RESUMEN
The bacterial lacZ gene encoding for beta-galactosidase (beta-gal) is a common reporter gene used in transgenic mice. Nonetheless, the absence of fluorigenic substrates usable in live animals greatly hampered the non-invasive follow-up of this reporter gene expression. We used far-red fluorescence for imaging beta-Gal expression in live cells in vitro or in vivo. The 9H-(1,3-dichloro-9,9-dimethylacridin- 2-one-7-yl) beta-D-galactopyranoside substrate was used to monitor beta-Gal expression as a reporter of tumor growth, or of the physiological levels of an endogenous gene or of gene transfer in lung. A quantitative evaluation of this method as well as a comparison of its sensitivity with Firefly Luciferase-based bioluminescence was also performed. In vivo measurements showed that 10(3) beta-Gal tumor cells located under the skin were detectable. In deeper organs like lung, as little as 5 ng of beta-Gal or Luciferase enzymes per mg of proteins were measured, confirming that both techniques reached similar sensibilities. Nonetheless, quantitative comparison of beta-Gal levels measured with far-red imaging or with a standardized enzymatic evaluation after killing revealed that the 2D-fluorescent reflectance imaging method is submitted to a color-dependent disparity of the organs and cannot supply quantitative measurements but that a simple correction can be applied.
Asunto(s)
Genes Reporteros , Terapia Genética/métodos , Operón Lac , Luciferasas/genética , Neoplasias/terapia , Animales , Expresión Génica , Marcadores Genéticos , Luminiscencia , Proteínas Luminiscentes , Ratones , Ratones Transgénicos , Microscopía Fluorescente/métodos , Transfección/métodos , beta-Galactosidasa/análisisRESUMEN
AIMS: Pancreatic islets can be lost early following allotransplantation from oxidative stress. Antioxidant enzyme overexpression could confer a beneficial effect on islets exposed to reactive oxygen species (ROS) and nitrogen species. Here, we tested the effect of MnTMPyP, a superoxide dismutase/catalase mimetic. METHODS: INS-1 insulin-secreting cells or human islets were cultured with MnTMPyP and exposed to a superoxide donor (the hypoxanthine/xanthine oxidase (HX/XO) system), a nitric oxide donor [3-morpholinosydnonimine (SIN-1)] or menadione. Viability of INS-1 cells was assessed by WST-1 colorimetric assay and FACS analysis (Live/Dead test). ROS production was determined using fluorescent probes. Islet viability was estimated by WST-1 assay and endocrine function by static incubation. RESULTS: Following MnTMPyP treatment, ROS production in INS-1 cells was reduced by 4- to 20-fold upon HX/XO challenge and up to 2-fold upon SIN-1 stress. This phenomenon correlated with higher viability measured by WST-1 or Live/Dead test. MnTMPyP preserved islet viability upon exposure to SIN-1 or menadione but not upon an HX/XO challenge. Similarly, decrease in insulin secretion tended to be less pronounced in MnTMPyP-treated islets than in control islet when exposed to SIN-1, but no changes were noticed during an HX/XO stress. CONCLUSIONS: MnTMPyP was able to improve the viability of INS-1 cells and human islets exposed to oxidative challenges in vitro. Protection of INS-1 cells could be as high as 90%. This agent is therefore potentially attractive in situations involving the overproduction of ROS, such as islet transplantation.
Asunto(s)
Islotes Pancreáticos/fisiología , Metaloporfirinas/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Manganeso , Ratas , Especies Reactivas de Oxígeno/metabolismo , Vasodilatadores/farmacologíaRESUMEN
DNA microarrays allow simultaneous measurement of the expression of several thousand genes in a biological specimen. This technique represents a major advance in the analysis of tumour biopsies. It may be used to refine the anatomical- pathological diagnosis at a molecular level and thus lead to better diagnostic and prognostic classification and improved therapeutic decisions.
Asunto(s)
Neoplasias Pulmonares/diagnóstico , Análisis de Secuencia por Matrices de Oligonucleótidos , Humanos , Neoplasias Pulmonares/genética , Oncología Médica/métodos , Neumología/métodosRESUMEN
A prospective screening program, including CT, autofluorescent bronchoscopy, biopsies and bronchial lavage (BL) collection, was initiated with the specific goal of identifying biomarkers for the early detection of non-small cell lung cancer. We report and discuss the results of p16, DAPK, MGMT, FHIT and APC methylation analysis in the 126 first patients: 77 at high risk of cancer and 49 followed up after primary cancer resection. Positive results were found in 49% of BLs, 53% in current smokers and 43% in former smokers. In presence of peripheral tumours, only 38% of BLs were abnormal versus 73% in presence of central tumours, 50% in presence of preneoplasic lesions and 47% in absence of lesions. FHIT methylation was an early event, observed in one-third of the BLs from patients with or without lesions as well as in tumours. APC methylation was a late event observed in 33% of tumours but rarely in BLs. p16 was methylated in 17% of BLs but in 48% of tumours; DAPK in 15% of BL and 22% of tumours. MGMT methylation was rare. Among patients followed up after cancer surgery, 14 were in remission with normalised BL, whereas three had positive BLs and relapsed with a central tumour. Thus, gene methylation in BL might help to detect central tumours but a CT is crucial for peripheral cancer detection.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Metilación de ADN , Genes Relacionados con las Neoplasias , Neoplasias Pulmonares/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Lavado Broncoalveolar , Carcinoma de Pulmón de Células no Pequeñas/patología , Femenino , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/diagnóstico , Regiones Promotoras Genéticas , Estudios Prospectivos , Sensibilidad y Especificidad , Fumar/efectos adversos , Tomografía Computarizada por Rayos XRESUMEN
Mesenchymal stem cells (MSC) have recently been used successfully in humans to control severe graft-versus-host disease. However, the mechanisms involved in their immunomodulatory effects remain a matter of debate. Here, we show that MSC are unable to activate allogeneic T cells even in the presence of T-cell growth factors. We then found that MSC inhibit T-cell proliferation triggered either by allogeneic, mitogenic or antigen-specific stimuli. Interestingly, MSC inhibit T-cell proliferation by inducing apoptosis of activated T cells, but have no effect on resting T cells. Furthermore, we show that this apoptosis could be related to the conversion of tryptophan into kynurenine by indoleamine 2,3-dioxygenase expressed by MSC in the presence of IFNgamma. Moreover, we show that the inhibitory effect of MSC is neither abrogated nor modified during expansion in culture or after irradiation. Together, these results bring new insight to the mechanisms of immunosuppression induced by MSC and might help to develop their clinical use controlling immune-related adverse effects in humans.
Asunto(s)
Apoptosis/inmunología , Células Madre Mesenquimatosas/inmunología , Linfocitos T/inmunología , Muerte Celular/inmunología , Humanos , Interferón gamma/farmacología , Leucocitos Mononucleares/inmunología , Células Madre Mesenquimatosas/enzimología , Triptófano Oxigenasa/biosíntesis , Triptófano Oxigenasa/inmunologíaRESUMEN
UNLABELLED: Complete staging (extensive marrow investigation and meta-iodo benzylguanine (MIBG) scan) is considered as mandatory both at diagnosis and after chemotherapy for assessment of metastases in neuroblastomas. However the correlation between bone marrow invasion and uptake of MIBG at metastatic sites remains unclear. This study investigates whether MIBG alone is sufficiently sensitive to make these procedures redundant. PATIENTS AND METHODS: 20 children over one year of age, with histologically proven metastatic neuroblastoma were studied. Extensive bone marrow assessment and MIBG bone scan performed both at diagnosis and after completion of induction chemotherapy were reviewed. RESULTS: At diagnosis metastases were detected by marrow investigation alone in 2, MIBG alone in 2 and both procedures in 16. After induction chemotherapy metastases were detected by only marrow investigation in 2, by only MIBG in 3, by both procedures in 6 patients, and by none in 9. CONCLUSIONS: Whether marrow investigations and MIBG scan explore the same phenomenon remains unclear. However it appears that marrow disease that is histologically detectable may remain MIBG negative both at diagnosis and after treatment. Both procedures are still justified at time of diagnosis and evaluation of response.
Asunto(s)
3-Yodobencilguanidina , Examen de la Médula Ósea/métodos , Neoplasias de la Médula Ósea/diagnóstico , Neoplasias Óseas/diagnóstico , Neuroblastoma/diagnóstico , Radiofármacos , Neoplasias de la Médula Ósea/diagnóstico por imagen , Neoplasias de la Médula Ósea/secundario , Neoplasias Óseas/diagnóstico por imagen , Neoplasias Óseas/secundario , Niño , Preescolar , Humanos , Ilion/diagnóstico por imagen , Lactante , Estadificación de Neoplasias , Neuroblastoma/diagnóstico por imagen , Neuroblastoma/tratamiento farmacológico , Cintigrafía , Estudios RetrospectivosRESUMEN
OBJECTIVES: A large fraction of an islet graft can be lost early following allotransplantation from various non specific mechanisms including oxidative stress. Overexpression of antioxidant enzymes could confer a beneficial effect on islets exposed to reactive oxygen and nitrogen species. We examined the viability of beta cells driven to overexpress glutathione peroxidase (GPx) and exposed to a superoxide donor (hypoxanthine/xanthine oxidase HX/XO) and a nitric oxide donor (3-morpholinosydnonimine SIN-1). METHODS: Cultured INS-1 rat-derived insulin-secreting cells were transfected by an E1-deleted adenovirus carrying GPx cDNA (AdGPx). Additional experiments were performed with an adenovector carrying Cu/Zn superoxide dismutase cDNA (AdSOD). Cellular viability was tested by the WST-1 colorimetric assay and functionality by static incubation. RESULTS: AdGPx increased GPx activity within 48 hours from 0 (untransfected cells) to 60 +/- 11 U/g (cells transfected at an MOI of 25: 1). GPx overexpression significantly reduced cytotoxicity induced by HX/XO from 10.81 +/- 1.41 to 5.42 +/- 2.62% at 10 mU/ml and from 61.19 +/- 4.17 to 52.9 +/- 4.39% at 20 mU/ml (p=0.0002, transfected cells vs control cells). Doses of SIN-1 from 600 to 1000 micromol/l resulted in cytotoxicity ranging from 17.66 +/- 3.48 to 45.97 +/- 6.48% in control cells and from 5.65 +/- 1.37 to 35.80 +/- 5.59% in AdGPx transfected cells (p=0.015). The combination of AdGPx and AdSOD did not exhibit any synergistic cytoprotective effect. Control cells exposed to a HX/XO stress exhibited a reduction in glucose-theophylline stimulated insulin secretion by half, while stressed GPx overexpressing-cells maintained the same insulin secretion level than non-stressed cells. CONCLUSIONS: Adenoviral-induced overexpression of GPx enhances the resistance of a rat beta cell line to both reactive oxygen (ROS) and reactive nitrogen species (RNS) cytotoxicity. Transposition of these findings to human islet transplantation with a clinically-relevant procedure deserves further investigations.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Glutatión Peroxidasa/genética , Insulina/metabolismo , Molsidomina/análogos & derivados , Estrés Oxidativo/fisiología , Adenoviridae , Animales , Bovinos , ADN Complementario/genética , Inhibidores Enzimáticos/farmacología , Glucosa/farmacología , Glutatión Peroxidasa/metabolismo , Hipoxantina/farmacología , Secreción de Insulina , Insulinoma , Molsidomina/farmacología , Neoplasias Pancreáticas , Ratas , Proteínas Recombinantes/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Transfección , Células Tumorales Cultivadas , Xantina Oxidasa/farmacologíaRESUMEN
VP22, a structural protein from herpes simplex virus type I, exhibits the unique property of intercellular trafficking. This protein is exported from primary expressing cells and subsequently imported into neighbouring cells. This property is conserved when VP22 is genetically fused to a protein, making it a promising tool to enhance the delivery of a gene product. We chose to study the intercellular transport and biological effect of a fusion protein between the putative tumour suppressor gene p27(Kip1) and VP22. We show that in vitro, P27VP22 is able to spread as efficiently as VP22. Functionality of the P27VP22 protein was demonstrated by its ability to inhibit cyclin/CDK2 complexes activity. In proliferation and clonogenicity assays, transfection with the P27VP22 plasmid resulted in a stronger cell growth inhibition when compared to transfection with the p27(Kip1) vector. In vivo, sub cutaneous tumours established in nude mice were injected with naked DNA encoding P27 or P27VP22. Our results show that P27VP22 can spread in vivo and that injections of the P27VP22 plasmid resulted in a significantly greater antitumour activity than injections of the P27 plasmid. This study confirms the usefulness of VP22-mediated delivery and suggests that P27VP22 may have applications in cancer gene therapy.
Asunto(s)
Proteínas de Ciclo Celular , Terapia Genética/métodos , Proteínas Inmediatas-Precoces , Neoplasias Experimentales/terapia , Proteínas Recombinantes de Fusión/genética , Transfección/métodos , Proteínas Supresoras de Tumor , Proteínas Virales , Animales , Antígeno CD11b/metabolismo , Células COS , Caspasa 3 , Caspasas/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Femenino , Células HeLa , Humanos , Ratones , Ratones Desnudos , Proteínas Reguladoras y Accesorias ViralesRESUMEN
The mechanisms involved in p53-mediated cell death remain controversial. In the present study, we investigated this cell death pathway by stably transfecting the p53-null H358 cell line with a tetracycline-dependent wild type p53-expressing vector. Restoration of p53 triggered a G(2)/M cell cycle arrest and enhanced BAX protein expression, without inducing apoptosis or potentiating the cytotoxic effect of etoposide, vincristine, and cis-platinum. Accordingly, overexpression of BAX in H358 cells, through stable transfection of a tetracycline-regulated expression vector, did not induce cell death. Interestingly, the methylxanthine caffeine (4 mm) promoted the translocation of BAX from the cytosol to the mitochondria. In the setting of an overexpression of BAX, caffeine induced a conformational change of the protein and apoptosis. The consequences of caffeine were independent of its cell cycle-related activities. All together, caffeine synergizes with p53 for inducing cell death through a cell cycle-independent mechanism, involving mitochondrial translocation and conformational change of BAX protein.
Asunto(s)
Apoptosis , Cafeína/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas/metabolismo , Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Western Blotting , Muerte Celular , Línea Celular , Cisplatino/farmacología , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Etopósido/farmacología , Genes p53/genética , Humanos , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Plásmidos/metabolismo , Conformación Proteica , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Vincristina/farmacología , Xantinas/farmacología , Proteína X Asociada a bcl-2RESUMEN
We have studied the occurrence and association of 11q deletions with other chromosomal imbalances in Stage 4 neuroblastomas. To this purpose we have performed comparative genomic hybridization (CGH) analysis on 50 Stage 4 neuroblastomas and these data were analyzed together with those from 33 previously published cases. We observed a high incidence of 11q deletion in Stage 4 neuroblastoma without MYCN amplification (59%) whereas 11q loss was only observed in 15% of neuroblastomas with MYCN-amplification (p = 0.0002) or 11% of cases with 1p deletion detected by CGH (p = 0.0001). In addition, 11q loss showed significant positive correlation with 3p loss (p = 0.0002). Event-free survival was poor and not significantly different for patients with or without 11q deletion. Our study provides further evidence that Stage 4 neuroblastomas with 11q deletions represent a distinct genetic subgroup that typically shows no MYCN-amplification nor 1p deletion. Moreover, it shows that neuroblastomas with 11q deletion also often present 3p deletion. This genetic subgroup shows a similar poor prognosis as MYCN amplified 4 neuroblastomas.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 11 , Genoma Humano , Neuroblastoma/genética , Hibridación de Ácido Nucleico , Adolescente , Niño , Preescolar , Aberraciones Cromosómicas , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 3 , Supervivencia sin Enfermedad , Femenino , Humanos , Hibridación Fluorescente in Situ , Lactante , Masculino , Modelos Genéticos , Estudios Multicéntricos como Asunto , Mutación , Metástasis de la Neoplasia , Neuroblastoma/diagnóstico , Neuroblastoma/mortalidad , Pronóstico , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
p53 gene therapy can induce tumor regression, but the low efficacy of in vivo gene transfer has greatly hampered the mechanistic analysis of this antitumoral activity. We therefore used a p53-null human NSCLC cell line in which we reintroduced the wild-type p53 gene under control of a tetracycline-dependent promoter. P53 induction provokes cell cycle arrest in G0/G1 and G2/M phase, an up-regulation of p21, a down-regulation of cyclin B1 and appearance of senescence features without down-regulation of human telomerase reverse transcriptase. No detectable morphological changes of apoptosis nor procaspase-3 activation are observed. In subcutaneous tumors grafted in nude mice, the induction of p53 expression leads to a complete and longlasting tumor regression in 28 days which is associated with cell cycle arrest, but not detectable apoptosis nor inhibition of angiogenesis. These results show that irreversible cell cycle arrest is sufficient to elicit tumor regression after p53 gene transfer in p53-deficient tumor cells.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/terapia , Genes p53 , Terapia Genética/métodos , Neoplasias Pulmonares/terapia , Animales , Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclo Celular/genética , Senescencia Celular/genética , Femenino , Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Microscopía Fluorescente , Transfección , Células Tumorales CultivadasRESUMEN
Immunotherapy with interleukin 2 (IL-2) is not an effective anti-cancer treatment in the majority of patients with renal cell carcinoma (RCC), suggesting that the activation of cytotoxic T cells or NK cells may be impaired in vivo in these patients. The production of immunosuppressive factors by RCC was investigated. Using immunohistochemistry, IL-10 was detectable in 10 of 21 tumour samples tested. IL-10 was undetectable in the supernatant of cell lines derived from these RCCs. However, these cell lines or their conditioned medium (RCC CM), but not normal renal epithelial cells adjacent to the RCC or breast carcinoma cell lines, were found to induce IL-10, as well as prostaglandin E2 (PGE2) and tumour necrosis factor (TNF)alpha production by autologous or allogeneic peripheral blood mononuclear cells (PBMCs) and monocytes. IL-10 production induced by RCC CM was found to be dependent on TNF-alpha and PGE2 since an anti-TNF-alpha antibody (Ab) inhibited 40-70% of IL-10 production by monocytes, and the combination of anti-TNF-alpha Ab and indomethacin, an inhibitor of PGE2 production, inhibited 80-94% of RCC CM-induced IL-10 production by monocytes. The RCC CM of the five cell lines tested were found to induce a down-regulation of the expression of HLA-DR and CD86, as well as a strong inhibition of mannose receptor-dependent endocytosis by monocytes. The blockade of HLA-DR and CD86 expression was partially abrogated by indomethacin and anti-IL-10 Ab respectively, and completely abrogated by an anti-TNF-alpha Ab. The inhibition of mannose receptor-dependent endocytosis was partially abrogated by an anti-IL-10 Ab and completely abrogated by an anti-TNF-alpha Ab. These results indicate that RCCs induce IL-10, PGE2 and TNF-alpha production by monocytes, which down-regulate the expression of cell-surface molecules involved in antigen presentation as well as their endocytic capacity.
Asunto(s)
Carcinoma de Células Renales/inmunología , Dinoprostona/biosíntesis , Interleucina-10/biosíntesis , Neoplasias Renales/inmunología , Monocitos/inmunología , Secuencia de Bases , Medios de Cultivo Condicionados , Cartilla de ADN , Dinoprostona/fisiología , Ensayo de Inmunoadsorción Enzimática , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/fisiologíaAsunto(s)
Ensayos Clínicos como Asunto/estadística & datos numéricos , Terapia Genética/estadística & datos numéricos , Neoplasias/terapia , Sistema de Registros/estadística & datos numéricos , Antígenos de Neoplasias/genética , Citocinas/administración & dosificación , Citocinas/genética , Resistencia a Antineoplásicos/genética , Técnicas de Transferencia de Gen , Genes Supresores de Tumor , Marcadores Genéticos , Vectores Genéticos/administración & dosificación , Antígenos HLA/genética , Antígenos HLA/uso terapéutico , Humanos , Neoplasias/genética , Neoplasias/inmunología , Profármacos/uso terapéuticoRESUMEN
The escape of malignant cells from the immune response against the tumor may result from a defective differentiation or function of professional antigen-presenting cells (APC), ie, dendritic cells (DC). To test this hypothesis, the effect of human renal cell carcinoma cell lines (RCC) on the development of DC from CD34(+) progenitors was investigated in vitro. RCC cell lines were found to release soluble factors that inhibit the differentiation of CD34(+) cells into DC and trigger their commitment towards monocytic cells (CD14(+)CD64(+)CD1a-CD86(-)CD80(-)HLA-D Rlow) with a potent phagocytic capacity but lacking APC function. RCC CM were found to act on the two distinct subpopulations emerging in the culture at day 6 ([CD14(+)CD1a-] and [CD14(-)CD1a+]) by inhibiting the differentiation into DC of [CD14(+)CD1a-] precursors and blocking the acquisition of APC function of the [CD14(-)CD1a+] derived DC. Interleukin-6 (IL-6) and macrophage colony-stimulating factor (M-CSF) were found to be responsible for this phenomenon: antibodies against IL-6 and M-CSF abrogated the inhibitory effects of RCC CM; and recombinant IL-6 and/or M-CSF inhibited the differentiation of DC similarly to RCC CM. The inhibition of DC differentiation by RCC CM was preceeded by an induction of M-CSF receptor (M-CSFR; CD115) and a loss of granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR; CD116) expression at the surface of CD34(+) cells, two phenomenon reversed by anti-IL-6/IL-6R and anti-M-CSF antibodies, respectively. Finally, a panel of tumor cell lines producing IL-6 and M-CSF induced similar effects. Taken together, the results suggest that the inhibition of DC development could represent a frequent mechanism by which tumor cells will escape immune recognition.
Asunto(s)
Antígenos CD34 , Carcinoma de Células Renales/metabolismo , Células Dendríticas/fisiología , Interleucina-6/fisiología , Factor Estimulante de Colonias de Macrófagos/fisiología , Anticuerpos/farmacología , Antígenos CD34/metabolismo , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Relación Dosis-Respuesta a Droga , Factores de Crecimiento Endotelial/inmunología , Factores de Crecimiento Endotelial/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunofenotipificación , Interleucina-6/inmunología , Interleucina-6/farmacología , Linfocinas/inmunología , Linfocinas/farmacología , Factor Estimulante de Colonias de Macrófagos/inmunología , Factor Estimulante de Colonias de Macrófagos/farmacología , Receptor de Factor Estimulante de Colonias de Macrófagos/biosíntesis , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The first protocol of gene therapy was started in 1990 for two children with adenosine deaminase deficiency. In 1997, the field largely widened, with 2100 patients treated: 10 per cent suffer from hereditary disease, cystic fibrosis mainly, and 90 per cent acquired disease, mainly cancers or HIV infection. No toxicity of gene therapy has been observed for the patients or their environment. A psychological barrier has been crossed which might allow an easing of the regulations. Industrial firms are heavily engaged in this field and are defining the rules of production and use. After in vivo administration to patients, the gene is expressed but the vectorisation is still very weak. Gene therapy should thus be limited to indications with a well-established clinical and scientific rationale.
Asunto(s)
Enfermedades Genéticas Congénitas/terapia , Terapia Genética , Niño , Infecciones por VIH/terapia , Humanos , Neoplasias/terapia , Inmunodeficiencia Combinada Grave/terapiaRESUMEN
The prognosis of pediatric neuroblastoma depends both on clinical presentation and on certain cellular and molecular characteristics. At the present time, two hypotheses can be drawn to explain both clinical and biological heterogeneity. In the first hypothesis, neuroblastoma progresses from early to late clinical stages through a classical multistep process linked to an accumulation of molecular abnormalities. In the second hypothesis, neuroblastoma represents an heterogeneous group of unrelated diseases, where most of stages I and II or stage IVS neuroblastomas can rather be considered as benign tumors, and stage IV neuroblastoma as a true malignant proliferation. To ascertain relevant biological factors for the prognosis of the disease, it is uppermost important that all investigators agree on biological criteria for analysis when neuroblastoma tissue is available in screened and unscreened populations. This paper reviews the biological tools available for prognosis in neuroblastoma, the priority for analysis of biological markers according to reliability, feasibility, and reproducibility of analysis procedure, and the conditions of tissue storage for further analysis of these biological markers. The standardized biological evaluation of neuroblastoma will allow, first, to collect sufficient data for multivariate analysis of prognostic factors and, second, to better define the putative links between various forms of the disease.
Asunto(s)
Biomarcadores de Tumor , Neuroblastoma/genética , Biomarcadores de Tumor/metabolismo , Biopsia/métodos , Médula Ósea , Preescolar , Cromosomas Humanos Par 1/genética , Amplificación de Genes , Eliminación de Gen , Genes myc/genética , Marcadores Genéticos , Humanos , Receptores de Hialuranos/metabolismo , Lactante , Proteínas de Neoplasias/metabolismo , Neuroblastoma/sangre , Neuroblastoma/patología , Ploidias , Pronóstico , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor trkA , Receptores de Factor de Crecimiento Nervioso/metabolismoAsunto(s)
Terapia Genética/métodos , Inmunoterapia/métodos , Interleucina-2/genética , Neoplasias/terapia , Animales , Carcinoma de Células Renales/terapia , Protocolos Clínicos , Femenino , Técnicas de Transferencia de Gen , Humanos , Interleucina-2/biosíntesis , Neoplasias Renales/terapia , Neoplasias Mamarias Experimentales/terapia , Melanoma/terapia , Ratones , Neoplasias/genética , Neoplasias/inmunologíaAsunto(s)
Neoplasias/genética , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Apoptosis , Sustancias de Crecimiento/genética , Humanos , Incidencia , Metástasis de la Neoplasia , Neoplasias/metabolismo , Neoplasias/patología , Oncogenes/fisiología , Lesiones Precancerosas/diagnóstico , PronósticoRESUMEN
We developed a new vector for gene targeting of neuroblastoma (NB) cells, based on the utilization o a monoclonal antibody (chCE7) covalently linked to polylysine (PL). In the presence of chloroquine, chCE7-PL-DNA complexes transfected NB cells as efficiently as DOTAP, transfectam, TF-X50, or lipofectamine. This was demonstrated by transfection of the luciferase or beta-galactosidase reporter genes in three different NB cell lines. This transfection was specific, since it was inhibited in the presence of competing unconjugated chCE7 antibody (Ab), and was not observed in cell lines negative for the CE7 antigen. We tested the potential biological activity of a plasmid coding for gamma-interferon (gamma IFN) transfected with chCE7-PL. HLA ABC expression on NB cells was induced after transfection with pCMV-gamma IFN at a higher level than after incubation with 1000 IU/ml of purified gamma IFN. Moreover, these HLA ABC-positive NB cells were able to activate autologous cytotoxic T lymphocytes in vitro. Thus chCE7-PL is able to target a plasmid to NB cells and to allow the expression of the transfected gene in a biologically active form.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Neuroblastoma/genética , Transfección , Especificidad de Anticuerpos , Citotoxicidad Inmunológica , Antígenos HLA-D/metabolismo , Humanos , Interferón gamma/inmunología , Neuroblastoma/inmunología , Polilisina , Células Tumorales CultivadasRESUMEN
A pilot study of neuroblastoma mass screening was initiated in January 1990 in the Rhône French district. The expected number of births per year is 26,000. The study is designed for a 5-year period with three major goals: 1) measurement of the compliance rate of a voluntary test at 4 months of age; 2) evaluation of the technical value of high-pressure liquid chromatography (HPLC) as a screening method; and 3) detailed biological characterization of all detected tumors. 61,551 children were screened between May 1, 1990 and December 31, 1993. Participation was 69% in 1990, 81.5% in 1991, and over 83% in 1992. HPLC was a satisfactory assay method. The number of clinical examinations required for positive tests as defined in the protocol is 1 per 3,621 tests. The false positive rate is 1 per 3,583 tests. Eight neuroblastomas were discovered by-screening (one stage I, three stage II, one stage III, three stage IVs). All are alive and well but were good prognosis cases according to the main prognostic factors. Five patients were discovered before screening (so called Halo effect): one stage I, one stage III, three stage IVs. One died of disease and four are alive in complete remission after treatment. Two patients were false negative (one stage III with N-myc amplification, one stage IV with bad prognosis features) and three cases of neuroblastoma were missed because of noncompliance with the screening program. This pilot study concludes on the feasibility of a mass screening program in France. The estimated cumulative incidence of neuroblastoma at 3 years is 1 per 4,375 living births and overdiagnosis is probable. All the detected cases were of good prognosis and the false negative ones were poor prognosis cases.