Harm's way: polyglutamine repeats and the activation of an apoptotic pathway.
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Apoptosis↗
Biomedical subjects
D R Green
Publications and source records attributed to D R Green.
Mitochondria and apoptosis: HQ or high-security prison?
Whether we view the mitochondria as the headquarters for the leader of a crack suicide squad or as a prison for the leader of a militant coup, the role of the mitochondria in the apoptotic process is now well established. During apoptosis the integrity of the mitochondria is breeched, the mitochondrial transmembrane potential drops, the electron transport chain is disrupted. and proteins from the mitochondrial intermembrane space (MIS) such as cytochrome c are released into the cytosol, although not necessarily in that order. In the cytosol, cytochrome c forms part of a proteinaceous complex that directly activates caspase-9, one of the apical enzymes responsible for the dismantling of the cell. In this way a mitochondrial factor which is normally locked away from the rest of the cell can directly trigger apoptosis. The need to regulate the release of cytochrome c suggests that the mitochondria may be the decision center for whether a cell lives or dies. Various hypotheses have been formulated to explain how proteins of the MIS are released and how this process is regulated. These include the Bcl-2-regulated opening of a permeability transition pore or an increase in mitochondrial transmembrane potential followed by outer membrane rupture. It remains to be clarified which mitochondria specific events are essential for apoptosis and which are merely consequences of apoptosis.
Animals↗
T cells are just dying to accept grafts.
Two papers suggest that apoptosis of alloreactive T cells is required for induction of peripheral transplantation tolerance. These findings raise new questions about how lymphocytes reach from beyond the grave to influence the immune response (pages 1298-1302 and 1303-1307).
Apoptosis↗
Characterization of Apt- cell lines exhibiting cross-resistance to glucocorticoid- and Fas-mediated apoptosis.
Apoptosis induction by staurosporine, ceramide, and Fas stimulation was investigated in the mouse thymoma cell line W7.2 and a panel of dexamethasone (dex)-resistant W7.2 mutant cell lines, Apt3.8, Apt4.8 and Apt5.8, and a Bcl-2 transfected W7.2 cell line (Wbcl2). While W7. 2 cells were found to be sensitive to these apoptosis inducers, the Apt- mutants and Wbcl2 cells were shown to be resistant to some or all of the treatments. Specifically, all three Apt- mutants and Wbcl2 cells were found to be resistant to ceramide and Fas-mediated apoptosis, whereas, Apt4.8 and Apt5.8 were sensitive to staurosporine-induced apoptosis under conditions in which Apt3.8 and Wbcl2 cells were resistant. Measurements of caspase activity and cytochrome c release in cytosolic extracts of dex and staurosporine-treated cells indicated that the recessive Apt- mutations effect steps upstream of mitochondrial dysfunction. Steady-state RNA levels of apoptosis-associated gene transcripts showed that the observed differential resistance of the Apt- cell lines could not be explained by altered expression of numerous Bcl-2 or Fas related genes. Transient transfection of human Fas gene coding sequences into the Apt- mutants and Wbcl2 cells did not induce apoptosis, even though these same cell lines were sensitive to ectopic expression of the FADD and caspase 8 genes. Taken together, these data provide genetic evidence for the existence of shared components in the dex- and Fas-mediated apoptotic pathways in W7.2 cells.
Animals↗
Fas ligand, death gene.
The concept of death genes goes back to the early days of programmed cell death, when a researcher's model system was required to be dependent on transcription of the dying cell in order to qualify as apoptosis. In 1987 Andrew Wyllie,1 one of the pioneers of cell death research, outlined four 'cardinal elements' of apoptosis: one of which was a requirement for macromolecular synthesis. In the following years the complexity of the apoptotic process has become evident and while it is now clear that apoptosis does not have to rely on gene expression, the idea of death genes remains. Induction of an apoptotic cascade via activation of caspases, selective release of mitochondrial proteins and further activation of caspases, can be stimulated by engagement of the Fas surface molecule via membrane bound or soluble forms of Fas ligand (FasL). The FasL gene, which is often transcriptionally inactive, becomes activated in many forms of transcription/translation dependent apoptosis. Here we will discuss FasL as a candidate death gene.
Apoptosis↗
Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to Fas ligand induction and cell death.
The JNK pathway modulates AP-1 activity. While in some cells it may have proliferative and protective roles, in neuronal cells it is involved in apoptosis in response to stress or withdrawal of survival signals. To understand how JNK activation leads to apoptosis, we used PC12 cells and primary neuronal cultures. In PC12 cells, deliberate JNK activation is followed by induction of Fas ligand (FasL) expression and apoptosis. JNK activation detected by c-Jun phosphorylation and FasL induction are also observed after removal of either nerve growth factor from differentiated PC12 cells or KCl from primary cerebellar granule neurons (CGCs). Sequestation of FasL by incubation with a Fas-Fc decoy inhibits apoptosis in all three cases. CGCs derived from gld mice (defective in FasL) are less sensitive to apoptosis caused by KCl removal than wild-type neurons. In PC12 cells, protection is also conferred by a c-Jun mutant lacking JNK phosphoacceptor sites and a small molecule inhibitor of p38 mitogen-activated protein kinase and JNK, which inhibits FasL induction. Hence, the JNK-to-c-Jun-to-FasL pathway is an important mediator of stress-induced neuronal apoptosis.
Animals↗
Autospecific gammadelta thymocytes that escape negative selection find sanctuary in the intestine.
alphabeta or gammadelta thymocytes whose T-cell receptors (TCRs) recognize endogenously expressed antigens (Ag) are autospecific and, thus, potentially self-reactive. In the thymus, such T cells are eliminated during T-cell development through a process known as negative selection. As a model of negative selection of gammadelta T cells, we have used G8 gammadelta-T cell transgenic mice, which express a gammadelta TCR that recognizes the nonpolymorphic MHC class I TL(b) molecule. Here, we demonstrate that negative selection of autospecific gammadelta T cells is almost complete in the adult thymus but is markedly attenuated in the neonatal thymus. A consequence of this attenuated negative selection is that potentially self-reactive gammadelta thymocytes are allowed to escape negative selection, undergo extrathymic differentiation, and find sanctuary in the intestinal epithelium. Interestingly, the ability of these potentially self-reactive gammadelta T cells to find sanctuary requires both the intestinal epithelial environment and the extrathymic presence of the self-Ag. The implications of these findings on the development and persistence of autoreactive T cells in autoimmune disease are discussed.
Aging↗
The regulation of apoptotic cell death.
Apoptosis is a fundamental biological phenomenon in which the death of a cell is genetically and biochemically regulated. Different molecules are involved in the regulation of the apoptotic process. Death receptors, coupled to distinct members of the caspases as well as other adapter molecules, are involved in the initiation of the stress signals (The Indictment). Members of the Bcl-2 family control at the mitochondrial level the decision between life and death (The Judgement). The effector caspases are responsible for all morphological and biochemical changes related to apoptosis including the "eat-me" signals perceived by phagocytes and neighboring cells (The Execution). Finally, apoptosis would have little biological significance without the recognition and removal of the dying cells (The Burial).
Apoptosis↗
Granzyme B mimics apical caspases. Description of a unified pathway for trans-activation of executioner caspase-3 and -7.
Granzyme B (GrB) is predicted to trigger apoptosis by activating preferred caspases, but the zymogens that are directly processed by the granzyme and the requirements for these interactions remain unclarified. We examined this dilemma by comparing the kinetics and pattern of GrB-mediated activation of the executioner caspase-7 in vitro and in vivo. GrB rapidly activates procaspase-7 in vitro by cleaving between the large and small subunits leaving the propeptide intact. During GrB-mediated apoptosis, the caspase-7 propeptide is removed and cleavage occurs between the subunits. Strikingly, caspase-7 is unprocessed in caspase-3-deficient MCF-7 cells exposed to GrB but is rapidly activated when the cells are solubilized. Transfection with caspase-3 restores the removal of the caspase-7 propeptide and the capacity of GrB to subsequently activate the caspase. The data suggest that GrB activates caspase-3, which then removes the propeptide of caspase-7 allowing activation by GrB. Thus GrB initiates the death pathway by processing the accessible caspase-3, and the caspase-7 propeptide regulates trans-activation of the zymogen by granzyme. As a consequence, two proteases, caspase-3 and GrB, are required to activate procaspase-7.
Apoptosis↗
Pro-caspase-3 is a major physiologic target of caspase-8.
The apoptotic signal triggered by ligation of members of the death receptor family is promoted by sequential activation of caspase zymogens. We show here that in a purified system, the initiator caspases-8 and -10 directly process the executioner pro-caspase-3 with activation rates (kcat/Km) of 8.7 x 10(5) and 2.8 x 10(5) M-1 s-1, respectively. These rates are of sufficient magnitude to indicate direct processing in vivo. Differentially processed forms of caspase-3 that accumulate during its activation have similar rates of activation, activities, and specificities. The pattern and rate of caspase-8 induced activation of pro-caspase-3 in cytosolic extracts was the same as in a purified system. Moreover, immunodepletion of a putative intermediary in the pathway to activation, pro-caspase-9, was without consequence. Taken together these data demonstrate that the initiator caspase-8 can directly activate pro-caspase-3 without the requirement for an accelerator. The in vitro data thus help to deconvolute previous in vivo transfection studies which have debated the role of a direct versus indirect transmission of the apoptotic signal generated by ligation of death receptors.
Apoptosis↗
Apoptotic pathways: the roads to ruin.
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Animals↗
Mitochondria and apoptosis.
A variety of key events in apoptosis focus on mitochondria, including the release of caspase activators (such as cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, and participation of pro- and antiapoptotic Bcl-2 family proteins. The different signals that converge on mitochondria to trigger or inhibit these events and their downstream effects delineate several major pathways in physiological cell death.
Animals↗
Bcr-Abl exerts its antiapoptotic effect against diverse apoptotic stimuli through blockage of mitochondrial release of cytochrome C and activation of caspase-3.
Bcr-Abl expression in leukemic cells is known to exert a potent effect against apoptosis due to antileukemic drugs, but its mechanism has not been elucidated. Recent reports have indicated that a variety of apoptotic stimuli cause the preapoptotic mitochondrial release of cytochrome c (cyt c) into cytosol, which mediates the cleavage and activity of caspase-3 involved in the execution of apoptosis. Whether Bcr-Abl exerts its antiapoptotic effect upstream to the cleavage and activation of caspase-3 or acts downstream by blocking the ensuing degradation of substrates resulting in apoptosis, has been the focus of the present studies. In these, we used (1) the human acute myelogenous leukemia (AML) HL-60 cells that are stably transfected with the bcr-abl gene (HL-60/Bcr-Abl) and express p185 Bcr-Abl; and (2) the chronic myelogenous leukemia (CML)-blast crisis K562 cells, which have endogenous expression of p210 Bcr-Abl. Exposure of the control AML HL-60 cells to high-dose Ara-C (HIDAC), etoposide, or sphingoid bases (including C2 ceramide, sphingosine, or sphinganine) caused the accumulation of cyt c in the cytosol, loss of mitochondrial membrane potential (MMP), and increase in the reactive oxygen species (ROS). These preapoptotic events were associated with the cleavage and activity of caspase-3, resulting in the degradation of poly (adenosine diphosphate [ADP]-ribose) polymerase (PARP) and DNA fragmentation factor (DFF), internucleosomal DNA fragmentation, and morphologic features of apoptosis. In contrast, in HL-60/Bcr-Abl and K562 cells, these apoptotic stimuli failed to cause the cytosolic accumulation of cyt c and other associated mitochondrial perturbations, as well as the failure to induce the activation of caspase-3 and apoptosis. While the control HL-60 cells showed high levels of Bcl-2 and barely detectable Bcl-xL, HL-60/Bcr-Abl cells expressed high levels of Bcl-xL and undetectable levels of Bcl-2, a pattern of expression similar to the one in K562 cells. Bax and caspase-3 expressions were not significantly different between HL-60/Bcr-Abl or K562 versus HL-60 cells. These findings indicate that Bcr-Abl expression blocks apoptosis due to diverse apoptotic stimuli upstream by preventing the cytosolic accumulation of cyt c and other preapoptotic mitochondrial perturbations, thereby inhibiting the activation of caspase-3 and execution of apoptosis.
Antineoplastic Agents↗
Modification of phosphatidylinositol 3-kinase SH2 domain binding properties by Abl- or Lck-mediated tyrosine phosphorylation at Tyr-688.
In cells expressing the oncogenic Bcr-Abl tyrosine kinase, the regulatory p85 subunit of phosphatidylinositol 3-kinase is phosphorylated on tyrosine residues. We report that this phosphorylation event is readily catalyzed by the Abl and Lck protein-tyrosine kinases in vitro, by Bcr-Abl or a catalytically activated Lck-Y505F in co-transfected COS cells, and by endogenous kinases in transfected Jurkat T cells upon triggering of their T cell antigen receptor. Using these systems, we have mapped a major phosphorylation site to Tyr-688 in the C-terminal SH2 domain of p85. Tyrosine phosphorylation of p85 in vitro or in vivo was not associated with detectable change in the enzymatic activity of the phosphatidylinositol 3-kinase heterodimer, but correlated with a strong reduction in the binding of some, but not all, phosphoproteins to the SH2 domains of p85. This provides an additional candidate to the list of SH2 domains regulated by tyrosine phosphorylation and may explain why association of phosphatidylinositol 3-kinase with some cellular ligands is transient or of lower stoichiometry than anticipated.
Fusion Proteins, bcr-abl↗
Fas ligand- mediated killing by intestinal intraepithelial lymphocytes. Participation in intestinal graft-versus-host disease.
In vitro studies have demonstrated that intestinal intraepithelial lymphocytes (IEL) are constitutively cytotoxic; however, the mechanism and target of their cytotoxicity are unknown. Apoptosis of intestinal epithelial cells (IEC) and an increase in IEL numbers are classical signs of intestinal graft-versus-host disease (GVHD), although whether IEL can mediate IEC apoptosis directly in GVHD is unclear. Recent evidence suggests that target epithelial organ injury observed in GVHD is predominantly Fas-mediated; therefore, we investigated the possibility that IEL induce apoptosis of IEC through a Fas-mediated mechanism. Here, we demonstrate that the IEL isolated from normal mice readily display potent Fas ligand (FasL)-mediated killing activity after CD3 stimulation, and that IEC express Fas, suggesting that IEC are potential targets for FasL-mediated killing by IEL. In vitro, IEL isolated from GVHD mice have markedly increased FasL-mediated killing potential and are spontaneously cytolytic toward host-derived tumor cells predominantly through a Fas-mediated pathway. In vivo transfer of IEL isolated from GVHD mice induced significantly more IEC apoptosis in F1 wild-type mice than in Fas-defective F1lpr mice. Thus, these results demonstrate that FasL-mediated death of IEC by IEL is a major mechanism of IEC apoptosis seen in GVHD.
Animals↗
Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization.
Mitochondrial cytochrome c, which functions as an electron carrier in the respiratory chain, translocates to the cytosol in cells undergoing apoptosis, where it participates in the activation of DEVD-specific caspases. The apoptosis inhibitors Bcl-2 or Bcl-xL prevent the efflux of cytochrome c from mitochondria. The mechanism responsible for the release of cytochrome c from mitochondria during apoptosis is unknown. Here, we report that cytochrome c release from mitochondria is an early event in the apoptotic process induced by UVB irradiation or staurosporine treatment in CEM or HeLa cells, preceding or at the time of DEVD-specific caspase activation and substrate cleavage. A reduction in mitochondrial transmembrane potential (Deltapsim) occurred considerably later than cytochrome c translocation and caspase activation, and was not necessary for DNA fragmentation. Although zVAD-fmk substantially blocked caspase activity, a reduction in Deltapsim and cell death, it failed to prevent the passage of cytochrome c from mitochondria to the cytosol. Thus the translocation of cytochrome c from mitochondria to cytosol does not require a mitochondrial transmembrane depolarization.
Amino Acid Chloromethyl Ketones↗
The c-Jun N-terminal kinase cascade plays a role in stress-induced apoptosis in Jurkat cells by up-regulating Fas ligand expression.
T lymphocytes undergo apoptosis in response to cellular stress, including UV exposure and gamma irradiation. However, the mechanism by which stress stimuli induce apoptosis is not well understood. While stress stimuli induce the activation of the c-Jun N-terminal kinase (JNK) pathway, it is not clear whether the JNK cascade is activated as a result of cell death or whether the cascade participates in inducing apoptosis. Using a Jurkat T cell line transfected with dominant active (DA)-mitogen-activated protein kinase kinase kinase (MEKK1) in a tetracycline-regulated expression system, we found that expression of DA-MEKK1 results in the apoptosis of Jurkat cells in parallel with prolonged JNK activation. Moreover, DA-MEKK1 induced Fas ligand (FasL) cell surface and mRNA expression, as well as FasL promoter activation. Interference with Fas/FasL interaction prevented DA-MEKK1-mediated apoptosis. In comparing the effect of different stress stimuli to DA-MEKK1, we found that UV, gamma irradiation, and anisomycin prolonged JNK activation in parallel with FasL expression and onset of cell death. In addition, these stimuli also enhance cell surface expression of FasL. Interference with Fas/FasL interactions inhibited anisomycin but not UV- or gamma irradiation-induced apoptosis. Our data show that while the JNK pathway contributes to stress-induced apoptosis in T lymphocytes by regulating FasL expression, not all stress stimuli use the same cell death pathway.
Apoptosis↗