Showing papers on "Apoptosis published in 2001"

Proliferation, cell cycle and apoptosis in cancer

Abstract: Beneath the complexity and idiopathy of every cancer lies a limited number of 'mission critical' events that have propelled the tumour cell and its progeny into uncontrolled expansion and invasion One of these is deregulated cell proliferation, which, together with the obligate compensatory suppression of apoptosis needed to support it, provides a minimal 'platform' necessary to support further neoplastic progression Adroit targeting of these critical events should have potent and specific therapeutic consequences

SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase

Abstract: Jun N-terminal kinase (JNK) is a stress-activated protein kinase that can be induced by inflammatory cytokines, bacterial endotoxin, osmotic shock, UV radiation, and hypoxia. We report the identification of an anthrapyrazolone series with significant inhibition of JNK1, -2, and -3 (Ki = 0.19 μM). SP600125 is a reversible ATP-competitive inhibitor with >20-fold selectivity vs. a range of kinases and enzymes tested. In cells, SP600125 dose dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-γ, TNF-α, and prevented the activation and differentiation of primary human CD4 cell cultures. In animal studies, SP600125 blocked (bacterial) lipopolysaccharide-induced expression of tumor necrosis factor-α and inhibited anti-CD3-induced apoptosis of CD4+ CD8+ thymocytes. Our study supports targeting JNK as an important strategy in inflammatory disease, apoptotic cell death, and cancer.

The expanding role of mitochondria in apoptosis

Abstract: The initial insight into the genetic basis of apoptosis, or programmed cell death, was gained from ingenious studies of the roundworm Caenorhabditis elegans (for review, see Horvitz 1999). These studies revealed a linear pathway whereby the products of two genes, designated Ced-3 andCed-4, were necessary and sufficient to trigger the perfectly timed and orchestrated death of 131 preordained cells during development. The relevance of this pathway to higher animals was established by the discovery of apparent mammalian orthologs of these genes and the demonstration that the mammalian Ced-3-related genes encode proteases (designated caspases) whose activities are responsible for the morphological changes characteristic of apoptosis (for review, see Hengartner 2000). The complexity of the apoptotic program began to increase with the discovery of Bcl-2, a gene whose product causes resistance to apoptosis in lymphocytes (Vaux et al. 1988; McDonnell et al. 1989). Bcl-2 was shown to correct partially the phenotype of a C. elegans mutation in Ced-9, a cell survival gene that functions upstream of Ced-4 and Ced-3 (Vaux et al. 1992). This finding suggested an apparent one-for-one correlation between the C. elegans and mammalian proand antiapoptotic pathways. However, this correlation did not explain two observations made in mammalian cells. First, the Bcl-2 protein was found on the membrane of mitochondria, which were not implicated in C. elegans apoptosis; and second, apoptotic changes could be produced in Xenopus laevis oocyte extracts only when a membrane fraction enriched in mitochondria was present (Hockenberry et al. 1990; Newmeyer et al. 1994). The complex role of mitochondria in mammalian cell apoptosis came into focus when biochemical studies identified several mitochondrial proteins that are able to activate cellular apoptotic programs directly (Liu et al. 1996; Susin et al. 1999; Du et al. 2000; Verhagen et al. 2000; Li et al. 2001). Normally, these proteins reside in the intermembrane space of mitochondria. In response to a variety of apoptotic stimuli, they are released to the cytosol and/or the nucleus. They promote apoptosis either by activating caspases and nucleases or by neutralizing cytosolic inhibitors of this process. A complex picture has emerged in which mitochondrial and cytosolic proapoptotic proteins interact with antiapoptotic proteins with each cell’s life or death hanging in the balance. This review summarizes the recent data on the expanding and complex role of mitochondria in apoptosis.

Gadd153 Sensitizes Cells to Endoplasmic Reticulum Stress by Down-Regulating Bcl2 and Perturbing the Cellular Redox State

Abstract: gadd153, also known as chop, is a highly stress-inducible gene that is robustly expressed following disruption of homeostasis in the endoplasmic reticulum (ER) (so-called ER stress). Although all reported types of ER stress induce expression of Gadd153, its role in the stress response has remained largely undefined. Several studies have correlated Gadd153 expression with cell death, but a mechanistic link between Gadd153 and apoptosis has never been demonstrated. To address this issue we employed a cell model system in which Gadd153 is constitutively overexpressed, as well as two cell lines in which Gadd153 expression is conditional. In all cell lines, overexpression of Gadd153 sensitized cells to ER stress. Investigation of the mechanisms contributing to this effect revealed that elevated Gadd153 expression results in the down-regulation of Bcl2 expression, depletion of cellular glutathione, and exaggerated production of reactive oxygen species. Restoration of Bcl2 expression in Gadd153-overexpressing cells led to replenishment of glutathione and a reduction in levels of reactive oxygen species, and it protected cells from ER stress-induced cell death. We conclude that Gadd153 sensitizes cells to ER stress through mechanisms that involve down-regulation of Bcl2 and enhanced oxidant injury.

Endonuclease G is an apoptotic DNase when released from mitochondria

Abstract: Nucleosomal fragmentation of DNA is a hallmark of apoptosis (programmed cell death), and results from the activation of nucleases in cells undergoing apoptosis. One such nuclease, DNA fragmentation factor (DFF, a caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD)), is capable of inducing DNA fragmentation and chromatin condensation after cleavage by caspase-3 (refs 2,3,4). However, although transgenic mice lacking DFF45 or its caspase cleavage site have significantly reduced DNA fragmentation, these mice still show residual DNA fragmentation and are phenotypically normal. Here we report the identification and characterization of another nuclease that is specifically activated by apoptotic stimuli and is able to induce nucleosomal fragmentation of DNA in fibroblast cells from embryonic mice lacking DFF. This nuclease is endonuclease G (endoG), a mitochondrion-specific nuclease that translocates to the nucleus during apoptosis. Once released from mitochondria, endoG cleaves chromatin DNA into nucleosomal fragments independently of caspases. Therefore, endoG represents a caspase-independent apoptotic pathway initiated from the mitochondria.

Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death

Abstract: Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosome-a caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation. Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodies-the very first wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.

Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I

Abstract: The execution phase of apoptosis is characterized by marked changes in cell morphology that include contraction and membrane blebbing. The actin-myosin system has been proposed to be the source of contractile force that drives bleb formation, although the biochemical pathway that promotes actin-myosin contractility during apoptosis has not been identified. Here we show that the Rho effector protein ROCK I, which contributes to phosphorylation of myosin light-chains, myosin ATPase activity and coupling of actin-myosin filaments to the plasma membrane, is cleaved during apoptosis to generate a truncated active form. The activity of ROCK proteins is both necessary and sufficient for formation of membrane blebs and for re-localization of fragmented DNA into blebs and apoptotic bodies.

Endothelial Apoptosis as the Primary Lesion Initiating Intestinal Radiation Damage in Mice

Abstract: Gastrointestinal (GI) tract damage by chemotherapy or radiation limits their efficacy in cancer treatment. Radiation has been postulated to target epithelial stem cells within the crypts of Lieberkuhn to initiate the lethal GI syndrome. Here, we show in mouse models that microvascular endothelial apoptosis is the primary lesion leading to stem cell dysfunction. Radiation-induced crypt damage, organ failure, and death from the GI syndrome were prevented when endothelial apoptosis was inhibited pharmacologically by intravenous basic fibroblast growth factor (bFGF) or genetically by deletion of the acid sphingomyelinase gene. Endothelial, but not crypt, cells express FGF receptor transcripts, suggesting that the endothelial lesion occurs before crypt stem cell damage in the evolution of the GI syndrome. This study provides a basis for new approaches to prevent radiation damage to the bowel.

ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis

Abstract: Apoptosis signal‐regulating kinase (ASK) 1 is activated in response to various cytotoxic stresses including TNF, Fas and reactive oxygen species (ROS) such as H2O2, and activates c‐Jun NH2‐terminal kinase (JNK) and p38. However, the roles of JNK and p38 signaling pathways during apoptosis have been controversial. Here we show that by deleting ASK1 in mice, TNF‐ and H2O2‐induced sustained activations of JNK and p38 are lost in ASK1 −/− embryonic fibroblasts, and that ASK1 −/− cells are resistant to TNF‐ and H2O2‐induced apoptosis. TNF‐ but not Fas‐induced apoptosis requires ROS‐dependent activation of ASK1–JNK/p38 pathways. Thus, ASK1 is selectively required for TNF‐ and oxidative stress‐induced sustained activations of JNK/p38 and apoptosis.

The mitochondrion in apoptosis: how pandora's box opens

Abstract: There is widespread agreement that mitochondria have a function in apoptosis, but the mechanisms behind their involvement remain controversial Here we suggest that opening of a multiprotein complex called the mitochondrial permeability transition pore complex is sufficient (and, usually, necessary) for triggering apoptosis

Erythropoietin prevents neuronal apoptosis after cerebral ischemia and metabolic stress

Abstract: Erythropoietin (EPO) promotes neuronal survival after hypoxia and other metabolic insults by largely unknown mechanisms. Apoptosis and necrosis have been proposed as mechanisms of cellular demise, and either could be the target of actions of EPO. This study evaluates whether antiapoptotic mechanisms can account for the neuroprotective actions of EPO. Systemic administration of EPO (5,000 units/kg of body weight, i.p.) after middle-cerebral artery occlusion in rats dramatically reduces the volume of infarction 24 h later, in concert with an almost complete reduction in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling of neurons within the ischemic penumbra. In both pure and mixed neuronal cultures, EPO (0.1–10 units/ml) also inhibits apoptosis induced by serum deprivation or kainic acid exposure. Protection requires pretreatment, consistent with the induction of a gene expression program, and is sustained for 3 days without the continued presence of EPO. EPO (0.3 units/ml) also protects hippocampal neurons against hypoxia-induced neuronal death through activation of extracellular signal-regulated kinases and protein kinase Akt-1/protein kinase B. The action of EPO is not limited to directly promoting cell survival, as EPO is trophic but not mitogenic in cultured neuronal cells. These data suggest that inhibition of neuronal apoptosis underlies short latency protective effects of EPO after cerebral ischemia and other brain injuries. The neurotrophic actions suggest there may be longer-latency effects as well. Evaluation of EPO, a compound established as clinically safe, as neuroprotective therapy in acute brain injury is further supported.

A novel influenza A virus mitochondrial protein that induces cell death

Abstract: While searching for alternative reading-frame peptides encoded by influenza A virus that are recognized by CD8+ T cells, we found an abundant immunogenic peptide encoded by the +1 reading frame of PB1. This peptide derives from a novel conserved 87-residue protein, PB1-F2, which has several unusual features compared with other influenza gene products in addition to its mode of translation. These include its absence from some animal (particularly swine) influenza virus isolates, variable expression in individual infected cells, rapid proteasome-dependent degradation and mitochondrial localization. Exposure of cells to a synthetic version of PB1-F2 induces apoptosis, and influenza viruses with targeted mutations that interfere with PB1-F2 expression induce less extensive apoptosis in human monocytic cells than those with intact PB1-F2. We propose that PB1-F2 functions to kill host immune cells responding to influenza virus infection.

Growth retardation and increased apoptosis in mice with homozygous disruption of the akt1 gene

Abstract: The serine/threonine kinase Akt has been implicated in the control of cell survival and metabolism. Here we report the disruption of the most ubiquitously expressed member of the akt family of genes, akt1, in the mouse. Akt1−/− mice are viable but smaller when compared to wild-type littermates. In addition, the life span of Akt1−/− mice, upon exposure to genotoxic stress, is shorter. However, Akt1−/− mice do not display a diabetic phenotype. Increased spontaneous apoptosis in testes, and attenuation of spermatogenesis is observed in Akt1−/− male mice. Increased spontaneous apoptosis is also observed in the thymi of Akt1−/− mice, and Akt1−/− thymocytes are more sensitive to apoptosis induced by γ-irradiation and dexamethasone. Finally, Akt1−/− mouse embryo fibroblasts (MEFs) are more susceptible to apoptosis induced by TNF, anti-Fas, UV irradiation, and serum withdrawal.

Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing

Abstract: Increased phosphorylation of myosin light chain (MLC) is necessary for the dynamic membrane blebbing that is observed at the onset of apoptosis. Here we identify ROCK I, an effector of the small GTPase Rho, as a new substrate for caspases. ROCK I is cleaved by caspase-3 at a conserved DETD1113/G sequence and its carboxy-terminal inhibitory domain is removed, resulting in deregulated and constitutive kinase activity. ROCK proteins are known to regulate MLC-phosphorylation, and apoptotic cells exhibit a gradual increase in levels of phosphorylated MLC concomitant with ROCK I cleavage. This phosphorylation, as well as membrane blebbing, is abrogated by inhibition of caspases or ROCK proteins, but both processes are independent of Rho activity. We also show that expression of active truncated ROCK I induces cell blebbing. Thus, activation of ROCK I by caspase-3 seems to be responsible for bleb formation in apoptotic cells.

Heat-shock protein 70 antagonizes apoptosis-inducing factor.

Abstract: Heat-shock protein 70 (Hsp70) has been reported to block apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome, the Apaf-1/cytochrome c/caspase-9 activation complex [1,2]. Here we show that overexpression of Hsp70 protects Apaf-1-/- cells against death induced by serum withdrawal, indicating that Apaf-1 is not the only target of the anti-apoptotic action of Hsp70. We investigated the effect of Hsp70 on apoptosis mediated by the caspase-independent death effector apoptosis inducing factor (AIF), which is a mitochondrial intermembrane flavoprotein [3,4]. In a cell-free system, Hsp70 prevented the AIF-induced chromatin condensation of purified nuclei. Hsp70 specifically interacted with AIF, as shown by ligand blots and co-immunoprecipitation. Cells overexpressing Hsp70 were protected against the apoptogenic effects of AIF targeted to the extramitochondrial compartment. In contrast, an anti-sense Hsp70 complementary DNA, which reduced the expression of endogenous Hsp70, increased sensitivity to the lethal effect of AIF. The ATP-binding domain of Hsp70 seemed to be dispensable for inhibiting cell death induced by serum withdrawal, AIF binding and AIF inhibition, although it was required for Apaf-1 binding. Together, our data indicate that Hsp70 can inhibit apoptosis by interfering with target proteins other than Apaf-1, one of which is AIF.

NF-κB Signals Induce the Expression of c-FLIP

Abstract: Activation of the transcription factor NF-κB is a major effector of the inducible resistance to death receptor-mediated apoptosis. Previous evidence indicates that the combined transcriptional activation of TRAF-1, TRAF-2, IAP-1, and IAP-2 is required to suppress cell death by tumor necrosis factor (TNF). Here we show that NF-κB activation upregulates the caspase 8 inhibitor FLIP, resulting in increased resistance to Fas ligand (FasL) or TNF. Restoration of either the full-length 55-kDa long form of FLIP or an alternatively spliced short form of FLIP in NF-κB null cells inhibits TNF- and FasL-induced cell death efficiently, whereas the expression of IAP or TRAF family members only partially rescues cells from death. Resistance to either FasL- or TNF-induced apoptosis is overcome when cells are incubated in the presence of the protein synthesis inhibitor cycloheximide. This treatment leads to the rapid downregulation of FLIP but not to that of TRAF2. Our findings suggest that FLIP is an important mediator of NF-κB-controlled antiapoptotic signals.

Inhibition of JNK activation through NF-κB target genes

Abstract: The proinflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) regulates immune responses, inflammation and programmed cell death (apoptosis). The ultimate fate of a cell exposed to TNF-alpha is determined by signal integration between its different effectors, including IkappaB kinase (IKK), c-Jun N-terminal protein kinase (JNK) and caspases. Activation of caspases is required for apoptotic cell death, whereas IKK activation inhibits apoptosis through the transcription factor NF-kappaB, whose target genes include caspase inhibitors. JNK activates the transcription factor c-Jun/AP-1, as well as other targets. However, the role of JNK activation in apoptosis induced by TNF-alpha is less clear. It is unknown whether any crosstalk occurs between IKK and JNK, and, if so, how it affects TNF-alpha-induced apoptosis. We investigated this using murine embryonic fibroblasts that are deficient in either the IKKbeta catalytic subunit of the IKK complex or the RelA/p65 subunit of NF-kappaB. Here we show that in addition to inhibiting caspases, the IKK/NF-kappaB pathway negatively modulates TNF-alpha-mediated JNK activation, partly through NF-kappaB-induced X-chromosome-linked inhibitor of apoptosis (XIAP). This negative crosstalk, which is specific to TNF-alpha signalling and does not affect JNK activation by interleukin-1 (IL-1), contributes to inhibition of apoptosis.

An Anti-apoptotic Protein Human Survivin is a Direct Inhibitor of Caspase-3 and -7

Abstract: Survivin, an apoptosis inhibitor/cell-cycle regulator, is critically required for suppression of apoptosis and ensuring normal cell division in the G2/M phase of the cell cycle. It is highly expressed in a cell cycle-regulated manner and localizes together with caspase-3 on microtubules within centrosomes. Whether survivin is a physiologically relevant caspase inhibitor has been unclear due to the difficulties with obtaining correctly folded survivin and finding the right conditions for inhibition assay. In this study, recombinant, active human survivin was expressed in Escherichia coli and purified to homogeneity. The protein, existing as a homodimer in solution, binds caspase-3 and -7 tightly with dissociation constants of 20.9 and 11.5 nM, respectively, when evaluated by surface plasmon resonance spectroscopy. Consistently, survivin potently inhibits the cleavage of a physiological substrate poly(ADP-ribose) polymerase and an artificial tetrapeptide by caspase-3 and -7 in vitro with apparent inhibition constants of 36.0 and 16.5 nM, respectively. The data suggest that sequestering caspase-3 and -7 in inhibited states on microtubules is at least one mechanism of survivin in the suppression of default apoptosis in the G2/M phase. The localization of survivin on microtubules, which is essential for its function, should increase the protective activity at the action site.