RESUMEN
BACKGROUND: Prognostic scoring systems are used to estimate the risk of mortality from metastatic renal cell carcinoma (mRCC). Outcomes from different therapies may vary within each risk group. These survival algorithms have been applied to assess outcomes in patients receiving T-cell checkpoint inhibitory immunotherapy and tyrosine kinase inhibitor therapy, but have not been applied extensively to patients receiving high dose interleukin-2 (HD IL-2) immunotherapy. METHODS: Survival of 810 mRCC patients treated from 2006 to 2017 with high dose IL-2 (aldesleukin) and enrolled in the PROCLAIMSM registry data base was assessed utilizing the International Metastatic RCC Database Consortium (IMDC) risk criteria. Median follow-up is 23.4 months (mo.) (range 0.2-124 mo.). Subgroup evaluations were performed by separating patients by prior or no prior therapy, IL-2 alone, or therapy subsequent to IL-2. Some patients were in two groups. We will focus on the 356 patients who received IL-2 alone, and evaluate outcome by risk factor categories. RESULTS: Among the 810 patients, 721 were treatment-naïve (89%) and 59% were intermediate risk. Overall, of the 249 patients with favorable risk, the median overall survival (OS) is 63.3 mo. and the 2-year OS is 77.6%. Of 480 patients with intermediate risk, median OS is 42.4 mo., 2-year OS 68.2%, and of 81 patients with poor risk, median OS 14 mo., 2-year OS 40.4%. Among those who received IL-2 alone (356 patients), median OS is 64.5, 57.6, and 14 months for favorable, intermediate and poor risk categories respectively. Two year survival among those treated only with HD IL-2 is 73.4, 63.7 and 39.8%, for favorable, intermediate and poor risk categories respectively. CONCLUSIONS: Among mRCC patients treated with HD IL-2, all risk groups have median and 2-year survival consistent with recent reports of checkpoint or targeted therapies for mRCC. Favorable and intermediate risk (by IMDC) patients treated with HD IL-2 have longer OS compared with poor risk patients, with most durable OS observed in favorable risk patients. Favorable risk patients treated with HD IL-2 alone have a 2-year OS of 74%. These data continue to support a recommendation for HD IL-2 for patients with mRCC who meet eligibility criteria. TRIAL REGISTRATION: PROCLAIM, NCT01415167 was registered with ClinicalTrials.gov on August 11, 2011, and initiated for retrospective data collection until 2006, and prospective data collection ongoing since 2011.
Asunto(s)
Antineoplásicos/administración & dosificación , Carcinoma de Células Renales/tratamiento farmacológico , Interleucina-2/administración & dosificación , Neoplasias Renales/tratamiento farmacológico , Anciano , Antineoplásicos/uso terapéutico , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Interleucina-2/uso terapéutico , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Metástasis de la Neoplasia , Estudios Prospectivos , Estudios Retrospectivos , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
We describe 20 patients with myeloma and 1 with primary amyloidosis from 15 centres, all with advanced renal failure, most of whom had PBSC mobilised using plerixafor following previous failed mobilisation by conventional means (plerixafor used up-front for 4 patients). For 15 patients, the plerixafor dose was reduced to 0.16 mg/kg/day, with a subsequent dose increase in one case to 0.24 mg/kg/day. The remaining six patients received a standard plerixafor dosage at 0.24 mg/kg/day. Scheduling of plerixafor and apheresis around dialysis was generally straightforward. Following plerixafor administration, all patients underwent apheresis. A median CD34+ cell dose of 4.6 × 10(6) per kg was achieved after 1 (n=7), 2 (n=10), 3 (n=3) or 4 (n=1) aphereses. Only one patient failed to achieve a sufficient cell dose for transplant: she subsequently underwent delayed re-mobilisation using G-CSF with plerixafor 0.24 mg/kg/day, resulting in a CD34+ cell dose of 2.12 × 10(6)/kg. Sixteen patients experienced no plerixafor toxicities; five had mild-to-moderate gastrointestinal symptoms that did not prevent apheresis. Fifteen patients have progressed to autologous transplant, of whom 12 remain alive without disease progression. Two patients recovered endogenous renal function post autograft, and a third underwent successful renal transplantation. Plerixafor is highly effective in mobilising PBSC in this difficult patient group.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Movilización de Célula Madre Hematopoyética/métodos , Compuestos Heterocíclicos/administración & dosificación , Mieloma Múltiple/terapia , Trasplante de Células Madre de Sangre Periférica , Insuficiencia Renal/terapia , Adulto , Anciano , Fármacos Anti-VIH/efectos adversos , Bencilaminas , Eliminación de Componentes Sanguíneos , Ciclamas , Relación Dosis-Respuesta a Droga , Femenino , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Movilización de Célula Madre Hematopoyética/efectos adversos , Compuestos Heterocíclicos/efectos adversos , Humanos , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Mieloma Múltiple/complicaciones , Diálisis Renal , Insuficiencia Renal/complicaciones , Trasplante Autólogo , Trasplante HomólogoRESUMEN
Genetically modified dendritic cell (DC) vaccines expressing tumor-associated antigens are currently used for cancer immunotherapy. Peripheral blood (PB) monocyte precursors are a relatively convenient source of DCs for use in clinical studies, but are often contaminated by lymphocytes. The current study was conducted to examine the impact of T-lymphocyte contamination on genetically modified DC product. PB monocyte-derived DCs were efficiently transduced (75-95%) with an HIV-1-based self-inactivating lentiviral vector encoding a model antigen, the enhanced green fluorescent protein (eGFP). The lymphocyte-free DC culture transduced with Lenti-eGFP showed stable expression of eGFP without measurable decline in viability. In contrast, the eGFP-positive DCs disappeared rapidly in transduced DC cultures containing lymphocyte contaminants, concurrent with detectable activation and expansion of T-lymphocytes. Upon antigen recall, these T cells elicited major histocompatability complex-restricted antigen-specific cytotoxicity against eGFP-positive autologous DCs and mitogen-stimulated T lymphoblasts, mainly through the perforin-mediated pathway. In summary, this study demonstrate that the relative purity of DC cultures could determine the persistence of gene-modified DC, which may affect the induction of effective immune responses by DC vaccination strategies.
Asunto(s)
Vacunas contra el Cáncer , Células Dendríticas , Terapia Genética/métodos , Linfocitos , Neoplasias/terapia , Traslado Adoptivo , Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes/genética , VIH-1/genética , Humanos , Linfocitos T Reguladores/inmunología , Transducción Genética/métodosRESUMEN
Genetic modification of human lymphocytes is being employed in strategies to correct enzyme deficiencies, encode cytokines and to redirect lymphocytes to antigenic targets other than those encoded by their endogenous T cell receptor. However, expression of transgenes in primary lymphocytes is generally low. Reasoning that vector modification may lead to increased transgene expression and subsequent increases in function, we have performed two retroviral vector modifications and report their effect on the functional expression in primary lymphocytes. A chimeric receptor specific for the colon carcinoma-associated antigen, EGP40, was initially incorporated into the retroviral vector LXSN. In this vector, receptor expression is driven by the Moloney murine leukemia virus LTR, and neomycin phosphotransferase expression driven by the SV40 promoter. Replacement of SV40 with an internal ribosomal entry site (IRES) increased the transgene activity of a mouse T cell line and human PBL as judged by increased cytokine release in response to antigen positive target cells. A further increase in transgene function was generated by the additional incorporation of a splice acceptor motif into the construct. Human PBL transduced with vector incorporating both IRES and intron were consistently more effective at lysing antigen positive colorectal carcinoma cells.
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Antígenos de Neoplasias/genética , Moléculas de Adhesión Celular/genética , Neoplasias del Colon/patología , Terapia Genética/métodos , Vectores Genéticos/genética , Inmunoterapia/métodos , Adulto , Animales , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Neoplasias del Colon/inmunología , Citotoxicidad Inmunológica , Molécula de Adhesión Celular Epitelial , Regulación de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Humanos , Inmunofenotipificación , Interferón gamma/biosíntesis , Ratones , Virus de la Leucemia Murina de Moloney/genética , Sitios de Empalme de ARN , Transducción Genética , Transgenes/genética , Células Tumorales CultivadasRESUMEN
Anti-CD3/anti-CD28 monoclonal antibody-coactivated T cells (COACTs) proliferate, secrete tumoricidal cytokines, and mediate non-major histocompatibility complex (MHC)-restricted cytotoxicity. This phase I study was done to determine the safety, maximum tolerated dose, technical limits of expansion, and modulation of immune functions in cancer patients given COACTs. Coactivated T cells were produced by stimulating peripheral blood mononuclear cells (PBMCs) with OKT3 anti-CD3 and 9.3 (anti-CD28)-coated beads in the presence of 100 IU interleukin (IL)-2 per milliliter for 14 days. The beads were removed after 4 days of culture. Ten courses of COACTs were given to eight patients with renal cell (1), ovarian (2), breast (1), and colorectal (4) carcinomas; two patients received two courses of COACTs. Patients were given up to 10 x 10 9 COACTs twice a week for 3 weeks without dose-limiting toxicities. Patients at the first and second dose levels received a mean total of 17.6 and 42.4 x 10 9 COACTs, respectively. After 14 days of culture, the COACTs contained a mean of 57.5% CD4+ cells and 42.5% CD8+ cells, exhibited non-MHC-restricted cytotoxicity, and produced significant amounts of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and granulocyte macrophage colony-stimulating factor (GM-CSF). Infusions were safe and induced measurable serum levels of IFNgamma, TNFalpha, and IL-4 in two patients. Peripheral blood mononuclear cells from patients who received COACTs secreted higher amounts of IFNgamma and GM-CSF on in vitro anti-CD3/anti-CD28 restimulation than PBMCs obtained before immunotherapy. The detection of cytokines in patient sera and enhanced in vitro production of cytokines by anti-CD3/anti-CD28-stimulated patient PBMCs after COACT infusions suggest that COACTs were modulating immune responses in cancer patients.
Asunto(s)
Antígenos CD28/inmunología , Complejo CD3/inmunología , Inmunoterapia Adoptiva , Neoplasias/inmunología , Neoplasias/terapia , Linfocitos T/inmunología , Adulto , Anciano , Células Cultivadas , Citocinas/biosíntesis , Citotoxicidad Inmunológica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Humanos , Inmunofenotipificación , Interferón gamma/biosíntesis , Interferón gamma/sangre , Interleucina-4/biosíntesis , Interleucina-4/sangre , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
During Drosophila visual system development, photoreceptors R7 and R8 project axons to targets in distinct layers of the optic lobe. We show here that the LAR receptor tyrosine phosphatase is required in the eye for correct targeting of R7 axons. In LAR mutants, R7 axons initially project to their correct target layer, but then retract to the R8 target layer. This targeting defect can be fully rescued by transgenic expression of LAR in R7, and partially rescued by expression of LAR in R8. The phosphatase domains of LAR are required for its activity in R7, but not in R8. These data suggest that LAR can act both as a receptor in R7, and as a ligand provided by R8. Genetic interactions implicate both Enabled and Trio in LAR signal transduction.
Asunto(s)
Axones/fisiología , Drosophila , Proteínas del Tejido Nervioso , Células Fotorreceptoras de Invertebrados/ultraestructura , Proteínas Tirosina Fosfatasas , Receptores de Superficie Celular/fisiología , Animales , Drosophila/genética , Ojo/ultraestructura , Femenino , Expresión Génica , Masculino , Mosaicismo , Mutación , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Receptores de Superficie Celular/genética , Proteínas Recombinantes , Retina/ultraestructura , Transducción de Señal , Transfección , Transgenes , Vías Visuales/crecimiento & desarrollo , Vías Visuales/metabolismoRESUMEN
Proteins of the Hedgehog (Hh) family act as important developmental signals in a variety of species [1]. Hh proteins are synthesized as full-length precursors that are autocatalytically cleaved by their C-terminal domains to release the signaling N-terminal domains [2]. The addition of a cholesterol molecule to the C terminus of the signaling domain is concomitant with cleavage [3]. Vertebrate Sonic hedgehog (Shh) proteins have also been shown to acquire a fatty acid chain on the N-terminal cysteine of this domain [4], which is required for a subset of their in vivo functions [5, 6]. A mutation of the corresponding cysteine in Drosophila Hh transforms it into a dominant-negative protein [6]. We have identified a novel gene, sightless (sit), which is required for the activity of Drosophila Hh in the eye and wing imaginal discs and in embryonic segmentation. sit acts in the cells that produce Hh, but does not affect hh transcription, Hh cleavage, or the accumulation of Hh protein. sit encodes a conserved transmembrane protein with homology to a family of membrane-bound acyltransferases. The Sit protein could act by acylating Hh or by promoting other modifications or trafficking events necessary for its function.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas de Drosophila , Proteínas de Insectos/metabolismo , Acilación , Aciltransferasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , Drosophila/genética , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Regulación de la Expresión Génica , Genes de Insecto , Proteínas Hedgehog , Proteínas de Insectos/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Células Fotorreceptoras de Invertebrados/crecimiento & desarrollo , Células Fotorreceptoras de Invertebrados/metabolismo , Homología de Secuencia de Aminoácido , Alas de Animales/crecimiento & desarrollo , Alas de Animales/metabolismoRESUMEN
The inherited human disease tuberous sclerosis, characterized by hamartomatous tumors, results from mutations in either TSC1 or TSC2. We have characterized mutations in the Drosophila Tsc1 and Tsc2/gigas genes. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions.
Asunto(s)
Ciclo Celular/fisiología , Genes Supresores de Tumor/genética , Células Fotorreceptoras de Invertebrados/citología , Proteínas/genética , Proteínas Represoras/genética , Esclerosis Tuberosa/genética , Animales , Tamaño de la Célula , Ciclina A/metabolismo , Ciclina E/metabolismo , ADN/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Femenino , Citometría de Flujo , Colorantes Fluorescentes/metabolismo , Proteínas Fluorescentes Verdes , Humanos , Inmunohistoquímica , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Fenotipo , Células Fotorreceptoras de Invertebrados/fisiología , Células Fotorreceptoras de Invertebrados/ultraestructura , Ploidias , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Esclerosis Tuberosa/fisiopatología , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de TumorRESUMEN
The posteriorly expressed signaling molecules Hedgehog and Decapentaplegic drive photoreceptor differentiation in the Drosophila eye disc, while at the anterior lateral margins Wingless expression blocks ectopic differentiation. We show here that mutations in axin prevent photoreceptor differentiation and lead to tissue overgrowth and that both these effects are due to ectopic activation of the Wingless pathway. In addition, ectopic Wingless signaling causes posterior cells to take on an anterior identity, reorienting the direction of morphogenetic furrow progression in neighboring wild-type cells. We also show that signaling by Decapentaplegic and Hedgehog normally blocks the posterior expression of anterior markers such as Eyeless. Wingless signaling is not required to maintain anterior Eyeless expression and in combination with Decapentaplegic signaling can promote its downregulation, suggesting that additional molecules contribute to anterior identity. Along the dorsoventral axis of the eye disc, Wingless signaling is sufficient to promote dorsal expression of the Iroquois gene mirror, even in the absence of the upstream factor pannier. However, Wingless signaling does not lead to ventral mirror expression, implying the existence of ventral repressors.
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Proteínas Adaptadoras Transductoras de Señales , Tipificación del Cuerpo , Proteínas Portadoras/metabolismo , Proteínas de Drosophila , Drosophila/embriología , Ojo/embriología , Proteínas Proto-Oncogénicas/metabolismo , Animales , Proteína Axina , Proteínas Hedgehog , Proteínas de Insectos , Modelos Biológicos , Transducción de Señal , Factores de Transcripción , Proteína Wnt1RESUMEN
The Drosophila Hedgehog protein and its vertebrate counterpart Sonic hedgehog are required for a wide variety of patterning events throughout development. Hedgehog proteins are secreted from cells and undergo autocatalytic cleavage and cholesterol modification to produce a mature signaling domain. This domain of Sonic hedgehog has recently been shown to acquire an N-terminal acyl group in cell culture. We have investigated the in vivo role that such acylation might play in appendage patterning in mouse and Drosophila; in both species Hedgehog proteins define a posterior domain of the limb or wing. A mutant form of Sonic hedgehog that cannot undergo acylation retains significant ability to repattern the mouse limb. However, the corresponding mutation in Drosophila Hedgehog renders it inactive in vivo, although it is normally processed. Furthermore, overexpression of the mutant form has dominant negative effects on Hedgehog signaling. These data suggest that the importance of the N-terminal cysteine of mature Hedgehog in patterning appendages differs between species.
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Tipificación del Cuerpo , Proteínas de Drosophila , Extremidades/embriología , Proteínas de Insectos/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Transactivadores , Acilación , Animales , Cisteína/genética , Drosophila/embriología , Evolución Molecular , Proteínas Hedgehog , Humanos , Ratones , Mutación , Fenotipo , Polidactilia/etiología , Transducción de Señal , Especificidad de la Especie , Alas de Animales/embriologíaAsunto(s)
Ojo/embriología , Animales , Tipificación del Cuerpo , Diferenciación Celular/genética , Drosophila , Inducción Embrionaria , Evolución Molecular , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Proteínas del Ojo , Regulación del Desarrollo de la Expresión Génica , Genes Reguladores , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Morfogénesis , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Células Fotorreceptoras/metabolismo , Proteínas Represoras , VertebradosRESUMEN
We have identified mutations in two genes, blind spot and kohtalo, that encode Drosophila homologues of human TRAP240 and TRAP230, components of a large transcriptional coactivation complex homologous to the yeast Mediator complex. Loss of either blind spot or kohtalo has identical effects on the development of the eye-antennal disc. Eye disc cells mutant for either gene can express decapentaplegic and atonal in response to Hedgehog signaling, but they maintain inappropriate expression of these genes and fail to differentiate further. Mutant cells in the antennal disc lose expression of Distal-less and misexpress eyeless, suggesting a partial transformation towards the eye fate. blind spot and kohtalo are not required for cell proliferation or survival, and their absence cannot be rescued by activation of the Hedgehog or Notch signaling pathways. These novel and specific phenotypes suggest that TRAP240 and TRAP230 act in concert to mediate an unknown developmental signal or a combination of signals.
Asunto(s)
Proteínas de Drosophila , Drosophila/embriología , Extremidades/embriología , Proteínas del Ojo/metabolismo , Proteínas de Insectos/metabolismo , Proteínas Nucleares/química , Células Fotorreceptoras de Invertebrados/embriología , Transactivadores , Secuencia de Aminoácidos , Animales , Diferenciación Celular , División Celular , Clonación Molecular , Drosophila/metabolismo , Proteínas del Ojo/química , Proteínas del Ojo/genética , Proteínas Hedgehog , Inmunohistoquímica , Hibridación in Situ , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/fisiología , Complejo Mediador , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Morfogénesis , Mutación/genética , Fenotipo , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/metabolismo , Subunidades de Proteína , ARN Mensajero/análisis , ARN Mensajero/genética , Receptores Notch , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de TranscripciónRESUMEN
The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific downstream target genes. We report here that the product of the trithorax group gene osa is required to repress such genes in the absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes present and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression.
Asunto(s)
Proteínas de Ciclo Celular , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Drosophila/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/metabolismo , Animales , Proteínas del Dominio Armadillo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Cromatina/ultraestructura , Citosol/metabolismo , Proteínas de Unión al ADN/genética , Drosophila/embriología , Embrión no Mamífero , Elementos de Facilitación Genéticos , Femenino , Regulación del Desarrollo de la Expresión Génica , Histona Desacetilasa 1 , Histona Desacetilasas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Masculino , Mutación , Factores del Dominio POU , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transactivadores/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba , Proteína Wnt1RESUMEN
Cells in the morphogenetic furrow of the Drosophila eye disc undergo a striking shape change immediately prior to their neuronal differentiation. We have isolated mutations in a novel gene, act up (acu), that is required for this shape change. acu encodes a homolog of yeast cyclase-associated protein, which sequesters monomeric actin; we show that acu is required to prevent actin filament polymerization in the eye disc. In contrast, profilin promotes actin filament polymerization, acting epistatically to acu. However, both acu and profilin are required to prevent premature Hedgehog-induced photoreceptor differentiation ahead of the morphogenetic furrow. These findings suggest that dynamic changes in actin filaments alter cell shape to control the movement of signals that coordinate a wave of differentiation.
Asunto(s)
Actinas/metabolismo , Proteínas Contráctiles , Proteínas de Drosophila , Ojo/metabolismo , Genes Letales , Proteínas de Insectos/metabolismo , Células Fotorreceptoras de Invertebrados/citología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Diferenciación Celular , Tamaño de la Célula , Drosophila/embriología , Drosophila/genética , Drosophila/metabolismo , Drosophila/fisiología , Ojo/embriología , Femenino , Proteínas Hedgehog , Humanos , Proteínas de Insectos/genética , Masculino , Ratones , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Morfogénesis , Polímeros , Profilinas , Homología de Secuencia de AminoácidoRESUMEN
We have previously shown that the Ste20 kinase encoded by misshapen (msn) functions upstream of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase module in Drosophila. msn is required to activate the Drosophila JNK, Basket (Bsk), to promote dorsal closure of the embryo. A mammalian homolog of Msn, Nck interacting kinase, interacts with the SH3 domains of the SH2-SH3 adapter protein Nck. We now show that Msn likewise interacts with Dreadlocks (Dock), the Drosophila homolog of Nck. dock is required for the correct targeting of photoreceptor axons. We have performed a structure-function analysis of Msn in vivo in Drosophila in order to elucidate the mechanism whereby Msn regulates JNK and to determine whether msn, like dock, is required for the correct targeting of photoreceptor axons. We show that Msn requires both a functional kinase and a C-terminal regulatory domain to activate JNK in vivo in Drosophila. A mutation in a PXXP motif on Msn that prevents it from binding to the SH3 domains of Dock does not affect its ability to rescue the dorsal closure defect in msn embryos, suggesting that Dock is not an upstream regulator of msn in dorsal closure. Larvae with only this mutated form of Msn show a marked disruption in photoreceptor axon targeting, implicating an SH3 domain protein in this process; however, an activated form of Msn is not sufficient to rescue the dock mutant phenotype. Mosaic analysis reveals that msn expression is required in photoreceptors in order for their axons to project correctly. The data presented here genetically link msn to two distinct biological events, dorsal closure and photoreceptor axon pathfinding, and thus provide the first evidence that Ste20 kinases of the germinal center kinase family play a role in axonal pathfinding. The ability of Msn to interact with distinct classes of adapter molecules in dorsal closure and photoreceptor axon pathfinding may provide the flexibility that allows it to link to distinct upstream signaling systems.
Asunto(s)
Axones/metabolismo , Proteínas de Drosophila , Células Fotorreceptoras de Invertebrados/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Animales , Drosophila/embriología , Embrión no Mamífero , Ojo/embriología , Ojo/inervación , Ojo/metabolismo , Ganglios de Invertebrados/embriología , Ganglios de Invertebrados/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Prolina , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Relación Estructura-Actividad , Quinasas p21 Activadas , Dominios Homologos srcRESUMEN
Transduction with chimeric T-cell receptor genes can be used to redirect primary T lymphocytes to recognize specific antigens (Ags), including ovarian and breast cancer Ags. To extend this approach to colon cancer we report here redirection of T cells using a chimeric receptor recognizing the colon cancer-associated Ag EGP40. Chimeric T cell receptors were constructed by ligating single-chain genes of either of two EGP40-specific monoclonal antibodies (CO17.1 A, GA733) to the Fc receptor gamma-signaling chain. Retroviral vectors incorporating these constructs were used to transduce a murine T-cell line and human peripheral blood lymphocytes. These modified T cells were analyzed for specific recognition of colon cancer lines by measuring cytokine release and lytic activity against tumor targets. Murine lymphocytes transduced with the chimeric receptor based on GA733, but not CO17.1A, released cytokine specifically in response to EGP40-expressing colon cancer cell lines. Recognition of colon cancer targets by murine lymphocytes was blocked by the addition of GA733 antibody or soluble EGP40 Ag, confirming that colon cancer recognition is based on specific chimeric receptor-Ag interaction. Human lymphocytes transduced with chimeric GA733 specifically recognized colon carcinoma cells in cytokine release assays and lysed EGP40-expressing tumor cells. Genetic modification of T cells can be used to redirect T cells against EGP40-expressing tumor cells. The expression of chimeric GA733 in the autologous lymphocytes of patients may provide a source of tumor-reactive cells with therapeutic application against colon cancer.
Asunto(s)
Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transfección/inmunología , Células 3T3 , Adulto , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/metabolismo , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Neoplasias del Colon/genética , Neoplasias del Colon/terapia , Molécula de Adhesión Celular Epitelial , Humanos , Células K562 , Ratones , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de IgG/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Linfocitos T/virología , Células Tumorales CultivadasRESUMEN
The dorsoventral midline of the Drosophila eye disc is a source of signals that stimulate growth of the eye disc, define the point at which differentiation initiates, and direct ommatidial rotation in opposite directions in the two halves of the eye disc. This boundary region seems to be established by the genes of the iroquois complex, which are expressed in the dorsal half of the disc and inhibit fringe expression there. Fringe controls the activation of Notch and the expression of its ligands, with the result that Notch is activated only at the fringe expression boundary at the midline. The secreted protein Wingless activates the dorsal expression of the iroquois genes. We show here that pannier, which encodes a GATA family transcription factor expressed at the dorsal margin of the eye disc from embryonic stages on, acts upstream of wingless to control mirror and fringe expression and establish the dorsoventral boundary. Loss of pannier function leads to the formation of an ectopic eye field and the reorganization of ommatidial polarity, and ubiquitous pannier expression can abolish the eye field. Pannier is thus the most upstream element yet described in dorsoventral patterning of the eye disc.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/embriología , Ojo/embriología , N-Acetilglucosaminiltransferasas , Proteínas Proto-Oncogénicas/fisiología , Factores de Transcripción/fisiología , Animales , Tipificación del Cuerpo , Cruzamientos Genéticos , Drosophila melanogaster/genética , Embrión no Mamífero/fisiología , Femenino , Genes de Insecto , Proteínas de Insectos/genética , Masculino , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Factores de Transcripción/genética , Proteína Wnt1RESUMEN
The Drosophila BMP homolog DPP can function as a morphogen, inducing multiple cell fates across a developmental field. However, it is unknown how graded levels of extracellular DPP are interpreted to organize a sharp boundary between different fates. Here we show that opposing DPP and EGF signals set the boundary for an ovarian follicle cell fate. First, DPP regulates gene expression in the follicle cells that will create the operculum of the eggshell. DPP induces expression of the enhancer trap reporter A359 and represses expression of bunched, which encodes a protein similar to the mammalian transcription factor TSC-22. Second, DPP signaling indirectly regulates A359 expression in these cells by downregulating expression of bunched. Reduced bunched function restores A359 expression in cells that lack the Smad protein MAD; ectopic expression of BUNCHED suppresses A359 expression in this region. Importantly, reduction of bunched function leads to an expansion of the operculum and loss of the collar at its boundary. Third, EGF signaling upregulates expression of bunched. We previously demonstrated that the bunched expression pattern requires the EGF receptor ligand GURKEN. Here we show that activated EGF receptor is sufficient to induce ectopic bunched expression. Thus, the balance of DPP and EGF signals sets the boundary of bunched expression. We propose that the juxtaposition of cells with high and low BUNCHED activity organizes a sharp boundary for the operculum fate.
Asunto(s)
Proteínas de Drosophila , Drosophila/crecimiento & desarrollo , Drosophila/genética , Factor de Crecimiento Epidérmico/genética , Genes de Insecto , Proteínas de Insectos/genética , Ovario/crecimiento & desarrollo , Factor de Crecimiento Transformador alfa , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Cartilla de ADN/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Operón Lac , Datos de Secuencia Molecular , Mutación , Ovario/citología , Transducción de Señal , Factores de Crecimiento Transformadores/genéticaRESUMEN
The yeast SWI/SNF complex and its Drosophila and mammalian homologs are thought to control gene expression by altering chromatin structure, but the mechanism and specificity of this process are not fully understood. The Drosophila osa gene, like yeast SWI1, encodes an AT-rich interaction (ARID) domain protein. We present genetic and biochemical evidence that Osa is a component of the Brahma complex, the Drosophila homolog of SWI/SNF. The ARID domain of Osa binds DNA without sequence specificity in vitro, but it is sufficient to direct transcriptional regulatory domains to specific target genes in vivo. Endogenous Osa appears to promote the activation of some of these genes. We show evidence that some Brahma-containing complexes do not contain Osa and that Osa is not required to localize Brahma to chromatin. These data suggest that Osa modulates the function of the Brahma complex.