RESUMEN
AIMS: Low-grade serous carcinomas (LGSCs) and their precursors serous borderline tumours (SBTs) characteristically harbour mutations in BRAF, KRAS or NRAS but rarely in TP53, whereas high-grade serous carcinomas (HGSCs) are characterised by frequent TP53 mutations but rare BRAF, KRAS or NRAS mutations. In a small subset of cases, LGSCs and/or SBTs develop into high-grade tumours, including HGSCs and poorly differentiated carcinomas (PDCs). Here, we sought to define the repertoire of somatic genetic alterations in low-grade serous tumours and synchronous or metachronous high-grade adnexal carcinomas. METHODS AND RESULTS: DNA extracted from five SBTs/LGSCs and synchronous or metachronous HGSCs/PDCs and matched normal tissue was subjected to massively parallel sequencing targeting all exons and selected non-coding regions of 341 cancer-related genes. The low-grade and high-grade tumours from a given case were related, and shared mutations and copy number alterations. Progression from low-grade to high-grade lesions was observed, and involved the acquisition of additional mutations and/or copy number alterations, or shifts from subclonal to clonal mutations. Only two (an HGSC and a PDC) of the five high-grade tumours investigated harboured TP53 mutations, whereas NRAS and KRAS hotspot mutations were seen in two HGSCs and one HGSC, respectively. CONCLUSIONS: Our results suggest that progression from SBT to HGSC may take place in a subset of cases, and that at least some of the rare HGSCs lacking TP53 mutations may be derived from a low-grade serous precursor.
Asunto(s)
Biomarcadores de Tumor/genética , Cistadenocarcinoma Seroso/genética , Cistadenoma Seroso/genética , Neoplasias de los Genitales Femeninos/genética , Adulto , Anciano , Anciano de 80 o más Años , Cistadenocarcinoma Seroso/patología , Cistadenoma Seroso/patología , Progresión de la Enfermedad , Femenino , Neoplasias de los Genitales Femeninos/patología , Humanos , Persona de Mediana Edad , Clasificación del Tumor , Neoplasias Primarias Múltiples/patología , Neoplasias Primarias Secundarias/patologíaRESUMEN
Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra leading to disabling deficits. Dopamine neuron grafts may provide a significant therapeutic advance over current therapies. We have generated midbrain dopamine neurons from human embryonic stem cells and manufactured large-scale cryopreserved dopamine progenitors for clinical use. After optimizing cell survival and phenotypes in short-term studies, the cell product, MSK-DA01, was subjected to an extensive set of biodistribution, toxicity, and tumorigenicity assessments in mice under GLP conditions. A large-scale efficacy study was also performed in rats with the same lot of cells intended for potential human use and demonstrated survival of the grafted cells and behavioral amelioration in 6-hydroxydopamine lesioned rats. There were no adverse effects attributable to the grafted cells, no obvious distribution outside the brain, and no cell overgrowth or tumor formation, thus paving the way for a future clinical trial.
Asunto(s)
Dopamina , Células Madre Embrionarias Humanas , Animales , Diferenciación Celular , Neuronas Dopaminérgicas , Mesencéfalo , Ratones , Ratas , Distribución TisularRESUMEN
PURPOSE: To define the pattern of Rb expression relative to cell cycle position and cell type in the developing human retina. METHODS: Cryosections of fetal week 11-18 retinas were immunostained for Rb and cell cycle- or cell type-specific markers. RESULTS: Rb was prominent in retinal progenitor cells (RPCs) expressing the cyclin D1, cyclin A, and cytoplasmic cyclin B markers of G1, S, and early to mid G2 phases, but not in RPCs expressing the phosphohistone H3 marker of late G2 and M. Rb was not detected in the earliest postmitotic ganglion, amacrine, horizontal, and bipolar cell precursors migrating away from the ventricular layer, but was detected as such cells underwent further differentiation. Among photoreceptors, Rb was not detected in the earliest RXRgamma(+) cone precursors or in the earliest Nrl(+) rod precursors, but subsequently rose to high levels in cones and to low levels in rods. Rb was prominent at the time when Müller glia exit the cell cycle and was generally expressed in a pattern complementary to p27(Kip1). CONCLUSIONS: Rb exhibits cell cycle-specific expression in RPCs, with loss in late G2-M and restoration in G1. Rb is re-expressed after postmitotic ganglion, amacrine, horizontal, and bipolar cell precursors migrate away from the ventricular layer; after the appearance of early cone and rod markers; but coinciding with Müller glia cell cycle withdrawal. The results suggest that Rb does not mediate the initial proliferative arrest of retinal neurons, but may indirectly induce arrest in RPCs or maintain an arrest in postmitotic precursors.
Asunto(s)
Ciclo Celular/fisiología , Retina/embriología , Proteína de Retinoblastoma/metabolismo , Células Amacrinas/citología , Células Amacrinas/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Ciclina D , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Ciclinas/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Edad Gestacional , Humanos , Microscopía Confocal , Fenotipo , Células Fotorreceptoras de Vertebrados/citología , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/citología , Retina/metabolismo , Células Bipolares de la Retina/citología , Células Bipolares de la Retina/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Células Horizontales de la Retina/citología , Células Horizontales de la Retina/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
PURPOSE: To determine the efficacy of inducing p53-mediated cell death in retinoblastoma cells by Nutlin 3A, a small molecule HDM2 inhibitor. METHODS: Retinoblastoma cell lines WERI-RB-1 and Y79 were treated with Nutlin 3A. Cell viability assays, Western blot analyses, confocal microscopy, and flow cytometry were performed to measure cell survival, p53 protein levels, activation of downstream targets, and apoptosis. To determine whether the effects of Nutlin 3A were p53-dependent, cell viability assays were performed on Y79 cells expressing short interfering RNA (siRNA) against p53. RESULTS: Nutlin 3A induced cell death in Y79 and WERI-RB-1 in the 5- to 10-microM dose range. Treated cells demonstrated increased protein levels of p53 and the p53 targets p21 and HDM2. Phosphorylation of p53-serine-15, a marker for activation of p53 via genotoxic mechanisms, was absent. Y79 cells expressing siRNA against p53 demonstrated resistance to Nutlin 3A. CONCLUSIONS: Nutlin 3A induced p53-mediated apoptosis in a dose-dependent, nongenotoxic fashion in 2 retinoblastoma cell lines. CLINICAL RELEVANCE: Nutlin 3A is effective against retinoblastoma cells in a nongenotoxic manner. Because the mutagenic effects of radiation and chemotherapy may increase risks of secondary tumor formation, targeted p53 activation may be a safer alternative treatment for retinoblastoma in the future.
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Apoptosis/efectos de los fármacos , Imidazoles/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Neoplasias de la Retina/patología , Retinoblastoma/patología , Proteína p53 Supresora de Tumor/metabolismo , Anexina A5/metabolismo , Western Blotting , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Microscopía Confocal , Fosforilación , ARN Interferente Pequeño/farmacología , Neoplasias de la Retina/metabolismo , Retinoblastoma/metabolismo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Regulación hacia ArribaRESUMEN
Neovascular age-related macular degeneration is a leading cause of irreversible vision loss in the Western world. Cytokine-targeted therapies (such as anti-vascular endothelial growth factor) are effective in treating pathologic ocular angiogenesis, but have not led to a durable effect and often require indefinite treatment. Here, we show that Nutlin-3, a small molecule antagonist of the E3 ubiquitin protein ligase MDM2, inhibited angiogenesis in several model systems. We found that a functional p53 pathway was essential for Nutlin-3-mediated retinal antiangiogenesis and disruption of the p53 transcriptional network abolished the antiangiogenic activity of Nutlin-3. Nutlin-3 did not inhibit established, mature blood vessels in the adult mouse retina, suggesting that only proliferating retinal vessels are sensitive to Nutlin-3. Furthermore, Nutlin-3 inhibited angiogenesis in nonretinal models such as the hind limb ischemia model. Our work demonstrates that Nutlin-3 functions through an antiproliferative pathway with conceivable advantages over existing cytokine-targeted antiangiogenesis therapies.
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Inhibidores de la Angiogénesis/farmacología , Imidazoles/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Piperazinas/farmacología , Vasos Retinianos/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Miembro Posterior/irrigación sanguínea , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Isquemia/tratamiento farmacológico , Degeneración Macular/tratamiento farmacológico , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Ratas , Vasos Retinianos/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The benefits of inhibiting vascular endothelial growth factor (VEGF) signaling in cancer patients are predominantly attributed to effects on tumor endothelial cells. Targeting non-endothelial stromal cells to further impact tumor cell growth and survival is being pursued through the inhibition of additional growth factor pathways important for the survival and/or proliferation of these cells. However, recent data suggest that VEGF receptor (VEGFR)-specific inhibitors may target lymphatic vessels and pericytes in addition to blood vessels. Here, in fact, we demonstrate that DC101 (40 mg/kg, thrice a week), an antibody specific to murine VEGFR2, significantly reduces all three of these stromal components in subcutaneous (SKRC-29) and orthotopic (786-O-LP) models of renal cell carcinoma (RCC) established in nu/nu athymic mice. Sunitinib (40 mg/kg, once daily), a receptor tyrosine kinase inhibitor of VEGFR2 and other growth factor receptors, also caused significant loss of tumor blood vessels in RCC models but had weaker effects than DC101 on pericytes and lymphatic vessels. In combination, sunitinib did not significantly add to the effects of DC101 on tumor blood vessels, lymphatic vessels, or pericytes. Nevertheless, sunitinib increased the effect of DC101 on tumor burden in the SKRC-29 model, perhaps related to its broader specificity. Our data have important implications for combination therapy design, supporting the conclusion that targeting VEGFR2 alone in RCC has the potential to have pleiotropic effects on tumor stroma.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Indoles/farmacología , Neoplasias Renales/tratamiento farmacológico , Pirroles/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/patología , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Femenino , Humanos , Factor 1 Inducible por Hipoxia/biosíntesis , Indoles/uso terapéutico , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/patología , Vasos Linfáticos/efectos de los fármacos , Vasos Linfáticos/patología , Ratones , Ratones Desnudos , Mutación , Trasplante de Neoplasias , Neovascularización Patológica , Pericitos/efectos de los fármacos , Pericitos/patología , Pirroles/uso terapéutico , Células del Estroma/efectos de los fármacos , Células del Estroma/patología , Sunitinib , Carga Tumoral , Células Tumorales Cultivadas , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaAsunto(s)
Aparato Lagrimal/metabolismo , Ratones/metabolismo , Modelos Biológicos , Lágrimas/metabolismo , Animales , Canales de Cloruro/fisiología , Técnicas de Placa-Clamp , Canales de Potasio/fisiología , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12RESUMEN
Cancer patients receiving epidermal growth factor receptor (EGFR) antibody therapy often experience an acneiform rash of uncertain etiology in skin regions rich in pilosebaceous units. Currently, this condition is treated symptomatically with very limited, often anecdotal success. Here, we show that a monoclonal antibody targeting murine EGFR, ME1, caused a neutrophil-rich hair follicle inflammation in mice, similar to that reported in patients. This effect was preceded by the appearance of lipid-filled hair follicle distensions adjacent to enlarged sebaceous glands. The cytokine tumor necrosis factor-alpha (TNFalpha), localized immunohistochemically to this affected region of the pilosebaceous unit, was specifically up-regulated by ME1 in skin but not in other tissues examined. Moreover, skin inflammation was reduced by cotreatment with the TNFalpha signaling inhibitor, etanercept, indicating the involvement of TNFalpha in this inflammatory process. Interleukin-1, a cytokine that frequently acts in concert with TNFalpha, is also involved in this process given the efficacy of the interleukin-1 antagonist Kineret. Our results provide a mechanistic framework to develop evidence-based trials for EGFR antibody-induced skin rash in patients with cancer.
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Anticuerpos Monoclonales/efectos adversos , Dermatitis/prevención & control , Receptores ErbB/inmunología , Interleucina-1/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/farmacología , Anticuerpos Monoclonales/uso terapéutico , Dermatitis/etiología , Dermatitis/patología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Receptores ErbB/antagonistas & inhibidores , Etanercept , Exantema/inducido químicamente , Exantema/prevención & control , Femenino , Humanos , Inmunoglobulina G/farmacología , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1/genética , Interleucina-1/metabolismo , Ratones , Ratones SCID , Receptores del Factor de Necrosis Tumoral , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/efectos de los fármacos , Piel/metabolismo , Piel/patología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In glands such as the liver and pancreas, gap junctions containing connexin 26 and 32 (Cx26 and Cx32, respectively) couple the secretory cells. Uncoupling these junctions compromises the secretory function of these glands. Lacrimal glands also contain extensive arrays of gap junctions consisting of Cx26 and Cx32. We wanted to determine the role of these junctions in fluid secretion. In Cx32-deficient mice, immunocytochemistry showed that, in the male lacrimal gland, the remaining Cx26 was found evenly distributed in the membrane whereas there was little in the membranes of female glands. Western blot analysis of Cx26 showed that female Cx32-deficient mice expressed Cx26. Patch-clamp analyses of acinar cell coupling showed that the cell pairs from male glands were coupled whereas those from female glands were not. Stimulated fluid production by the glands from Cx32-deficient mice was abnormally low in female glands compared with controls at low topical doses of carbachol. The protein secretory response to different doses of carbachol was the same in all animals. These data suggest that gap junctions are essential for optimal fluid secretion in lacrimal glands.