RESUMO
BACKGROUND: Regulatory T cells (Treg) represent a promising target for novel treatment strategies in patients with inflammatory/allergic diseases. A soluble derivate of the Treg surface molecule glycoprotein A repetitions predominant (sGARP) has strong anti-inflammatory and regulatory effects on human cells in vitro as well as in vivo through de novo induction of peripheral Treg. The aim of this study was to investigate the immunomodulatory function of sGARP and its possible role as a new therapeutic option in allergic diseases using a humanized mouse model. METHODS: To analyze the therapeutic effects of sGARP, adult NOD/Scidγc(-/-) (NSG) mice received peripheral blood mononuclear cells (PBMC) derived from allergic patients with sensitization against birch allergen. Subsequently, allergic inflammation was induced in the presence of Treg alone or in combination with sGARP. RESULTS: In comparison with mice that received Treg alone, additional treatment with sGARP reduced airway hyperresponsiveness (AHR), influx of neutrophils and macrophages into the bronchoalveolar lavage (BAL), and human CD45(+) cells in the lungs. Furthermore, the numbers of mucus-producing goblet cells and inflammatory cell infiltrates were reduced. To elucidate whether the mechanism of action of sGARP involves the TGF-ß receptor pathway, mice additionally received anti-TGF-ß receptor II (TGF-ßRII) antibodies. Blocking the signaling of TGF-ß through TGF-ßRII abrogated the anti-inflammatory effects of sGARP, confirming its essential role in inhibiting the allergic inflammation. CONCLUSION: Induction of peripheral tolerance via sGARP is a promising potential approach to treat allergic airway diseases.
Assuntos
Inflamação/etiologia , Inflamação/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Hipersensibilidade Respiratória/etiologia , Hipersensibilidade Respiratória/metabolismo , Adulto , Alérgenos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Tolerância Imunológica , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Inflamação/patologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Hipersensibilidade Respiratória/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
The MTS1 gene on chromosome 9p21 encodes the p16 inhibitor of cyclinD/Cdk-4 complexes, and is deleted or mutated in a variety of tumour types. We found allelic deletions of 9p21-p22 in 85% of pancreatic adenocarcinomas. Analysis of MTS1 in pancreatic carcinomas (27 xenografts and 10 cell lines) showed homozygous deletions in 15 (41%) and sequence changes in 14 (38%). These included eight point mutations (four nonsense, two missense and two splice site mutations) and six deletions/insertions, all accompanied by loss of the wild-type allele. Sequencing of MTS1 from primary tumours confirmed the mutations. Coexistent inactivations of both MTS1 and p53 was common and suggests that abnormal regulation of cyclin-dependent kinases may play an important role in the biology of pancreatic carcinoma.
Assuntos
Adenocarcinoma/genética , Proteínas de Transporte/genética , Neoplasias Pancreáticas/genética , Sequência de Bases , Deleção Cromossômica , Cromossomos Humanos Par 9 , Inibidor p16 de Quinase Dependente de Ciclina , Deleção de Genes , Genes p53 , Humanos , Dados de Sequência Molecular , Mutação , Células Tumorais CultivadasRESUMO
Chromosome deletions are the most common genetic events observed in cancer. These deletions are generally thought to reflect the existence of a tumour suppressor gene within the lost region. However, when the lost region does not precisely coincide with a hereditary cancer locus, identification of the putative tumour suppressor gene (target of the deletion) can be problematic. For example, previous studies have demonstrated that chromosome 18q is lost in over 60% of colorectal as well as in other cancers, but the lost region could not be precisely determined. Here we present a rigorous strategy for mapping and evaluating allelic deletions in sporadic tumours, and apply it to the evaluation of chromosome 18 in colorectal cancers. Using this approach, we define a minimally lost region (MLR) on chromosome 18q21, which contains at least two candidate tumour suppressor genes, DPC4 and DCC. The analysis further suggested genetic heterogeneity, with DPC4 the deletion target in up to a third of the cases and DCC or a neighbouring gene the target in the remaining tumours.
Assuntos
Cromossomos Humanos Par 18 , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA , Genes Supressores de Tumor , Transativadores , Proteínas Supressoras de Tumor , Alelos , Animais , Sequência de Bases , Moléculas de Adesão Celular/genética , Mapeamento Cromossômico , Receptor DCC , Análise Mutacional de DNA , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Proteínas/genética , Receptores de Superfície Celular , Proteína Smad4 , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
BACKGROUND: Perturbations in the expression profiles of microRNAs (miRNAs) have been reported for a variety of different cancers. Differentially expressed miRNAs have not been systematically evaluated in basal cell carcinoma (BCC) of the skin. OBJECTIVES: To initiate a microarray-based miRNA profiling study to identify specific miRNA candidates that are differentially expressed in BCC. METHODS: Patients with BCC (n = 7) were included in this study. Punch biopsies were harvested from the tumour centre (lesional, n = 7) and from adjacent nonlesional skin (intraindividual control, n = 7). Microarray-based miRNA expression profiles were obtained on an Agilent platform using miRBase 16 screening for 1205 Homo sapiens (hsa)-miRNA candidates. To validate the microarray data, the expression of seven dysregulated miRNAs was measured by TaqMan quantitative real-time reverse transcription polymerase chain reaction. RESULTS: We identified 16 significantly upregulated (hsa-miR-17, hsa-miR-18a, hsa-miR-18b, hsa-miR-19b, hsa-miR-19b-1*, hsa-miR-93, hsa-miR-106b, hsa-miR-125a-5p, hsa-miR-130a, hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa-miR-182, hsa-miR-455-3p, hsa-miR-455-5p and hsa-miR-542-5p) and 10 significantly downregulated (hsa-miR-29c, hsa-miR-29c*, hsa-miR-139-5p, hsa-miR-140-3p, hsa-miR-145, hsa-miR-378, hsa-miR-572, hsa-miR-638, hsa-miR-2861 and hsa-miR-3196) miRNAs in BCC compared with nonlesional skin. Data mining revealed connections to many tumour-promoting pathways, such as the Hedgehog and the mitogen-activated protein kinase/extracellular signal-regulated kinase signalling cascades. CONCLUSIONS: This study identified several miRNA candidates that may play a role in the molecular pathogenesis of BCC.
Assuntos
Carcinoma Basocelular/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , MicroRNAs/genética , RNA Neoplásico/genética , Neoplasias Cutâneas/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma Basocelular/metabolismo , Estudos de Casos e Controles , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Cutâneas/metabolismoRESUMO
Families with both melanoma and pancreatic cancer are extremely rare and some are affected with the autosomal dominant inherited familial atypical multiple mole melanoma-pancreatic cancer (FAMMM-PC) syndrome. The phenotypic and genotypic expressions of such pancreatic cancer-melanoma prone families are not well defined. The National Case Collection of Familial Pancreatic Cancer of the Deutsche Krebshilfe includes 110 pancreatic cancer families, 18 of which (16%) show an association of pancreatic cancer and melanoma. These 18 families were analysed regarding their phenotype and the prevalence of germline mutations in the candidate genes CDKN2A, BRCA2, CHEK2, NOD2, ARL11 and Palladin (PALLD). There were two types of families: five families with the FAMMM-PC phenotype and 13 PC/melanoma families without the multiple mole phenotypes (PCMS). The prevalences of PC and melanoma in the two types of families were similar. The prevalence of other tumour types, especially breast carcinoma, was higher (11%) in PCMS- than in FAMMM-PC families (2.4%, p = 0.02). CDKN2A mutations were identified in 2 of 18 (11%) PCMS families. A cosegregating BRCA2 mutation was detected in one PCMS family without breast cancer. None of the reported germline mutations in the NOD2, Palladin, ARL11 or CHEK2 genes were detected in either type of family. In conclusion, families with an accumulation of PC and melanoma show a large variety of phenotypic expression, which is not always consistent with the FAMMM-PC phenotype. More PC/melanoma-prone families need to be analysed to clarify whether such families represent variations of the FAMMM-PC syndrome or two distinct hereditary cancer syndromes.
Assuntos
Predisposição Genética para Doença , Melanoma/genética , Neoplasias Pancreáticas/genética , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína BRCA2/genética , Análise Mutacional de DNA , Família , Feminino , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Polimorfismo Genético , Neoplasias Cutâneas/patologia , Adulto JovemRESUMO
BACKGROUND: Pilot studies were suggestive for a role of clonal T cells in the pathogenesis of systemic sclerosis (SSc). OBJECTIVES: To investigate the presence of clonal T cells in both peripheral blood and skin of a large collection of patients with SSc. METHODS: Polymerase chain reaction and high-resolution capillary electrophoresis for detecting T-cell clonality were performed in a series of 126 patients with SSc. RESULTS: Seventy-seven (61%) of 126 patients had clonal T cells in their peripheral blood. In contrast, a clonal T-cell population was present in only four of 29 (14%) age-matched healthy controls (P = 0.03). Older patients were more likely to have clonal T cells than younger patients with SSc (P < 0.0001). Clonal T cells were more commonly detected in the blood of patients with limited cutaneous SSc (48 of 65 patients, 74%) than in those with diffuse cutaneous disease (29 of 61, 48%; P = 0.0002). Lesional skin specimens from 20 of 44 patients (45%) had detectable clonal T-cell populations. There was no correlation between the presence of circulating clonal T cells and lesional clonal T cells, sex, disease duration, extent of skin sclerosis, digital ulcers, organ involvement (e.g. interstitial lung disease, kidney disease, oesophagus involvement), treatment of SSc, or autoantibody profile. CONCLUSIONS: Many patients with SSc have expanded clonal T cells in their peripheral blood and skin. These clonal T cells could play a critical role in the pathogenesis of SSc, especially in limited cutaneous disease.
Assuntos
Escleroderma Sistêmico/imunologia , Pele/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Imunidade Celular , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Reação em Cadeia da Polimerase , Escleroderma Sistêmico/genética , Adulto JovemRESUMO
About 90 percent of human pancreatic carcinomas show allelic loss at chromosome 18q. To identify candidate tumor suppressor genes on 18q, a panel of pancreatic carcinomas were analyzed for convergent sites of homozygous deletion. Twenty-five of 84 tumors had homozygous deletions at 18q21.1, a site that excludes DCC (a candidate suppressor gene for colorectal cancer) and includes DPC4, a gene similar in sequence to a Drosophila melanogaster gene (Mad) implicated in a transforming growth factor-beta (TGF-beta)-like signaling pathway. Potentially inactivating mutations in DPC4 were identified in six of 27 pancreatic carcinomas that did not have homozygous deletions at 18q21.1. These results identify DPC4 as a candidate tumor suppressor gene whose inactivation may play a role in pancreatic and possibly other human cancers.
Assuntos
Cromossomos Humanos Par 18 , Proteínas de Ligação a DNA , Genes Supressores de Tumor , Neoplasias Pancreáticas/genética , Proteínas/genética , Transativadores , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Divisão Celular , Mapeamento Cromossômico , Deleção de Genes , Expressão Gênica , Marcadores Genéticos , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Transplante de Neoplasias , Neoplasias Pancreáticas/patologia , Proteínas/química , Proteínas/fisiologia , Transdução de Sinais , Proteína Smad4 , Fator de Crescimento Transformador beta/fisiologia , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is known for its very poor overall prognosis. Accurate early diagnosis and new therapeutic modalities are therefore urgently needed. We used 377 feature microRNA (miRNA) arrays to investigate miRNA expression in normal pancreas, chronic pancreatitis, and PDAC tissues as well as PDAC-derived cell lines. A pancreatic miRNome was established comparing the data from normal pancreas with a reference set of 33 human tissues. The expression of miR-216 and -217 and lack of expression of miR-133a were identified as characteristic of pancreas tissue. Unsupervised clustering showed that the three pancreatic tissues types can be classified according to their respective miRNA expression profiles. We identified 26 miRNAs most prominently misregulated in PDAC and a relative quantitative reverse transcriptase-polymerase chain reaction index using only miR-217 and -196a was found to discriminate normal pancreas, chronic pancreatitis and cancerous tissues, establishing a potential utility for miRNAs in diagnostic procedures. Lastly, comparing differentially expressed genes from PDAC with predicted miRNA target genes for the top 26 miRNAs, we identified potential novel links between aberrant miRNA expression and known target genes relevant to PDAC biology. Our data provides novel insights into the miRNA-driven pathophysiological mechanisms involved in PDAC development and offers new candidate targets to be exploited both for diagnostic and therapeutic strategies.
Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , MicroRNAs/análise , Neoplasias Pancreáticas/genética , Adenocarcinoma/etiologia , Biomarcadores , Carcinoma Ductal Pancreático/etiologia , Linhagem Celular Tumoral , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Pâncreas/metabolismo , Neoplasias Pancreáticas/etiologia , Pancreatite Crônica/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Patients with chronic obstructive pulmonary disease (COPD) demonstrate a limited exercise capacity. It is unknown whether muscle fiber atrophy and subsequent decrease in force production contributes to this functional limitation. Therefore, the purpose of this investigation was to determine whether emphysema-induced muscle fiber atrophy leads to a reduction in locomotory muscle force production. Maximal muscle force production and fiber cross-sectional area were measured in the almost exclusively fast-twitch extensor digitorium longus muscles at 4 and 8 months following saline (control, n=8/time period) or elastase (emphysema, n=15/time period) instillation in the lungs of hamsters. Excised lung volume increased 145 and 161% with emphysema at 4 and 8 months, respectively (both P<0.01). Muscle mass, maximal force, and fiber cross-section were unaltered at 4 months. However, absolute mass (-15%) and fiber cross-sectional area (-18%) were reduced at 8 months (both P<0.01). Surprisingly, maximal force was preserved in emphysema animals. These data demonstrate that maximal muscle force may be preserved in the face of emphysema-induced fiber atrophy.
Assuntos
Enfisema/fisiopatologia , Contração Muscular/fisiologia , Força Muscular/fisiologia , Músculo Esquelético/fisiopatologia , Atrofia Muscular/fisiopatologia , Animais , Cricetinae , Modelos Animais de Doenças , Enfisema/induzido quimicamente , Enfisema/complicações , Seguimentos , Masculino , Mesocricetus , Fibras Musculares Esqueléticas/classificação , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Tamanho do Órgão , Elastase PancreáticaRESUMO
There are currently no models of exercise that recruit and train muscles, such as the rat spinotrapezius, that are suitable for transmission intravital microscopic investigation of the microcirculation. Recent experimental evidence supports the concept that running downhill on a motorized treadmill recruits the spinotrapezius muscle of the rat. Based on these results, we tested the hypothesis that 6 wk of downhill running (-14 degrees grade) for 1 h/day, 5 days/wk, at a speed of up to 35 m/min, would 1) increase whole body peak oxygen uptake (Vo(2 peak)), 2) increase spinotrapezius citrate synthase activity, and 3) reduce the fatigability of the spinotrapezius during electrically induced 1-Hz submaximal tetanic contractions. Trained rats (n = 6) elicited a 24% higher Vo(2 peak) (in ml.min(-1).kg(-1): sedentary 58.5 +/- 2.0, trained 72.7 +/- 2.0; P < 0.001) and a 41% greater spinotrapezius citrate synthase activity (in mumol.min(-1).g(-1): sedentary 14.1 +/- 0.7, trained 19.9 +/- 0.9; P < 0.001) compared with sedentary controls (n = 6). In addition, at the end of 15 min of electrical stimulation, trained rats sustained a greater percentage of the initial tension than their sedentary counterparts (control 34.3 +/- 3.1%, trained 59.0 +/- 7.2%; P < 0.05). These results demonstrate that downhill running is successful in promoting training adaptations in the spinotrapezius muscle, including increased oxidative capacity and resistance to fatigue. Since the spinotrapezius muscle is commonly used in studies using intravital microscopy to examine microcirculatory function at rest and during contractions, our results suggest that downhill running is an effective training paradigm that can be used to investigate the mechanisms for improved microcirculatory function following exercise training in health and disease.
Assuntos
Músculo Esquelético/fisiologia , Condicionamento Físico Animal/fisiologia , Animais , Citrato (si)-Sintase/fisiologia , Feminino , Microcirculação , Fadiga Muscular/fisiologia , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/enzimologia , Consumo de Oxigênio/fisiologia , Ratos , Ratos Sprague-Dawley , Corrida/fisiologiaRESUMO
Pancreatic adenocarcinoma is the fifth leading cause of cancer death in the United States. Mutations in the K-ras oncogene occur in 85% of pancreatic adenocarcinomas and have also been identified in 75% of pancreatic ducts with mucinous cell hyperplasia seen in association with chronic pancreatitis. We identified K-ras mutations in 65% of duct lesions associated not only with chronic pancreatitis but also with pancreatic adenocarcinoma and distal common bile duct carcinoma (cholangiocarcinoma). These observations make K-ras a potential candidate for a gene-based diagnostic test. Indeed, K-ras mutations have been demonstrated in the pancreatic secretions of patients with pancreatic carcinoma and pancreatic intraductal neoplasia. We analyzed stool specimens for mutated K-ras sequences using a plaque hybridization assay in patients with pancreatic adenocarcinoma, cholangiocarcinoma, and chronic pancreatitis. K-ras mutations were detected in stool specimens from 6 of 11 patients with pancreatic adenocarcinoma, from 2 of 3 patients with cholangiocarcinoma, and from 1 of 3 patients with chronic pancreatitis. The K-ras mutations found in stool specimens from patients with pancreatic carcinoma were identical to those in the primary cancer in five cases. Mutations found in the stool specimens from one patient with pancreatic cancer, one patient with chronic pancreatitis, and two patients with cholangiocarcinoma were the same as those identified in pancreatic ductal mucinous cell hyperplasia lesions present in the resected pancreas specimens. Our data suggest that the K-ras mutations originating from cells of pancreatic adenocarcinomas and from cells shed by abnormal pancreatic duct epithelium can be detected in the stool. These results support the further exploration of stool K-ras analysis as a potential screening assay for the early detection of pancreatic adenocarcinoma and precursor lesions such as pancreatic ductal mucinous cell hyperplasia.
Assuntos
Adenocarcinoma/genética , Códon/genética , Fezes , Genes ras/genética , Mutação/genética , Neoplasias Pancreáticas/genética , Doença Aguda , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Colangiocarcinoma/genética , Humanos , Hiperplasia/genética , Pessoa de Meia-Idade , Dados de Sequência Molecular , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Pancreatite/genéticaRESUMO
A candidate tumor suppressor gene, DPC4, located at 18q21.1, has recently been shown to be inactivated in half of pancreatic adenocarcinomas. The close developmental relationship of the pancreas and biliary tract prompted us to determine the role of DPC4 in the multistep carcinogenesis of biliary tract carcinoma. A search for mutations in the genomic sequence of the highly conserved COOH-terminal domain of DPC4 (exons 8-11) was performed by single-strand conformational polymorphism analysis. Five of 32 (16%) primary biliary tract carcinomas had point mutations in the DPC4 sequence. Interestingly, inactivation of DPC4 was especially common in carcinomas originating from the common bile duct (four of eight specimens analyzed), suggesting an important role for DPC4 in the development of this subtype of biliary tract tumor.
Assuntos
Neoplasias do Sistema Biliar/genética , Carcinoma/genética , Proteínas de Ligação a DNA , Transativadores/genética , Sequência de Aminoácidos , DNA de Neoplasias/genética , Genes Supressores de Tumor , Humanos , Dados de Sequência Molecular , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína Smad4RESUMO
We identified a homozygous deletion in a pancreatic carcinoma (DPC) that localized to a 1-cM region at chromosome 13q12.3, which lay within the 6-cM locus of familial breast cancer susceptibility (BRCA-2). Here we present a physical map of the region, consisting of YAC, PAC, and cosmid contigs. The YAC contig comprises 16 clones that together span the entire BRCA2 region. The PAC contig comprises 22 clones that together span the DPC region. Seventy cosmid clones were localized within and near the DPC region. Thirty-five sequence-tagged sites were defined and localized within the map. The map indicates the size of the DPC region to be near 250 kb, and provides mapped and cloned resources for the search for the putative tumor suppressor gene(s) in the region.
Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 13 , DNA de Neoplasias/genética , Neoplasias Pancreáticas/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Clonagem Molecular , Cosmídeos , Primers do DNA/química , Biblioteca Genômica , Humanos , Dados de Sequência Molecular , Sitios de Sequências RotuladasRESUMO
The Rb/p16 tumor-suppressive pathway is abrogated frequently in human tumors, either through inactivation of the Rb or p16INK4a/CDKN2/MTS1 tumor-suppressor proteins, or through alteration or overexpression of the cyclin D1 or cyclin-dependent kinase 4 oncoproteins. We reported previously that the p16 gene was genetically inactivated in 82% of pancreatic carcinomas. Nearly half of these inactivations were by intragenic mutation of p16, and the remainder were by homozygous deletion of the gene. Here, we analyzed pancreatic carcinomas for additional mechanisms by which the Rb/p16 pathway might be inactivated. Transcriptional silencing of the p16 gene in association with methylation of its 5'-CpG island was examined by methylation-specific PCR in 18 pancreatic carcinomas. Nine of these were known to harbor an intragenic mutation in p16, and nine had a wild-type p16 coding sequence. Seven of the 18 tumors were hypermethylated, and all 7 were p16 wild-type (P = 0.001). Complete silencing of transcription from methylated wild-type gene sequences was demonstrated. Immunohistochemical analysis revealed normal expression levels of the Rb protein in all carcinomas studied. None of the carcinomas had genomic amplification of the cyclin D1 or CDK4 genes, and none had mutation of the p16-binding domain of CDK4. An additional p16 mutation was identified. In total, the Rb/p16 pathway was abrogated in 49 of the 50 carcinomas (98%) studied, all through inactivation of the p16 gene. Similar results were obtained in an independently analyzed series of 19 pancreatic carcinomas. These data demonstrate the central role of the Rb/p16 pathway in the development of pancreatic carcinoma.
Assuntos
Proteínas de Transporte/genética , Neoplasias Pancreáticas/genética , Proteína do Retinoblastoma/genética , Proteínas de Transporte/metabolismo , Cromossomos Humanos Par 13 , Inibidor p16 de Quinase Dependente de Ciclina , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Heterozigoto , Humanos , Mutação , Neoplasias Pancreáticas/metabolismo , Proteína do Retinoblastoma/metabolismo , Células Tumorais CultivadasRESUMO
During tumorigenesis, positive selection is exerted upon those tumor cells that alter rate-limiting regulatory pathways. A corollary of this principle is that mutation of one gene abrogates the need for alteration of another gene in the same pathway and also that the coexistence in a single tumor of mutations in different genes implies their involvement in distinct tumor-suppressive pathways. We studied 42 pancreatic adenocarcinomas for genetic alterations in the K-ras oncogene and the p16, p53, and DPC4 tumor suppressor genes. All of them had the K-ras gene mutated. Thirty-eight % of the tumors had four altered genes, another 38% had three altered genes, 15% had two altered genes, and 8% of the tumors had one altered gene. Interestingly, we noted a high concordance of DPC4 and p16 inactivations (P = 0.007), suggesting that the genetic inactivation of p16 increases the selective advantage of subsequent mutation in DPC4. No statistically significant association was identified between the alteration of these cancer genes and pathological or clinical parameters. This type of multigenic analysis in human tumors may serve to substantiate experimental tumor models and thus increase our understanding of the truly physiologically relevant tumor-suppressive pathways that are abrogated during human tumorigenesis.
Assuntos
Adenocarcinoma/genética , Proteínas de Ligação a DNA , Genes Supressores de Tumor , Neoplasias Pancreáticas/genética , Proteína BRCA2 , Proteínas de Transporte/genética , Inibidor p16 de Quinase Dependente de Ciclina , Análise Mutacional de DNA , DNA de Neoplasias/genética , Feminino , Genes p53 , Genes ras , Heterozigoto , Humanos , Masculino , Proteínas de Neoplasias/genética , Deleção de Sequência , Proteína Smad4 , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
p53 and MTS1 are known to be mutationally inactivated in pancreatic adenocarcinoma. Other tumor suppressor genes are likely also to play a role. To define chromosomal arms which may harbor additional tumor suppressor genes, we performed an extensive allelotype on pancreatic cancer utilizing a xenograft enrichment technique. Eighty-eight percent (28/32) of primary tumors gave rise to xenografts. Eighteen cases were used in a PCR-based allelotype using 283 polymorphic markers, over 2800 informative assays, and an average coverage of 4.1 informative markers per chromosomal arm per case. Highly frequent allelic loss (> 60%) was seen at chromosomes 1p, 9p, 17p, and 18q. Moderately frequent allelic loss (40-60%) was seen at 3p, 6p, 6q, 8p, 10q, 12q, 13q, 18p, 21q, and 22q. The average fractional allelic loss was 0.36. Allelic and sequence stability was demonstrated among 64 parallel and second-passage xenografts derived from 12 cases of pancreatic adenocarcinoma with the ascertainment of over 3000 single alleles. The findings were confirmed in primary tumors. In only two instances were discrepancies revealed between the allelic loss data obtained from corresponding parallel xenografts, probably due to the xenografting of minor subpopulations, reflecting genetic heterogeneity of the primary tumor.
Assuntos
Adenocarcinoma/genética , Neoplasias Pancreáticas/genética , Alelos , Animais , Proteínas de Transporte/genética , Mapeamento Cromossômico , Inibidor p16 de Quinase Dependente de Ciclina , DNA de Neoplasias/genética , Genes Supressores de Tumor , Heterozigoto , Camundongos , Camundongos Nus , Transplante de Neoplasias , Mutação Puntual , Deleção de Sequência , Transplante HeterólogoRESUMO
Absolute genetic differences between neoplastic and nonneoplastic cells can be discerned at sites of homozygous deletions. These deletions are of critical interest because they might be useful in the identification of defective biochemical pathways in neoplastic cells, and subsequently for the development of new treatment strategies in human cancer. We identified an area at 18q21.1 involved by homozygous deletions in 30% of pancreatic carcinomas. To characterize the homozygous deletions, we constructed a detailed physical map of nearly 2 Mb, containing yeast artificial chromosomes, P1-derived artificial chromosomes, cosmids and 24 sequence-tagged sites. The homozygously deleted are contained a new candidate tumor-suppressor gene (DPC4). To date, 23 (64%) of 35 pancreatic carcinomas carry at least one homozygous deletion at a published locus. The study of the total gene content of these loci, facilitated by the sequence-tagged site markers and maps of these regions, should help to reveal the absolute biochemical differences between neoplastic and nonneoplastic cells for a common human tumor.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 18 , Deleção de Genes , Neoplasias Pancreáticas/genética , Alelos , Sequência de Bases , Cromossomos Artificiais de Levedura/genética , Clonagem Molecular , Cosmídeos/genética , Genes Supressores de Tumor , Homozigoto , Humanos , Dados de Sequência MolecularRESUMO
We recently identified a novel tumor-suppressor gene, DPC4, at chromosome 18q21.1 and found that both alleles of DPC4 were inactivated in nearly one-half of the pancreatic carcinomas. Here, we analyzed 338 tumors, originating from 12 distinct anatomic sites, for alterations in the DPC4 gene. Sixty-four specimens were selected for the presence of the allelic loss of 18q and were further analyzed for DPC4 sequence alterations. An alteration of the DPC4 gene sequence was identified in one of eight breast carcinomas and one of eight ovarian carcinomas. These results indicate that whereas DPC4 inactivation is prevalent in pancreatic carcinoma (48%), it is distinctly uncommon (< 10%) in the other tumor types examined. The tissue restriction of alterations in DPC4, as in many other tumor-suppressor genes, emphasizes the complexity of rate-limiting checkpoints in human tumorigenesis.
Assuntos
Cromossomos Humanos Par 18 , Proteínas de Ligação a DNA , Genes Supressores de Tumor , Neoplasias/genética , Proteínas/genética , Transativadores , DNA de Neoplasias/genética , Deleção de Genes , Heterozigoto , Humanos , Mutação Puntual , Proteína Smad4 , Células Tumorais CultivadasRESUMO
Tumors of the endocrine pancreas are extremely rare, and molecular mechanisms leading to their development are not well understood. A candidate tumor suppressor gene, DPC4, located at 18q21, has recently been shown to be inactivated in half of pancreatic adenocarcinoma xenografts. The close anatomical relationship of the exocrine and endocrine pancreas prompted us to determine the role of DPC4 in the tumorigenesis of 25 pancreatic islet cell tumors (11 insulinomas, nine non-functioning endocrine carcinomas, three gastrinomas, two vipomas). A mutation screening of the highly conserved COOH-terminal domain of DPC4 (exons 8-11) was performed by single-strand conformational variant (SSCP) analysis and a PCR-based deletion assay. Five of nine (55%) non-functioning endocrine pancreatic carcinomas revealed either point mutations, small intragenic deletions or homozygous deletion of DPC4 sequences compared to none of the insulinomas, gastrinomas or vipomas. These results suggest that DPC4 is an important target gene promoting tumorigenesis of non-functioning neuroendocrine pancreatic carcinomas.
Assuntos
Adenoma de Células das Ilhotas Pancreáticas/genética , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/genética , Genes Supressores de Tumor , Mutação , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Transativadores/genética , Análise Mutacional de DNA , Gastrinoma/genética , Humanos , Insulinoma/genética , Perda de Heterozigosidade , Mutação Puntual , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Deleção de Sequência , Proteína Smad4 , Vipoma/genéticaRESUMO
We recently identified DPC4/Smad4 as a candidate tumor suppressor gene mutated or lost in one half of pancreatic carcinomas and in a subset of colon and biliary tract carcinomas. DPC4 plays a key role in signal transduction of the TGF-beta superfamily of molecules and inactivation of TGF-beta mediated growth inhibition is supposed to be the driving force for DPC4 inactivation in human tumors. However, DPC4 mediated tumor suppression by reconstitution of defective cells has not yet been reported. Here we show suppression of tumorigenicity in nude mice by stable reexpression of DPC4 in SW480 colon carcinoma cells. In vitro growth of DPC4-transfected cells was not affected and resistance towards TGF-beta mediated growth inhibition was retained. Instead, cells exhibited morphological alterations and adhesion and spreading were accelerated. These phenotypic changes were associated with reduced expression levels of the endogenous urokinase-type plasminogen activator (uPA) and plasminogen-activator-inhibitor-1 (PAI-1) genes, the products of which are implicated in the control of cell adhesion and invasion. In patients, high expression levels of uPA and PAI-1 correlate with poor prognosis. Thus, reduced expression of uPA and PAI-1 is consistent with suppression of tumorigenicity in DPC4 reconstituted cells. These results demonstrate DPC4's tumor suppressive function and suggest a potential role for DPC4 as a modulator of cell adhesion and invasion.