RESUMO
Immune-checkpoint inhibitors (ICI) now represent the standard of care for several cancer types. In pre-clinical models, absence of an intact gut microbiome negatively impacted ICI efficacy and these findings permitted to unravel the importance of the commensal microbiota in immuno-oncology. Recently, multiple clinical studies including more than 1800 patients in aggregate demonstrated the negative predictive impact of treatments with broad-spectrum antibiotics (ATB) on cancer patients receiving ICI. Altogether, these results have led to the hypothesis that ATB-induced dysbiosis might influence the clinical response through the modulation of the gut microbiome. Controversy still remains, as ATB treatment might simply constitute a surrogate marker of unfit or immunodeficient patients. In this review, we summarize recent publications addressing the impact of the gut microbiome on ICI efficacy, discuss currently available data on the effect of ATB administered in different time-frames respect to ICI initiation, and finally, evoke the therapeutic implications of these findings.
Assuntos
Antibacterianos/efeitos adversos , Antineoplásicos Imunológicos/efeitos adversos , Disbiose/mortalidade , Microbioma Gastrointestinal/efeitos dos fármacos , Imunoterapia/mortalidade , Neoplasias/mortalidade , Disbiose/tratamento farmacológico , Disbiose/microbiologia , Microbioma Gastrointestinal/imunologia , Humanos , Imunoterapia/efeitos adversos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Prognóstico , Taxa de SobrevidaRESUMO
BACKGROUND: Tumor-infiltrating lymphocytes (TILs) represent a prognostic factor for survival in primary breast cancer (BC). Nonetheless, neoepitope load and TILs cytolytic activity are modest in BC, compromising the efficacy of immune-activating antibodies, which do not yet compete against immunogenic chemotherapy. PATIENTS AND METHODS: We analyzed by functional flow cytometry the immune dynamics of primary and metastatic axillary nodes [metastatic lymph nodes (mLN)] in early BC (EBC) after exposure to T-cell bispecific antibodies (TCB) bridging CD3ε and human epidermal growth factor receptor 2 (HER2) or Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5 (CEACAM5), before and after chemotherapy. Human leukocyte antigen (HLA) class I loss was assessed by whole exome sequencing and immunohistochemistry. One hundred primary BC, 64 surrounding 'healthy tissue' and 24 mLN-related parameters were analyzed. RESULTS: HLA loss of heterozygosity was observed in EBC, at a clonal and subclonal level and was associated with regulatory T cells and T-cell immunoglobulin and mucin-domain-3 expression restraining the immuno-stimulatory effects of neoadjuvant chemotherapy. TCB bridging CD3ε and HER2 or CEACAM5 could bypass major histocompatibility complex (MHC) class I loss, partially rescuing T-cell functions in mLN. CONCLUSION: TCB should be developed in BC to circumvent low MHC/peptide complexes.
Assuntos
Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Antígenos de Histocompatibilidade Classe I/genética , Linfócitos do Interstício Tumoral/imunologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Seguimentos , Variação Genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Linfonodos/imunologia , Linfonodos/patologia , Metástase Linfática , Terapia Neoadjuvante , Invasividade Neoplásica , Prognóstico , Estudos Prospectivos , Receptor ErbB-2/metabolismoRESUMO
The tumor microenvironment (TME) is an integral part of cancer. Recognition of the essential nature of the TME in cancer evolution has led to a shift from a tumor cell-centered view of cancer development to the concept of a complex tumor ecosystem that supports tumor growth and metastatic dissemination. Accordingly, novel targets within the TME have been uncovered that can help direct and improve the actions of various cancer therapies, notably immunotherapies that work by potentiating host antitumor immune responses. Here, we review the composition of the TME, how this attenuates immunosurveillance, and discuss existing and potential strategies aimed at targeting cellular and molecular TME components.
Assuntos
Imunidade Celular , Imunoterapia , Neoplasias/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Humanos , Neoplasias/imunologia , Neoplasias/patologiaRESUMO
BACKGROUND: Cisplatin-resistant non-small cell lung cancer (NSCLC) cells are often characterized by alterations in vitamin B-related metabolic processes, including the overexpression and hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1) and the downregulation of pyridoxal kinase (PDXK), correlating with elevated apoptosis resistance. Low PDXK expression is an established negative prognostic factor in NSCLC. PATIENTS AND METHODS: We determined by immunohistochemistry the expression of PARP1 and the level of its product, poly(ADP-ribose) (PAR), in two independent cohorts of patients with resected NSCLC. RESULTS: Intratumoral high levels (above median) of PAR (but not PARP1 protein levels) had a negative prognostic impact in both the training (92 stage I subjects) and validation (133 stage I and II subjects) cohorts, as determined by univariate and multivariate analyses. The simultaneous assessment of PAR and PDXK protein levels improved risk stratification. CONCLUSION: NSCLC patients with high intratumoral PARP1 activity (i.e. elevated PAR levels above median) and low PDXK expression (below median) had a dismal prognosis, while patients with low PARP1 activity and high PDXK expression had a favorable outcome. Altogether, these results underscore the clinical potential and possible therapeutic relevance of these biomarkers.
Assuntos
Biomarcadores Tumorais/biossíntese , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Poli(ADP-Ribose) Polimerases/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Seguimentos , Humanos , Líquido Intracelular/metabolismo , Masculino , Pessoa de Meia-Idade , Poli(ADP-Ribose) Polimerase-1 , Poli Adenosina Difosfato Ribose/biossíntese , PrognósticoRESUMO
Nuclear DNA damage and ligation of plasma-membrane death receptors have long been recognized as initial triggers of apoptosis that induce mitochondrial membrane permeabilization (MMP) and/or the direct activation of caspases. Accumulating evidence suggests that other organelles, including the endoplasmic reticulum (ER), lysosomes and the Golgi apparatus, are also major points of integration of pro-apoptotic signalling or damage sensing. Each organelle possesses sensors that detect specific alterations, locally activates signal transduction pathways and emits signals that ensure inter-organellar cross-talk. The ER senses local stress through chaperones, Ca2+-binding proteins and Ca2+ release channels, which might transmit ER Ca2+ responses to mitochondria. The ER also contains several Bcl-2-binding proteins, and Bcl-2 has been reported to exert part of its cytoprotective effect within the ER. Upon membrane destabilization, lysosomes release cathepsins that are endowed with the capacity of triggering MMP. The Golgi apparatus constitutes a privileged site for the generation of the pro-apoptotic mediator ganglioside GD3, facilitates local caspase-2 activation and might serve as a storage organelle for latent death receptors. Intriguingly, most organelle-specific death responses finally lead to either MMP or caspase activation, both of which might function as central integrators of the death pathway, thereby streamlining lysosome-, Golgi- or ER-elicited responses into a common pathway.
Assuntos
Apoptose , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Animais , Núcleo Celular , Dano ao DNA , Endossomos/metabolismo , OrganelasRESUMO
Apoptotic DNA degradation has been thought to be a cell-autonomous process. Recent evidence suggests that heterophagic recognition and engulfment of dying cells by non-apoptotic cells may be critical for the activation and/or action of apoptogenic DNases.
Assuntos
Apoptose/genética , Fragmentação do DNA/fisiologia , Timo/citologia , Animais , Marcação In Situ das Extremidades CortadasRESUMO
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.
Assuntos
Apoptose/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Flavoproteínas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Membrana/metabolismo , Proteínas/metabolismo , Animais , Fator de Indução de Apoptose , Fator Apoptótico 1 Ativador de Proteases , Núcleo Celular/ultraestrutura , Sobrevivência Celular , Células Cultivadas , Cromatina/fisiologia , Cromatina/ultraestrutura , Meios de Cultura Livres de Soro , Flavoproteínas/genética , Proteínas de Membrana/genética , Camundongos , Proteínas/antagonistas & inibidores , Proteínas/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Mammalian cells respond to stress by accumulating or activating a set of highly conserved proteins known as heat-shock proteins (HSPs). Several of these proteins interfere negatively with apoptosis. We show that the small HSP known as Hsp27 inhibits cytochrome-c-mediated activation of caspases in the cytosol. Hsp27 does not interfere with granzyme-B-induced activation of caspases, nor with apoptosis-inducing factor-mediated, caspase-independent, nuclear changes. Hsp27 binds to cytochrome c released from the mitochondria to the cytosol and prevents cytochrome-c-mediated interaction of Apaf-1 with procaspase-9. Thus, Hsp27 interferes specifically with the mitochondrial pathway of caspase-dependent cell death.
Assuntos
Apoptose , Grupo dos Citocromos c/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Neoplasias/metabolismo , Fator de Indução de Apoptose , Caspases/metabolismo , Citosol/metabolismo , Citosol/fisiologia , Ativação Enzimática , Flavoproteínas/metabolismo , Proteínas de Choque Térmico HSP27 , Humanos , Proteínas de Membrana/metabolismo , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Células U937RESUMO
In many instances, permeabilization of mitochondrial membranes is a rate-limiting step of apoptotic or necrotic cell demise. This has important consequences for the pathophysiology of cell death, as well as for its pharmacological control.
Assuntos
Apoptose/fisiologia , Membranas Intracelulares/fisiologia , Mitocôndrias/fisiologia , Doença , Modelos Biológicos , PermeabilidadeRESUMO
Immunogenic cell death, characterized by calreticulin exposure on the surface of the dying cell, release of the nuclear protein high mobility group box 1 (HMGB1), and release of ATP, enables stimulation of the immune system. We outlined the importance of this kind of cell death for the success of some anticancer chemotherapies. However, defects in the immunogenic cell death signalling pathway can lead to therapeutic failure, apparently because anticancer immune responses must contribute to the efficacy of chemotherapeutic regimens. These defects can be related to the therapy, the tumour cell, the host or the tumour-host interface. It is necessary to characterize these defects to restore and improve the efficacy of anticancer chemotherapies.
Assuntos
Antineoplásicos/farmacologia , Morte Celular/imunologia , Resistencia a Medicamentos Antineoplásicos/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Antineoplásicos/uso terapêutico , HumanosRESUMO
Activation of human thymocytes and pre-B cells via the CD3/T cell receptor (TCR) complex or the IgM/B cell receptor complex, respectively, results in apoptotic cell death. Similarly, cross-linking of the activation marker CD69, which belongs to the natural killer complex, causes apoptosis of lipopolysaccharide-preactivated monocytes. Here we show that pertussis toxin (PTX) inhibits the activation-induced apoptosis of these three cell types, though it fails to prevent the programmed cell death that follows exposure of cells to the synthetic glucocorticoid dexamethasone (thymocytes, pre-B cells) or to interleukin 4 (monocytes). The capacity of pertussis toxin to suppress activation-induced death is not due to quenching of the activation signal, because thymocytes exposed to PTX are still capable of mobilizing Ca2+ after TCR-alpha/beta cross-linking and proliferate in response to costimulation with PTX and CD3/TCR ligation. The apoptosis-inhibitory effect of PTX depends on the presence of an intact adenosine diphosphate (ADP)-ribosylating moiety, since a mutant pertussis toxin molecule that lacks enzymatic activity, but still possesses the membrane translocating activity, fails to interfere with activation-induced cell death. A toxin that induces a different spectrum of ADP ribosylation than PTX, cholera toxin, fails to inhibit apoptosis. To suppress apoptosis, the intact PTX holotoxin must be added to cells before the lethal activation step; its addition 30 min after initial activation remains without effect on apoptosis. These data unravel a PTX sensitive signal transduction event that intervenes during an early step of activation-induced cell death of immune cells.
Assuntos
Apoptose/efeitos dos fármacos , Ativação Linfocitária , Monócitos/efeitos dos fármacos , Toxina Pertussis , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Linfócitos T/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologia , Células Cultivadas , Humanos , Monócitos/fisiologia , Poli(ADP-Ribose) Polimerases/fisiologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/fisiologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/fisiologiaRESUMO
Poly- and oligoclonal T cell stimuli like anti-CD3 epsilon monoclonal antibody or Staphylococcus aureus enterotoxin B (SEB), injected at doses that per se are not lethal, provoke acute death within less than 24 h, provided that endogenous glucocorticoids (GC) are depleted by adrenalectomy or by injection of saturating amounts of the GC receptor antagonist RU-38486 (mifepristone). Pharmacological doses of the GC agonist dexamethasone (DEX) alter the in vivo response of splenic V beta 8+ T cells to SEB, thus impeding the expansion of such cells and causing their rapid (3 d) clonal deletion. In contrast, coadministration of RU-38486 counteracts a SEB-induced early (12 h) reduction of V beta 8+CD4+ and V beta 8+CD8+ spleen cells. In vivo T cell stimulation by injection of bacterial superantigen induces a rapid (peak at 90-120 min) increase in corticosterone serum levels, suggesting that endogenous GC might control early T cell activation. Accordingly, kinetic studies revealed that RU-38486 has to be administered within 2 h after superantigen administration to exert its lethal effect. Similarly, exogenous GC must be injected during this critical phase (2 h) to rescue animals from acute death induced by coinjection of SEB and D-galactosamine (GalN). Adrenalectomy, injection of RU-38486 and priming with GalN per se provoke the programmed death of peripheral CD4+ and CD8+ T cells. Thus, three manipulations that sensitize mice for the lethal effect of T cell stimulation also exert a proapoptotic effect on peripheral T cells. In synthesis, endogenous and exogenous GC regulate T cell responses and determine the propensity of peripheral T cells to undergo apoptosis.
Assuntos
Glucocorticoides/fisiologia , Ativação Linfocitária , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Antígenos/imunologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Células Clonais , Dexametasona/farmacologia , Enterotoxinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mifepristona/farmacologia , Baço/citologia , Staphylococcus aureus/metabolismo , Linfócitos T/efeitos dos fármacosRESUMO
Bcl-2 belongs to a family of apoptosis-regulatory proteins which incorporate into the outer mitochondrial as well as nuclear membranes. The mechanism by which the proto-oncogene product Bcl-2 inhibits apoptosis is thus far elusive. We and others have shown previously that the first biochemical alteration detectable in cells undergoing apoptosis, well before nuclear changes become manifest, is a collapse of the mitochondrial inner membrane potential (delta psi m), suggesting the involvement of mitochondrial products in the apoptotic cascade. Here we show that mitochondria contain a pre-formed approximately 50-kD protein which is released upon delta psi m disruption and which, in a cell-free in vitro system, causes isolated nuclei to undergo apoptotic changes such as chromatin condensation and internucleosomal DNA fragmentation. This apoptosis-inducing factor (AIF) is blocked by N-benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (Z-VAD.fmk), an antagonist of interleukin-1 beta-converting enzyme (ICE)-like proteases that is also an efficient inhibitor of apoptosis in cells. We have tested the effect of Bcl-2 on the formation, release, and action of AIF. When preventing mitochondrial permeability transition (which accounts for the pre-apoptotic delta psi m disruption in cells), Bcl-2 hyperexpressed in the outer mitochondrial membrane also impedes the release of AIF from isolated mitochondria in vitro. In contrast, Bcl-2 does not affect the formation of AIF, which is contained in comparable quantities in control mitochondria and in mitochondria from Bcl-2-hyperexpressing cells. Furthermore, the presence of Bcl-2 in the nuclear membrane does not interfere with the action of AIF on the nucleus, nor does Bcl-2 hyperexpression protect cells against AIF. It thus appears that Bcl-2 prevents apoptosis by favoring the retention of an apoptogenic protease in mitochondria.
Assuntos
Apoptose/efeitos dos fármacos , Endopeptidases/farmacologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Compartimento Celular , Núcleo Celular/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Permeabilidade , Inibidores de Proteases/farmacologiaRESUMO
Under physiological conditions, the vast majority of T cells differentiate in the thymus, an organ that provides an optimal microenvironment for T cell maturation and shapes the T cell repertoire via positive and negative selection processes. In the present report, we demonstrate that neonatal thymectomy of CBA/H mice results in a diminution of T cells in peripheral lymphoid organs (spleen, lymph nodes), but is followed by a marked transient (12 wk) increase in Thy-1+ CD3+ cells in the peritoneal cavity. These cells exhibit predominantly a double-negative (CD4-CD8-) phenotype among which products of the T cell receptor (TCR) V beta 11 gene family (i.e., an I-E-reactive TCR normally deleted in I-E-bearing CBA/H mice) are selectively overexpressed. This observation suggests that, under athymic conditions, T cell differentiation and/or accumulation may occur in the peritoneal cavity. Intraperitoneal inoculation of an interleukin 2 (IL-2) vaccinia virus construct that releases high titers of human IL-2 in vivo induces conversion of these double-negative T cells to either CD4+ CD8- or CD4- CD8+ single positives, and allows in vitro stimulation of TCR V beta 11-bearing cells with a clonotypic anti-V beta antibody. Since IL-2 induces autoimmune manifestations (DNA autoantibodies, rheumatoid factors, and interstitial nephritis) in thymectomized CBA/H mice, but not in sham-treated littermates, this lymphokine is likely to enhance the autoaggressive function of T cells that bear forbidden, potentially autoreactive TCR gene products and that are normally deleted in the thymus.
Assuntos
Doenças Autoimunes/imunologia , Imunidade Celular/efeitos dos fármacos , Interleucina-2/farmacologia , Receptores de Antígenos de Linfócitos T/fisiologia , Subpopulações de Linfócitos T/imunologia , Animais , Animais Recém-Nascidos/imunologia , Líquido Ascítico/imunologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos CBA , Baço/imunologia , TimectomiaRESUMO
According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1beta converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (DeltaPsim). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the DeltaPsim is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the DeltaPsim collapse and subsequent apoptosis. Cytosols from anti-Fas-treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their DeltaPsim. In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.
Assuntos
Apoptose/imunologia , Ceramidas/farmacologia , Cisteína Endopeptidases/imunologia , Receptor fas/imunologia , Apoptose/efeitos dos fármacos , Caspase 1 , Cisteína Endopeptidases/metabolismo , Ativação Enzimática , Citometria de Fluxo , Humanos , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Células Tumorais CultivadasRESUMO
In a number of experimental systems, the early stage of the apoptotic process, i.e., the stage that precedes nuclear disintegration, is characterized by the breakdown of the inner mitochondrial transmembrane potential (delta psi m). This delta psi m disruption is mediated by the opening of permeability transition (PT) pores and appears to be critical for the apoptotic cascade, since it is directly regulated by Bcl-2 and since mitochondria induced to undergo PT in vitro become capable of inducing nuclear chromatinolysis in a cell-free system of apoptosis. Here, we addressed the question of which apoptotic events are secondary to mitochondrial PT. We tested the effect of a specific inhibitor of PT, bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, on a prototypic model of apoptosis glucocorticoid-induced thymocyte death. In addition to abolishing the apoptotic delta psi m disruption, BA prevents a number of phenomena linked to apoptosis: depletion of nonoxidized glutathione, generation of reactive oxygen species, translocation of NF kappa B, exposure of phosphatidylserine residues on the outer plasma membrane, cytoplasmic vacuolization, chromatin condensation, and oligonucleosomal DNA fragmentation. BA is also an efficient inhibitor of p53-dependent thymocyte apoptosis induced by DNA damage. These data suggest that a number of apoptotic phenomena are secondary to PT. In addition, we present data indicating that apoptotic delta psi m disruption is secondary to transcriptional events. These data connect the PT control point to the p53- and ICE/ Ced 3-regulated control points of apoptosis and place PT upstream of nuclear and plasma membrane features of PCD.
Assuntos
Apoptose , Membranas Intracelulares/fisiologia , Mitocôndrias/fisiologia , Animais , Apoptose/efeitos dos fármacos , Ácido Bongcréquico/farmacologia , Dexametasona/farmacologia , Feminino , Citometria de Fluxo , Membranas Intracelulares/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Permeabilidade/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
In a number of experimental systems in which lymphocyte depletion was induced by apoptosis-inducing manipulations, no apoptotic morphology and ladder-type DNA fragmentation were detected among freshly isolated peripheral lymphocytes ex vivo. Here we report that one alteration that can be detected among splenocytes stimulated with lymphocyte-depleting doses of dexamethasone (DEX) in vivo is a reduced uptake of 3,3'dihexyloxacarbocyanine iodide (DiOC6[3]), a fluorochrome which incorporates into cells dependent upon their mitochondrial transmembrane potential (delta psi m). In contrast, ex vivo isolated splenocytes still lacked established signs of programmed cell death (PCD):DNA degradation into high or low molecular weight fragments, ultrastructural changes of chromatin arrangement and endoplasmatic reticulum, loss in viability, or accumulation of intracellular peroxides. Moreover, no changes in cell membrane potential could be detected. A reduced delta psi m has been observed in response to different agents inducing lymphoid cell depletion in vivo (superantigen and glucocorticoids [GC]), in mature T and B lymphocytes, as well as their precursors. DEX treatment in vivo, followed by cytofluorometric purification of viable delta psi mlow splenic T cells ex vivo, revealed that this fraction of cells is irreversibly committed to undergoing DNA fragmentation. Immediately after purification neither delta psi mlow, nor delta psi mhigh cells, exhibit detectable DNA fragmentation. However, after short-term culture (37 degrees C, 1 h) delta psi mlow cells show endonucleolysis, followed by cytolysis several hours later. Incubation of delta psi mlow cells in the presence of excess amount of the GC receptor antagonist RU38486 (which displaces DEX from the GC receptor), cytokines that inhibit DEX-induced cell death, or cycloheximide fails to prevent cytolysis. The antioxidant, N-acetylcysteine, as well as linomide, an agent that effectively inhibits DEX or superantigen-induced lymphocyte depletion in vivo, also stabilize the DiOC6(3) uptake. In contrast, the endonuclease inhibitor, aurintricarboxylic acid acts at later stages of apoptosis and only retards the transition from the viable delta psi mlow to the nonviable fraction. Altogether, these data suggest a sequence of PCD-associated events in which a reduction in delta psi m constitutes an obligate irreversible step of ongoing lymphocyte death, preceding other alterations of cellular physiology, and thus allowing for the ex vivo assessment of PCD.
Assuntos
Apoptose , Linfócitos/fisiologia , Mitocôndrias/fisiologia , Animais , Ácido Aurintricarboxílico/farmacologia , Células Cultivadas , DNA Mitocondrial/fisiologia , Dexametasona/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Superantígenos/fisiologiaRESUMO
Anucleate cells can be induced to undergo programmed cell death (PCD), indicating the existence of a cytoplasmic PCD pathway that functions independently from the nucleus. Cytoplasmic structures including mitochondria have been shown to participate in the control of apoptotic nuclear disintegration. Before cells exhibit common signs of nuclear apoptosis (chromatin condensation and endonuclease-mediated DNA fragmentation), they undergo a reduction of the mitochondrial transmembrane potential (delta psi m) that may be due to the opening of mitochondrial permeability transition (PT) pores. Here, we present direct evidence indicating that mitochondrial PT constitutes a critical early event of the apoptotic process. In a cell-free system combining purified mitochondria and nuclei, mitochondria undergoing PT suffice to induce chromatin condensation and DNA fragmentation. Induction of PT by pharmacological agents augments the apoptosis-inducing potential of mitochondria. In contrast, prevention of PT by pharmacological agents impedes nuclear apoptosis, both in vitro and in vivo. Mitochondria from hepatocytes or lymphoid cells undergoing apoptosis, but not those from normal cells, induce disintegration of isolated Hela nuclei. A specific ligand of the mitochondrial adenine nucleotide translocator (ANT), bongkreik acid, inhibits PT and reduces apoptosis induction by mitochondria in a cell-free system. Moreover, it inhibits the induction of apoptosis in intact cells. Several pieces of evidence suggest that the proto-oncogene product Bcl-2 inhibits apoptosis by preventing mitochondrial PT. First, to inhibit nuclear apoptosis, Bcl-2 must be localized in mitochondrial but not nuclear membranes. Second, transfection-enforced hyperexpression of Bcl-2 directly abolishes the induction of mitochondrial PT in response to a protonophore, a pro-oxidant, as well as to the ANT ligand atractyloside, correlating with its apoptosis-inhibitory effect. In conclusion, mitochondrial PT appears to be a critical step of the apoptotic cascade.
Assuntos
Apoptose , Mitocôndrias/metabolismo , Animais , Atractilosídeo/metabolismo , Núcleo Celular/patologia , Sistema Livre de Células , Células Cultivadas , Membranas Intracelulares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/enzimologia , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias Hepáticas/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Dilatação Mitocondrial , Permeabilidade , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2RESUMO
Syncytia arising from the fusion of cells expressing a lymphotropic HIV type 1-encoded envelope glycoprotein complex (Env) with cells expressing the CD4/CXC chemokine receptor 4 complex spontaneously undergo cell death. Here we show that this process is accompanied by caspase activation and signs of mitochondrial membrane permeabilization (MMP), including the release of intermembrane proteins such as cytochrome c (Cyt-c) and apoptosis-inducing factor (AIF) from mitochondria. In Env-induced syncytia, caspase inhibition did not suppress AIF- and Cyt-c translocation, yet it prevented all signs of nuclear apoptosis. Translocation of Bax to mitochondria led to MMP, which was inhibited by microinjected Bcl-2 protein or bcl-2 transfection. Bcl-2 also prevented the subsequent nuclear chromatin condensation and DNA fragmentation. The release of AIF occurred before that of Cyt-c and before caspase activation. Microinjection of AIF into syncytia sufficed to trigger rapid, caspase-independent Cyt-c release. Neutralization of endogenous AIF by injection of an antibody prevented all signs of spontaneous apoptosis occurring in syncytia, including the Cyt-c release and nuclear apoptosis. In contrast, Cyt-c neutralization only prevented nuclear apoptosis, and did not affect AIF release. Our results establish that the following molecular sequence governs apoptosis of Env-induced syncytia: Bax-mediated/Bcl-2-inhibited MMP --> AIF release --> Cyt-c release --> caspase activation --> nuclear apoptosis.
Assuntos
Apoptose/fisiologia , Antígenos CD4/fisiologia , Caspases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Produtos do Gene env/metabolismo , Células Gigantes/virologia , HIV-1/fisiologia , Mitocôndrias/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Fusão Celular , Técnicas de Cocultura , Genes env , Células Gigantes/citologia , Células Gigantes/fisiologia , Células HeLa , Humanos , Membranas Intracelulares/fisiologia , Cinética , Permeabilidade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , TransfecçãoRESUMO
Early in programmed cell death (apoptosis), mitochondrial membrane permeability increases. This is at least in part due to opening of the permeability transition (PT) pore, a multiprotein complex built up at the contact site between the inner and the outer mitochondrial membranes. The PT pore has been previously implicated in clinically relevant massive cell death induced by toxins, anoxia, reactive oxygen species, and calcium overload. Here we show that PT pore complexes reconstituted in liposomes exhibit a functional behavior comparable with that of the natural PT pore present in intact mitochondria. The PT pore complex is regulated by thiol-reactive agents, calcium, cyclophilin D ligands (cyclosporin A and a nonimmunosuppressive cyclosporin A derivative), ligands of the adenine nucleotide translocator, apoptosis-related endoproteases (caspases), and Bcl-2-like proteins. Although calcium, prooxidants, and several recombinant caspases (caspases 1, 2, 3, 4, and 6) enhance the permeability of PT pore-containing liposomes, recombinant Bcl-2 or Bcl-XL augment the resistance of the reconstituted PT pore complex to pore opening. Mutated Bcl-2 proteins that have lost their cytoprotective potential also lose their PT modulatory capacity. In conclusion, the PT pore complex may constitute a crossroad of apoptosis regulation by caspases and members of the Bcl-2 family.