RESUMEN
The French Cancer Plan 2003-2007 has made translational research central to its research programme, to ensure the care-research continuum and the quickest application possible for the most recent discoveries, for the patients' benefit. This is a new field of research, still little-known or ill-understood. A working group, composed of physicians and researchers from academic research and industrial research, sought to define translational research in cancerology and define the issues at stake in it. Translational research needs to develop in close connection with the patients in order to enable a bi-directional flow of knowledge from cognitive research toward medical applications and from observations made on patients toward cognitive research. Placed under the aegis of the French National Cancer Institute and Leem Research, the group has put forth a strategy for implementing translational research in cancerology in France to make it attractive, competitive and efficient and to foster the development of public-private partnerships.
Asunto(s)
Investigación Biomédica/organización & administración , Difusión de Innovaciones , Neoplasias/terapia , Investigación Biomédica/normas , Francia , Humanos , Comunicación Interdisciplinaria , Modelos Animales , Neoplasias/genética , Participación del Paciente/métodosRESUMEN
Prostaglandin E(2) plays multiple roles both in the physiology and the physiopathology of human brain, which are not completely understood. We have identified in a subset of human glioblastoma multiforme (GBM) tumors, the most common form of adult brain cancer, an increased expression of mPGES-1, the enzyme which catalyses the isomerization of PGH(2) into PGE(2) downstream of cyclooxygenase 2 (COX-2). The sensitivity of primary cultures of GBM to apoptosis was augmented by the overexpression of mPGES-1, whereas the knockdown of its expression by shRNA decreased the apoptotic threshold in vitro and stimulated tumor growth in vivo. Adding extracellular PGE(2) in the culture medium failed to reproduce mPGES-1 effect on the cell viability in vitro. However, the intracellular injection of PGE(2) induced a dose-dependent apoptosis in GBM cultures, which was dependent on the presence of Bax, a pro-apoptotic protein. We show that PGE(2) physically associates with Bax, triggering its apoptotic-like change in conformation and its subsequent association with mitochondria. Our results raise questions about the role of PGE(2) in the control of apoptosis and in its potential impact in central nervous system pathologies.
Asunto(s)
Apoptosis , Neoplasias Encefálicas/metabolismo , Dinoprostona/biosíntesis , Glioblastoma/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Dinoprostona/farmacología , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Masculino , Ratones , Ratones Desnudos , Prostaglandina-E Sintasas , Tasa de Supervivencia , Proteína X Asociada a bcl-2/químicaRESUMEN
To analyze the involvement of structured water (bound to macromolecules) in apoptosis-induced mitochondrial outer-membrane permeability, we compared the dynamics of water protons from nuclear magnetic resonance (NMR) data in apoptotic liver mitochondria with that of control mitochondria incubated in vitro with free Ca(2+) (opening of the permeability transition pore, PTP) or with Bax alpha. Our results demonstrate that water molecules in apoptotic mitochondria exhibit an accelerated translational motion of structured water common with that induced by the opening of the PTP, but limited in amplitude. On the other hand, no significant quantitative change in structured water was observed in apoptotic mitochondria, a phenomenon also observed with Bax alpha-induced permeability. We conclude that the changes observed in the different water phases differ both quantitatively and qualitatively during the opening of the PTP and the Bax alpha-induced permeability, and that the apoptotic mitochondria exhibit mixed properties between these model situations.
Asunto(s)
Apoptosis/fisiología , Calcio/farmacología , Membranas Mitocondriales/fisiología , Proteína X Asociada a bcl-2/farmacología , Animales , Western Blotting , Espectroscopía de Resonancia Magnética , Microscopía Electrónica , Mitocondrias Hepáticas/fisiología , Mitocondrias Hepáticas/ultraestructura , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/ultraestructura , Dilatación Mitocondrial , Permeabilidad/efectos de los fármacos , Ratas , Temperatura , Canales Aniónicos Dependientes del Voltaje/efectos de los fármacos , Canales Aniónicos Dependientes del Voltaje/farmacología , Canales Aniónicos Dependientes del Voltaje/fisiología , Agua/fisiologíaRESUMEN
The release of holocytochrome c (cyt c) from mitochondria into the cytosol is reportedly a landmark of the execution phase of apoptosis. As shown here, the P-glycoprotein- (P-gp) expressing K562/ADR cell line (but not the parental K562 cell line) exhibits both cytosolic and mitochondrial cyt c in the absence of any signs of apoptosis. K562/ADR cells were found to be relatively resistant to a variety of different inducers of apoptosis, and blocking the P-gp did not reverse this resistance. The release of cyt c in non-apoptotic K562/ADR cells was not accompanied by that of any other mitochondrial apoptogenic protein, such as AIF or Smac/DIABLO, and was inhibited by Bcl-2 over expression. In addition, using a cell-free system, we show that mitochondria isolated from K562/ADR cells spontaneously released cyt c. These data suggest that cyt c release may be compatible with the preservation of mitochondrial integrity and function, as well as cell proliferation.
Asunto(s)
Citocromos c/metabolismo , Citosol/metabolismo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Leucemia/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Apoptosis , Proteínas Reguladoras de la Apoptosis , Western Blotting , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células K562 , Microscopía Confocal , Microscopía Electrónica , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
We tested whether rat and human MPO have similar antigenic determinants using 36 human MPO-ANCA positive sera, one mouse anti-rat MPO and four mouse anti-human MPO monoclonal reagents. Purified rat and human MPO were used in ELISA, with or without crossinhibition by preincubation with human MPO or irrelevant antigen in the liquid phase. Only one human MPO ANCA positive serum exhibited significant binding in rat MPO ELISA. This binding was poorly inhibited by preincubation with human MPO in the liquid phase, but was conserved after adsorption of non specific anti-rat activity in a chromatography column. Three mouse anti-human MPO IgG monoclonal antibodies did not recognize rat MPO. Only one mouse anti-human MPO IgA monoclonal antibody bound to rat MPO. This binding was poorly inhibited by preincubation with human MPO (35% at 2 micro g/ml). Conversely, the mouse anti-rat MPO monoclonal did not bind human MPO. We have concluded that: (1) Most human MPO-ANCA recognize antigenic determinants on human MPO which are absent on rat MPO. Therefore, human auto-antibodies bind to epitopes which recently appeared after species evolution; (2) Inversely, the mouse anti-rat MPO monoclonal do not bind human MPO. Therefore, rat MPO epitopes have been altered during species evolution; (3) Mice injected with human MPO preferentially develop antibodies against xeno-epitopes which are not present in rodents. Therefore, human MPO may not be the best antigen to raise ANCA in animal models and (4) A comparison of the amino acid sequences of rat and human MPO may help elucidate the major antigenic epitopes.
Asunto(s)
Epítopos/análisis , Peroxidasa/inmunología , Animales , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Anticuerpos Monoclonales/inmunología , Unión Competitiva , Ensayo de Inmunoadsorción Enzimática/métodos , Evolución Molecular , Humanos , Inmunoglobulina A/metabolismo , Inmunoglobulina G/metabolismo , Ratas , Especificidad de la EspecieAsunto(s)
Neoplasias del Colon/inmunología , Carbohidratos de la Dieta/administración & dosificación , Fructosa/administración & dosificación , Oligosacáridos/administración & dosificación , Linfocitos T/inmunología , Animales , Neoplasias del Colon/epidemiología , Neoplasias del Colon/prevención & control , Incidencia , Ratones , Ratones Endogámicos C57BLRESUMEN
Apoptosis dysfunction in metastases has been suggested to participate in their poor response to conventional anticancer treatments. To address this question, we have analyzed the sensitivity to cell death induced by non-steroid anti-inflammatory drug, Sulindac, the most common drug used in colon cancer chemotherapy, 5-fluorouracil (5-FU) and the short chain fatty acid, butyrate (Bu) in cell lines derived from a primary colorectal tumor (ALT-I) as well as the liver (ALT-F) and the lymph-node (ALT-G) metastases. We have previously shown both in vitro by analyzing anchorage-independent cell proliferation and in vivo by subcutaneous injection into athymic nude mice that the ALT-F and ALT-G cells were more tumorigenic than the primary ALT-I cells. All these cell lines, derived from an untreated patient, were highly resistant to apoptosis induced by 5-FU and Sulindac but were sensitive to Bu-induced apoptosis. The resistance to apoptosis was, as quantified by the induction of caspase activity and the relative percentage of apoptotic cells, higher in the metastatic cell lines, than in the ALT cell line. When compared to the primary tumor, more anti-apoptotic bcl-2 and less pro-apoptotic bax were expressed in the liver and lymph node metastatic cell lines. Quite remarkably, the expression of bax was up-regulated during Bu-treatment, a feature that could explain its powerful pro-apoptotic activity.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Butiratos/farmacología , Neoplasias del Colon/patología , Fluorouracilo/farmacología , Neoplasias Hepáticas/secundario , Metástasis Linfática/patología , Anexina A5/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Western Blotting , Neoplasias del Colon/metabolismo , Resistencia a Antineoplásicos , Células HT29/efectos de los fármacos , Células HT29/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulindac/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
We have identified, using site-directed mutagenesis, a proline located at position 13 of Baxalpha (Bax) as crucial for the maintenance of its cytosolic conformation. The substitution of this proline by a valine results in a strong binding of Bax to mitochondria and to conformational changes monitored by a decreased sensitivity of Bax to mild proteolysis and the enhancement of its oligomerization state. Deletion of the C-terminus of Bax does not modify its intracellular localization. On the other hand, the pro-apoptotic activity of Bax is enhanced by a deletion of the C-terminus in the absence of the N-terminus but is decreased in its presence. These results suggest that both extremities functionally interact to control the activity but not the subcellular localization of Bax.
Asunto(s)
Compartimento Celular , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/metabolismo , Citosol/metabolismo , Membranas Intracelulares/metabolismo , Mitocondrias Hepáticas/metabolismo , Datos de Secuencia Molecular , Mutación , Prolina/genética , Prolina/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteína X Asociada a bcl-2RESUMEN
Dendritic cells (DC) have been shown to be efficient antigen-presenting cells (APC) and, as such, could be considered ideal candidates for cancer immunotherapy. Immature DC (iDC) efficiently capture surrounding antigens; however, only mature DC (mDC) prime naive T lymphocytes. Clinical trials using DC-based tumor vaccines have achieved encouraging, but limited, success, possibly due to the use of immature or incompletely mature DC. Thus, it was apparent that a method capable of generating large numbers of fully functional iDC, their pulsing with desired form of tumor antigens and the subsequent complete and reproducible maturation of iDC is needed. Therefore, we compared two different methods of producing large numbers of iDC. Both protocols yielded comparable numbers of cells with an iDC phenotype with phagocytic function. We next determined which of the clinically applicable activators could induce the complete and reproducible maturation of DC, in order to define the most suitable combination for future clinical trials. Only a combination of TNFalpha + Poly (I:C), or a previously described cytokine cocktail of TNFalpha + IL-1beta + IL-6 + prostaglandin E2, induced the complete activation of the whole DC population, as assessed by the cell surface expression of CD83 and costimulatory molecules. The matured DC were functionally superior to iDC in their ability to stimulate the proliferation of allogeneic lymphocytes and autologous keyhole limpet hemocyanin (KLH)-specific T lymphocytes. Furthermore, only the combination of TNFalpha + Poly (L:C) activated DC to produce large amounts of biologically active p70 IL-12. Thus DC maturation by TNFalpha + Poly (I:C) could efficiently bias T cell response towards Th1 response. Implementation of our results into clinical protocols used for DC generation could be beneficial for future immunotherapy trials.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Dendríticas/citología , Interleucina-12/metabolismo , Apoptosis , Diferenciación Celular , División Celular , Supervivencia Celular , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Dinoprostona/farmacología , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Hemocianinas/inmunología , Humanos , Inmunofenotipificación , Interleucina-1/farmacología , Interleucina-12/química , Interleucina-13/farmacología , Interleucina-6/farmacología , Prueba de Cultivo Mixto de Linfocitos , Melanoma/patología , Fagocitosis , Poli I-C/farmacología , Estructura Terciaria de Proteína , Proteínas Recombinantes/farmacología , Reproducibilidad de los Resultados , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
RATIONALE AND OBJECTIVES: The elimination of hepatocyte-directed particulate contrast agents has not been studied in the same detail as particles eliminated mainly by the mononuclear phagocyte system. The aim of the present study was to elucidate the fate of these particles by a multidisciplinary approach. METHODS: After intravenous injection of AMI-HS particles directed to the hepatocytes, rats were killed and cytological studies, by both electron microscopy and histochemistry, and spectroscopic studies of the bile were performed. The data were compared with a dynamic magnetic resonance study of the heart and liver. RESULTS: The particles were rapidly cleared from the blood by Kupffer cells and hepatocytes and then found first in the vascular and later in the biliary pole of the hepatocytes. After 24 hours, a relaxometric characterization of the bile showed the presence of unchanged particles in the bile. CONCLUSIONS: These results show the capacity of the liver to excrete unchanged AMI-HS particles directly into the bile.
Asunto(s)
Bilis/metabolismo , Medios de Contraste/farmacocinética , Hepatocitos/metabolismo , Imagen por Resonancia Magnética , Animales , Bilis/química , Transporte Biológico , Medios de Contraste/administración & dosificación , Inyecciones Intravenosas , Hierro/análisis , Hígado/diagnóstico por imagen , Hígado/patología , Espectroscopía de Resonancia Magnética , Masculino , Microscopía Electrónica , Compuestos Orgánicos , Ratas , UltrasonografíaRESUMEN
We have analyzed the expression of the anti-apoptotic proteins bcl-2, bcl-xl and that of bax, a pro-apoptotic protein, in human WHO grade II astrocytomas (LGA) and WHO grade IV glioblastoma multiforme (GBM). Tumors were obtained immediately after surgical resection and were analyzed by immunohistochemistry (IHC), laser confocal microscopy (LCM) and immunoblots. Both IHC and immunoblot analysis indicated that the expression of bcl-xl was not significantly different between LGA and GBM. IHC indicated that the expression of bcl-2 was inversely correlated to the grade of the tumors (i.e more cells were bcl-2 positive in LGA than in GBM) while the expression of bax was unaffected by the grade of the tumor. In contrast, immunoblots revealed a parallel increase in the expression of bcl-2 and bax from the low to high grade tumor, suggesting a co-regulation of the expression of these two proteins during tumoral progression. Confocal analyses provide us with another possible level of complexicity in the regulation of apoptosis in these tumors, as these markers exhibited different subcellular localizations: bcl-2 was strictly associated with mitochondria and bcl-xl was present in both cytosolic and mitochondrial compartments while bax was found essentially in the cytosol of the tumoral cells. Taken together, our data suggest that the role of bcl-2 related proteins could be regulated at different levels in human astrocytomas (expression, subcellular localization, antigen exposure ...) which should be studied by different techniques.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas/genética , Adulto , Anciano , Apoptosis/genética , Astrocitoma/química , Astrocitoma/mortalidad , Astrocitoma/patología , Neoplasias Encefálicas/química , Neoplasias Encefálicas/mortalidad , Femenino , Regulación Neoplásica de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/análisis , Glioblastoma/química , Glioblastoma/mortalidad , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Microscopía Confocal , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Análisis de Supervivencia , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
The appearance of multidrug-resistant (MDR) proteins or the acquisition of a defective apoptotic programme are major drawbacks in the treatment of cancers since both induce a resistance to classical chemotherapy. However, a link between the two mechanisms has not, as yet, been clearly established. In this study, HL-60 cells cultured in the continual presence of a sub-lethal dose of doxorubicin (dox; HL-60/Dox) were used as a model to study acquired chemoresistance. During the induction of chemoresistance, the appearance of a functional P-glycoprotein (P-gp), in addition to the expression of anti-apoptotic Bcl-2, Bcl-XL and pro-apoptotic Bax proteins was assessed. Parental cells which are sensitive to dox, have no P-gp activity and express Bcl-2 and Bax. After 4 weeks of treatment, a functional P-gp was detected in HL-60/Dox cells. In addition, the synthesis of Bcl-2 appeared to be replaced by Bcl-XL while that of Bax remained unchanged. These cells were also resistant to apoptosis induced by both P-gp and non-P-gp substrates. This inability to induce apoptosis could have resulted from the induction of the expression of the inhibitor of apoptosis protein (XIAP). Our data show that acquired chemoresistance could involve a parallel induction of P-gp and an impairment of the apoptotic pathway.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Apoptosis , Células HL-60/efectos de los fármacos , Proteínas , Caspasa 3 , Caspasas/metabolismo , Citarabina/farmacología , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Activación Enzimática , Citometría de Flujo , Fluoresceínas/farmacología , Humanos , Péptido Hidrolasas/metabolismo , Fenotipo , Probenecid/farmacología , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Inhibidora de la Apoptosis Ligada a X , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
On the basis of studies using the Min mouse model of colon carcinogenesis, we have recently proposed that a fibre-like food (short-chain fructo-oligosaccharides, sc-FOS) fermented in the colon may stimulate a mechanism of cancer immunosurveillance. In the present paper, we have investigated the expression of cytokines as potential effector molecules. Interleukin (IL-)4, IL-5, IL-13, IL-15 and interferon (INF)-gamma mRNAs were detected by a multi-probe ribonuclease protection assay in C57BL/6J and Min mouse colons. IL-15 mRNA expression was significantly amplified (P=0.01) by the sc-FOS-enriched diet in the colon of Min mice.
Asunto(s)
Colon/inmunología , Colon/metabolismo , Fibras de la Dieta/administración & dosificación , Genes APC , Interleucina-15/biosíntesis , Oligosacáridos/administración & dosificación , ARN Mensajero/biosíntesis , Animales , Citocinas/biosíntesis , Dieta , Interleucina-15/genética , Ratones , Ratones Endogámicos C57BL , Ratones MutantesRESUMEN
PURPOSE: To demonstrate the involvement of the various renal structures in acute tubular necrosis (ATN). MATERIAL AND METHODS: In 15 rats, using a T1-/T2-weighted sequence, either gadodiamide alone, or gadodiamide in combination with sprodiamide (a susceptibility agent) were used to enhance the various anatomical substrates of the kidney. The results were compared to those of pathological verification. RESULTS: Experimentally induced ATN of the rat kidney causes profound changes in the medulla, leaving the cortex largely intact. The difference between the normal cortex and the partially necrotic outer medulla, on the one hand, and the papillary region, was significantly enhanced with the combination, whereas a larger region composed of the inner and outer medulla was enhanced after the gadolinium chelate alone. CONCLUSION: The results varied considerably between the two procedures; the double contrast demonstrated a clear difference between the inner and outer medulla, and the gadolinium chelate alone demonstrated a clear difference between the medulla and the cortex. These results demonstrated a clear difference in the compartmentalization between the inner and outer medullary regions, providing complementary information about the pathological condition of the kidney.
Asunto(s)
Medios de Contraste/administración & dosificación , Disprosio , Gadolinio DTPA , Necrosis Tubular Aguda/diagnóstico , Túbulos Renales/patología , Imagen por Resonancia Magnética/métodos , Compuestos Organometálicos , Ácido Pentético , Animales , Diagnóstico Diferencial , Combinación de Medicamentos , Gadolinio DTPA/administración & dosificación , Inyecciones Intravenosas , Médula Renal/patología , Masculino , Compuestos Organometálicos/administración & dosificación , Ácido Pentético/administración & dosificación , Ácido Pentético/análogos & derivados , Ratas , Ratas Endogámicas BNRESUMEN
BACKGROUND: Dietary fibres have been proposed as protective agents against colon cancer but results of both epidemiological and experimental studies are inconclusive. AIMS: Hypothesising that protection against colon cancer may be restricted to butyrate producing fibres, we investigated the factors needed for long term stable butyrate production and its relation to susceptibility to colon cancer. METHODS: A two part randomised blinded study in rats, mimicking a prospective study in humans, was performed using a low fibre control diet (CD) and three high fibre diets: starch free wheat bran (WB), type III resistant starch (RS), and short chain fructo-oligosaccharides (FOS). Using a randomised block design, 96 inbred rats were fed for two, 16, 30, or 44 days to determine the period of adaptation to the diets, fermentation profiles, and effects on the colon, including mucosal proliferation on day 44. Subsequently, 36 rats fed the same diets for 44 days were injected with azoxymethane and checked for aberrant crypt foci 30 days later. RESULTS: After fermentation had stabilised (44 days), only RS and FOS produced large amounts of butyrate, with a trophic effect in the large intestine. No difference in mucosal proliferation between the diets was noted at this time. In the subsequent experiment one month later, fewer aberrant crypt foci were present in rats fed high butyrate producing diets (RS, p=0.022; FOS, p=0.043). CONCLUSION: A stable butyrate producing colonic ecosystem related to selected fibres appears to be less conducive to colon carcinogenesis.
Asunto(s)
Butiratos/metabolismo , Colon/metabolismo , Neoplasias del Colon/prevención & control , Fibras de la Dieta/administración & dosificación , Animales , Azoximetano , Carcinógenos , Colon/patología , Fermentación , Mucosa Intestinal/patología , Oligosacáridos/administración & dosificación , Ratas , Ratas Endogámicas , Almidón/administración & dosificaciónRESUMEN
The expression of bcl-2-related proteins has been shown to be a key element in tumoral malignancy. The degradation of the extracellular matrix (ECM) by specialized matrix metalloproteinases (MMPs) is another major step in tumor invasion and metastasis. We have examined, in a rat glioma cell line A15A5, the effect of the stable transfection of human bcl-2, bax and bcl-xl on MMPs expression. Using a zymographic assay, we found that all transfected cell lines expressed a gelatinase activity which is predominantly associated with MMP-9. In bcl-2 and bcl-xl transfected cells, the transcription of MMP-9 was decreased compared to that of control or bax transfected cells. In addition, in bax transfected A15A5, we observed a down regulation of TIMP-1, the inhibitor of MMP-9. These results suggest that the ratio between MMP-9 and its inhibitor TIMP-1 is tightly controlled in cells overexpressing bcl-2 related proteins (i.e., high ratio in bax transfected A15A5 and low ratio in bcl-2 transfected A15A5). However, MMPs secreted by bcl-2 transfected cells were still capable of hydrolyzing FasL present on human lymphocytes. Our results suggest that the expression of bcl-2 related proteins could participate in the regulation of MMP-9/TIMP-1 in gliomas.
Asunto(s)
Metaloproteinasas de la Matriz/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Apoptosis , Proteína Ligando Fas , Glioma/genética , Glioma/metabolismo , Glioma/patología , Humanos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
INTRODUCTION: Interleukin-2 (IL-2) and sodium butyrate allow rats to be cured of peritoneal carcinomatosis from colon cancer. We performed a phase I trial of IL-2 and high-dose arginine butyrate (ArgB) in patients with advanced metastatic colorectal cancer. PATIENTS AND METHODS: From April to July 1997, six patients were included in the trail; they had a median age of 52 years, four had a performance status of 0, two had a performance status of 1 with normal biological functions. All patients had received at least two prior lines of chemotherapy. A fixed dose of 18 MIU/m2 IL-2,was administered by subcutaneous injection and ArgB was delivered via continuous intravenous infusion on days 1-6 with escalating doses starting at 2 g kg(-1) day(-1). RESULTS: The planned dose escalation was not possible because of toxicities. A daily ArgB dose of 2 g/kg was delivered for nine cycles. Level 2 (4 g/kg) could not be delivered in three of the six patients because of liver toxicity. The dose-limiting toxicities were fatigue and liver function disturbances. The maximum tolerated dose for ArgB was 3 g kg(-1) day(-1), in combination with IL-2 at 12 MIU m2 day(-1). No clinical response was seen. Pharmacokinetic analysis showed large intra- and interindividual variations. CONCLUSION: This schedule with a high dose of ArgB proved to be highly toxic with liver insufficiency. We will be running another trial with lower doses of ArgB calculated from the schedule used in the experimental model, starting at a dose of 20 mg kg(-1) day(-1) for ArgB and 200000 UI kg(-1) day(-1) IL-2, every 8 h.
Asunto(s)
Arginina/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Interleucina-2/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Anciano , Animales , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/patología , Femenino , Humanos , Infusiones Intravenosas , Inyecciones Subcutáneas , Interleucina-2/inmunología , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Ratas , Resultado del TratamientoRESUMEN
We have shown previously that rats can be cured from induced peritoneal colon carcinomatosis by injections of apoptotic bodies derived from tumor cells and interleukin 2. This curative treatment generated a tumor-specific cytotoxic T-cell response associated with a humoral response. Autoantibodies from sera of cured rats strongly recognized a Mr 67,000 protein from apoptotic bodies and weakly reacted with a protein of Mr approximately 97,000 in PROb parental cells. We now show that these autoantibodies are directed against BARD1, originally identified as a protein interacting with the product of the breast cancer gene 1, BRCA1. We demonstrate that the Mr 67,000 antigen is a cleaved form of BARD1 present in apoptotic bodies derived from rat and human colon and mammary carcinoma cell lines. Moreover, we show that the cleavage site of BARD1 is located NH2 terminally but downstream of the RING domain essential for BARD1 and BRCA1 protein interaction. In vitro studies using [35S]methionine-labeled human BARD1 and apoptotic cellular extracts derived from SW48 carcinoma cells indicate that BARD1 proteolysis occurs at an early stage of apoptosis and in a cell cycle-dependent manner. This hydrolysis is inhibited by EGTA, and the calpain inhibitor I, N-acetyl-leu-leu-norleucinal, but not by several caspases inhibitors, suggesting that BARD1 is hydrolyzed by the calcium-dependent cysteine proteases, calpains. Thus, the highly immunogenic form of cleaved BARD1 could contribute to the antitumoral response mediated by apoptotic bodies.
Asunto(s)
Apoptosis , Autoantígenos/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/metabolismo , Autoantígenos/química , Proteína BRCA1/metabolismo , Western Blotting , Neoplasias de la Mama/metabolismo , Calpaína/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Ciclo Celular , Fraccionamiento Celular , Clonación Molecular , Neoplasias del Colon/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , ADN Complementario/metabolismo , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Biblioteca de Genes , Humanos , Leupeptinas/farmacología , Neoplasias Mamarias Animales/metabolismo , Ratones , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
The interaction of the anti-apoptotic members of the Bcl-2 family with mitochondria, through their hydrophobic C-terminus, has been proposed to play a crucial role in the execution phase of apoptosis. We report here that a substitution of the C-terminal end of pro-apoptotic bax by that of anti-apoptotic bcl-xL (baxCxL) does not modify its association with mitochondria in human and rat cells or in Saccharomyces cerevisiae. In addition, while bax sensitizes these cells to apoptotic stimuli, the construct baxCxL does not affect the apoptotic response in transfected cells. These results suggest that the C-terminus of bax plays an important role in apoptosis independently of its membrane addressing/targeting mechanism.