RESUMEN

PURPOSE: Clinical development of cancer drugs has a low success rate. Prognostic and predictive biomarkers using minimally invasive approaches hold promise for increasing the probability of success by enabling disease characterization, patient selection and early detection of drug treatment effect. Enumeration and molecular characterization of circulating tumor cells (CTC) may address some of these needs, and thus were evaluated for utility in a Phase I solid tumor clinical study. EXPERIMENTAL DESIGN: Blood samples for CTC analysis were obtained from 24 cancer patients in a multi-center all-comer Phase I study of MEDI-575, a novel anti-PDGFRα antibody. Samples were taken at screening and analyzed for enumeration of CTC using the CellSearch(®) platform and for molecular characterization using a novel quantitative RT-PCR assay. RESULTS: Fifty-nine percent of the patients showed at least 1 CTC per 7.5 ml of blood at baseline. Progression-free survival (PFS) and overall survival (OS) of patients with 0 CTCs at baseline were longer than PFS and Os for patients with 1-3 and >3 CTCs (8.8 versus 1.4 and 1.3 months PFS, P = 0.02; 9.0 vs 7.4 and 3.5 months OS, P = 0.20, respectively). Patients with 0 CTC showed a greater percentage of stable disease than the other 2 groups with 1-3 and >3 CTCs (57% vs 29% and 0%). The multimarker qRT-PCR method detected CTC in 40% of the patients, and 80% of these patients were positive for pre-selected drug target genes. CONCLUSION: CTC enumeration of patients in an all-comer study is feasible and may allow for patient stratification for PFS and Os to evaluate the clinical response of investigational agents. Gene expression profiling of isolated CTC may provide a means for molecular characterization of selected tumor targets.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Neoplasias/sangre , Neoplasias/metabolismo , Células Neoplásicas Circulantes , Adulto , Anciano , Supervivencia sin Enfermedad , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Probabilidad , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Factores de Tiempo

RESUMEN

BACKGROUND: Targeting the cell-surface receptor EphA2, which is highly expressed in some solid tumors, is a novel approach for cancer therapy. We aimed to evaluate the safety profile, maximum tolerated dose (MTD), pharmacokinetics, and antitumor activity of MEDI-547, an antibody drug conjugate composed of the cytotoxic drug auristatin (toxin) linked to a human anti-EphA2 monoclonal antibody (1C1), in patients with solid tumors relapsed/refractory to standard therapy. METHODS: In this phase 1, open-label study with planned dose-escalation and dose-expansion cohorts, patients received a 1-h intravenous infusion of MEDI-547 (0.08 mg/kg) every 3 weeks. RESULTS: Six patients received 0.08 mg/kg; all discontinued treatment. Dose escalation was not pursued. The study was stopped before cohort 2 enrollment due to treatment-related bleeding and coagulation events (hemorrhage-related, n = 3; epistaxis, n = 2). Therefore, lower doses were not explored and an MTD could not be selected. The most frequently reported treatment-related adverse events (AEs) were increased liver enzymes, decreased hemoglobin, decreased appetite, and epistaxis. Three patients (50%) experienced treatment-related serious AEs, including conjunctival hemorrhage, pain (led to study drug discontinuation), liver disorder, and hemorrhage. Best response included progressive disease (n = 5; 83.3%) and stable disease (n = 1; 16.7%). Minimal or no dissociation of toxin from 1C1 conjugate occurred in the blood. Serum MEDI-547 concentrations decreased rapidly, ~70% by 3 days post-dose. No accumulation of MEDI-547 was observed at 0.08 mg/kg upon administration of a second dose 3 weeks following dose 1. CONCLUSIONS: The safety profile of MEDI-547 does not support further clinical investigation in patients with advanced solid tumors.

Asunto(s)

Aminobenzoatos/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias/tratamiento farmacológico , Anciano , Aminobenzoatos/efectos adversos , Anticuerpos Monoclonales/efectos adversos , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Resistencia a Antineoplásicos , Epistaxis/inducido químicamente , Femenino , Hemorragia/inducido químicamente , Humanos , Persona de Mediana Edad , Neoplasias/metabolismo , Receptor EphA2/inmunología

RESUMEN

PURPOSE: To conduct a phase I dose-escalation trial assessing safety and response of recombinant immunotoxin moxetumomab pasudotox (CAT-8015, HA22) in chemotherapy-resistant hairy cell leukemia (HCL). PATIENTS AND METHODS: Eligible patients had relapsed/refractory HCL after ≥ two prior therapies and required treatment because of abnormal blood counts. Patients received moxetumomab pasudotox 5 to 50 µg/kg every other day for three doses (QOD ×3), with up to 16 cycles repeating at ≥ 4-week intervals if patients did not experience disease progression or develop neutralizing antibodies. RESULTS: Twenty-eight patients were enrolled, including three patients each at 5, 10, 20, and 30 µg/kg, four patients at 40 µg/kg, and 12 patients at 50 µg/kg QOD ×3 for one to 16 cycles each (median, four cycles). Dose-limiting toxicity was not observed. Two patients had transient laboratory abnormalities consistent with grade 2 hemolytic uremic syndrome with peak creatinine of 1.53 to 1.66 mg/dL and platelet nadir of 106,000 to 120,000/µL. Drug-related toxicities in 25% to 64% of the 28 patients included (in decreasing frequency) grade 1 to 2 hypoalbuminemia, aminotransferase elevations, edema, headache, hypotension, nausea, and fatigue. Of 26 patients evaluable for immunogenicity, 10 patients (38%) made antibodies neutralizing more than 75% of the cytotoxicity of 1,000 ng/mL of immunotoxin, but this immunogenicity was rare (5%) after cycle 1. The overall response rate was 86%, with responses observed at all dose levels, and 13 patients (46%) achieved complete remission (CR). Only 1 CR lasted less than 1 year, with the median disease-free survival time not yet reached at 26 months. CONCLUSION: Moxetumomab pasudotox at doses up to 50 µg/kg QOD ×3 has activity in relapsed/refractory HCL and has a safety profile that supports further clinical development for treatment of this disease.

Asunto(s)

Toxinas Bacterianas/uso terapéutico , Exotoxinas/uso terapéutico , Inmunoterapia/métodos , Leucemia de Células Pilosas/terapia , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Adulto , Anciano , Toxinas Bacterianas/efectos adversos , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos , Exotoxinas/efectos adversos , Femenino , Humanos , Inmunoterapia/efectos adversos , Leucemia de Células Pilosas/inmunología , Leucemia de Células Pilosas/patología , Masculino , Persona de Mediana Edad , Factores de Tiempo , Resultado del Tratamiento

RESUMEN

Targeted therapies with monoclonal antibodies have been increasingly incorporated into the treatment for both lymphoid and myeloid hematological malignancies. Rituximab, the first approved monoclonal antibody for the treatment of cancer, has revolutionized our approach to the management of chronic lymphocytic leukemia and non-Hodgkin's lymphoma. However, there is still an unmet medical need for novel therapeutic approaches, especially for patients in the relapsed/refractory setting. Therapeutic agents with specificity against different surface antigens on malignant B cells hold promise for improving clinical outcome in these patients. Throughout the last decade, CD22, a B-cell-restricted phosphoglycoprotein of the immunoglobulin superfamily, has gained considerable interest as a therapeutic target for B-cell-directed therapies. Several novel therapeutic agents that selectively target CD22 are being developed as an alternative approach for cancer treatment. This review summarizes the current knowledge of CD22 and discusses the rationale for targeting CD22 in B-cell malignancies with immunotherapeutic agents. This review also describes some of the most promising investigational anti-CD22 agents for the treatment of B-cell malignancies.

Asunto(s)

Anticuerpos Monoclonales/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma de Células B/tratamiento farmacológico , Terapia Molecular Dirigida , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Humanos

RESUMEN

Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS) are clinically similar disorders characterized by microvascular thrombosis, hemolysis, thrombocytopenia, and end-organ damage. Although they may present with overlapping symptoms, multiple etiologies have been proposed for these thrombotic microangiopathies (TMA). Chemotherapy-induced TMA, which has been described with the use of mitomycin, gemcitabine, and other drugs, has a poor prognosis. Recently, reports of TMA associated with targeted cancer agents have surfaced in the literature. We discuss the clinical presentation, outcome, and etiology of TMA reported with the use of immunotoxins, monoclonal antibodies, and tyrosine kinase inhibitors. A search of PubMed and meeting abstracts was conducted for cases of TMA with the use of targeted cancer agents. The defining symptoms, laboratory values, time to onset, and patient outcomes were compiled. Consistent definitions of TMA and grading of severity in these cases are lacking. However, presentation of TMA in these cases revealed the importance of monitoring for renal toxicity, hemolysis, and thrombocytopenia. Patient outcomes seem to differ from those seen in cases of chemotherapy-induced TMA and may reflect a different underlying etiology. Little is known about the pathogenesis of TMA with targeted cancer agents. In contrast to chemotherapy-induced TMA, partial to full reversibility may be a common outcome. However, further research is warranted into optimal management of patients diagnosed with TMA following treatment with targeted agents.

Asunto(s)

Antineoplásicos/efectos adversos , Microangiopatías Trombóticas/inducido químicamente , Animales , Humanos , Neoplasias/complicaciones , Microangiopatías Trombóticas/diagnóstico , Microangiopatías Trombóticas/terapia

RESUMEN

PURPOSE: We have previously reported on the safety and immunologic response of a poxvirus-based vaccine encoding prostate-specific antigen (PSA) used in combination with radiation therapy in patients with localized prostate cancer. We hypothesized that a "metronomic" dose of interleukin 2 (IL-2) as a biological adjuvant would cause less toxicity while maintaining immunologic response. EXPERIMENTAL DESIGN: Eighteen patients with localized prostate cancer were treated in a single-arm trial using previously established doses of vaccine and radiation therapy. The vaccine used was a recombinant vaccinia virus engineered to encode PSA admixed with a recombinant vaccinia encoding the costimulatory molecule B7.1, followed by booster vaccinations with a recombinant fowlpox vector expressing PSA. Patients received a total of eight planned vaccination cycles, once every 4 weeks, with granulocyte-macrophage colony-stimulating factor given on days 1 to 4 and interleukin 2 (IL-2) at a dose of 0.6 MIU/M2 given from days 8 to 21 after each vaccination. Definitive external beam radiation therapy was initiated after the third vaccination cycle. Patients were evaluated for safety and immunologic response. Toxicity and immunologic activity were compared with the previously reported regimen containing a higher dose of IL-2. RESULTS: Seventeen of 18 patients received all eight cycles of vaccine with IL-2. Five of eight HLA-A2+ patients evaluated had an increase in PSA-specific T cells of > or =3-fold. Toxicities were generally mild, with only seven vaccination cycles of 140 given resulting in grade 3 toxicities possibly attributable to IL-2. CONCLUSIONS: Metronomic-dose IL-2 in combination with vaccine and radiation therapy is safe, can induce prostate-specific immune responses, and has immunologic activity similar to low-dose IL-2, with markedly reduced toxicities.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Vacunas contra el Cáncer/administración & dosificación , Interleucina-2/administración & dosificación , Antígeno Prostático Específico/inmunología , Neoplasias de la Próstata/terapia , Adyuvantes Inmunológicos/efectos adversos , Anciano , Vacunas contra el Cáncer/efectos adversos , Vacunas contra el Cáncer/inmunología , Terapia Combinada , Citometría de Flujo , Virus de la Viruela de las Aves de Corral/genética , Vectores Genéticos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/efectos adversos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Humanos , Interleucina-2/efectos adversos , Interleucina-2/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Masculino , Persona de Mediana Edad , Neoplasias de la Próstata/inmunología , Radioterapia , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Virus Vaccinia/genética

RESUMEN

PURPOSE: To describe the considerations leading to marketing approval of ixabepilone in combination with capecitabine and as monotherapy for the treatment of advanced breast cancer that is refractory to other chemotherapies. EXPERIMENTAL DESIGN: Data from one randomized multicenter trial comparing combination therapy with ixabepilone and capecitabine to capecitabine alone were analyzed for support of the combination therapy indication. For monotherapy, a single-arm trial of ixabepilone was analyzed. Supporting data came from an additional single-arm combination therapy study and two single-arm monotherapy studies. RESULTS: In patients with metastatic or locally advanced breast cancer who had disease progression on or following an anthracycline and a taxane, ixabepilone plus capecitabine showed an improvement in progression-free survival compared with capecitabine alone {median progression-free survival, 5.7 [95% confidence interval (95% CI), 4.8-6.7] versus 4.1 (95% CI, 3.1-4.3) months, stratified log-rank P < 0.0001; hazard ratio, 0.69 (95% CI, 0.58-0.83)}. As monotherapy for patients who had disease progression on or following an anthracycline, a taxane, and capecitabine, ixabepilone as monotherapy showed a 12% objective response rate by independent blinded review and 18% by investigator assessment. The major toxicities from ixabepilone therapy were peripheral neuropathy and myelosuppression, particularly neutropenia. CONCLUSIONS: On October 16, 2007, the Food and Drug Administration approved ixabepilone for injection in combination with capecitabine or as monotherapy for the treatment of patients with advanced breast cancer who have experienced disease progression on previous chemotherapies.

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos , Anciano , Neoplasias de la Mama/mortalidad , Capecitabina , Desoxicitidina/administración & dosificación , Desoxicitidina/efectos adversos , Desoxicitidina/análogos & derivados , Supervivencia sin Enfermedad , Epotilonas/administración & dosificación , Epotilonas/efectos adversos , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/efectos adversos , Fluorouracilo/análogos & derivados , Humanos , Persona de Mediana Edad , Metástasis de la Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico

RESUMEN

The transforming growth factor-beta (TGF-beta) pathway plays dual roles in cancer, inhibiting epithelial cell growth under normal physiologic conditions, but promoting invasion and metastasis once growth inhibitory responses are lost. Two recent papers show that TGF-beta receptor III is the most common TGF-beta pathway component downregulated in prostate cancer. Here, we discuss the implications of these findings and what it may mean about the biology of this disease.

Asunto(s)

Regulación hacia Abajo , Inhibinas/metabolismo , Neoplasias de la Próstata , Proteoglicanos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Testículo/metabolismo , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Transforming growth factor (TGF)-beta1 activity has been shown to increase vascular endothelial barrier permeability, which is believed to precede several pathologic conditions, including pulmonary edema and vessel inflammation. In endothelial monolayers, TGF-beta1 increases permeability, and a number of studies have demonstrated the alteration of cell-cell contacts by TGF-beta1. We hypothesized that focal adhesion complexes also likely contribute to alterations in endothelial permeability. We examined early signal transduction events associated with rapid changes in monolayer permeability and the focal adhesion complex of bovine pulmonary artery endothelial cells. Western blotting revealed rapid tyrosine phosphorylation of focal adhesion kinase (FAK) and Src kinase in response to TGF-beta1; inhibition of both of these kinases using pp2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), ameliorates TGF-beta1-induced monolayer permeability. Activation of FAK/Src requires activation of the epidermal growth factor receptor downstream of the TGF-beta receptors, and is blocked by the epidermal growth factor receptor inhibitor AG1478. Immunohistochemistry showed that actin and the focal adhesion proteins paxillin, vinculin, and hydrogen peroxide-inducible clone-5 (Hic-5) are rearranged in response to TGF-beta1; these proteins are released from focal adhesion complexes. Rearrangement of paxillin and vinculin by TGF-beta1 is not blocked by the FAK/Src inhibitor, pp2, or by SB431542 inhibition of the TGF-beta type I receptor, anaplastic lymphoma kinase 5; however, pp1 (4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which inhibits both type I and type II TGF-beta receptors, does block paxillin and vinculin rearrangement. Hic-5 protein rearrangement requires FAK/Src activity. Together, these results suggest that TGF-beta1-induced monolayer permeability involves focal adhesion and cytoskeletal rearrangement through both FAK/Src-dependent and -independent pathways.

Asunto(s)

Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Quinasa de Linfoma Anaplásico , Animales , Bovinos , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Receptores ErbB/genética , Proteína-Tirosina Quinasas de Adhesión Focal/antagonistas & inhibidores , Adhesiones Focales/efectos de los fármacos , Adhesiones Focales/metabolismo , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Paxillin/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras , Activación Transcripcional/efectos de los fármacos , Vinculina/metabolismo

RESUMEN

Using a yeast two-hybrid screen, we found that SNIP1 (Smad nuclear-interacting protein 1) associates with c-Myc, a key regulator of cell proliferation and transformation. We demonstrate that SNIP1 functions as an important regulator of c-Myc activity, binding the N terminus of c-Myc through its own C terminus, and that SNIP1 enhances the transcriptional activity of c-Myc both by stabilizing it against proteosomal degradation and by bridging the c-Myc/p300 complex. These effects of SNIP1 on c-Myc likely contribute to synergistic effects of SNIP1, c-Myc, and H-Ras in inducing formation of foci in an in vitro transformation assay and also in supporting anchorage-independent growth. The significant association of SNIP1 and c-Myc staining in a non-small cell lung cancer tissue array is further evidence that their activities might be linked and suggests that SNIP1 might be an important modulator of c-Myc activity in carcinogenesis.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transcripción Genética , Línea Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Proto-Oncogénicas c-myc/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Sensibilidad y Especificidad , Análisis de Matrices Tisulares , Técnicas del Sistema de Dos Híbridos

RESUMEN

Transforming growth factor-beta (TGF-beta) is the prototypical member of a family of growth factors that play important roles in normal development and human diseases. We identified the gene for fibroblast growth factor-binding protein 1 (FGF-BP1) as being significantly repressed following TGF-beta treatment. FGF-BP1 is an extracellular matrix bound protein that enhances fibroblast growth factor (FGF) signaling. We demonstrate here that TGF-beta signaling significantly represses FGF-BP1 expression in mesenchymal and neural crest cells undergoing in vitro smooth muscle differentiation. Analysis of the downstream signaling pathways shows that Smad2/3 are crucial for efficient FGF-BP1 repression by TGF-beta. Furthermore, we identified a novel element in the region from -785 to -782 bp of the FGF-BP1 promoter, which represents a known binding site for Hypermethylation in Cancer-1 (Hic-1), necessary for repression of FGF-BP1 by TGF-beta. These data define the molecular mechanism of transcriptional repression of an important target of TGF-beta signaling during angiogenesis.

Asunto(s)

Proteínas de la Matriz Extracelular/fisiología , Silenciador del Gen/fisiología , Regiones Promotoras Genéticas/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Animales , Secuencia de Bases , Sitios de Unión , Diferenciación Celular , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel , Músculo Liso/citología , Músculo Liso/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

We recently reported that transforming growth factor (TGF)-beta induced the neural crest stem cell line Monc-1 to differentiate into a spindle-like contractile smooth muscle cell (SMC) phenotype and that Smad signaling played an important role in this phenomenon. In addition to Smad signaling, other pathways such as mitogen-activated protein kinase (MAPK), phosphoinositol-3 kinase, and RhoA have also been shown to mediate TGF-beta actions. The objectives of this study were to examine whether these signaling pathways contribute to TGF-beta-induced SMC development and to test whether Smad signaling cross-talks with other pathway(s) during SMC differentiation induced by TGF-beta. We demonstrate here that RhoA signaling is critical to TGF-beta-induced SMC differentiation. RhoA kinase (ROCK) inhibitor Y27632 significantly blocks the expression of multiple SMC markers such as smooth muscle alpha-actin, SM22alpha, and calponin in TGF-beta-treated Monc-1 cells. In addition, Y27632 reversed the cell morphology and abolished the contractility of TGF-beta-treated cells. RhoA signaling was activated as early as 5 min following TGF-beta addition. Dominant negative RhoA blocked nuclear translocation of Smad2 and Smad3 because of the inhibition of phosphorylation of both Smads and inhibited Smad-dependent SBE promoter activity, whereas constitutively active RhoA significantly enhanced SBE promoter activity. Consistent with these results, C3 exotoxin, an inhibitor of RhoA activation, significantly attenuated SBE promoter activity and inhibited Smad nuclear translocation. Taken together, these data point to a new role for RhoA as a modulator of Smad activation while regulating TGF-beta-induced SMC differentiation.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Proteína Smad2/fisiología , Proteína smad3/fisiología , Factor de Crecimiento Transformador beta/farmacología , Proteína de Unión al GTP rhoA/metabolismo , Amidas/farmacología , Animales , Línea Celular , Ratones , Ratones Endogámicos C3H , Relajantes Musculares Centrales/farmacología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Piridinas/farmacología

RESUMEN

During vascular development, nascent endothelial networks are invested with a layer of supporting cells called pericytes in capillaries or smooth muscle in larger vessels. The cellular lineage of smooth muscle precursors and factors responsible for regulating their differentiation remain uncertain. In vivo, cells derived from the multipotent neural crest can give rise to vascular smooth muscle in parts of the head and also the cardiac outflow tract. Although transforming growth factor-beta (TGF-beta) has previously been shown to induce some smooth muscle markers from primary cultures of neural crest stem cells, the extent of the differentiation induced was not clear. In this study, we demonstrate that TGF-beta can induce many of the markers and characteristics of vascular smooth muscle from a neural crest stem cell line, Monc-1. Within 3 days of in vitro treatment, TGF-beta induces multiple smooth muscle-specific markers, while downregulating epithelial markers present on the parent cells. Treatment with TGF-beta also induces a contractile phenotype that responds to the muscarinic agonist carbachol and is not immediately reversed on TGF-beta withdrawal. Examination of the signaling pathways involved revealed that TGF-beta activation of Smad2 and Smad3 appear to be essential for the observed differentiation. Taken together, this system provides a novel model of smooth muscle differentiation that reliably recapitulates the process observed in vivo and allows for dissection of the pathways and processes involved in this process.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Miocitos del Músculo Liso/citología , Cresta Neural/efectos de los fármacos , Transactivadores/fisiología , Factor de Crecimiento Transformador beta/farmacología , Actinas/biosíntesis , Actinas/genética , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada/citología , Línea Celular Transformada/efectos de los fármacos , Medios de Cultivo/farmacología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Marcación de Gen , Ratones , Ratones Endogámicos C3H , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/genética , Contracción Muscular , Proteínas Musculares/biosíntesis , Proteínas Musculares/genética , Cresta Neural/citología , Interferencia de ARN , ARN Interferente Pequeño/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Proteína Smad2 , Proteína smad3 , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Transfección , Factor de Crecimiento Transformador beta/antagonistas & inhibidores

RESUMEN

Smad proteins transduce signals from transforming growth factor-beta (TGF-beta) superfamily ligands to regulate the expression of target genes. In order to identify novel partners of Smad proteins in transcriptional regulation, we performed a two-hybrid screen using Smad5, a protein that is activated predominantly by bone morphogenetic protein (BMP) signaling. We identified an interaction between Smad5 and suppressor of variegation 3-9 homolog 2 (Suv39h2), a chromatin modifier enzyme. Suv39h proteins are histone methyltransferases that methylate histone H3 on lysine 9, resulting in transcriptional repression or silencing of target genes. Biochemical studies in mammalian cells demonstrated that Smad5 binds to both known mammalian isoforms of Suv39h proteins, and that Smad proteins activated by the TGF-beta signaling pathway, Smad2 and Smad3, do not bind with significant affinity. Functional studies using the muscle creatine kinase (MCK) promoter, which is suppressed by BMP signaling, demonstrate that Suv39h proteins and Smads cooperate to repress promoter activity. These data suggest a model where association of Smad proteins with Suv39h methyltransferases can repress or silence genes involved in developmental processes, and argues that inefficient gene repression may result in the alteration of the differentiated phenotype. Thus, examination of the Smad-Suv interaction may provide insight into the mechanism of phenotypic determination mediated by BMP signaling.

Asunto(s)

Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Unión al ADN/metabolismo , Silenciador del Gen , N-Metiltransferasa de Histona-Lisina/metabolismo , Transactivadores/metabolismo , Animales , Diferenciación Celular , Línea Celular , Creatina Quinasa/genética , Forma MM de la Creatina-Quinasa , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Isoenzimas/genética , Ligandos , Ratones , Mioblastos/citología , Mioblastos/metabolismo , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Metiltransferasas , Estructura Terciaria de Proteína , Transducción de Señal , Proteínas Smad , Transcripción Genética , Activación Transcripcional , Transfección , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Smad proteins transduce signals from transforming growth factor-beta (TGF-beta) receptors and regulate transcription of target genes. TGF-beta is implicated in the regulation of the smooth muscle cell specific gene SM22alpha, but little is known about how Smads are involved in SM22alpha gene transcription. In this report, we demonstrate that TGF-beta activation of the SM22alpha promoter is Smad dependent in C3H10T1/2 cells, BALB 3T3 cells and neural crest Monc-1 cells. We find that the promoter region from -162 to +41 is sufficient to up-regulate the reporter gene upon TGF-beta induction. Smad3, Smad1 and Smad4 are found in TGF-beta inducible complexes that bind to a region containing a Smad binding site (SBS) and a medea box. Both the SBS and medea box are necessary for complex formation and are functionally important. Smad4 is limiting for TGF-beta induction, and Smad3, but not Smad1, significantly contributes to maximal activation. Time course luciferase assays and time course gel mobility shift assays reveal that the Smad3/4 complex is largely responsible for the immediate response of the SM22alpha promoter to TGF-beta induction, and also contributes to the maximal promoter activity. We further demonstrate that AP-1 elements contribute to induction of the SM22alpha promoter by TGF-beta.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Regulación de la Expresión Génica/efectos de los fármacos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Proteínas Musculares/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Smad , Proteína Smad1 , Proteína smad3 , Proteína Smad4 , Transactivadores/genética , Factor de Transcripción AP-1/metabolismo , Transcripción Genética

RESUMEN

BACKGROUND: The intracellular signaling events of the bone morphogenetic proteins (BMPs) involve the R-Smad family members Smad1, Smad5, Smad8 and the Co-Smad, Smad4. Smads are currently considered to be DNA-binding transcriptional modulators and shown to recruit the master transcriptional co-activator CBP/p300 for transcriptional activation. SNIP1 is a recently discovered novel repressor of CBP/p300. Currently, the detailed molecular mechanisms that allow R-Smads and Co-Smad to co-operatively modulate transcription events are not fully understood. RESULTS: Here we report a novel physical and functional link between Smad1 and the 26S proteasome that contributes to Smad1- and Smad4-mediated transcriptional regulation. Smad1 forms a complex with a proteasome beta subunit HsN3 and the ornithine decarboxylase antizyme (Az). The interaction is enhanced upon BMP type I receptor activation and occur prior to the incorporation of HsN3 into the mature 20S proteasome. Furthermore, BMPs trigger the translocation of Smad1, HsN3 and Az into the nucleus, where the novel CBP/p300 repressor protein SNIP1 is further recruited to Smad1/HsN3/Az complex and degraded in a Smad1-, Smad4- and Az-dependent fashion. The degradation of the CBP/p300 repressor SNIP1 is likely an essential step for Smad1-, Smad4-mediated transcriptional activation, since increased SNIP1 expression inhibits BMP-induced gene responses. CONCLUSIONS: Our studies thus add two additional important functional partners of Smad1 into the signaling web of BMPs and also suggest a novel mechanism for Smad1 and Smad4 to co-modulate transcription via regulating proteasomal degradation of CBP/p300 repressor SNIP1.

Asunto(s)

Proteínas Morfogenéticas Óseas/metabolismo , Cisteína Endopeptidasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Complejos Multienzimáticos/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta , Animales , Western Blotting , Proteína Morfogenética Ósea 2 , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1 , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/farmacología , Células COS , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Cisteína Endopeptidasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mutación , Pruebas de Precipitina , Complejo de la Endopetidasa Proteasomal , Unión Proteica/efectos de los fármacos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Proteínas Smad , Proteína Smad1 , Transactivadores/genética , Transactivadores/metabolismo , Transcripción Genética , Células Tumorales Cultivadas , Técnicas del Sistema de Dos Híbridos

RESUMEN

The zebrafish mutant violet beauregarde (vbg) can be identified at two days post-fertilization by an abnormal circulation pattern in which most blood cells flow through a limited number of dilated cranial vessels and fail to perfuse the trunk and tail. This phenotype cannot be explained by caudal vessel abnormalities or by a defect in cranial vessel patterning, but instead stems from an increase in endothelial cell number in specific cranial vessels. We show that vbg encodes activin receptor-like kinase 1 (Acvrl1; also known as Alk1), a TGFbeta type I receptor that is expressed predominantly in the endothelium of the vessels that become dilated in vbg mutants. Thus, vbg provides a model for the human autosomal dominant disorder, hereditary hemorrhagic telangiectasia type 2, in which disruption of ACVRL1 causes vessel malformations that may result in hemorrhage or stroke. Movies available on-line

Asunto(s)

Receptores de Activinas/genética , Receptores de Activinas/metabolismo , Vasos Sanguíneos/embriología , Cabeza/irrigación sanguínea , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Vasos Sanguíneos/anomalías , Circulación Cerebrovascular , Mapeo Cromosómico , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Cabeza/embriología , Humanos , Datos de Secuencia Molecular , Mutación , Fosfoproteínas/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteínas Smad , Proteína Smad5 , Proteína Smad8 , Telangiectasia Hemorrágica Hereditaria/genética , Telangiectasia Hemorrágica Hereditaria/fisiopatología , Transactivadores/metabolismo , Pez Cebra/embriología

Asunto(s)

Proteínas de Unión al ADN/genética , Ratones Noqueados/genética , Transactivadores/genética , Animales , Femenino , Masculino , Ratones , Proteínas Smad , Proteína Smad1