs during apoptosis. Ascorbate, particularly in the presence of hydrogen peroxide, increased the levels of fragmentation induced by OP. As well as undergoing fragmentation, the DNA from nuclei was also found to contain 8-hydroxydeoxyguanosine, which indicates attack (oxidation) by the hydroxyl radical. Complementary experiments in vitro involving ESR determinations of hydroxyl radical formation and measurements of DNA oxidation under biomimetic conditions demonstrated that Cu2+, but not Fe3+, forms a complex with either OP or NC (but not the other complexing agents tested) that stimulates hydroxyl radical formation and DNA damage in the presence of hydrogen peroxide and ascorbate. It is therefore proposed that OP in the nuclei incubations binds to Cu2+, which exists naturally in chromosomes, forming a complex that promotes hydroxyl-radical-dependent DNA fragmentation. These findings demonstrate the promotion of hydroxyl-radical-mediated DNA damage by endogenous Cu2+ and, perhaps more significantly, demonstrate that the internucleosomal DNA 'laddering' that is often used as an indicator of apoptosis may also result from DNA fragmentation by non-enzymic processes.

Animals↗
PubMed1996

Signal transduction pathways in apoptosis.

Emerging evidence indicates that apoptosis is regulated by some of the same signal transduction pathways previously implicated in other physiological cellular responses, including alterations in intracellular Ca2+ compartmentalization, activation of protein kinases and phosphatases, alteratios in pH and oxidative stress. Interestingly, signals that promote apoptosis in one model can suppress cell death in another, indicating that cellular responses are determined by the intrinsic programming of the cell in question. This review will summarize current knowledge of the signal transduction pathways regulating apoptosis and discuss how they may be coupled to components of the molecular machinery for cell death.

Animals↗
PubMed1996

Apoptosis--molecular mechanisms and biomedical implications.

Apoptosis is a distinct form of cell death of importance in tissue development and homeostasis and in several diseases. This review summarizes current knowledge about the regulation and molecular mechanisms of apoptosis and discusses the potential role of disregulated apoptosis in several major diseases. Finally, we speculate that modulation of apoptosis may be a target in future drug therapy.

Apoptosis↗
PubMed1996

P2x purinoceptor is not important in thymocyte apoptosis.

Since the P2X purinoceptor has been shown to be homologous with RP-2, a gene activated in thymocytes undergoing apoptosis after glucocorticoid treatment, we have investigated the possible role of P2X purinoceptors in thymocyte apoptosis. We report that P2X purinoceptor mRNA is expressed both in untreated rat thymocytes and thymocytes treated with the synthetic glucocorticoid, 6 alpha-methylprednisolone, in a dose that can induce apoptosis. Furthermore, the P2X receptor agonist, extracellular ATP, was unable to trigger apoptosis in rat thymocytes and had no effect on methylprednisolone-induced DNA fragmentation. Finally, the P2X purinoceptor antagonist, suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, had no inhibitory effects on DNA fragmentation caused by methylprednisolone. Thus, our data demonstrate that P2X purinoceptors are not directly involved in glucocorticoid-mediated thymocyte apoptosis.

Adenosine Triphosphate↗
PubMed1996

Cytoskeletal breakdown and apoptosis elicited by NO donors in cerebellar granule cells require NMDA receptor activation.

We have recently demonstrated that nitric oxide (NO) donors can trigger either apoptosis or necrosis of neurons as a function of the intensity of the exposure. Here, we show that the apoptosis induced by the NO donors S-nitrosocysteine (SNOC) or S-nitroso-N-acetyl-penicillamine (SNAP) in cultured cerebellar granule cells (CGCs) depends on NMDA receptor (NMDA-R) activation leading to intracellular Ca2+ overload. Early dissolution of actin filaments followed by breakdown of microtubules and nuclear lamins preceded the appearance of typical apoptotic features. NO donors induced tyrosine nitration in neurons, in a small population of contaminating astrocytes, and in cultures of cerebellar astroglial cells. However, astrocytes neither displayed cytoskeletal alterations nor underwent apoptosis. Competitive and uncompetitive NMDA receptor antagonists, such as D-aminophosphonovaleric acid and MK-801, did not influence tyrosine nitration but prevented the accumulation of intracellular Ca2+, cytoskeletal breakdown, and apoptosis induced by either SNOC or SNAP in CGCs. Taken together, these data strongly suggest that Ca2+ influx through NMDA-R-gated ion channels is a critical event in CGC apoptosis induced by NO donors.

Animals↗
PubMed1996

Short exposures to tunicamycin induce apoptosis in SV40-transformed but not in normal human fibroblasts.

When SV40-transformed fibroblasts (line 90VAVI) were exposed to tunicamycin, an inhibitor of N-linked glycosylation, an extensive cell death occured compared with untransformed fibroblasts. A considerable cell loss was obtained within 24 h after tunicamycin addition, and after 72 h there were hardly any virus-transformed cells alive. A 2-h pulse treatment with tunicamycin was found to be almost as effective as a continuous 48-h treatment in killing the cells. Even such a short exposure as 7 min resulted in a drastically decreased cell viability (54%). The morphology of the dying tunicamycin-treated 90VAVI cells suggested that they were undergoing apoptosis. This was also supported by the appearance of nuclear condensation, as assayed by propidium iodide uptake, which was detectable within 2 h after tunicamycin addition. Furthermore, analysis of DNA from tunicamycin-treated 90VAVI cells by field inversion gel electrophoresis revealed DNA degradation into 50 kbp fragments within 2 h, and conventional agarose gel electrophoresis showed 'DNA laddering', indicating internucleosomal DNA cleavage, detectable after 36 h. Together with the finding that tunicamycin within seconds caused an elevation of [Ca2+]i, a well documented early feature of apoptosis in many experimental systems, these results strongly suggest that tunicamycin-induced cell death in 90VAVI is due to apoptosis. The short tunicamycin exposure required to trigger cell death in 90VAVI indicates that the apoptotic process is irreversibly induced soon after its addition. It seems unlikely that the pool of one or several specific N-linked glycoproteins could be depleted during such a short period. Instead the overall accumulation of unglycosylated proteins in ER might contribute to the apoptotic response in 90VAVI. Tunicamycin also killed and induced DNA degradation in the breast cancer cell line MDA-231.

Apoptosis↗
PubMed1996

Intracellular redox changes during apoptosis.

In the current paradigm for apoptotic cell death, the activity of a family of proteases related to interleukin 1-beta converting enzyme (ICE) orchestrates the multiple downstream events (such as cell shrinkage and chromatin degradation) that comprise apoptosis. A variety of stimuli can induce this type of cell death. One of the most reproducible inducers is mild oxidative stress, although it is unclear how an oxidative stimulus activates ICE-like proteases. Oxidative modification of proteins and lipids have also been observed in cells undergoing apoptosis in response to non-oxidative stimuli, suggesting that intracellular oxidation may be a general feature of the effector phase of apoptosis. However, attempts to consistently detect a requirement for reactive oxygen species in apoptosis have been inconclusive. Recent experiments revealing that apoptosis is typically accompanied by a depletion of intracellular reduced glutathione (GSH) are also discussed. In JURKATT lymphocytes treated with antibodies to the Fas/APO-1 surface receptor, this depletion results from an accelerated efflux of the reduced thiol rather than any intracellular oxidation. As GSH is the most abundant cytosolic reductant, we propose that its efflux may provide a non-oxidative mechanism by which the reducing environment of apoptotic cells is lost. An increase in oxidative damage to proteins and lipids would then result even in the absence of an increase in the production of oxidants. This may explain the seemingly contradictory findings that increased oxidative stress is not required for apoptosis even though antioxidants often inhibit the process and peroxidised products accumulate in apoptotic cells.

Journal Article↗
PubMed1996

The role of calcium in the regulation of apoptosis.

The recognition that apoptosis is regulated by an evolutionarily conserved set of polypeptides from the nematode Caenorhabditis elegans to humans suggests that a conserved set of biochemical mechanisms may also he involved in the response. Work from a number of independent laboratories suggests that alterations in cytosolic Ca2+ homeostasis represent one such candidate mechanism, and molecular targets for Ca2+ are now being identified. This review will summarize what is known about the role of Ca2+ in the regulation of apoptosis and discuss how Ca2+ might interact with some of the other biochemical signals implicated in cell death.

Animals↗
PubMed1995

Constitutive nuclear NF kappa B/rel DNA-binding activity of rat thymocytes is increased by stimuli that promote apoptosis, but not inhibited by pyrrolidine dithiocarbamate.

Rat thymocytes spontaneously undergo apoptotic death in cell culture, and are also sensitive to the induction of apoptosis by various stimuli. We show that unstimulated thymocytes constitutively express a p50-containing nuclear factor kappa B (NF kappa B)/rel DNA-binding activity in their nuclei. When the cells were fractionated by density-gradient centrifugation this activity was found to be most pronounced in immature CD4+8+ thymocytes, the cell population that undergoes selection by apoptosis in vivo and that is most sensitive to external inducers of apoptosis in vitro. The intensity of the NF kappa B/rel protein-DNA complex was significantly enhanced 30 min after exposing thymocytes to methylprednisolone or etoposide, two agents well known to induce apoptosis in these cells. Expression of this DNA-binding activity therefore correlates with the subsequent occurrence of apoptosis. By analogy to other systems, it has been suggested that antioxidants such as pyrrolidine dithiocarbamate (PDTC) inhibit thymocyte apoptosis by preventing the activation of an NF kappa B/rel transcription factor. However, we have found that etoposide induces a very similar enhancement of the NF kappa B/rel DNA-binding activity in the presence or absence of PDTC, despite a pronounced inhibition of apoptotic DNA fragmentation in the former situation. Dithiocarbamates therefore do not exert their anti-apoptotic activity in thymocytes by inhibiting the activation of this transcription factor.

Animals↗
PubMed1995

Dithiocarbamates induce apoptosis in thymocytes by raising the intracellular level of redox-active copper.

Dithiocarbamates are metal-chelating compounds that can exert either pro-oxidant or antioxidant effects in different situations. They have recently been found to potently inhibit apoptotic cell death, an activity attributed to their antioxidant action. However, when thymocytes were exposed to pyrrolidine dithiocarbamate, an oxidation of the glutathione pool occurred within 90 min. Longer incubation resulted in cell shrinkage, chromatin fragmentation, glutathione depletion, and eventual cell lysis, which is typical of apoptosis in these cells. These changes were inhibited by inclusion of non-permeable metal chelators in the incubation medium, suggesting that pyrrolidine dithiocarbamate exerts its toxic effect by transporting a redox-active metal into the cell. This was directly confirmed when sustained 8-fold elevations of intracellular copper were detected after addition of pyrrolidine dithiocarbamate. In agreement with this, supplementation of the incubation medium with submicromolar concentrations of copper significantly potentiated pyrrolidine dithiocarbamate toxicity. We conclude that pyrrolidine dithiocarbamate exerts a powerful pro-oxidant effect on thymocytes due to its ability to transport external redox-active copper into cells. The resulting increase in glutathione disulfide may also explain the temporary anti-apoptotic activity of this compound described in other systems.

Animals↗