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OBJECTIVES: Mutation analysis by massive parallel sequencing (MPS) is routinely performed in the clinical management of lung cancer in Sweden. We describe the clinical and mutational profiles of lung cancer patients subjected to the first 1.5 years of treatment predictive MPS testing in an autonomous regional health care region. METHODS: Tumors from all patients with lung cancer who had an MPS test from January 2015 to June 2016 in the Skåne health care region in Sweden (1.3 million citizens) were included. Six hundred eleven tumors from 599 patients were profiled using targeted sequencing with a 26-gene exon-focused panel. Data on disease patterns and characteristics of the patients subjected to testing were assembled, and correlations between mutational profiles and clinical features were analyzed. RESULTS: MPS with the 26-gene panel revealed alterations in 92% of the 611 lung tumors, with the most frequent mutations detected in the nontargetable genes TP53 (62%) and KRAS (37%). Neither KRAS nor TP53 mutations were associated with disease pattern, chemotherapy response, progression-free survival, or overall survival in advanced-stage disease treated with platinum-based doublet chemotherapy as a first-line treatment. Among targetable genes, EGFR driver mutations were detected in 10% of the tumors, and BRAF p.V600 variants in 2.3%. For the 71 never smokers (12%), targetable alterations (EGFR mutations, BRAF p.V600, MET exon 14 skipping, or ALK/ROS1 rearrangement) were detected in 59% of the tumors. CONCLUSION: Although the increasing importance of MPS as a predictor of response to targeted therapies is indisputable, its role in prognostics or as a predictor of clinical course in nontargetable advanced stage lung cancer requires further investigation.
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Precision medicine requires accurate multi-gene clinical diagnostics. We describe the implementation of an Illumina TruSight Tumor (TST) clinical NGS diagnostic framework and parallel validation of a NanoString RNA-based ALK, RET, and ROS1 gene fusion assay for combined analysis of treatment predictive alterations in non-small cell lung cancer (NSCLC) in a regional healthcare region of Sweden (Scandinavia). The TST panel was clinically validated in 81 tumors (99% hotspot mutation concordance), after which 533 consecutive NSCLCs were collected during one-year of routine clinical analysis in the healthcare region (~90% advanced stage patients). The NanoString assay was evaluated in 169 of 533 cases. In the 533-sample cohort 79% had 1-2 variants, 12% >2 variants and 9% no detected variants. Ten gene fusions (five ALK, three RET, two ROS1) were detected in 135 successfully analyzed cases (80% analysis success rate). No ALK or ROS1 FISH fusion positive case was missed by the NanoString assay. Stratification of the 533-sample cohort based on actionable alterations in 11 oncogenes revealed that 66% of adenocarcinomas, 13% of squamous carcinoma (SqCC) and 56% of NSCLC not otherwise specified harbored ≥1 alteration. In adenocarcinoma, 10.6% of patients (50.3% if including KRAS) could potentially be eligible for emerging therapeutics, in addition to the 15.3% of patients eligible for standard EGFR or ALK inhibitors. For squamous carcinoma corresponding proportions were 4.4% (11.1% with KRAS) vs 2.2%. In conclusion, multiplexed NGS and gene fusion analyses are feasible in NSCLC for clinical diagnostics, identifying notable proportions of patients potentially eligible for emerging molecular therapeutics.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias Pulmonares/genética , Mutación , Proteínas de Fusión Oncogénica/genética , Análisis de Secuencia de ADN/métodos , Quinasa de Linfoma Anaplásico , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Medicina de Precisión , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Tirosina Quinasas Receptoras/genética , Análisis de Secuencia de ARN , SueciaRESUMEN
BACKGROUND: Histopathological diagnosis is important for prognostication and choice of treatment in patients with cancer in the lung. Metastases to the lungs are common and need to be distinguished from primary lung cancer. Furthermore, cases with synchronous or metachronous primary lung cancers (although infrequent) are often handled differently than cases with lung cancer with intrapulmonary metastasis or relapse, respectively. In some cases, morphology and immunohistochemical staining is not sufficient for certain diagnosis. METHODS: The present study included six cases where molecular genetic analysis in form of pyrosequencing or targeted next-generation sequencing was of value for certain diagnosis of selected tumours in the lung. RESULTS: Two of the included cases were rare metastases to the lung; colorectal cancer with IHC profile consistent with primary lung cancer and malignant adenomyoepithelioma of the breast, respectively, where molecular genetic analysis was of aid for proving the relationship to the primary tumour. The other four cases were multiple lung adenocarcinomas where molecular genetic analysis was of aid to distinguish between intrapulmonary metastasis and synchronous tumour. CONCLUSIONS: Comparison of molecular genetic profile may be an important tool for determination of relationship between tumours in some situations and should always be considered in unclear cases. Further studies on concordance and discordance of molecular genetic profiles between spatially or temporally different tumours with common origin may be helpful for improved diagnostics of pulmonary tumours.
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Adenocarcinoma/genética , Adenomioepitelioma/genética , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias Colorrectales/genética , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias Pulmonares/genética , Análisis de Secuencia de ADN , Adenocarcinoma/patología , Adenocarcinoma/terapia , Adenocarcinoma del Pulmón , Adenomioepitelioma/secundario , Adenomioepitelioma/terapia , Anciano , Biopsia , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/terapia , Diagnóstico Diferencial , Femenino , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Masculino , Persona de Mediana Edad , Fenotipo , Valor Predictivo de las Pruebas , TranscriptomaRESUMEN
Multicolor flow cytometry (MFC) and real-time quantitative PCR (RQ-PCR) are important independent techniques to determine minimal residual disease (MRD) in acute myeloid leukemia (AML). MFC is the standard method, but may be unreliable. Therefore, MFC-based determination of MRD with an RQ-PCR-based approach targeting the nucleophosmin 1 (NPM1) type A mutation was set out to compare. Since most current NPM1 RQ-PCR MRD protocols suffer from clear definitions of quantifiability, we sought to define quantifiability in a reproducible and standardized manner. The limit of quantifiability of our RQ-PCR protocol for the NPM1 type A mutation varied between 0.002% and 0.04% residual leukemic cells depending on the features of the standard curve for each PCR experiment. The limit of detection was close to 0.001% leukemic cells. The limit of detection by MFC ranged from 0.01% to 1% depending on the phenotype of the leukemic cells as compared with non-leukemic bone marrow cells. Forty-five MRD samples from 15 patients using both NPM1 mutation specific RQ-PCR and MFC were analyzed. In 32 of the 45 samples (71%), an MRD-signal could be detected with RQ-PCR. A quantifiable NPM1 mutation signal was found in 15 samples (33%) (range 0.003%-2.6% leukemic cells). By contrast, only two follow-up samples (4%) showed residual leukemic cells (0.04% and 0.3%, respectively) by MFC. Thus, RQ-PCR of the NPM1 type A mutation was more sensitive and reliable than MFC for determination of MRD, which might have clinical implications. © 2016 Wiley Periodicals, Inc.
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Leucemia Mieloide Aguda/diagnóstico , Neoplasia Residual/diagnóstico , Proteínas Nucleares/genética , Adulto , Anciano , Femenino , Citometría de Flujo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Mutación , Neoplasia Residual/genética , Neoplasia Residual/patología , Proteínas Nucleares/aislamiento & purificación , Nucleofosmina , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Myxothiazol is a respiratory chain complex III (CIII) inhibitor that binds to the ubiquinol oxidation site Qo of CIII. It blocks electron transfer from ubiquinol to cytochrome b and thus inhibits CIII activity. It has been utilized as a tool in studies of respiratory chain function in in vitro and cell culture models. We developed a mouse model of biochemically induced and reversible CIII inhibition using myxothiazol. We administered myxothiazol intraperitoneally at a dose of 0.56 mg/kg to C57Bl/J6 mice every 24 h and assessed CIII activity, histology, lipid content, supercomplex formation, and gene expression in the livers of the mice. A reversible CIII activity decrease to 50% of control value occurred at 2 h post-injection. At 74 h only minor histological changes in the liver were found, supercomplex formation was preserved and no significant changes in the expression of genes indicating hepatotoxicity or inflammation were found. Thus, myxothiazol-induced CIII inhibition can be induced in mice for four days in a row without overt hepatotoxicity or lethality. This model could be utilized in further studies of respiratory chain function and pharmacological approaches to mitochondrial hepatopathies.
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Antifúngicos/efectos adversos , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Hígado/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Animales , Modelos Animales de Enfermedad , Complejo III de Transporte de Electrones/metabolismo , Hígado/metabolismo , Hígado/patología , Metacrilatos/efectos adversos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Tiazoles/efectos adversosRESUMEN
BACKGROUND: Podocalyxin-like 1 (PODXL) is a cell-adhesion glycoprotein and stem cell marker that has been associated with an aggressive tumour phenotype and adverse outcome in several cancer types. We recently demonstrated that overexpression of PODXL is an independent factor of poor prognosis in colorectal cancer (CRC). The aim of this study was to validate these results in two additional independent patient cohorts and to examine the correlation between PODXL mRNA and protein levels in a subset of tumours. METHOD: PODXL protein expression was analyzed by immunohistochemistry in tissue microarrays with tumour samples from a consecutive, retrospective cohort of 270 CRC patients (cohort 1) and a prospective cohort of 337 CRC patients (cohort 2). The expression of PODXL mRNA was measured by real-time quantitative PCR in a subgroup of 62 patients from cohort 2. Spearman's Rho and Chi-Square tests were used for analysis of correlations between PODXL expression and clinicopathological parameters. Kaplan Meier analysis and Cox proportional hazards modelling were applied to assess the relationship between PODXL expression and time to recurrence (TTR), disease free survival (DFS) and overall survival (OS). RESULTS: High PODXL protein expression was significantly associated with unfavourable clinicopathological characteristics in both cohorts. In cohort 1, high PODXL expression was associated with a significantly shorter 5-year OS in both univariable (HR = 2.28; 95% CI 1.43-3.63, p = 0.001) and multivariable analysis (HR = 2.07; 95% CI 1.25-3.43, p = 0.005). In cohort 2, high PODXL expression was associated with a shorter TTR (HR = 2.93; 95% CI 1.26-6.82, p = 0.013) and DFS (HR = 2.44; 95% CI 1.32-4.54, p = 0.005), remaining significant in multivariable analysis, HR = 2.50; 95% CI 1.05-5.96, p = 0.038 for TTR and HR = 2.11; 95% CI 1.13-3.94, p = 0.019 for DFS.No significant correlation could be found between mRNA levels and protein expression of PODXL and there was no association between mRNA levels and clinicopathological parameters or survival. CONCLUSIONS: Here, we have validated the previously demonstrated association between immunohistochemical expression of PODXL and poor prognosis in CRC in two additional independent patient cohorts. The results further underline the potential utility of PODXL as a biomarker for more precise prognostication and treatment stratification of CRC patients.
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Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/química , Sialoglicoproteínas/análisis , Anciano , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/genética , Distribución de Chi-Cuadrado , Estudios de Cohortes , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Pronóstico , Modelos de Riesgos Proporcionales , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Sialoglicoproteínas/biosíntesis , Sialoglicoproteínas/genética , Estadísticas no Paramétricas , Resultado del TratamientoRESUMEN
BACKGROUND & AIMS: Liver is a target organ in many mitochondrial disorders, especially if the complex III assembly factor BCS1L is mutated. To reveal disease mechanism due to such mutations, we have produced a transgenic mouse model with c.232A>G mutation in Bcs1l, the causative mutation for GRACILE syndrome. The homozygous mice develop mitochondrial hepatopathy with steatosis and fibrosis after weaning. Our aim was to assess cellular mechanisms for disease onset and progression using metabolomics. METHODS: With mass spectrometry we analyzed metabolite patterns in liver samples obtained from homozygotes and littermate controls of three ages. As oxidative stress might be a mechanism for mitochondrial hepatopathy, we also assessed H(2)O(2) production and expression of antioxidants. RESULTS: Homozygotes had a similar metabolic profile at 14 days of age as controls, with the exception of slightly decreased AMP. At 24 days, when hepatocytes display first histopathological signs, increases in succinate, fumarate and AMP were found associated with impaired glucose turnover and beta-oxidation. At end stage disease after 30 days, these changes were pronounced with decreased carbohydrates, high levels of acylcarnitines and amino acids, and elevated biogenic amines, especially putrescine. Signs of oxidative stress were present in end-stage disease. CONCLUSIONS: The findings suggest an early Krebs cycle defect with increases of its intermediates, which might play a role in disease onset. During disease progression, carbohydrate and fatty acid metabolism deteriorate leading to a starvation-like condition. The mouse model is valuable for further investigations on mechanisms in mitochondrial hepatopathy and for interventions.
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Complejo III de Transporte de Electrones/deficiencia , Hígado/metabolismo , Chaperonas Moleculares/genética , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Monofosfato/metabolismo , Animales , Antioxidantes/metabolismo , Complejo III de Transporte de Electrones/genética , Fumaratos/metabolismo , Peróxido de Hidrógeno/metabolismo , Espectrometría de Masas , Ratones , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Mutación , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Ácido Succínico/metabolismoRESUMEN
UNLABELLED: Mitochondrial dysfunction is an important cause for neonatal liver disease. Disruption of genes encoding oxidative phosphorylation (OXPHOS) components usually causes embryonic lethality, and thus few disease models are available. We developed a mouse model for GRACILE syndrome, a neonatal mitochondrial disease with liver and kidney involvement, caused by a homozygous BCS1L mutation (232A>G). This gene encodes a chaperone required for incorporation of Rieske iron-sulfur protein (RISP) into complex III of respiratory chain. Homozygous mutant mice after 3 weeks of age developed striking similarities to the human disease: growth failure, hepatic glycogen depletion, steatosis, fibrosis, and cirrhosis, as well as tubulopathy, complex III deficiency, lactacidosis, and short lifespan. BCS1L was decreased in whole liver cells and isolated mitochondria of mutants at all ages. RISP incorporation into complex III was diminished in symptomatic animals; however, in young animals complex III was correctly assembled. Complex III activity in liver, heart, and kidney of symptomatic mutants was decreased to 20%, 40%, and 40% of controls, respectively, as demonstrated with electron flux kinetics through complex III. In high-resolution respirometry, CIII dysfunction resulted in decreased electron transport capacity through the respiratory chain under maximum substrate input. Complex I function, suggested to be dependent on a functional complex III, was, however, unaffected. CONCLUSION: We present the first viable model of complex III deficiency mimicking a human mitochondrial disorder. Incorporation of RISP into complex III in young homozygotes suggests another complex III assembly factor during early ontogenesis. The development of symptoms from about 3 weeks of age provides a convenient time window for studying the pathophysiology and treatment of mitochondrial hepatopathy and OXPHOS dysfunction in general.
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Complejo III de Transporte de Electrones/deficiencia , Hepatopatías/genética , Enfermedades Mitocondriales/genética , Chaperonas Moleculares/genética , Mutación/genética , ATPasas Asociadas con Actividades Celulares Diversas , Acidosis Láctica/genética , Animales , Colestasis/genética , Modelos Animales de Enfermedad , Complejo III de Transporte de Electrones/metabolismo , Retardo del Crecimiento Fetal/genética , Hemosiderosis/genética , Homocigoto , Errores Innatos del Metabolismo/genética , Ratones , Ratones Mutantes , Fosforilación Oxidativa , Aminoacidurias Renales/genéticaRESUMEN
Regulating the choice between neural stem cell maintenance versus differentiation determines growth and size of the developing brain. Here we identify TGF-beta signaling as a crucial factor controlling these processes. At early developmental stages, TGF-beta signal activity is localized close to the ventricular surface of the neuroepithelium. In the midbrain, but not in the forebrain, Tgfbr2 ablation results in ectopic expression of Wnt1/beta-catenin and FGF8, activation of Wnt target genes, and increased proliferation and horizontal expansion of neuroepithelial cells due to shortened cell-cycle length and decreased cell-cycle exit. Consistent with this phenotype, self-renewal of mutant neuroepithelial stem cells is enhanced in the presence of FGF and requires Wnt signaling. Moreover, TGF-beta signal activation counteracts Wnt-induced proliferation of midbrain neuroepithelial cells. Thus, TGF-beta signaling controls the size of a specific brain area, the dorsal midbrain, by antagonizing canonical Wnt signaling and negatively regulating self-renewal of neuroepithelial stem cells.
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Diferenciación Celular , Mesencéfalo/citología , Mesencéfalo/fisiología , Transducción de Señal , Células Madre/citología , Células Madre/fisiología , Factor de Crecimiento Transformador beta/fisiología , Proteína Wnt1/fisiología , Animales , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/fisiología , Humanos , Mesencéfalo/embriología , Ratones , Células Neuroepiteliales/citología , Células Neuroepiteliales/fisiología , Neuronas/citología , Neuronas/fisiología , Especificidad de Órganos , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
BACKGROUND: The FFAR1 receptor is expressed mainly in pancreatic beta cells and is activated by medium to long chain free fatty acids (FFAs), as well as by thiazolidinediones, resulting in elevated Ca(2+) concentrations and promotion of insulin secretion. These properties suggest that FFAR1 could be a mediator of lipotoxicity and a potential candidate gene for Type 2 diabetes (T2D). We therefore investigated whether variations at the FFAR1 locus are associated with T2D and beta cell function. METHODOLOGY/PRINCIPAL FINDINGS: We re-sequenced the FFAR1 region in 96 subjects (48 healthy and 48 T2D individuals) and found 13 single nucleotide polymorphisms (SNPs) 8 of which were not previously described. Two SNPs located in the upstream region of the FFAR1 gene (rs1978013 and rs1978014) were chosen and genotyped in 1929 patients with T2D and 1405 healthy control subjects. We observed an association of rs1978013 and rs1978014 with insulinogenic index in males (p = 0.024) and females (p = 0.032), respectively. After Bonferroni corrections, no association with T2D was found in the case-control material, however a haplotype consisting of the T-G alleles conferred protection against T2D (p = 0.0010). CONCLUSIONS/SIGNIFICANCE: Variation in the FFAR1 gene may contribute to impaired beta cell function in T2D.
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Diabetes Mellitus Tipo 2/genética , Islotes Pancreáticos/fisiopatología , Receptores Acoplados a Proteínas G/genética , Anciano , Secuencia de Bases , Estudios de Casos y Controles , Cartilla de ADN , Diabetes Mellitus Tipo 2/fisiopatología , Femenino , Genotipo , Prueba de Tolerancia a la Glucosa , Humanos , Islotes Pancreáticos/fisiología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
In bacterial meningitis, chemokines lead to recruitment of polymorphonuclear leucocytes (PMN) into the CNS. At the site of infection in the subarachnoid space, PMN release reactive oxygen species, reactive nitrogen intermediates (RNI) and interleukin-1beta (IL-1beta). Although these immune factors assist in clearance of bacteria, they also result in neuronal injury associated with meningitis. Transforming growth factor beta (TGFbeta) is a potent deactivator of PMN and macrophages since TGFbeta suppresses the production of ROI, RNI and IL-1. Here, we report that the deletion of the TGFbeta receptor II gene in PMN enhances PMN recruitment into the CNS of mice with Streptococcus pneumoniae meningitis. This was associated with more efficient clearance of bacteria, and almost complete prevention of intracerebral necrotizing vasculitis. Differences in PMN in the CNS of infected control mice and mice lacking TGFbeta receptor II were not explained by altered expression of chemokines acting on PMN. Instead, TGFbeta was found to impair the expression of L (leucocyte)-selectin on PMN from control mice but not from mice lacking TGFbeta receptor II. L-selectin is known to be essential for PMN recruitment in bacterial meningitis. We conclude that defective TGFbeta signalling in PMN is beneficial in bacterial meningitis by ameliorating migration of PMN and bacterial clearance.
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Eliminación de Gen , Meningitis Neumocócica/genética , Neutrófilos/fisiología , Receptores de Factores de Crecimiento Transformadores beta/genética , Vasculitis del Sistema Nervioso Central/genética , Animales , Hemorragia Cerebral/inmunología , Quimiotaxis de Leucocito/inmunología , Modelos Animales de Enfermedad , Inmunidad Innata/inmunología , Selectina L/análisis , Macrófagos/inmunología , Macrófagos/metabolismo , Meningitis Neumocócica/inmunología , Ratones , Ratones Noqueados , Microglía/inmunología , Microglía/metabolismo , Neutrófilos/inmunología , Fagocitos/fisiología , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Vasculitis del Sistema Nervioso Central/inmunología , Vasculitis del Sistema Nervioso Central/prevención & controlRESUMEN
Smooth muscle cell (SMC)-specific deletion of transforming growth factor beta (TGF-beta) signaling would help elucidate the mechanisms through which TGF-beta signaling contributes to vascular development and disease. We attempted to generate mice with SMC-specific deletion of TGF-beta signaling by mating mice with a conditional ("floxed") allele for the type II TGF-beta receptor (tgfbr2flox) to mice with SMC-targeted expression of Cre recombinase. We bred male mice transgenic for smooth muscle myosin heavy chain (SMMHC)-Cre with females carrying tgfbr2flox. Surprisingly, SMMHC-Cre mice recombined tgfbr2flox at low levels in SMC and at high levels in the testis. Recombination of tgfbr2flox in testis correlated with high-level expression of SMMHC-Cre in testis and germline transmission of tgfbr2null. In contrast, mice expressing Cre from a SM22alpha promoter (SM22-Cre) efficiently recombined tgfbr2flox in vascular and visceral SMC and the heart, but not in testis. Use of the R26R reporter allele confirmed that Cre-mediated recombination in vascular SMC was inefficient for SMMHC-Cre mice and highly efficient for SM22-Cre mice. Breedings that introduced the SM22-Cre allele into tgfbr2flox/flox zygotes in order to generate adult mice that are hemizygous for SM22-Cre and homozygous for tgfbr2flox- and would have conversion of tgfbr2flox/flox to tgfbr2null/null in SMC-produced no live SM22-Cre : tgfbr2flox/flox pups (P<0.001). We conclude: (1) "SMC-targeted" Cre lines vary significantly in specificity and efficiency of Cre expression; (2) TGF-beta signaling in the subset of cells that express SM22alpha is required for normal development; (3) generation of adult mice with absent TGF-beta signaling in SMC remains a challenge.
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Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Alelos , Animales , Linaje de la Célula , Cruzamientos Genéticos , Femenino , Regulación del Desarrollo de la Expresión Génica , Homocigoto , Integrasas/genética , Masculino , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso Vascular/irrigación sanguínea , Músculo Liso Vascular/embriología , Cadenas Pesadas de Miosina/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Recombinación Genética , Transducción de Señal , Testículo/embriología , Testículo/metabolismoRESUMEN
We have taken advantage of the Cre/lox system to generate a mouse model with inducible deficiency of transforming growth factor beta receptor II (TbetaRII). Using this approach, transforming growth factor beta (TGF-beta) signaling deficiency can be restricted to the hematopoietic system by bone marrow transplantation. Mice that received transplants with TbetaRII-/- bone marrow develop a lethal inflammatory disorder closely resembling that of TGF-beta1-null mice. Previous in vitro studies have suggested multiple roles for TGF-beta in T-cell development, including proliferation, apoptosis, and differentiation. We used our transplantation model to ask whether T-cell development is normal in the absence of TGF-beta signaling. The findings show for the first time in vivo and in fetal thymus organ culture (FTOC) that TGF-beta is not required for thymocytes to differentiate along the entire pathway of thymic T-cell development, as defined by the expression patterns of CD4, CD8, CD25, and CD44. In contrast to previous investigations, no increase of thymocyte apoptosis was observed. However, TbetaRII-deficient CD8+ thymocytes displayed a 2-fold increase in proliferation rate, as determined by bromodeoxyuridine (BrdU) incorporation in vivo. These results reinforce the importance of TGF-beta as an immune regulator critical for T-cell function.
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Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Timo/citología , Timo/metabolismo , Animales , Trasplante de Médula Ósea , Proliferación Celular , Ratones , Ratones Noqueados , Técnicas de Cultivo de Órganos , Proteínas Serina-Treonina Quinasas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Specific inactivation of TGFbeta signaling in neural crest stem cells (NCSCs) results in cardiovascular defects and thymic, parathyroid, and craniofacial anomalies. All these malformations characterize DiGeorge syndrome, the most common microdeletion syndrome in humans. Consistent with a role of TGFbeta in promoting non-neural lineages in NCSCs, mutant neural crest cells migrate into the pharyngeal apparatus but are unable to acquire non-neural cell fates. Moreover, in neural crest cells, TGFbeta signaling is both sufficient and required for phosphorylation of CrkL, a signal adaptor protein implicated in the development of DiGeorge syndrome. Thus, TGFbeta signal modulation in neural crest differentiation might play a crucial role in the etiology of DiGeorge syndrome.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Síndrome de DiGeorge/etiología , Cresta Neural/fisiología , Proteínas Nucleares/metabolismo , Transducción de Señal/fisiología , Células Madre/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Síndrome de DiGeorge/genética , Síndrome de DiGeorge/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Noqueados , Cresta Neural/citología , Proteínas Nucleares/genética , Fosforilación , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Development of the eye depends partly on the periocular mesenchyme derived from the neural crest (NC), but the fate of NC cells in mammalian eye development and the signals coordinating the formation of ocular structures are poorly understood. RESULTS: Here we reveal distinct NC contributions to both anterior and posterior mesenchymal eye structures and show that TGFbeta signaling in these cells is crucial for normal eye development. In the anterior eye, TGFbeta2 released from the lens is required for the expression of transcription factors Pitx2 and Foxc1 in the NC-derived cornea and in the chamber-angle structures of the eye that control intraocular pressure. TGFbeta enhances Foxc1 and induces Pitx2 expression in cell cultures. As in patients carrying mutations in PITX2 and FOXC1, TGFbeta signal inactivation in NC cells leads to ocular defects characteristic of the human disorder Axenfeld-Rieger's anomaly. In the posterior eye, NC cell-specific inactivation of TGFbeta signaling results in a condition reminiscent of the human disorder persistent hyperplastic primary vitreous. As a secondary effect, retinal patterning is also disturbed in mutant mice. CONCLUSION: In the developing eye the lens acts as a TGFbeta signaling center that controls the development of eye structures derived from the NC. Defective TGFbeta signal transduction interferes with NC-cell differentiation and survival anterior to the lens and with normal tissue morphogenesis and patterning posterior to the lens. The similarity to developmental eye disorders in humans suggests that defective TGFbeta signal modulation in ocular NC derivatives contributes to the pathophysiology of these diseases.
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Anomalías del Ojo/etiología , Cresta Neural/fisiología , Transducción de Señal , Células Madre/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Movimiento Celular , Células Cultivadas , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Cristalino/metabolismo , Ratones , Ratones Mutantes , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ratas , Células Madre/citología , Factores de Transcripción , Proteína del Homeodomínio PITX2RESUMEN
Transforming growth factor beta (TGF-beta) is a potent inhibitor of hepatocyte proliferation in vitro and is suggested to be a key negative regulator of liver growth. To directly address the role of TGF-beta signaling in liver regeneration in vivo, the TGF-beta type II receptor gene (Tgfbr2) was selectively deleted in hepatocytes by crossing "floxed" Tgfbr2 conditional knockout mice with transgenic mice expressing Cre under control of the albumin promoter. Hepatocytes isolated from liver-specific Tgfbr2 knockout (R2LivKO) mice were refractory to the growth inhibitory effects of TGF-beta1. The peak of DNA synthesis after 70% partial hepatectomy occurred earlier (36 vs. 48 hours) and was 1.7-fold higher in R2LivKO mice compared with controls. Accelerated S-phase entry by proliferating R2LivKO hepatocytes coincided with the hyperphosphorylation of Rb protein and the early upregulation of cyclin D1 and cyclin E. However, by 120 hours after partial hepatectomy, hepatocyte proliferation was back to baseline in both control and R2LivKO liver. Regenerating R2LivKO liver showed evidence of increased signaling by activin A and persistent activity of the Smad pathway. Blockage of activin A signaling by the specific inhibitor follistatin resulted in increased hepatocyte proliferation at 120 hours, particularly in R2LivKO livers. In conclusion, TGF-beta regulates G(1) to S phase transition of hepatocytes, but intact signaling by TGF-beta is not required for termination of liver regeneration. Increased signaling by activin A may compensate to regulate liver regeneration when signaling through the TGF-beta pathway is abolished, and may be a principal factor in the termination of liver regeneration.
Asunto(s)
Regeneración Hepática/fisiología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/fisiología , Activinas/antagonistas & inhibidores , Activinas/fisiología , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , ADN/biosíntesis , Proteínas de Unión al ADN/fisiología , Folistatina/farmacología , Hepatocitos/citología , Subunidades beta de Inhibinas/antagonistas & inhibidores , Subunidades beta de Inhibinas/fisiología , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Transducción de Señal/efectos de los fármacos , Proteínas Smad , Transactivadores/fisiología , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1RESUMEN
The ribosomal protein S19 (RPS19) is located in the small (40S) subunit and is one of 79 ribosomal proteins. The gene encoding RPS19 is mutated in approximately 25% of patients with Diamond-Blackfan anemia, which is a rare congenital erythroblastopenia. Affected individuals present with decreased numbers or the absence of erythroid precursors in the bone marrow, and associated malformations of various organs are common. We produced C57BL/6J mice with a targeted disruption of murine Rps19 to study its role in erythropoiesis and development. Mice homozygous for the disrupted Rps19 were not identified as early as the blastocyst stage, indicating a lethal effect. In contrast, mice heterozygous for the disrupted Rps19 allele have normal growth and organ development, including that of the hematopoietic system. Our findings indicate that zygotes which are Rps19(-/-) do not form blastocysts, whereas one normal Rps19 allele in C57BL/6J mice is sufficient to maintain normal ribosomal and possibly extraribosomal functions.
Asunto(s)
Implantación del Embrión/fisiología , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Anemia de Diamond-Blackfan/genética , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Femenino , Marcación de Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Embarazo , Células Madre/fisiologíaRESUMEN
Enforced expression of Hoxb4 dramatically increases the regeneration of murine hematopoietic stem cells (HSCs) after transplantation and enhances the repopulation ability of human severe combined immunodeficiency (SCID) repopulating cells. Therefore, we asked what physiologic role Hoxb4 has in hematopoiesis. A novel mouse model lacking the entire Hoxb4 gene exhibits significantly reduced cellularity in spleen and bone marrow (BM) and a subtle reduction in red blood cell counts and hemoglobin values. A mild reduction was observed in the numbers of primitive progenitors and stem cells in adult BM and fetal liver, whereas lineage distribution was normal. Although the cell cycle kinetics of primitive progenitors was normal during endogenous hematopoiesis, defects in proliferative responses of BM Lin(-) Sca1(+) c-kit(+) stem and progenitor cells were observed in culture and in vivo after the transplantation of BM and fetal liver HSCs. Quantitative analysis of mRNA from fetal liver revealed that a deficiency of Hoxb4 alone changed the expression levels of several other Hox genes and of genes involved in cell cycle regulation. In summary, the deficiency of Hoxb4 leads to hypocellularity in hematopoietic organs and impaired proliferative capacity. However, Hoxb4 is not required for the generation of HSCs or the maintenance of steady state hematopoiesis.
Asunto(s)
Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Animales , Antimetabolitos/farmacología , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/fisiología , Recuento de Células , División Celular/fisiología , Femenino , Trasplante de Tejido Fetal , Fluorouracilo/farmacología , Regulación del Desarrollo de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/trasplante , Hígado/citología , Hígado/embriología , Hígado/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , EmbarazoRESUMEN
Studies in vitro implicate transforming growth factor beta (TGF-beta) as a key regulator of hematopoiesis with potent inhibitory effects on progenitor and stem cell proliferation. In vivo studies have been hampered by early lethality of knock-out mice for TGF-beta isoforms and the receptors. To directly assess the role of TGF-beta signaling for hematopoiesis and hematopoietic stem cell (HSC) function in vivo, we generated a conditional knock-out model in which a disruption of the TGF-beta type I receptor (T beta RI) gene was induced in adult mice. HSCs from induced mice showed increased proliferation recruitment when cultured as single cells under low stimulatory conditions in vitro, consistent with an inhibitory role of TGF-beta in HSC proliferation. However, induced T beta RI null mice show normal in vivo hematopoiesis with normal numbers and differentiation ability of hematopoietic progenitor cells. Furthermore HSCs from T beta RI null mice exhibit a normal cell cycle distribution and do not differ in their ability long term to repopulate primary and secondary recipient mice following bone marrow transplantation. These findings challenge the classical view that TGF-beta is an essential negative regulator of hematopoietic stem cells under physiologic conditions in vivo.
Asunto(s)
Receptores de Activinas Tipo I/fisiología , Células Madre Hematopoyéticas/citología , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Factor de Crecimiento Transformador beta/fisiología , Receptores de Activinas Tipo I/deficiencia , Receptores de Activinas Tipo I/genética , Animales , Trasplante de Médula Ósea , Ciclo Celular , Diferenciación Celular , División Celular , Células Cultivadas , Hematopoyesis , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de SeñalRESUMEN
Recent studies in mouse models deficient in transforming growth factor beta (TGF-beta) signaling have documented TGF-beta as one of the major regulators of immune function. TGF-beta1-null animals demonstrated massive autoimmune inflammation affecting multiple organs, but attempts to transfer the phenotype to normal animals by bone marrow transplantation only resulted in minor inflammatory lesions. We wanted to ask whether a lethal inflammatory phenotype would develop following transplantation of bone marrow deficient for the TGF-beta type II receptor (TbetaRII) gene to normal recipient animals. The TbetaRII-null mutation would generate a cell autonomous phenotype that cannot be reverted by the influence of endocrine or paracrine TGF-beta derived from the recipient animal. We have generated conditional knockout mice in which the TbetaRII gene is disrupted upon induction with interferon-alphabeta or polyI:polyC. We show that induction of TbetaRII gene disruption in these mice by polyI:polyC results in a lethal inflammatory disease. Importantly, bone marrow from conditional knockout mice transferred to normal recipent mice caused a similar lethal inflammation, regardless of whether induction of TGF-beta receptor deficiency occurred in donor animals before, or in recipient animals after transplantation. These results show that TGF-beta signaling deficiency within cells of hematopoietic origin is sufficient to cause a lethal inflammatory disorder in mice. This animal model provides an important tool to further clarify the pathogenic mechanisms in animals deficient for TGF-beta signaling and the importance of TGF-beta to regulate immune functions.