RESUMEN
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.
Asunto(s)
Apoptosis , Grupo Citocromo c/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Neoplasias/metabolismo , Factor Inductor de la Apoptosis , Caspasas/metabolismo , Citosol/metabolismo , Citosol/fisiología , Activación Enzimática , Flavoproteínas/metabolismo , Proteínas de Choque Térmico HSP27 , Humanos , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Células U937RESUMEN
HSP27 form oligomeric structures up to 800 Kda. In cultured cells, the equilibrium between small and large oligomers shifted towards smaller oligomers when phosphorylated on serine residues. To further explore HSP27 structural organization and its repercussion in HSP27 antiapoptotic and tumorigenic properties, we transfected colon cancer REG cells with wild type HSP27 and two mutants in which the phosphorylatable serine residues have been replaced by alanine (to mimic the non phosphorylated protein) or aspartate (to mimic the phosphorylated protein). In growing cells, wild type and alanine mutant formed small and large oligomers and demonstrated antiapoptotic activity while aspartate mutant only formed small multimers and had no antiapoptotic activity. In a cell-free system, only large oligomeric structures interfered with cytochrome c-induced caspase activation, thereby inhibiting apoptosis. The inability of the aspartate mutant to form large oligomers and to protect tumor cells from apoptosis was overcome by growing the cells in vivo, either in syngeneic animals or nude mice. These observations were reproduced by culturing the cells at confluence in vitro. In conclusion (1) large oligomers are the structural organization of HSP27 required for its antiapoptotic activity and (2) cell-cell contacts induce the formation of large oligomers, whatever the status of phosphorylatable serines, thereby increasing cell tumorigenicity.
Asunto(s)
Apoptosis/fisiología , Neoplasias Colorrectales/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Neoplasias/química , Procesamiento Proteico-Postraduccional , Células Tumorales Cultivadas/metabolismo , Sustitución de Aminoácidos , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ácido Aspártico/química , Biopolímeros , Caspasa 3 , Caspasa 9 , Caspasas/metabolismo , Comunicación Celular , Recuento de Células , Sistema Libre de Células , Cisplatino/farmacología , Neoplasias Colorrectales/patología , Cumarinas/metabolismo , Etopósido/farmacología , Proteínas de Choque Térmico/metabolismo , Ratones , Peso Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/metabolismo , Oligopéptidos/metabolismo , Fosforilación , Fosfoserina/química , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Expression of inducible heat shock protein 70 (HSP70) in tumor cells has been proposed to enhance their immunogenicity. However, HSP70 has also been demonstrated to prevent tumor cell death, a key process for the development of tumor cell immunogenicity. In the present study, we investigated the influence of the HSP70 protein level on PRO colon cancer cell growth and immunogenicity in syngeneic BDIX rats and nude mice. These cells have a basal expression of HSP70 which can be substantially increased by heat shock. When injected subcutaneously in syngeneic animals, PRO cells do not induce any detectable immune response and give rise to progressive, metastatic and lethal tumors. Stable transfection of an anti-sense hsp70 cDNA in PRO cells (PRO-70AS cells) strongly decreased HSP70 expression and sensitized cell-free extracts to cytochrome c/dATP-mediated activation of caspases. Subcutaneous injection of PRO-70AS cells induced tumors that rapidly regressed in syngeneic rats while they grew normally in nude mice. Syngeneic rats injected with PRO-70AS cells became protected against a further challenge with PRO cells. The tumor-specific immune response induced by HSP70-depleted PRO-70AS cells was associated with an increased rate of cell death in vivo. These PRO-70AS cells were also more sensitive to NO-mediated, caspase-dependent, macrophage cytotoxicity in vivo. Altogether, these results indicate that reduced level of HSP70 expression in PRO- colon cancer cells results in the generation of a specific immune response by promoting cell death in vivo.
Asunto(s)
Neoplasias del Colon/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/fisiología , Animales , Apoptosis , División Celular , Línea Celular , Sistema Libre de Células , Neoplasias del Colon/patología , ADN Complementario/metabolismo , Immunoblotting , Macrófagos/metabolismo , Ratones , Plásmidos/metabolismo , Ratas , Factores de Tiempo , Transfección , Células Tumorales CultivadasRESUMEN
U937 leukemic cells treated for 24 h with 16 nM 12-O-tetradecanoylphorbol 13-acetate (TPA), that induces their macrophagic terminal differentiation, become resistant to etoposide-induced apoptosis. Exposure of undifferentiated U937 cells to 50 microM etoposide for 6 h, that triggers apoptosis in 80% cells, activates procaspase-2L, -3 and -8, induces the mitochondrial release of cytochrome c and decreases Mcl-1 expression without modifying Bcl-2, Bcl-xL and Bax protein levels. All these events are inhibited in TPA-differentiated U937 cells that are also resistant to vinblastine-induced and Fas-mediated cell death. Interestingly, these cells are not inherently resistant to apoptosis induction. Exposure of TPA-differentiated U937 cells to 0.8 microg/ml cycloheximide for 24 h, that triggers apoptosis in 50% cells, activates procaspase-2L, -3 and -8, induces the mitochondrial release of cytochrome c and decreases Bcl-xL expression without modifying Bcl-2, Mcl-1 and Bax protein levels. All these events are not observed in undifferentiated cells treated in similar conditions. These results indicate that the apoptotic pathway that involves the release of cytochrome c from mitochondria and the cleavage of procaspases remains functional in TPA-differentiated cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinógenos/farmacología , Acetato de Tetradecanoilforbol/farmacología , Antineoplásicos Fitogénicos/farmacología , Caspasa 2 , Caspasa 3 , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Cicloheximida/farmacología , Grupo Citocromo c/metabolismo , Precursores Enzimáticos/metabolismo , Etopósido/farmacología , Proteína Ligando Fas , Humanos , Glicoproteínas de Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células U937 , Vinblastina/farmacología , Proteína bcl-XRESUMEN
As shown here, mitochondria purified from different organs (liver, brain, kidney, spleen and heart) contain both pro-caspase-9 and the processed, mature form of caspase-9. Purified liver mitochondria release mature caspase-9 upon induction of permeability transition in vitro. This is accompanied by a discrete increase in the enzymatic cleavage of pro-caspase-9 substrates. We found that SHEP neuroblastoma cells constitutively contain pre-processed caspase-9 in their mitochondria, using a combination of subcellular fractionation and immunofluorescence with an antibody specific for the processed caspase. This is a cell type-specific phenomenon since HeLa cells mitochondria mainly contain pro-caspase-9 and comparatively little processed caspase-9. Upon introduction of apoptosis, mitochondrial pro-caspase-9 translocates to the cytosol and to the nucleus. This phenomenon is inhibited by transfection with Bcl-2. In synthesis, we report the unexpected finding that mitochondria can contain a pre-processed caspase isoform in non-apoptotic cells. Bcl-2-mediated regulation of mitochondrial membrane permeabilization may contribute to apoptosis control by preventing mitochondrial, pre-processed caspase-9 from interacting with its cytosolic activators.
Asunto(s)
Apoptosis/fisiología , Caspasas/fisiología , Mitocondrias/enzimología , Caspasa 9 , Caspasas/metabolismo , Núcleo Celular/enzimología , Citosol/enzimología , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Neuroblastoma , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Estaurosporina/farmacología , Células Tumorales CultivadasRESUMEN
Molecular and serologic methods usable for the biological diagnosis of Coltivirus infection are reported. We designed a multiplex reverse transcription-polymerase chain reaction system that allowed the simultaneous and specific amplification of three genomic segments from as little as 0.01 plaque-forming units. Another system in the S2 viral segment permitted the differential diagnosis of American and European viral isolates. We also discuss some improvements of previous ELISAs, and the results obtained with paired sera from Colorado tick fever (CTF) virus-infected individuals. Western blot analysis was developed that allowed the detection of antibodies to a 38-kD viral protein in all tested sera. It also enabled the detection of anti-CTF virus antibodies in ELISA-negative sera. Specific IgM antibodies against a synthetic viral peptide could be detected in sera at the acute stage of the infection. Together, these results should permit the diagnosis of Coltivirus infection at any stage of the pathology.
Asunto(s)
Fiebre por Garrapatas del Colorado/diagnóstico , Virus de la Fiebre por Garrapatas del Colorado/inmunología , Virus de la Fiebre por Garrapatas del Colorado/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Pruebas Serológicas/métodos , Virología/métodos , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Secuencia de Bases , Western Blotting/métodos , Western Blotting/estadística & datos numéricos , Línea Celular , Fiebre por Garrapatas del Colorado/inmunología , Fiebre por Garrapatas del Colorado/virología , Virus de la Fiebre por Garrapatas del Colorado/genética , Cricetinae , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/estadística & datos numéricos , Estudios de Evaluación como Asunto , Femenino , Humanos , Inmunoglobulina M/sangre , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , Sensibilidad y Especificidad , Pruebas Serológicas/estadística & datos numéricos , Virología/estadística & datos numéricosRESUMEN
Anticancer drugs can induce tumour cell death by apoptosis. The main pathway from specific damage induced by the drug to apoptosis involves activation of caspases in the cytosol by pro-apoptotic molecules such as cytochrome c released from the mitochondria. At least in some cell types, anticancer drugs also upregulate the expression of death receptors and sensitize tumour cells to their cognate ligands, which could be used to amplify the response to cytotoxic drugs. The Bcl-2 family of proteins, which includes anti- and pro-apoptotic molecules, regulates cell sensitivity at the mitochondrial level. Chemotherapeutic drugs modulate their expression (e.g. through p53-dependent gene transcription), their activity (e.g. by phosphorylation) and their subcellular localization (e.g. by translocation of pro-apoptotic proteins from the cytosol to the mitochondria). When interacting with tumour cells, anticancer drugs also activate lipid- and kinase-dependent signalling pathways that modulate the death response to specific damage. Protective pathways include activation of NF kappa B transcription factor, accumulation of heat shock proteins and activation of proteins involved in cell cycle regulation. The recent identification on these pathways to cell death has suggested several new strategies to improve the therapeutic efficacy of currently used anticancer drug regimens.
Asunto(s)
Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Citotoxinas/toxicidad , Neoplasias/tratamiento farmacológico , Humanos , Neoplasias/patología , Neoplasias/fisiopatología , Células Tumorales CultivadasRESUMEN
We have previously shown that the small heat shock protein HSP27 inhibited apoptotic pathways triggered by a variety of stimuli in mammalian cells. The present study demonstrates that HSP27 overexpression decreases U937 human leukemic cell sensitivity to etoposide-induced cytotoxicity by preventing apoptosis. As observed for Bcl-2, HSP27 overexpression delays poly(ADP-ribose)polymerase cleavage and procaspase-3 activation. In contrast with Bcl-2, HSP27 overexpression does not prevent etoposide-induced cytochrome c release from the mitochondria. In a cell-free system, addition of cytochrome c and dATP to cytosolic extracts from untreated cells induces the proteolytic activation of procaspase-3 in both control and bcl-2-transfected U937 cells but fails to activate procaspase-3 in HSP27-overexpressing cells. Immunodepletion of HSP27 from cytosolic extracts increases cytochrome c/dATP-mediated activation of procaspase-3. Overexpression of HSP27 also prevents procaspase-9 activation. In the cell-free system, immunodepletion of HSP27 increases LEDH-AFC peptide cleavage activity triggered by cytochrome c/dATP treatment. We conclude that HSP27 inhibits etoposide-induced apoptosis by preventing cytochrome c and dATP-triggered activity of caspase-9, downstream of cytochrome c release.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Grupo Citocromo c/fisiología , Precursores Enzimáticos/antagonistas & inhibidores , Etopósido/farmacología , Proteínas de Choque Térmico/fisiología , Caspasa 3 , Caspasa 9 , Nucleótidos de Desoxiadenina/fisiología , Resistencia a Antineoplásicos , Activación Enzimática , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Células U937RESUMEN
Exposure of U937 human leukemic cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces their differentiation into monocyte/macrophage-like cells. This terminal differentiation is associated with a resistant phenotype to apoptosis induced by the topoisomerase II inhibitor etoposide. The inhibition occurs upstream of the mitochondrial release of cytochrome c and the activation of procaspase-2, -3, -6, -7, -8, and -9. By using cell-free systems, it was demonstrated that the mitochondrial pathway to cell death that involves mitochondrial membrane depolarization, cytochrome c release and cytosolic activation of procaspases by cytochrome c/dATP remains functional in TPA-differentiated U937 cells. Accordingly, 2 drugs recently shown to target the mitochondria, namely lonidamine and arsenic trioxide, bypass the resistance of TPA-differentiated U937 cells to classical anticancer drugs. Cell death induced by the 2 compounds is associated with mitochondrial membrane depolarization, release of cytochrome c and Smac/Diablo from the mitochondria, activation of caspases, poly(ADP-ribose) polymerase cleavage and internucleosomal DNA fragmentation. Moreover, the decreased glutathione content associated with the differentiation process amplifies the ability of arsenic trioxide to activate the mitochondrial pathway to cell death. Similar results were obtained by comparing undifferentiated and TPA-differentiated human HL60 leukemic cells. These data demonstrate that mitochondria-targeting agents bypass the resistance to classical anticancer drugs induced by TPA-mediated leukemic cell differentiation. (Blood. 2001;97:3931-3940)