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1.
Nature ; 409(6817): 207-11, 2001 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-11196646

RESUMEN

Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Here we show that metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic programme in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss. We conclude that Apaf-1 is inactivated in metastatic melanomas, which leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumour type.


Asunto(s)
Apoptosis , Melanoma/metabolismo , Proteínas/metabolismo , Antineoplásicos/farmacología , Factor Apoptótico 1 Activador de Proteasas , Caspasa 9 , Caspasas/metabolismo , Cromosomas Humanos Par 12 , Clonación Molecular , Metilación de ADN , ADN de Neoplasias/metabolismo , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Genes p53 , Humanos , Pérdida de Heterocigocidad , Melanoma/patología , Melanoma/secundario , Mutación , Proteínas/genética , Células Tumorales Cultivadas
2.
Blood ; 96(12): 3922-31, 2000 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-11090079

RESUMEN

Recent studies have suggested that variations in levels of caspases, a family of intracellular cysteine proteases, can profoundly affect the ability of cells to undergo apoptosis. In this study, immunoblotting was used to examine levels of apoptotic protease activating factor-1 (Apaf-1) and procaspases-2, -3, -7, -8, and -9 in bone marrow samples (at least 80% leukemia) harvested before chemotherapy from adults with newly diagnosed acute myelogenous leukemia (AML, 42 patients) and acute lymphocytic leukemia (ALL, 18 patients). Levels of each of these polypeptides varied over a more than 10-fold range between specimens. In AML samples, expression of procaspase-2 correlated with levels of Apaf-1 (R(s) = 0.52, P <.02), procaspase-3 (R(s) = 0.56, P <.006) and procaspase-8 (R(s) = 0.64, P <.002). In ALL samples, expression of procaspases-7 and -9 was highly correlated (R(s) = 0.90, P <.003). Levels of these polypeptides did not correlate with prognostic factors or response to induction chemotherapy. In further studies, 16 paired samples (13 AML, 3 ALL), the first harvested before induction therapy and the second harvested at the time of leukemia regrowth, were also examined. There were no systematic alterations in levels of Apaf-1 or procaspases at relapse compared with diagnosis. These results indicate that levels of initiator caspases vary widely among different leukemia specimens but cast doubt on the hypothesis that this variation is a major determinant of drug sensitivity for acute leukemia in the clinical setting. (Blood. 2000;96:3922-3931)


Asunto(s)
Caspasas/metabolismo , Precursores Enzimáticos/metabolismo , Leucemia/diagnóstico , Enfermedad Aguda , Adolescente , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Factor Apoptótico 1 Activador de Proteasas , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/metabolismo , Células de la Médula Ósea/química , Células de la Médula Ósea/enzimología , Caspasas/efectos de los fármacos , Caspasas/inmunología , Estudios de Cohortes , Precursores Enzimáticos/efectos de los fármacos , Precursores Enzimáticos/inmunología , Células HL-60 , Humanos , Immunoblotting , Leucemia/metabolismo , Leucemia/terapia , Leucemia Mieloide/diagnóstico , Leucemia Mieloide/metabolismo , Leucemia Mieloide/terapia , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Pronóstico , Proteínas/efectos de los fármacos , Proteínas/inmunología , Proteínas/metabolismo
3.
Nat Cell Biol ; 2(11): 859-62, 2000 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11056544

RESUMEN

Oncogenes that promote cell-cycle progression also sensitize cells to agents that induce apoptosis, possibly by inactivating inhibitors that ordinarily provide protection against cell death. Here we show that the adenoviral oncogene E1A sensitizes cells to an anti-cancer drug by at least two pathways. One establishes a link between the drug and pro-apoptotic factors, but is not sufficient for sensitization without the second pathway, which suppresses inhibitors of apoptosis.


Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Apoptosis , Oncogenes , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Células Cultivadas , Grupo Citocromo c/metabolismo , Etopósido/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Células HeLa , Humanos , Células Jurkat , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Proteína X Asociada a bcl-2
4.
J Biol Chem ; 274(30): 21155-61, 1999 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-10409669

RESUMEN

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.


Asunto(s)
Apoptosis , Caspasas/metabolismo , Grupo Citocromo c/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Caspasa 3 , Cricetinae , Activación Enzimática , Células HL-60 , Humanos , Especificidad por Sustrato
5.
Proc Natl Acad Sci U S A ; 95(23): 13664-9, 1998 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-9811857

RESUMEN

Understanding how oncogenic transformation sensitizes cells to apoptosis may provide a strategy to kill tumor cells selectively. We previously developed a cell-free system that recapitulates oncogene dependent apoptosis as reflected by activation of caspases, the core of the apoptotic machinery. Here, we show that this activation requires a previously identified apoptosis-promoting complex consisting of caspase-9, APAF-1, and cytochrome c. As predicted by the in vitro system, preventing caspase-9 activation blocked drug-induced apoptosis in cells sensitized by E1A, an adenoviral oncogene. Oncogenes, such as E1A, appear to facilitate caspase-9 activation by several mechanisms, including the control of cytochrome c release from the mitochondria.


Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Apoptosis , Caspasas/metabolismo , Proteínas Oncogénicas/metabolismo , Factor Apoptótico 1 Activador de Proteasas , Caspasa 9 , Transformación Celular Neoplásica , Transformación Celular Viral , Células Cultivadas , Grupo Citocromo c/metabolismo , Activación Enzimática , Fibroblastos , Humanos , Proteínas/metabolismo
6.
Genes Dev ; 11(10): 1266-76, 1997 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-9171371

RESUMEN

Many genotoxic agents kill tumor cells by inducing apoptosis; hence, mutations that suppress apoptosis produce resistance to chemotherapy. Although directly activating the apoptotic machinery may bypass these mutations, how to achieve this activation in cancer cells selectively is not clear. In this study, we show that the drug-resistant 293 cell line is unable to activate components of the apoptotic machinery-the ICE-like proteases (caspases)-following treatment with an anticancer drug. Remarkably, extracts from untreated cells spontaneously activate caspases and induce apoptosis in a cell-free system, indicating that drug-resistant cells have not only the apoptotic machinery but also its activator. Comparing extracts from cells with defined genetic differences, we show that this activator is generated by the adenovirus E1A oncogene and is absent from normal cells. We provide preliminary characterization of this oncogene generated activity (OGA) and show that partially purified OGA activates caspases when added to extracts from untransformed cells. We suggest that agents that link OGA to caspases in cells would kill tumor cells otherwise resistant to conventional cancer therapy. As this killing relies on an activity generated by an oncogene, the effect of these agents should be selective for transformed cells.


Asunto(s)
Apoptosis/genética , Resistencia a Múltiples Medicamentos/genética , Oncogenes , Adenosina Trifosfato/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas E2 de Adenovirus/genética , Línea Celular , Sistema Libre de Células , Cromatografía por Intercambio Iónico , Cisteína Endopeptidasas/metabolismo , Activación Enzimática , Genes bcl-2 , Células HeLa , Humanos , Hidrólisis
7.
Proc Natl Acad Sci U S A ; 93(16): 8395-400, 1996 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-8710882

RESUMEN

Although proteases related to the interleukin 1 beta-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cell-free model system for apoptosis (S/M extracts). First, we compared the substrate specificities of two recombinant human IRPs, CPP32 and Mch2 alpha. Both enzymes cleaved poly-(ADP-ribose) polymerase, albeit with different efficiencies. Mch2 alpha also cleaved recombinant and nuclear lamin A at a conserved VEID decreases NG sequence located in the middle of the coiled-coil rod domain, producing a fragment that was indistinguishable from the lamin A fragment observed in S/M extracts and in apoptotic cells. In contrast, CPP32 did not cleave lamin A. The cleavage of lamin A by Mch2 alpha and by S/M extracts was inhibited by millimolar concentrations of Zn2+, which had a minimal effect on cleavage of poly (ADP-ribose) polymerase by CPP32 and by S/M extracts. We also found that N-(acetyltyrosinylvalinyl-N epsilon-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone, which derivatizes the larger subunit of active ICE, can affinity label up to five active IRPs in S/M extracts. Together, these observations indicate that the processing of nuclear proteins in apoptosis involves multiple IRPs having distinct preferences for their apoptosis-associated substrates.


Asunto(s)
Apoptosis , Caspasas , Ciclo Celular , Cisteína Endopeptidasas/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Afidicolina/farmacología , Caspasa 3 , Caspasa 6 , Núcleo Celular/enzimología , Humanos , Lamina Tipo A , Laminas , Datos de Secuencia Molecular , Péptidos/química , Poli(ADP-Ribosa) Polimerasas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Zinc/metabolismo
8.
Proc Natl Acad Sci U S A ; 92(20): 9042-6, 1995 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-7568069

RESUMEN

Although specific proteinases play a critical role in the active phase of apoptosis, their substrates are largely unknown. We previously identified poly(ADP-ribose) polymerase (PARP) as an apoptosis-associated substrate for proteinase(s) related to interleukin 1 beta-converting enzyme (ICE). Now we have used a cell-free system to characterize proteinase(s) that cleave the nuclear lamins during apoptosis. Lamin cleavage during apoptosis requires the action of a second ICE-like enyzme, which exhibits kinetics of cleavage and a profile of sensitivity to specific inhibitors that is distinct from the PARP proteinase. Thus, multiple ICE-like enzymes are required for apoptotic events in these cell-free extracts. Inhibition of the lamin proteinase with tosyllysine "chloromethyl ketone" blocks nuclear apoptosis prior to the packaging of condensed chromatin into apoptotic bodies. Under these conditions, the nuclear DNA is fully cleaved to a nucleosomal ladder. Our studies reveal that the lamin proteinase and the fragmentation nuclease function in independent parallel pathways during the final stages of apoptotic execution. Neither pathway alone is sufficient for completion of nuclear apoptosis. Instead, the various activities cooperate to drive the disassembly of the nucleus.


Asunto(s)
Apoptosis/fisiología , Endopeptidasas/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Línea Celular , Núcleo Celular/metabolismo , Sistema Libre de Células , Células HeLa , Humanos , Cinética , Laminas , Neoplasias Pulmonares , Datos de Secuencia Molecular , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/aislamiento & purificación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Proteasas/farmacología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Células Tumorales Cultivadas
9.
Nature ; 376(6535): 37-43, 1995 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-7596430

RESUMEN

The protease responsible for the cleavage of poly(ADP-ribose) polymerase and necessary for apoptosis has been purified and characterized. This enzyme, named apopain, is composed of two subunits of relative molecular mass (M(r)) 17K and 12K that are derived from a common proenzyme identified as CPP32. This proenzyme is related to interleukin-1 beta-converting enzyme (ICE) and CED-3, the product of a gene required for programmed cell death in Caenorhabditis elegans. A potent peptide aldehyde inhibitor has been developed and shown to prevent apoptotic events in vitro, suggesting that apopain/CPP32 is important for the initiation of apoptotic cell death.


Asunto(s)
Apoptosis/fisiología , Caspasas , Cisteína Endopeptidasas/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas del Helminto/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans , Caspasa 1 , Caspasa 3 , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/aislamiento & purificación , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas del Helminto/antagonistas & inhibidores , Humanos , Cinética , Espectrometría de Masas , Datos de Secuencia Molecular , Células Tumorales Cultivadas
10.
J Cell Sci Suppl ; 19: 41-9, 1995.
Artículo en Inglés | MEDLINE | ID: mdl-8655646

RESUMEN

Apoptotic cell death is characterized by a dramatic morphological transformation during which apparently healthy cells suddenly initiate a comprehensive program of motility changes and degradative activities that culminates in disassembly of the cell into membrane-enclosed vesicles. The mechanism of the cellular changes during this spectacular execution phase of apoptosis is just now yielding to biochemical analysis. In our laboratory, we have applied a novel in vitro system to the study of these events. In this system, nuclei isolated from healthy cells undergo the characteristic changes of apoptosis rapidly and synchronously. Using this system we have identified the first substrates for interleukin-1 beta-converting enzyme (ICE)-like proteinases during apoptotic execution. One of these, the nuclear enzyme poly (ADP-ribose) polymerase is cleaved very early in the apoptotic process. A second class of proteins, the nuclear lamins, is cleaved later in the pathway. Lamin cleavage requires a second ICE-related proteinase, and is essential for the complete dissolution of nuclei into apoptotic bodies. Studies with our cell-free extracts reveal that the various proteinases and nucleases that operate during the execution phase of apoptosis do so largely in independent parallel biochemical pathways. However, all of these pathways require the action of ICE-related proteinases for their initiation.


Asunto(s)
Apoptosis/fisiología , Núcleo Celular/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Secuencia de Aminoácidos , Animales , Caspasa 1 , Bovinos , Extractos Celulares , Núcleo Celular/enzimología , Cisteína Endopeptidasas/fisiología , Células HL-60 , Células HeLa , Humanos , Laminas , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasas/química
11.
Nature ; 371(6495): 346-7, 1994 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-8090205

RESUMEN

Recent studies suggest that proteases of the interleukin 1-beta-converting enzyme (ICE)/ced-3 family are involved in initiating the active phase of apoptosis. Here we identify a novel protease resembling ICE (prICE) that is active in a cell-free system that reproduces the morphological and biochemical events of apoptosis. prICE cleaves the nuclear enzyme poly(ADP-ribose) polymerase (PARP) at a tetrapeptide sequence identical to one of two ICE sites in pro-interleukin-1-beta. However, prICE does not cleave purified pro-interleukin-1-beta, and purified ICE does not cleave PARP, indicating that the two activities are distinct. Inhibition of prICE abolishes all manifestations of apoptosis in the extracts including morphological changes, cleavage of PARP and production of an oligonucleosomal ladder. These studies suggest that prICE might be pivotal in initiating the active phase of apoptosis in vitro and in intact cells.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Metaloendopeptidasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis , Caspasa 1 , Bovinos , Sistema Libre de Células , Pollos , Cisteína Endopeptidasas/química , Células HeLa , Humanos , Interleucina-1/metabolismo , Metaloendopeptidasas/química , Datos de Secuencia Molecular , Precursores de Proteínas/metabolismo
12.
J Cell Biol ; 123(1): 7-22, 1993 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-8408207

RESUMEN

We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn2+, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis.


Asunto(s)
Apoptosis/fisiología , Núcleo Celular/fisiología , Daño del ADN/fisiología , Mitosis/fisiología , Animales , Afidicolina/farmacología , Apoptosis/efectos de los fármacos , Fraccionamiento Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Sistema Libre de Células , Pollos , Cromosomas , Daño del ADN/efectos de los fármacos , Células HeLa , Humanos , Ratones , Membrana Nuclear/metabolismo , Nucleosomas/metabolismo , Protamina Quinasa/metabolismo , Fase S , Células Tumorales Cultivadas , Zinc/farmacología
13.
Cytometry ; 13(6): 649-52, 1992.
Artículo en Inglés | MEDLINE | ID: mdl-1451596

RESUMEN

A simple technique is suggested for the measurement of drop delay for flow sorting. While the flow cytometer was set to sort a fixed number of particles, the drop-delay setting was changed step by step, and at each step the HRP-coupled particles were sorted into a well of an immunoassay strip. Then the HRP activity of the sorted samples was revealed by routine methods. The maximum level of the enzyme activity shows the proper drop-delay setting. Determination of the drop-delay setting takes only a few minutes. The technique is independent of the type of flow cytometer and does not require any additional equipment.


Asunto(s)
Separación Celular/métodos , Citometría de Flujo/métodos , Animales , Bencimidazoles , Recuento de Células Sanguíneas/instrumentación , Recuento de Células Sanguíneas/métodos , Separación Celular/instrumentación , Citometría de Flujo/instrumentación , Fluoresceína-5-Isotiocianato , Peroxidasa de Rábano Silvestre , Microscopía Fluorescente , Nefelometría y Turbidimetría , Ranidae/sangre , Factores de Tiempo
14.
Exp Cell Res ; 195(1): 247-54, 1991 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-2055271

RESUMEN

Using flow cytometry we found that proliferation of Ehrlich ascites carcinoma (EAC) cells has been reversibly arrested in the second half of the G2 period at the plateau phase of tumor growth in vivo. The ratio of G2/G1 cells increased from 0.3 at 6 days post tumor inoculation to 2.5 at 16 days when up to 25-35% of EAC cells are in G2. It was shown that when ascites fluid removal was followed by transferral in culture, G2-blocked cells synchronously entered the G1 phase via mitosis. In the presence of ascites fluid in the culture medium, EAC cells progressed through G1 and S phases but accumulated in G2. Fetal bovine serum, beta-mercaptoethanol, and caffeine failed to release cells from the G2 block when added to ascites fluid in culture. It is concluded that neither nutrient depletion nor a lack of growth factors is responsible for the G2 arrest of EAC cells. We suggest that ascites fluid contains a factor(s) which potently interrupts the G2 phase of the cell cycle.


Asunto(s)
Carcinoma de Ehrlich/patología , Ciclo Celular , Animales , Ascitis/patología , ADN de Neoplasias/metabolismo , Citometría de Flujo , Técnicas In Vitro , Ratones , Índice Mitótico , Células Tumorales Cultivadas
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