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1.
Cell Death Differ ; 15(5): 859-66, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18239673

RESUMEN

Members of the inhibitor of apoptosis protein (IAP) family have demonstrated functions in cell death, cell signalling, cell migration and mitosis. Several of them are E3 enzymes in the ubiquitination of proteins that leads to their degradation by the proteosomal machinery. We previously reported that one of them, cellular inhibitor of apoptosis protein-1 (c-IAP1), migrated from the nucleus to the surface of the Golgi apparatus in cells undergoing differentiation. Here, we show that c-IAP1 is a client protein of the stress protein HSP90 beta. In three distinct cellular models, the two proteins interact and migrate from the nucleus to the cytoplasm along the differentiation process through a leptomycin B-sensitive pathway. Inhibition of HSP90 proteins by small chemical molecules and specific depletion of HSP90 beta isoform by siRNA both lead to auto-ubiquitination of c-IAP1 and its degradation by the proteasome machinery. This chaperone function of HSP90 towards c-IAP1 is specific of its beta isoform as specific depletion of HSP90alpha does not affect c-IAP1 content. Chemical inhibition of HSP90 or siRNA-mediated depletion of HSP90 beta both inhibit cell differentiation, which can be reproduced by siRNA-mediated depletion of c-IAP1. Altogether, these results suggest that HSP90 beta prevents auto-ubiquitination and degradation of its client protein c-IAP1, whose depletion would be sufficient to inhibit cell differentiation.


Asunto(s)
Diferenciación Celular/fisiología , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/metabolismo , Células Epiteliales/citología , Células Epiteliales/fisiología , Proteínas HSP90 de Choque Térmico/genética , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Macrófagos/citología , Macrófagos/fisiología , Isoformas de Proteínas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo
2.
Cell Death Differ ; 13(9): 1423-33, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16676004

RESUMEN

In healthy cells, cytochrome c (Cyt c) is located in the mitochondrial intermembrane/intercristae spaces, where it functions as an electron shuttle in the respiratory chain and interacts with cardiolipin (CL). Several proapoptotic stimuli induce the permeabilization of the outer membrane, facilitate the communication between intermembrane and intercristae spaces and promote the mobilization of Cyt c from CL, allowing for Cyt c release. In the cytosol, Cyt c mediates the allosteric activation of apoptosis-protease activating factor 1, which is required for the proteolytic maturation of caspase-9 and caspase-3. Activated caspases ultimately lead to apoptotic cell dismantling. Nevertheless, cytosolic Cyt c has been associated also to vital cell functions (i.e. differentiation), suggesting that its release not always occurs in an all-or-nothing fashion and that mitochondrial outer membrane permeabilization may not invariably lead to cell death. This review deals with the events involved in Cyt c release from mitochondria, with special attention to its regulation and final consequences.


Asunto(s)
Citocromos c/metabolismo , Mitocondrias/metabolismo , Regulación Alostérica , Animales , Apoptosis , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Calcio/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Permeabilidad de la Membrana Celular , Activación Enzimática , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Mitocondrias/enzimología , Membranas Mitocondriales/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo
3.
Handb Exp Pharmacol ; (172): 171-98, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16610360

RESUMEN

The highly conserved heat shock proteins (Hsps) accumulate in cells exposed to heat and a variety of other stressful stimuli. Hsps, that function mainly as molecular chaperones, allow cells to adapt to gradual changes in their environment and to survive in otherwise lethal conditions. The events of cell stress and cell death are linked and Hsps induced in response to stress appear to function at key regulatory points in the control of apoptosis. Hsps include anti-apoptotic and pro-apoptotic proteins that interact with a variety of cellular proteins involved in apoptosis. Their expression level can determine the fate of the cell in response to a death stimulus, and apoptosis-inhibitory Hsps, in particular Hsp27 and Hsp70, may participate in carcinogenesis. This review summarizes the apoptosis-regulatory function of Hsps.


Asunto(s)
Apoptosis , Proteínas de Choque Térmico/fisiología , Chaperonas Moleculares/fisiología , Animales , Chaperonina 60/fisiología , Proteínas del Choque Térmico HSP110/fisiología , Proteínas de Choque Térmico HSP27 , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas HSP90 de Choque Térmico/fisiología , Proteínas de Choque Térmico/antagonistas & inhibidores , Humanos , Proteínas de Neoplasias/fisiología , Neoplasias/tratamiento farmacológico , Neoplasias/etiología
4.
Int J Radiat Oncol Biol Phys ; 51(5): 1354-60, 2001 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-11728697

RESUMEN

PURPOSE: Nuclear factor-kappaB (NF-kappaB) has been implicated in anti-apoptotic gene transactivation, according to its transcriptional activity. The present study was designed to investigate whether constitutive NF-kappaB activity could modulate basal apoptosis and intrinsic radiosensitivity of KB head-and-neck carcinoma cell line and KB3 subline. The KB3 subline was more radiosensitive (SF2 = 0.48, alpha = 0.064) than the radioresistant KB parental cell line (SF2 = 0.80, alpha = 0.114). METHODS AND MATERIALS: Constitutive NF-kappaB DNA-binding activity was determined using electrophoretic mobility shift assay. Modulation of NF-kappaB activity was performed by exposing both cell lines to tumor necrosis factor alpha or dexamethasone. Apoptotic cell population was analyzed using flow cytometry (annexin V/propidium iodide). Radiosensitivity was assessed from determination of the surviving fraction at 2 Gy (SF2), and alpha and beta parameters were determined using the linear-quadratic model. RESULTS: Constitutive NF-kappaB activity was found to be significantly lower in KB3 than in KB. KB cell line exposure to dexamethasone significantly decreased NF-kappaB DNA-binding activity and, consequently, enhanced baseline apoptosis and radiosensitivity (alpha values: 0.114 vs. 0.052). Conversely, exposure of KB3 cells to tumor necrosis factor alpha increased NF-kappaB DNA-binding activity and resulted in a significant decrease (50%) in rate of apoptosis and in radiosensitivity (SF2 values: 0.48 vs. 0.63). CONCLUSIONS: Modulation of NF-kappaB DNA-binding activity influences baseline apoptosis and intrinsic radiosensitivity.


Asunto(s)
Apoptosis , Neoplasias de Cabeza y Cuello/radioterapia , FN-kappa B/fisiología , Tolerancia a Radiación , ADN/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Células Tumorales Cultivadas
5.
Int J Oncol ; 23(1): 81-7, 2003 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12792779

RESUMEN

5-Fluorouracil (5FU) exposure can lead to both G1/S arrest and apoptosis induction which are dependent of P53 induction. The human papilloma virus oncoproteins (HPV), E6 and E7, inactivate respectively P53 and Rb. P53 degradation by E6 protein, leads to lack of G1/S arrest after genotoxic stress. Overexpression of E7 protein prevents P53-induced G1/S arrest following DNA damage. However, few studies have described 5FU effect and efficacy on cancer cell lines presenting HPV 18 positive status. KB cell line and KB3 subline presented wild-type P53 status and difference in 5FU sensitivity. During 5FU exposure, P53 gene and protein expression was increased in both cell lines. E6 and E7 mRNA and protein expression was decreased in KB and KB3. P53 and E6 protein expressions were inversely correlated. 5FU exposure, induced a G1/S arrest which can be maintained or intensified by P53 via P21 induction expression. 5FU exposure has led to apoptosis induction related to P53 induction. In the present study, 5FU exposure was shown to induce G1/S arrest and apoptosis by P53-dependent molecular pathway, in HPV 18 positive cells.


Asunto(s)
Apoptosis , Proteínas de Unión al ADN , Resistencia a Antineoplásicos , Fluorouracilo/farmacología , Fase G1 , Proteínas Nucleares , Proteínas Oncogénicas Virales/biosíntesis , Fase S , Western Blotting , Bromodesoxiuridina/farmacología , Ciclo Celular , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , ADN/metabolismo , Daño del ADN , Citometría de Flujo , Humanos , Concentración 50 Inhibidora , Microscopía Fluorescente , Proteínas Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , ARN/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Proteína p53 Supresora de Tumor/metabolismo
6.
Cancer Radiother ; 8(5): 315-21, 2004 Oct.
Artículo en Francés | MEDLINE | ID: mdl-15561597

RESUMEN

NF-kappaB (Nuclear Factor-kappaB) was described for the first time in 1986 as a nuclear protein binding to the kappa immunoglobulin-light chain enhancer. Since then, NF-kappaB has emerged as an ubiquitous factor involved in the regulation of numerous important processes as diverse as immune and inflammatory responses, apoptosis and cell proliferation. These last two properties explain the implication of NF-kappaB in the tumorigenic process as well as the promise of a targeted therapeutic intervention. This review focuses on the current knowledge on NF-kappaB regulation and discusses the therapeutic potential of targeting NF-kappaB in cancer in particular during radiotherapy.


Asunto(s)
Factores Inmunológicos , FN-kappa B/fisiología , FN-kappa B/efectos de la radiación , Neoplasias/radioterapia , Antioxidantes/fisiología , Apoptosis/fisiología , Proliferación Celular , Supervivencia Celular/efectos de la radiación , Humanos , Neoplasias/etiología , Fosforilación , Inhibidores de Proteasoma , Tolerancia a Radiación , Radiación Ionizante
7.
Cell Death Differ ; 2008 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-25361076

RESUMEN

Members of the inhibitor of apoptosis protein (IAP) family have demonstrated functions in cell death, cell signalling, cell migration and mitosis. Several of them are E3 enzymes in the ubiquitination of proteins that leads to their degradation by the proteosomal machinery. We previously reported that one of them, cellular inhibitor of apoptosis protein-1 (c-IAP1), migrated from the nucleus to the surface of the Golgi apparatus in cells undergoing differentiation. Here, we show that c-IAP1 is a client protein of the stress protein HSP90ß. In three distinct cellular models, the two proteins interact and migrate from the nucleus to the cytoplasm along the differentiation process through a leptomycin B-sensitive pathway. Inhibition of HSP90 proteins by small chemical molecules and specific depletion of HSP90ß isoform by siRNA both lead to auto-ubiquitination of c-IAP1 and its degradation by the proteasome machinery. This chaperone function of HSP90 towards c-IAP1 is specific of its ß isoform as specific depletion of HSP90α does not affect c-IAP1 content. Chemical inhibition of HSP90 or siRNA-mediated depletion of HSP90ß both inhibit cell differentiation, which can be reproduced by siRNA-mediated depletion of c-IAP1. Altogether, these results suggest that HSP90ß prevents auto-ubiquitination and degradation of its client protein c-IAP1, whose depletion would be sufficient to inhibit cell differentiation.Cell Death and Differentiation advance online publication, 1 February 2008; doi:10.1038/sj.cdd.4402320.

8.
Cytometry ; 48(1): 6-13, 2002 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-12116375

RESUMEN

BACKGROUND: Bromodeoxyuridine (BrdU) cell cycle analysis using flow cytometry is of clinical interest for making treatment decisions or for predicting response and survival, through proliferation rate (labeling index or S-phase fraction) assessment or T(pot) calculation. Thymidylate synthase expression was tested in vitro, in vivo, and clinically as a prognostic factor for 5-fluorouracil (5FU) sensitivity. However, results were still controversial. Moreover, we had reported that 5FU sensitivity was related to the labeling index of untreated cell cultures. METHODS: We used six human cancer cell lines that exhibited a wide range of 5FU sensitivity. Cell cycle analysis was performed using flow cytometry monovariate propidium iodide (PI) analysis and bivariate distributions of BrdU incorporation versus DNA content. 5FU sensitivity was assayed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay. RESULTS: In all cell lines, 5FU exposure resulted in a statistically significant G(1)/S accumulation. No statistically significant relationship was seen between G(0)/G(1) delay determined by monovariate analysis and 5FU sensitivity. However, 5FU sensitivity was statistically correlated to the labeling index and G(1)/S subpopulation assessed with bivariate analysis using BrdU incorporation versus DNA content. CONCLUSIONS: Cellular proliferation parameters using BrdU incorporation are more informative than PI for in vitro 5FU sensitivity. Because BrdU incorporation could be assessed clinically, it could also be informative for 5FU clinical response prediction.


Asunto(s)
Antimetabolitos Antineoplásicos/toxicidad , Fluorouracilo/toxicidad , Fase G1/efectos de los fármacos , Fase S/efectos de los fármacos , Adenocarcinoma , Antimetabolitos , Neoplasias de la Mama , Bromodesoxiuridina , Colorantes , Femenino , Citometría de Flujo , Neoplasias de Cabeza y Cuello , Humanos , Neoplasias Pancreáticas , Propidio , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos
9.
Can J Physiol Pharmacol ; 80(7): 638-43, 2002 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12182321

RESUMEN

The effect of chemoresistance induction in radiosensitivity and cellular behavior after irradiation remains misunderstood. This study was designed to understand the relationship between radiation-induced cell cycle arrest, apoptosis, and radiosensitivity in KB cell line and KB3 subline selected after 5-fluorouracil (5FU) exposure. Exposure of KB cells to 5FU led to an increase in radiosensitivity. G2/M cell cycle arrest was observed in the two cell lines after irradiation. The radioresistant KB cell line reached the maximum arrest two hours before KB3. The cellular exit from this arrest was found to be related to the wild type p53 protein expression induction. After irradiation, only KB3 cell line underwent apoptosis. This apoptosis induction seemed to be independent of G2/M arrest exit, which was carried out later. The difference in radiosensitivity between KB and KB3 subline may result therefore from both a difference in apoptosis induction and a difference in G2/M arrest maximum duration. Moreover, 5FU exposure has led to an increase in constitutive p53 protein expression, which may be associated with an increase in basal apoptosis cell fraction. Given the existing correlation between radiosensitivity and the percentage of basal apoptosis, the constitutive p53 protein expression may be related to intrinsic radiosensitivity in our cellular model.


Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Fluorouracilo/farmacología , Genes p53/fisiología , Neoplasias de Cabeza y Cuello/patología , Fármacos Sensibilizantes a Radiaciones/farmacología , Anexina A5/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Línea Celular , Colorantes , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Fase G2/efectos de los fármacos , Fase G2/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Células KB , Mitosis/efectos de los fármacos , Mitosis/efectos de la radiación , Propidio
10.
Br J Cancer ; 83(10): 1380-6, 2000 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-11044365

RESUMEN

p53 tumour-suppressor gene is involved in cell growth control, arrest and apoptosis. Nevertheless cell cycle arrest and apoptosis induction can be observed in p53-defective cells after exposure to DNA-damaging agents such as 5-fluorouracil (5-FU) suggesting the importance of alternative pathways via p53-independent mechanisms. In order to establish relationship between p53 status, cell cycle arrest, Bcl-2/Bax regulation and 5-FU sensitivity, we examined p53 mRNA and protein expression and p53 protein functionality in wild-type (wt) and mutant (mt) p53 cell lines. p53 mRNA and p53 protein expression were determined before and after exposure to equitoxic 5-FU concentration in six human carcinoma cell lines differing in p53 status and displaying marked differences in 5-FU sensitivity, with IC(50)values ranging from 0.2-22.6 mM. 5-FU induced a rise in p53 mRNA expression in mt p53 cell lines and in human papilloma virus positive wt p53 cell line, whereas significant decrease in p53 mRNA expression was found in wt p53 cell line. Whatever p53 status, 5-FU altered p53 transcriptional and translational regulation leading to up-regulation of p53 protein. In relation with p53 functionality, but independently of p53 mutational status, after exposure to 5-FU equitoxic concentration, all cell lines were able to arrest in G1. No relationship was evidenced between G1 accumulation ability and 5-FU sensitivity. Moreover, after 5-FU exposure, Bax and Bcl-2 proteins regulation was under p53 protein control and a statistically significant relationship (r = 0.880, P = 0.0097) was observed between Bcl-2/Bax ratio and 5-FU sensitivity. In conclusion, whatever p53 status, Bcl-2 or Bax induction and Bcl-2/Bax protein ratio were correlated to 5-FU sensitivity.


Asunto(s)
Adenocarcinoma/genética , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Carcinoma/genética , Fluorouracilo/farmacología , Genes p53/genética , Neoplasias de Cabeza y Cuello/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas/análisis , Adenocarcinoma/patología , Neoplasias de la Mama/patología , Carcinoma/patología , Ciclo Celular , Relación Dosis-Respuesta a Droga , Femenino , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/patología , Humanos , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/biosíntesis , Células Tumorales Cultivadas/efectos de los fármacos , Regulación hacia Arriba , Proteína X Asociada a bcl-2
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