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1.
PLoS Comput Biol ; 18(6): e1010236, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35759459

RESUMEN

Microtubules and their post-translational modifications are involved in major cellular processes. In severe diseases such as neurodegenerative disorders, tyrosinated tubulin and tyrosinated microtubules are in lower concentration. We present here a mechanistic mathematical model of the microtubule tyrosination cycle combining computational modeling and high-content image analyses to understand the key kinetic parameters governing the tyrosination status in different cellular models. That mathematical model is parameterized, firstly, for neuronal cells using kinetic values taken from the literature, and, secondly, for proliferative cells, by a change of two parameter values obtained, and shown minimal, by a continuous optimization procedure based on temporal logic constraints to formalize experimental high-content imaging data. In both cases, the mathematical models explain the inability to increase the tyrosination status by activating the Tubulin Tyrosine Ligase enzyme. The tyrosinated tubulin is indeed the product of a chain of two reactions in the cycle: the detyrosinated microtubule depolymerization followed by its tyrosination. The tyrosination status at equilibrium is thus limited by both reaction rates and activating the tyrosination reaction alone is not effective. Our computational model also predicts the effect of inhibiting the Tubulin Carboxy Peptidase enzyme which we have experimentally validated in MEF cellular model. Furthermore, the model predicts that the activation of two particular kinetic parameters, the tyrosination and detyrosinated microtubule depolymerization rate constants, in synergy, should suffice to enable an increase of the tyrosination status in living cells.


Asunto(s)
Tubulina (Proteína) , Tirosina , Evaluación Preclínica de Medicamentos , Microtúbulos/química , Modelos Teóricos
2.
Breast Cancer Res Treat ; 156(2): 301-10, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26975189

RESUMEN

The objectives of this phase I/II study (NCT00140738) were to evaluate the safety and clinical activity of a cancer immunotherapeutic agent (recombinant HER2 protein (dHER2) and the immunostimulant AS15) in patients with HER2-overexpressing metastatic breast cancer (MBC). Forty HER2-positive MBC patients received up to 18 doses (12q2w, 6q3w) of dHER2 immunotherapeutic, as first- or second-line therapy following response to trastuzumab-based treatment as maintenance. Toxicity was graded by the Common Terminology Criteria for Adverse Events (CTCAE) and clinical activity was evaluated by target lesion assessment according to the Response Evaluation Criteria in Solid Tumors (RECIST). Immunogenicity was assessed. The dHER2 immunotherapeutic was well tolerated: grade 1/2 adverse events (AEs) were most common. No cardiac events were observed and one patient experienced an asymptomatic decrease of left ventricular ejection fraction below the normal range (47 %). Both humoral and cellular immunogenicity to the dHER2 antigen was observed. No patient discontinued the immunizations because of AEs but 35/40 withdrew prematurely, 34 because of disease progression (24/34 before or at the tumor assessment after dose 6). One patient achieved a complete response lasting 11 months and one patient had a partial response lasting 3.5 months. Ten patients experienced stable disease ≥26 weeks with 4/10 still in stable disease at the last tumor assessment after 47 weeks. Immunization of MBC patients with the dHER2 immunotherapeutic was associated with minimal toxicity and no cardiac events. Clinical activity was observed with two objective responses and prolonged stable disease for 10/40 patients.


Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias de la Mama/terapia , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/administración & dosificación , Trastuzumab/administración & dosificación , Adyuvantes Inmunológicos/efectos adversos , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/metabolismo , Supervivencia sin Enfermedad , Esquema de Medicación , Femenino , Humanos , Inmunoterapia , Persona de Mediana Edad , Receptor ErbB-2/genética , Proteínas Recombinantes/efectos adversos , Trastuzumab/uso terapéutico , Resultado del Tratamiento
3.
Breast Cancer Res Treat ; 156(2): 319-30, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26993131

RESUMEN

This Phase I dose-escalation study (NCT00058526) assessed the safety and immunogenicity of an anti-cancer immunotherapeutic (recombinant HER2 protein (dHER2) combined with the immunostimulant AS15) in patients with early-stage HER2-overexpressing breast cancer (BC). Sixty-one trastuzumab-naive patients with stage II-III HER2-positive BC received the dHER2 immunotherapeutic after surgical resection and adjuvant therapy. They were allocated into four cohorts receiving different doses of dHER2 (20, 100, 500 µg) combined with a fixed AS15 dose. Safety and immunogenicity (dHER2-specific antibody responses) were assessed. After completing the immunization schedule (three or six doses over 14 weeks) and a six-month follow-up, the patients were followed for 5 years for late toxicity, long-term immunogenicity, and clinical status. The immunizations were well tolerated, and increasing doses of dHER2 had no impact on the frequency or severity of adverse events. Few late toxicities were reported, and after 5 years 45/54 patients (83.3 %) were still alive, while 28/45 (62 %) with known disease status were disease free. Regarding the immunogenicity of the compound, a positive association was found between the dHER2 dose, the immunization schedule, and the prevalence of dHER2-specific humoral responses. Among the patients receiving the most intense immunization schedule with the highest dHER2 dose, 6/8 maintained their dHER2-specific antibody response 5 years after immunization. The dHER2 immunotherapeutic had an acceptable safety profile in early HER2-positive BC patients. dHER2-specific antibody responses were induced, with the rate of responders increasing with the dHER2 dose and the number and frequency of immunizations.


Asunto(s)
Neoplasias de la Mama/terapia , Factores Inmunológicos/administración & dosificación , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Proteínas Recombinantes/administración & dosificación , Regulación hacia Arriba , Adulto , Anciano , Neoplasias de la Mama/metabolismo , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Cálculo de Dosificación de Drogas , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Factores Inmunológicos/efectos adversos , Inmunoterapia , Persona de Mediana Edad , Proteínas Recombinantes/inmunología , Análisis de Supervivencia , Resultado del Tratamiento
4.
JCI Insight ; 9(3)2024 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-38165745

RESUMEN

The impairment of left ventricular (LV) diastolic function with an inadequate increase in myocardial relaxation velocity directly results in lower LV compliance, increased LV filling pressures, and heart failure symptoms. The development of agents facilitating the relaxation of human cardiomyocytes requires a better understanding of the underlying regulatory mechanisms. We performed a high-content microscopy-based screening in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using a library of 2,565 human miRNA mimics and measured relaxation kinetics via high-computing analyses of motion movies. We identified hsa-miR-548v, a primate-specific miRNA, as the miRNA producing the largest increase in relaxation velocities. This positive lusitropic effect was reproduced in engineered cardiac tissues generated with healthy and BRAF T599R mutant hiPSC-CMs and was independent of changes in calcium transients. Consistent with improvements in viscoelastic responses to mechanical stretch, RNA-Seq showed that hsa-miR-548v downregulated multiple targets, especially components of the mechanosensing machinery. The exogenous administration of hsa-miR-548v in hiPSC-CMs notably resulted in a significant reduction of ANKRD1/CARP1 expression and localization at the sarcomeric I-band. This study suggests that the sarcomere I-band is a critical control center regulating the ability of cardiomyocytes to relax and is a target for improving relaxation and diastolic dysfunction.


Asunto(s)
Cardiopatías , Células Madre Pluripotentes Inducidas , MicroARNs , Animales , Humanos , Cardiopatías/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , MicroARNs/metabolismo , Miocardio , Miocitos Cardíacos/metabolismo
5.
Bioinformatics ; 28(14): 1945-6, 2012 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-22586178

RESUMEN

MOTIVATION: High-throughput screening is a powerful technology principally used by pharmaceutical industries allowing the identification of molecules of interest within large libraries. Originally target based, cellular assays provide a way to test compounds (or other biological material such as small interfering RNA) in a more physiologically realistic in vitro environment. High-content screening (HCS) platforms are now available at lower cost, giving the opportunity for universities or research institutes to access those technologies for research purposes. However, the amount of information extracted from each experiment is multiplexed and hence difficult to handle. In such context, there is an important need for an easy-to-use, but still powerful software able to manage multidimensional screening data by performing adapted quality control and classification. HCS-analyzer includes: a user-friendly interface specifically dedicated to HCS readouts, an automated approach to identify systematic errors potentially occurring during screening and a set of tools to classify, cluster and identify phenotypes of interest among large and multivariate data. AVAILABILITY: The application, the C# .Net source code, as well as detailed documentation, are freely available at the following URL: http://hcs-analyzer.ip-korea.org.


Asunto(s)
Biología Computacional/métodos , Ensayos Analíticos de Alto Rendimiento , Programas Informáticos , Algoritmos , Análisis por Conglomerados , Industria Farmacéutica , ARN Interferente Pequeño , Interfaz Usuario-Computador
6.
Bioinformatics ; 28(2): 261-8, 2012 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-22121160

RESUMEN

MOTIVATION: High-throughput screening (HTS) is an important method in drug discovery in which the activities of a large number of candidate chemicals or genetic materials are rapidly evaluated. Data are usually obtained by measurements on samples in microwell plates and are often subjected to artefacts that can bias the result selection. We report here a novel edge effect correction algorithm suitable for RNA interference (RNAi) screening, because its normalization does not rely on the entire dataset and takes into account the specificities of such a screening process. The proposed method is able to estimate the edge effects for each assay plate individually using the data from a single control column based on diffusion model, and thus targeting a specific but recurrent well-known HTS artefact. This method was first developed and validated using control plates and was then applied to the correction of experimental data generated during a genome-wide siRNA screen aimed at studying HIV-host interactions. The proposed algorithm was able to correct the edge effect biasing the control data and thus improve assay quality and, consequently, the hit-selection step.


Asunto(s)
VIH/metabolismo , Interferencia de ARN , Algoritmos , Artefactos , Descubrimiento de Drogas , Estudio de Asociación del Genoma Completo , Humanos , ARN Interferente Pequeño/metabolismo
7.
PLoS One ; 18(4): e0283941, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37014877

RESUMEN

Intracellular accumulation of tau protein is a hallmark of Alzheimer's Disease and Progressive Supranuclear Palsy, as well as other neurodegenerative disorders collectively known as tauopathies. Despite our increasing understanding of the mechanisms leading to the initiation and progression of tau pathology, the field still lacks appropriate disease models to facilitate drug discovery. Here, we established a novel and modulatable seeding-based neuronal model of full-length 4R tau accumulation using humanized mouse cortical neurons and seeds from P301S human tau transgenic animals. The model shows specific and consistent formation of intraneuronal insoluble full-length 4R tau inclusions, which are positive for known markers of tau pathology (AT8, PHF-1, MC-1), and creates seeding competent tau. The formation of new inclusions can be prevented by treatment with tau siRNA, providing a robust internal control for use in qualifying the assessment of potential therapeutic candidates aimed at reducing the intracellular pool of tau. In addition, the experimental set up and data analysis techniques used provide consistent results in larger-scale designs that required multiple rounds of independent experiments, making this is a versatile and valuable cellular model for fundamental and early pre-clinical research of tau-targeted therapies.


Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Ratones , Animales , Humanos , Proteínas tau/genética , Proteínas tau/metabolismo , Ratones Transgénicos , Encéfalo/metabolismo , Tauopatías/metabolismo , Enfermedad de Alzheimer/patología , Neuronas/metabolismo , Descubrimiento de Drogas
8.
Med Sci (Paris) ; 37(3): 249-257, 2021 Mar.
Artículo en Francés | MEDLINE | ID: mdl-33739272

RESUMEN

The advent of the molecular biology and the completion of the human genome sequencing prompted the pharmaceutical industry to progressively implement target-centric drug discovery strategies. However, concerns regarding the research and development productivity during the last ten years, combined with technological developments in high-content screening, automation, image analysis and artificial intelligence triggered a renewed interest for the phenotypic drug discovery approaches. Target-centric and phenotypic approaches are more and more considered complementary, hence, positioning the target deconvolution on the critical path. This review analyzes the evolution of the target-centric and phenotypic approaches, focusing more specifically on the high-content screening and the target deconvolution technologies currently available.


TITLE: Du criblage à haut contenu à la déconvolution de cibles - Nouvelle donne pour les approches phénotypiques. ABSTRACT: L'avènement de la biologie moléculaire et l'achèvement du séquençage du génome humain ont conduit l'industrie pharmaceutique à progressivement implémenter des approches dites cible-centriques pour identifier les candidats médicaments. Cependant, la faible productivité de la recherche et du développement en ce début de millénaire, combinée aux évolutions technologiques dans des domaines tels que l'ingénierie cellulaire, le criblage à haut contenu, la robotique, l'analyse d'images et l'intelligence artificielle, ont nourri un fort regain d'intérêt pour les approches phénotypiques. De plus en plus fréquemment, les approches cible-centriques et phénotypiques sont considérées de façon complémentaire, positionnant ainsi les techniques de déconvolution1 de cible sur le chemin critique de la découverte et du développement de médicaments. Cette revue analyse l'évolution des approches cible-centriques versus phénotypiques, en se focalisant plus particulièrement sur le criblage à haut contenu et les différentes techniques de déconvolution de cible aujourd'hui disponibles.


Asunto(s)
Descubrimiento de Drogas/métodos , Humanos , Fenotipo , Investigación
9.
J Cheminform ; 13(1): 91, 2021 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-34819133

RESUMEN

With the development of advanced technologies in cell-based phenotypic screening, phenotypic drug discovery (PDD) strategies have re-emerged as promising approaches in the identification and development of novel and safe drugs. However, phenotypic screening does not rely on knowledge of specific drug targets and needs to be combined with chemical biology approaches to identify therapeutic targets and mechanisms of actions induced by drugs and associated with an observable phenotype. In this study, we developed a system pharmacology network integrating drug-target-pathway-disease relationships as well as morphological profile from an existing high content imaging-based high-throughput phenotypic profiling assay known as "Cell Painting". Furthermore, from this network, a chemogenomic library of 5000 small molecules that represent a large and diverse panel of drug targets involved in diverse biological effects and diseases has been developed. Such a platform and a chemogenomic library could assist in the target identification and mechanism deconvolution of some phenotypic assays. The usefulness of the platform is illustrated through examples.

10.
Ann Surg Oncol ; 17(6): 1530-8, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20177798

RESUMEN

BACKGROUND: The treatment of choice for elderly women with breast cancer remains controversial. This retrospective analysis of a cohort from a single institution was designed to evaluate whether such patients are really undertreated because of their age and to reappraise their usual management. METHODS: The characteristics of 538 patients aged > or = 70 years with operable breast cancer, treated between 1995 and 1999, were retrospectively analyzed comparing patients aged 70 to 75 years (group I, n = 288), 75 to 80 years (group II, n = 156), and > or = 80 years (group III, n = 94). Cause-specific survival, distant recurrence-free interval, and local control were estimated by the Kaplan-Meier method and compared by log rank test. Multivariate analysis used Cox regression. RESULTS: In group III, tumors were more frequently T2 than T1 (P < 0.0001) and estrogen receptor negative (P = 0.045) than in groups I and II. Surgery was performed in 94.6% of patients, breast-conserving in 72.1% (62% in group III; P = 0.0015) with axillary dissection in 89.2% (77% in group III; P = 0.0015); 100% received radiotherapy after lumpectomy (hypofractionated in 63% of group III; P < 0.0001). Adjuvant hormone therapy and chemotherapy were administered to 57 and 3.7% of patients, respectively. At 7 years, no difference in the three groups was observed for cause-specific survival (91% for group I, 89% for group II, 86% for group III) distant recurrence-free interval, and local control (>90%). CONCLUSIONS: Elderly patients with operable breast cancer who are completely and correctly treated with realistic treatment options that are based on surgery and adjuvant radiotherapy have a similar chance of being cured as younger patients.


Asunto(s)
Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Anciano Frágil , Mastectomía , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/radioterapia , Quimioterapia Adyuvante , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Francia , Hospitales Universitarios , Humanos , Estimación de Kaplan-Meier , Escisión del Ganglio Linfático , Mastectomía/métodos , Mastectomía/mortalidad , Mastectomía Segmentaria , Análisis Multivariante , Invasividad Neoplásica , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , Radioterapia Adyuvante , Estudios Retrospectivos , Resultado del Tratamiento
11.
Sarcoma ; 2009: 764379, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-20011664

RESUMEN

Purpose. An analysis of the clinicopathologic features and treatment of patients was performed to guide evaluation and management of postirradiation sarcoma. Patients and Methods. Between 1994 and 2001, 25 patients with postirradiation sarcoma were treated in one center with different chemotherapy, mainly in neoadjuvant setting (19). Tumors for which these patients received radiotherapy initially were mainly breast carcinoma (for 15 patients). The postirradiation sarcomas were of different histopathologic forms, most frequently osteosarcoma, leiomyosarcoma, and angiosarcoma. Results. Of the 25 patients, 19 were initially treated with chemotherapy. Nine of 19 pretreated patients achieved clinical partial response (RP = 47%). Leiomyosarcomas were good responders (3/4) and undifferentiated sarcoma (3/5). Responders were more often treated with MAID (6/8). Eight of the 9 responders underwent surgery. Two patients achieved complete histological response. Seven of the 9 good responders are alive with a median follow up of 24 months. For all treated patients, median follow up 24 months (6-84 months), overall survival and disease free survival were, respectively, 17/25 (68%), and 14/25 (56%). Conclusion. From our data, postirradiation sarcoma should not be managed differently from primary sarcoma. Chemotherapy has to be included in the treatment plan of postirradiation sarcoma, in future studies.

12.
Cancer Res ; 79(14): 3762-3775, 2019 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-31123088

RESUMEN

DNA damage checkpoint kinases ATR and WEE1 are among key regulators of DNA damage response pathways protecting cells from replication stress, a hallmark of cancer that has potential to be exploited for therapeutic use. ATR and WEE1 inhibitors are in early clinical trials and success will require greater understanding of both their mechanism of action and biomarkers for patient selection. Here, we report selective antitumor activity of ATR and WEE1 inhibitors in a subset of non-germinal center B-cell (GCB) diffuse large B-cell lymphoma (DLBCL) cell lines, characterized by high MYC protein expression and CDKN2A/B deletion. Activity correlated with the induction of replication stress, indicated by increased origin firing and retardation of replication fork progression. However, ATR and WEE1 inhibitors caused different amounts of DNA damage and cell death in distinct phases of the cell cycle, underlying the increased potency observed with WEE1 inhibition. ATR inhibition caused DNA damage to manifest as 53BP1 nuclear bodies in daughter G1 cells leading to G1 arrest, whereas WEE1 inhibition caused DNA damage and arrest in S phase, leading to earlier onset apoptosis. In vivo xenograft DLBCL models confirmed differences in single-agent antitumor activity, but also showed potential for effective ATR inhibitor combinations. Importantly, insights into the different inhibitor mechanisms may guide differentiated clinical development strategies aimed at exploiting specific vulnerabilities of tumor cells while maximizing therapeutic index. Our data therefore highlight clinical development opportunities for both ATR and WEE1 inhibitors in non-GCB DLBCL subtypes that represent an area of unmet clinical need. SIGNIFICANCE: ATR and WEE1 inhibitors demonstrate effective antitumor activity in preclinical models of DLBCL associated with replication stress, but new mechanistic insights and biomarkers of response support a differentiated clinical development strategy.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Replicación del ADN/efectos de los fármacos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Pirimidinonas/farmacología , Sulfóxidos/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Indoles , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Morfolinas , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Proto-Oncogénicas c-myc/genética , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Pirimidinonas/administración & dosificación , Sulfonamidas , Sulfóxidos/administración & dosificación , Ensayos Antitumor por Modelo de Xenoinjerto
13.
Curr Opin Pharmacol ; 42: 40-45, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30032033

RESUMEN

In recent years, questions about the sustainability of the current drug discovery process have triggered a revival of interest in phenotypic drug discovery approaches. This trend has clearly been amplified by the emergence of multiple cell-based assay technologies enabling a higher degree of translatability between in vitro conditions and physio-pathological situations, including induced pluripotent stem cells, three-dimensional models, co-culture and organ-on-a-chip systems, complemented by advances in gene editing technologies. Progress in High-Content Screening technology has also contributed to the recent excitement for phenotypic drug discovery approaches, bringing image-capture and processing, and data-analysis, to a level of content and throughput fully compatible with large scale drug discovery efforts. Nevertheless, implementation of HCS in discovery projects must be carefully considered, to ensure optimal performance and the generation of relevant data to enable the discovery of first-in-class medicines.


Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Preparaciones Farmacéuticas/química , Edición Génica/métodos , Humanos
14.
J Clin Invest ; 110(10): 1549-57, 2002 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12438452

RESUMEN

The efficacy of anti-tumor IgG reflects the balance between opposing signals mediated by activating and inhibitory Fc(gamma) receptors (Fc(gamma)Rs) expressed by effector cells. Here, we show that human malignant melanoma cells express the inhibitory low-affinity Fc(gamma) receptor Fc(gamma)RIIB1 in 40% of tested metastases. When melanoma cells were grafted in nude mice, a profound inhibition of Fc(gamma)RIIB1 tumor growth that required the intracytoplasmic region of the receptor was observed. IgG immune complexes (ICs) may be required for this inhibition, since sera from nude mice bearing tumors contained IgG that decreased the proliferation of Fc(gamma)RIIB1-positive cells in vitro, and tumor development of Fc(gamma)RIIB1-positive melanoma lines was not inhibited in antibody-defective severe combined immunodeficiency (SCID) mice. Passive immunization of SCID mice with anti-ganglioside G(D2) antibody resulted in significant inhibition of growth of Fc(gamma)RIIB1-positive tumors in an intracytoplasmic-dependent manner. Altogether, these data suggest that human melanoma cells express biologically active inhibitory Fc(gamma)RIIB1, which regulates their development upon direct interaction with anti-tumor antibodies. Therefore, Fc(gamma)R expression on human tumors may be one component of the efficacy of antibody-mediated therapies, and Fc(gamma)R-positive tumors could be the most sensitive candidates for such treatments.


Asunto(s)
Melanoma/inmunología , Receptores de IgG/metabolismo , Animales , Anticuerpos Antineoplásicos/sangre , División Celular , Humanos , Inmunización Pasiva , Técnicas In Vitro , Melanoma/patología , Melanoma/secundario , Melanoma/terapia , Ratones , Ratones Desnudos , Ratones SCID , Trasplante de Neoplasias , Receptores de IgG/química , Receptores de IgG/genética , Transfección , Trasplante Heterólogo , Células Tumorales Cultivadas
15.
Toxicol Sci ; 155(2): 444-457, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-28069985

RESUMEN

Cardiotoxicity is a common cause of attrition in preclinical and clinical drug development. Current in vitro approaches have two main limitations, they either are limited to low throughput methods not amendable to drug discovery or lack the physiological responses to allow an integrated risk assessment. A human 3D cardiac microtissue containing human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), cardiac endothelial cells and cardiac fibroblast were used to assess their suitability to detect drug induced changes in cardiomyocyte contraction. These cardiac microtissues, have a uniform size, spontaneously beat, lack a hypoxic core, and contain key markers of each cell type. Application of field stimulation and measurement of cardiac contraction confirm cardiac microtissues to be a suitable model to investigate drug-induced changes in cardiomyocyte contractility. Using a bespoke image acquisition work flow and optical flow analysis method to test 29 inotroptic and 13 non-inotroptic compounds in vivo We report that cardiac microtissues provide a high-throughput experimental model that is both able to detect changes in cardiac contraction with a sensitivity and specificity of 80 and 91%, respectively, and provide insight into the direction of the inotropic response. Allowing improved in vitro cardiac contractility risk assessment. Moreover, our data provide evidence of the detection of this liability at therapeutically relevant concentrations with a throughput amenable to drug discovery.


Asunto(s)
Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento/métodos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/ultraestructura , Células Cultivadas , Expresión Génica , Humanos
17.
Oncogene ; 24(47): 7094-104, 2005 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-16007141

RESUMEN

Specific HPV DNA sequences are associated with more than 90% of invasive carcinomas of the uterine cervix. Viral E6 and E7 oncogenes are key mediators in cell transformation by disrupting TP53 and RB pathways. To investigate molecular mechanisms involved in the progression of invasive cervical carcinoma, we performed a gene expression study on cases selected according to viral and clinical parameters. Using Coupled Two-Way Clustering and Sorting Points Into Neighbourhoods methods, we identified a 'cervical cancer proliferation cluster' composed of 163 highly correlated transcripts. Most of these transcripts corresponded to E2F pathway genes controlling cell division or proliferation, whereas none was known as TP53 primary target. The average expression level of the genes of this cluster was higher in tumours with an early relapse than in tumours with a favourable course (P = 0.026). Moreover, we found that E6/E7 mRNA expression level was positively correlated with the expression level of the cluster genes and with viral DNA load. These findings suggest that HPV E6/E7 expression level plays a key role in the progression of invasive carcinoma of the uterine cervix via the deregulation of cellular genes controlling tumour cell proliferation. HPV expression level may thus provide a biological marker useful for prognosis assessment and specific therapy of the disease.


Asunto(s)
Proliferación Celular , ADN Viral/genética , Proteínas de Unión al ADN/metabolismo , Invasividad Neoplásica/patología , Proteínas Oncogénicas Virales/metabolismo , Infecciones por Papillomavirus/genética , Neoplasias del Cuello Uterino/genética , Adulto , Anciano , Cuello del Útero/metabolismo , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Familia de Multigenes , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/virología , Carga Viral
18.
J Clin Oncol ; 23(35): 9008-21, 2005 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-16061912

RESUMEN

PURPOSE: To evaluate the toxicity, antitumoral effectiveness, and immunogenicity of repeated vaccinations with ALVAC miniMAGE-1/3, a recombinant canarypox virus containing a minigene encoding antigenic peptides MAGE-3(168-176) and MAGE-1(161-169), which are presented by HLA-A1 and B35 on tumor cells and can be recognized by cytolytic T lymphocytes (CTLs). MATERIALS AND METHODS: The vaccination schedule comprised four sequential injections of the recombinant virus, followed by three booster vaccinations with the MAGE-3(168-176) and MAGE-1(161-169) peptides. The vaccines were administered, both intradermally and subcutaneously, at 3-week intervals. RESULTS: Forty patients with advanced cancer were treated, including 37 melanoma patients. The vaccines were generally well tolerated with moderate adverse events, consisting mainly of transient inflammatory reactions at the virus injection sites. Among the 30 melanoma patients assessable for tumor response, a partial response was observed in one patient, and disease stabilization in two others. The remaining patients had progressive disease. Among the patients with stable or progressive disease, five showed evidence of tumor regression. A CTL response against the MAGE-3 vaccine antigen was detected in three of four patients with tumor regression, and in only one of 11 patients without regression. CONCLUSION: Repeated vaccination with ALVAC miniMAGE-1/3 is associated with tumor regression and with a detectable CTL response in a minority of melanoma patients. There is a significant correlation between tumor regression and CTL response. The contribution of vaccine-induced CTL in the tumor regression process is discussed in view of the immunologic events that could be analyzed in detail in one patient.


Asunto(s)
Vacunas contra el Cáncer/inmunología , Melanoma/terapia , Vacunas Virales/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/inmunología , Virus de la Viruela de los Canarios/inmunología , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Melanoma/inmunología , Antígenos Específicos del Melanoma , Persona de Mediana Edad , Proteínas de Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunología , Resultado del Tratamiento
19.
Leuk Lymphoma ; 47(6): 1023-8, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16840192

RESUMEN

Epidemiologic studies have reported an association between lymphoid neoplasia and melanoma. However, the clinical characteristics, medical history and outcome of patients presenting both diseases have not been clearly described. Patients who developed both lymphoma and melanoma at the Institut Curie between 1970 and 2005 were included in this retrospective study. Patient characteristics were analysed and a review of all previously published cases was then performed. The eight patients of our series and those derived from a review of the literature resulted in a population of 70 patients. The male/female sex ratio was greater than 1. Patients were older than 50 years. The mean interval to the second malignancy was 5 years and 13 years for lymphoma and melanoma, respectively. Most patients had an indolent B-cell lymphoma and localized melanoma. Frequent skin involvement was reported for T-cell lymphoma. Chemotherapy or external radiation therapy frequently preceded the second malignancy. Patients with lymphoma and melanoma should be closely monitored to detect the appearance of a second malignancy. Further studies are therefore warranted to elucidate this peculiar association.


Asunto(s)
Linfoma/complicaciones , Linfoma/diagnóstico , Melanoma/complicaciones , Melanoma/diagnóstico , Adulto , Anciano , Neoplasias del Ojo/complicaciones , Neoplasias del Ojo/diagnóstico , Femenino , Humanos , Linfoma/epidemiología , Masculino , Melanoma/epidemiología , Persona de Mediana Edad , Neoplasias Primarias Secundarias/diagnóstico , Estudios Retrospectivos , Neoplasias Cutáneas/complicaciones , Neoplasias Cutáneas/diagnóstico , Factores de Tiempo
20.
Sci Rep ; 6: 18940, 2016 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-26743172

RESUMEN

Ubiquitin signalling regulates most aspects of cellular life, thus deregulation of ubiquitylation has been linked with a number of diseases. E3 ubiquitin ligases provide substrate selectivity in ubiquitylation cascades and are therefore considered to be attractive targets for developing therapeutic molecules. In contrast to established drug target classes, such as protein kinases, GPCRs, hormone receptors and ion channels, ubiquitin drug discovery is in its early stages. This is, in part, due to the complexity of the ubiquitylation pathways and the lack of robust quantitative technologies that allow high-throughput screening of inhibitors. Here we report the development of a Ubiquitin Ligase Profiling system, which is a novel and generic cellular technology designed to facilitate identification of selective inhibitors against RING type E3 ubiquitin ligases. Utilization of this system requires a single co-transfection of cells with assay vectors, thereby enabling readout of E3 ubiquitin ligase catalytic activity within the cellular environment. Therefore, our robust high-throughput screening platform offers novel opportunities for the development of inhibitors against this difficult-to-target E3 ligase enzyme class.


Asunto(s)
Inhibidores Enzimáticos/farmacología , Vectores Genéticos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Descubrimiento de Drogas , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Nitrofuranos/farmacología , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sulfonas/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
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