RESUMO
Many conventional in vitro tests that are currently widely used for routine screening of chemicals have a sensitivity/specificity in the range between 60 % and 80 % for the detection of carcinogens. Most procedures were developed 30-40 years ago. In the last decades several assays became available which are based on the use of metabolically competent cell lines, improvement of the cultivation conditions and development of new endpoints. Validation studies indicate that some of these models may be more reliable for the detection of genotoxicants (i.e. many of them have sensitivity and specificity values between 80 % and 95 %). Therefore, they could replace conventional tests in the future. The bone marrow micronucleus (MN) assay with rodents is at present the most widely used in vivo test. The majority of studies indicate that it detects only 5-6 out of 10 carcinogens while experiments with transgenic rodents and comet assays seem to have a higher predictive value and detect genotoxic carcinogens that are negative in MN experiments. Alternatives to rodent experiments could be MN experiments with hen eggs or their replacement by combinations of new in vitro tests. Examples for promising candidates are ToxTracker, TGx-DDI, multiplex flow cytometry, γH2AX experiments, measurement of p53 activation and MN experiments with metabolically competent human derived liver cells. However, the realization of multicentric collaborative validation studies is mandatory to identify the most reliable tests.
Assuntos
Galinhas , Dano ao DNA , Animais , Carcinógenos , Ensaio Cometa , Feminino , Humanos , Testes para Micronúcleos , Testes de Mutagenicidade , Roedores , Sensibilidade e EspecificidadeRESUMO
The immunosuppressant rapamycin (RAPA) inhibits mammalian target of rapamycin (mTOR) functions and is applied after allogeneic bone marrow transplantation (BMT) to attenuate the development of graft-versus-host disease (GVHD), although the cellular targets of RAPA treatment are not well defined. Allogeneic T cells are the main drivers of GVHD, while immunoregulatory myeloid-derived suppressor cells (MDSCs) were recently identified as potent disease inhibitors. In this study, we analyzed whether RAPA prevents the deleterious effects of allogeneic T cells or supports the immunosuppressive functions of MDSCs in a BMT model with major histocompatibility complex (MHC) classes I and II disparities. RAPA treatment efficiently attenuated clinical and histological GVHD and strongly decreased disease-induced mortality. Although splenocyte numbers increased during RAPA treatment, the ratio of effector T cells to MDSCs was unaltered. However, RAPA treatment induced massive changes in the genomic landscape of MDSCs preferentially up-regulating genes responsible for uptake or signal transduction of lipopeptides and lipoproteins. Most importantly, MDSCs from RAPA-treated mice exhibited increased immunosuppressive potential, which was primarily inducible nitric oxide synthase (iNOS)-dependent. Surprisingly, RAPA treatment had no impact on the genomic landscape of T cells, which was reflected by unchanged expression of activation and exhaustion markers and cytokine profiles in T cells from RAPA-treated and untreated mice. Similarly, T cell cytotoxicity and the graft-versus-tumor effect were maintained as co-transplanted tumor cells were efficiently eradicated, indicating that the immunosuppressant RAPA might be an attractive approach to strengthen the immunosuppressive function of MDSCs without affecting T cell immunity.
Assuntos
Transplante de Medula Óssea , Doença Enxerto-Hospedeiro , Imunidade Celular/efeitos dos fármacos , Células Supressoras Mieloides/imunologia , Neoplasias Experimentais , Sirolimo/farmacologia , Linfócitos T/imunologia , Aloenxertos , Animais , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Doença Enxerto-Hospedeiro/prevenção & controle , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapiaAssuntos
Variações do Número de Cópias de DNA , Leucemia Linfocítica Crônica de Células B/genética , Rituximab/uso terapêutico , Proteína Supressora de Tumor p53/genética , Células Clonais/efeitos dos fármacos , Células Clonais/patologia , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Mutação , Rituximab/farmacologia , Resultado do TratamentoRESUMO
A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer.
Assuntos
Elementos Nucleotídeos Longos e Dispersos , Ribonucleoproteínas/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Animais , Linhagem Celular Tumoral , Cães , Endonucleases/genética , Endonucleases/metabolismo , Células HCT116 , Humanos , Células Madin Darby de Rim Canino , Melanoma Experimental , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Ribonucleoproteínas/metabolismo , Telomerase/biossíntese , Regulação para CimaRESUMO
Brown and white adipocytes have been shown to derive from different progenitors. In this study we sought to clarify the molecular differences between human brown and white adipocyte progenitors cells. To this end, we performed comparative gene array analysis on progenitor cells isolated from paired biopsies of deep and subcutaneous neck adipose tissue from individuals (n = 6) undergoing neck surgery. Compared with subcutaneous neck progenitors, cells from the deep neck adipose tissue displayed marked differences in gene expression pattern, including 355 differentially regulated (>1.5 fold) genes. Analysis of highest regulated genes revealed that STMN2, MME, ODZ2, NRN1 and IL13RA2 genes were specifically expressed in white progenitor cells, whereas expression of LRRC17, CNTNAP3, CD34, RGS7BP and ADH1B marked brown progenitor cells. In conclusion, progenitors from deep neck and subcutaneous neck adipose tissue are characterized by a distinct molecular signature, giving rise to either brown or white adipocytes. The newly identified markers may provide potential pharmacological targets facilitating brown adipogenesis.
Assuntos
Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Adipogenia/fisiologia , Regulação da Expressão Gênica/fisiologia , Células-Tronco/metabolismo , Gordura Subcutânea/metabolismo , Adipócitos Marrons/citologia , Adipócitos Brancos/citologia , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pescoço , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Células-Tronco/citologia , Gordura Subcutânea/citologiaRESUMO
The t(10;11)(p13-14;q14-21) translocation, giving rise to the CALM-AF10 fusion gene, is a recurrent chromosomal rearrangement observed in patients with poor prognosis acute myeloid leukemia (AML). Although splicing of the CALM-AF10 fusion transcripts has been described in AML patients, the contribution of different CALM and AF10 domains to in vivo leukemogenesis remains to be defined. We therefore performed detailed structure-function studies of the CALM-AF10 fusion protein. We demonstrate that fusion of the C-terminal 248 amino acids of CALM, which include the clathrin-binding domain, to the octapeptide motif-leucine-zipper (OM-LZ) domain of AF10 generated a fusion protein (termed CALM-AF10 minimal fusion (MF)), with strikingly enhanced transformation capabilities in colony assays, providing an efficient system for the expeditious assessment of CALM-AF10-mediated transformation. Leukemias induced by the CALM-AF10 (MF) mutant recapitulated multiple aspects of full-length CALM-AF10-induced leukemia, including aberrant Hoxa cluster upregulation, a characteristic molecular lesion of CALM-AF10 leukemias. In summary, this study indicates that collaboration of the clathrin-binding and the OM-LZ domains of CALM-AF10 is sufficient to induce AML. These findings further suggest that future approaches to antagonize CALM-AF10-induced transformation should incorporate strategies, which aim at blocking these key domains.
Assuntos
Clatrina/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Proteínas Monoméricas de Montagem de Clatrina/química , Fatores de Transcrição/químicaRESUMO
Autosomal-recessive hyper-IgE syndrome (AR-HIES) is a combined immunodeficiency recently found to be associated with mutations of DOCK8. Clinically, this disorder is characterized beside recurrent bacterial complications, in particular by an unusual susceptibility to extensive cutaneous viral complications and by a high risk for squamous cell carcinoma. Here, we report on lasting control over the disorder in two patients by hematopoietic cell transplantation (HCT). Both patients were suffering from extensive long-lasting cutaneous viral complications, in particular from disfiguring molluscum contagiosum infections, when treated at the age of 10 and 17 years. Donors were matched unrelated, and conditioning was carried out with a combination of fludarabine, melphalan and BM-targeted radioimmunotherapy. Both patients developed stable, full donor cell chimerism, with the exception of persistent low-IgA serum levels and the exception of normal immune functions. Over the course of several months, cutaneous manifestations of viral disease resolved completely and both patients remain clinically well and free of infectious complications at 4 and 2 years, respectively, after transplantation. This represents the first report indicating HCT to be curative in patients with AR-HIES, which should be considered early before life-threatening complications develop, which include malignancies.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Síndrome de Job/terapia , Adolescente , Criança , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Síndrome de Job/complicações , Molusco Contagioso/etiologia , Molusco Contagioso/terapia , Indução de Remissão , Resultado do TratamentoRESUMO
BACKGROUND: Deregulation of fibroblast growth factor receptor 3 (FGFR3) is involved in several malignancies. Its role in colorectal cancer has not been assessed before. METHODS: Expression of FGFR3 in human colorectal tumour specimens was analysed using splice variant-specific real-time reverse transcriptase PCR assays. To analyse the impact of FGFR3-IIIc expression on tumour cell biology, colon cancer cell models overexpressing wild-type (WT-3b and WT3c) or dominant-negative FGFR3 variants (KD3c and KD3b) were generated by either plasmid transfection or adenoviral transduction. RESULTS: Although FGFR3 mRNA expression is downregulated in colorectal cancer, alterations mainly affected the FGFR3-IIIb splice variant, resulting in an increased IIIc/IIIb ratio predominantly in a subgroup of advanced tumours. Overexpression of WT3c increased proliferation, survival and colony formation in all colon cancer cell models tested, whereas WT3b had little activity. In addition, it conferred sensitivity to autocrine FGF18-mediated growth and migration signals in SW480 cells with low endogenous FGFR3-IIIc expression. Disruption of FGFR3-IIIc-dependent signalling by dominant-negative FGFR3-IIIc or small interfering RNA-mediated FGFR3-IIIc knockdown resulted in inhibition of cell growth and induction of apoptosis, which could not be observed when FGFR3-IIIb was blocked. In addition, KD3c expression blocked colony formation and migration and distinctly attenuated tumour growth in SCID mouse xenograft models. CONCLUSION: Our data show that FGFR3-IIIc exerts oncogenic functions by mediating FGF18 effects in colorectal cancer and may constitute a promising new target for therapeutic interventions.
Assuntos
Movimento Celular , Neoplasias Colorretais/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Apoptose , Células CACO-2 , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
Recent advances in genome-wide single-nucleotide polymorphism (SNP) analyses have revealed previously unrecognized microdeletions and uniparental disomy (UPD) in a broad spectrum of human cancers. As acute myeloid leukemia (AML) represents a genetically heterogeneous disease, this technology might prove helpful, especially for cytogenetically normal AML (CN-AML) cases. Thus, we performed high-resolution SNP analyses in 157 adult cases of CN-AML. Regions of acquired UPDs were identified in 12% of cases and in the most frequently affected chromosomes, 6p, 11p and 13q. Notably, acquired UPD was invariably associated with mutations in nucleophosmin 1 (NPM1) or CCAAT/enhancer binding protein-alpha (CEBPA) that impair hematopoietic differentiation (P=0.008), suggesting that UPDs may preferentially target genes that are essential for proliferation and survival of hematopoietic progenitors. Acquired copy number alterations (CNAs) were detected in 49% of cases with losses found in two or more cases affecting, for example, chromosome bands 3p13-p14.1 and 12p13. Furthermore, we identified two cases with a cryptic t(6;11) as well as several non-recurrent aberrations pointing to leukemia-relevant regions. With regard to clinical outcome, there seemed to be an association between UPD 11p and UPD 13q cases with overall survival. These data show the potential of high-resolution SNP analysis for identifying genomic regions of potential pathogenic and clinical relevance in AML.
Assuntos
Dosagem de Genes , Leucemia Mieloide Aguda/genética , Polimorfismo de Nucleotídeo Único/genética , Dissomia Uniparental/genética , Adolescente , Adulto , Proteínas Estimuladoras de Ligação a CCAAT/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 6/genética , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Nucleofosmina , Adulto JovemRESUMO
BACKGROUND: CD147 (basigin, EMMPRIN) is a multifunctional, highly conserved glycoprotein enriched in pancreatic ductal adenocarcinomas (PDACs) which is associated with poor prognosis in many malignancies. The role of CD147 in pancreatic cancer, however, remains elusive. METHODS AND RESULTS: Silencing of CD147 by RNA interference (RNAi) reduced the proliferation rate of MiaPaCa2 and Panc1 cells. CD147 is required for the function and expression of the monocarboxylate transporters MCT1 and MCT4 that are expressed in human PDAC cells as demonstrated by real-time reverse transcription-PCR (RT-PCR) as well as immunohistology. MCT1 and MCT4 are the natural transporters of lactate, and MiaPaCa2 cells exhibited a high rate of lactate production, which is characteristic for the Warburg effect, an early hallmark of cancer that confers a significant growth advantage. Further induction of lactate production by sodium azide in MiaPaCa2 cells increased MCT1 as well as MCT4 expression. CD147 silencing inhibited the expression and function of MCT1 and MCT4 and resulted in an increased intracellular lactate concentration. Addition of exogenous lactate inhibited cancer cell growth in a dose-dependent fashion. In vivo, knock-down of CD147 in MiaPaCa2 cells by inducible short hairpin RNA (shRNA)-mediated CD147 silencing reduced invasiveness through the chorioallantoic membrane of chick embryos (CAM assay) and inhibited tumourigenicity in a xenograft model in nude mice. CONCLUSION: The function of CD147 as an ancillary protein that is required to sustain the expression and function of MCT1 and MCT4 is involved in the association of CD147 expression with the malignant potential of pancreatic cancer cells exhibiting the Warburg effect.
Assuntos
Basigina/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Simportadores/metabolismo , Animais , Basigina/genética , Western Blotting , Carcinoma Ductal Pancreático/patologia , Embrião de Galinha , Relação Dose-Resposta a Droga , Inativação Gênica , Glucose/metabolismo , Ácido Láctico/farmacologia , Camundongos , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/patologia , RNA Interferente Pequeno/genética , Regulação para CimaRESUMO
To enable detailed analyses of cell interactions in tumour development, new epithelial and mesenchymal cell lines were established from human hepatocellular carcinoma by spontaneous outgrowth in culture. We obtained several hepatocarcinoma (HCC)-, B-lymphoblastoid (BLC)-, and myofibroblastoid (MF)-lines from seven cases. In-depth characterisation included cell kinetics, genotype, tumourigenicity, expression of cell-type specific markers, and proteome patterns. Many functions of the cells of origin were found to be preserved. We studied the impact of the mesenchymal lines on hepatocarcinogenesis by in vitro assays. BLC- and MF-supernatants strongly increased the DNA replication of premalignant hepatocytes. The stimulation by MF-lines was mainly attributed to HGF secretion. In HCC-cells, MF-supernatant had only minor effects on cell growth but enhanced migration. MF-lines also stimulated neoangiogenesis through vEGF release. BLC-supernatant dramatically induced death of HCC-cells, which could be largely abrogated by preincubating the supernatant with TNFbeta-antiserum. Thus, the new cell lines reveal stage-specific stimulatory and inhibitory interactions between mesenchymal and epithelial tumour cells. In conclusion, the new cell lines provide unique tools to analyse essential components of the complex interplay between the microenvironment and the developing liver cancer, and to identify factors affecting proliferation, migration and death of tumour cells, neoangiogenesis, and outgrowth of additional malignancy.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Comunicação Celular , Neoplasias Hepáticas/fisiopatologia , Animais , Linhagem Celular Tumoral , Células Epiteliais , Humanos , Camundongos , RatosRESUMO
Fibroblast growth factor 5 (FGF5) is widely expressed in embryonic but scarcely in adult tissues. Here we report simultaneous overexpression of FGF5 and its predominant high-affinity receptor (FGFR1 IIIc) in astrocytic brain tumour specimens (N=49) and cell cultures (N=49). The levels of both ligand and receptor increased with enhanced malignancy in vivo and in vitro. Furthermore, secreted FGF5 protein was generally present in the supernatants of glioblastoma (GBM) cells. siRNA-mediated FGF5 downmodulation reduced moderately but significantly GBM cell proliferation while recombinant FGF5 (rFGF5) increased this parameter preferentially in cell lines with low endogenous expression levels. Apoptosis induction by prolonged serum starvation was significantly prevented by rFGF5. Moreover, tumour cell migration was distinctly stimulated by rFGF5 but attenuated by FGF5 siRNA. Blockade of FGFR1-mediated signals by pharmacological FGFR inhibitors or a dominant-negative FGFR1 IIIc protein inhibited GBM cell proliferation and/or induced apoptotic cell death. Moreover, rFGF5 and supernatants of highly FGF5-positive GBM cell lines specifically stimulated proliferation, migration and tube formation of human umbilical vein endothelial cells. In summary, we demonstrate for the first time that FGF5 contributes to the malignant progression of human astrocytic brain tumours by both autocrine and paracrine effects.
Assuntos
Comunicação Autócrina/fisiologia , Neoplasias Encefálicas/genética , Fator 5 de Crescimento de Fibroblastos/fisiologia , Glioblastoma/genética , Oncogenes , Comunicação Parácrina/fisiologia , Comunicação Autócrina/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Progressão da Doença , Fator 5 de Crescimento de Fibroblastos/genética , Fator 5 de Crescimento de Fibroblastos/farmacologia , Genes Dominantes/fisiologia , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Neovascularização Patológica/induzido quimicamente , Neovascularização Patológica/genética , Oncogenes/fisiologia , Comunicação Parácrina/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
Vaults are evolutionary highly conserved ribonucleoprotein (RNP) particles with a hollow barrel-like structure. They are 41 x 73 nm in size and are composed of multiple copies of three proteins and small untranslated RNA (vRNA). The main component of vaults represents the 110 kDa major vault protein (MVP), whereas the two minor vault proteins comprise the 193 kDa vault poly(ADP-ribose) polymerase (VPARP) and the 240 kDa telomerase-associated protein-1 (TEP1). Vaults are abundantly present in the cytoplasm of eukaryotic cells and they were found to be associated with cytoskeletal elements as well as occasionally with the nuclear envelope. Vaults and MVP have been associated with several cellular processes which are also involved in cancer development like cell motility and differentiation. Due to the over-expression of MVP (also termed lung resistance-related protein or LRP) in several P-glycoprotein (P-gp)-negative chemoresistant cancer cell lines, vaults have been linked to multidrug resistance (MDR). Accordingly, high levels of MVP were found in tissues chronically exposed to xenobiotics. In addition, the expression of MVP correlated with the degree of malignancy in certain cancer types, suggesting a direct involvement in tumor development and/or progression. Based on the finding that MVP binds several phosphatases and kinases including PTEN, SHP-2 as well as Erk, evidence is accumulating that MVP might be involved in the regulation of important cell signalling pathways including the PI3K/Akt and the MAPK pathways. In this review we summarize the current knowledge concerning the vault particle and discuss its possible cellular functions, focusing on the role of vaults in chemotherapy resistance.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Resistência a Múltiplos Medicamentos/fisiologia , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Ensaios Clínicos como Assunto/estatística & dados numéricos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Humanos , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/genéticaRESUMO
Upregulation of cyclooxygenase-2 (COX-2) and prostaglandin-dependent vascularisation in small adenomatous polyps is an essential part of colon carcinogenesis. To study the underlying cellular mechanisms, LT97 and Caco2 human colorectal tumour cells not expressing endogenous COX-2 were exposed to 1 microM prostaglandin E(2) (PGE(2)) in their medium. At 30 min after addition, expression of c-fos was stimulated 5-fold and 1.3-fold, respectively, depending on the activation of both extracellular signal-regulated kinase and p38. The amount of c-jun in nuclear extracts was increased 20% in LT97 cells. Expression of COX-2 was upregulated 1.7-fold in LT97 cells and 1.5-fold in Caco2 2 h after prostaglandin (PG) addition by a p38-mediated pathway. The known PGE(2) target gene vascular endothelial growth factor (VEGF) was not modulated. Effects of sustained PGE(2) production were studied in VACO235 cells that have high endogenous COX-2 and in LT97 cells infected with an adenovirus expressing COX-2. Prostaglandin E(2) secretion into the medium was 1-2 nM and 250 pM, respectively. Expression of both VEGF and c-fos was high in VACO235 cells. In LT97 cells, COX-2 upregulated c-fos expression and c-jun content in nuclear extracts 1.7- and 1.2-fold, respectively, in a PG-dependent way. This shows that exogenous PGE(2) as well as COX-2 overexpression affect signalling and gene expression in a way that enhances tumour progression.
Assuntos
Adenoma/genética , Neoplasias do Colo/genética , Dinoprostona/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Ciclo-Oxigenase 2/genética , Primers do DNA , Humanos , Reação em Cadeia da Polimerase , Prostaglandinas/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de SinaisRESUMO
The clinical management of pancreatic disease is often hampered by a lack of tissue diagnosis. Endoscopic pancreatography offers the opportunity to investigate exfoliated cells. However, the significance of mere cytological investigation is compromised by an insufficient sensitivity. The evaluation of the molecular background of carcinogenesis hopefully is capable of providing more sensitive diagnostic markers. The p16INK4a-/retinoblastoma tumour-suppressive pathway has been shown to be involved in the development of near to all pancreatic neoplasms. p14ARF is another tumour suppressor located in the immediate neighbourhood of p16INK4a. Promoter methylation has been demonstrated to be a major inactivating mechanism of both genes. We sought to further evaluate the role of the gene locus INK4a methylation status in the endoscopic differentiation of chronic inflammatory and neoplastic pancreatic disease. Pancreatic fluid specimens of 61 patients with either pancreatic carcinoma (PCA: 39), chronic pancreatitis (CP: 16) or a normal pancreatogram (NAD: 6) were retrieved. In order to detect methylation of either the p14ARF or the p16INK4a promoter a methylation-specific PCR protocol was applied. While 19 out of 39 patients with PCA showed p16 promoter methylation (49%), none of the 16 patients with CP revealed p16 promoter methylation. p14ARF methylation was found in a lower percentage of PCA specimens and in none of the samples of patients with CP. These results suggest a specific significance of INK4a for the development of malignant pancreatic disease. Our data further indicate a potential role for INK4a methylation as a diagnostic marker in the endoscopic differentiation of benign and malignant pancreatic disease.
Assuntos
Adenocarcinoma/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA , DNA de Neoplasias/análise , Pâncreas/metabolismo , Neoplasias Pancreáticas/genética , Pancreatite/genética , Proteína Supressora de Tumor p14ARF/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Bile/metabolismo , Estudos de Casos e Controles , Colangiopancreatografia Retrógrada Endoscópica , Doença Crônica , Primers do DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Mutação , Pâncreas/citologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Pancreatite/diagnóstico , Pancreatite/patologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genéticaRESUMO
PURPOSE: To examine the prevalence of DNA aneuploidy as a function of the extent of ulcerative colitis and to study the correlation of aneuploidy with clinical characteristics. Furthermore, the occurrence of aneuploidy and dysplasia during colonoscopic surveillance was studied in a subset of these patients. METHODS: By analyzing 5404 biopsy samples of 368 patients with ulcerative colitis, we have evaluated the importance of DNA ploidy measured by flow cytometry. We have also investigated the influence of extent (219 patients with extensive or total colitis vs. 149 patients with localized colitis) and duration of colitis on the development of dysplasia (patients with biopsy specimens that showed inflammation alone were compared with those with biopsy specimens that were equivocal or positive for dysplasia) and aneuploidy. Included was a subgroup of patients with ulcerative colitis and primary sclerosing cholangitis (n = 16). RESULTS: Aneuploidy was found in 8.7 percent (32/368) of all patients. The prevalence of aneuploidy increased by the extent of ulcerative colitis (2 percent localized, 6.8 percent extensive colitis, 14.9 percent total colitis). The frequency of aneuploidy was higher in patients with disease duration longer than 10 years (P = 0.007). Patients with ulcerative colitis and primary sclerosing cholangitis were more likely to develop aneuploidy (9/16, 56.3 percent vs. 14/120, 11.7 percent; P < 0.001) and dysplasia (4/16, 25 percent vs. 10/120, 8.3 percent; P = 0.06) than patients without primary sclerosing cholangitis. CONCLUSION: Because DNA aneuploidy represents an early alteration during neoplastic transformation in ulcerative colitis, flow cytometry is a valuable tool in the surveillance of those patients. Primary sclerosing cholangitis represents an additional risk factor for the development of DNA aneuploidy and dysplasia.
Assuntos
Colite Ulcerativa/patologia , Lesões Pré-Cancerosas/patologia , Aneuploidia , Biópsia , Ciclo Celular , Colangite Esclerosante/complicações , Estudos de Coortes , Colite Ulcerativa/complicações , Colite Ulcerativa/genética , Colonoscopia , Feminino , Citometria de Fluxo , Humanos , Masculino , Ploidias , Fatores de RiscoRESUMO
Patients with ulcerative colitis (UC) are prone to develop colorectal cancer which is related to the duration and extent of the disease. One of the earliest events in tumor progression is the development of aneuploidy. Aneuploidy is correlated with the grade of dysplasia which serves as a common but not always reproducible marker for the prediction of UC associated formation of cancer. We analyzed 48 biopsy samples from 5 patients with long-standing ulcerative colitis by comparative genomic hybridization (CGH). The majority of these samples represented premalignant stages which are not well characterized at the molecular level as yet. We compared biopsy samples from different colon locations in regard to chromosomal alterations, dysplasia status and DNA index. Besides chromosomal changes occurring only in certain patients in restricted areas of the colon we also detected amplifications and deletions which were common in all persons throughout the colon. The stage of dysplasia seems to have no influence on the number and appearance of chromosomal changes. Amplifications in 2, 3, 6, 9, 11, 12 and 15 were found in almost all cases. In dysplastic samples chromosomal regions 3, 6 and 11 revealed gains of DNA. Deletions were detected within 8q, 15, 18q, 20p and 22q. The affected chromosomal regions may contain yet unknown oncogenes or tumor suppressor genes participating in UC associated carcinogenesis. The conspicuous regions found in the CGH experiments allow the selective and detailed characterization at a molecular level.
Assuntos
Aberrações Cromossômicas/genética , Colite Ulcerativa/genética , Hibridização de Ácido Nucleico/métodos , Mapeamento Cromossômico , Colite Ulcerativa/patologia , DNA/análise , DNA/metabolismo , Progressão da Doença , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Estadiamento de NeoplasiasRESUMO
Overexpression of the major vault protein (MVP) has been linked to a multidrug resistance (MDR) phenotype. We describe a ubiquitously expressed MVP mRNA splice variant (long (L)-MVP) differing from the regular isoform (short (S)-MVP) within the 5'-leader. Only L-MVP mRNA contains a small upstream open reading frame which was proven to inhibit in vitro and in vivo MVP expression in cis. L-MVP represented an almost constant portion of total MVP mRNA in diverse normal tissues, but was more variable in malignant cell types. MDR sublines with altered MVP expression displayed changed S-MVP/L-MVP ratios as compared to their drug-sensitive counterparts. Our results suggest alternative splicing as one mechanism for regulation of MVP expression.
Assuntos
Processamento Alternativo , Fases de Leitura Aberta , RNA Mensageiro , Partículas de Ribonucleoproteínas em Forma de Abóbada/genética , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar , Resistência a Múltiplos Medicamentos/genética , Humanos , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
Cancer-related fibrin deposition and fibrinolysis were investigated by two-dimensional gel electrophoresis of human solid tumor and effusion specimen in addition to plasma samples. Fibrinogen gamma-chain dimer indicating fibrin deposition and plasmin-generated fibrinogen beta-chain fragments were identified in various solid tumor types by amino acid sequencing, mass spectrometry analysis and Western blotting. In tumor-associated effusions, these techniques allowed to observe plasmin-generated fragments of fibrinogen alpha, beta and gamma-chains in addition to elevated levels of acute-phase proteins. Similar observations were made in case of inflammation-associated effusions. No fibrin degradation product was observed in plasma samples, however, high amounts of fibrinogen gamma-chain dimer crosslinked by transglutaminase were detected in plasma from tumor patients, but not in plasma from controls and patients suffering acute infections and/or inflammations. This finding demonstrated that high transglutaminase activity may be associated with cancer. The presented data indicate that the amount of crosslinked fibrinogen gamma-chain dimer in plasma may correlate with tumor-associated fibrin deposition. The tumor-biological relevance of this potential marker protein is discussed.
Assuntos
Biomarcadores Tumorais/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Fibrinogênio/metabolismo , Neoplasias/diagnóstico , Biomarcadores Tumorais/sangue , Estudos de Casos e Controles , Dimerização , Eletroforese em Gel Bidimensional , Fibrina/metabolismo , Fibrinólise , Hemostasia , Humanos , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/metabolismo , Neoplasias/sangue , Neoplasias/metabolismo , Derrame Pleural Maligno/metabolismo , Proteoma/análise , Distribuição TecidualRESUMO
BACKGROUND: We evaluate the usefulness of screening for p53 and Ki-ras mutations in comparison with histological and flow cytometric findings. METHODS: We analyzed 1486 biopsy samples from 769 locations of 83 patients with long-standing ulcerative colitis enrolled in a surveillance program by means of histology, flow cytometry and SSCP analysis. As a control we used 66 biopsy samples of 16 patients with irritable bowel disease. RESULTS: With respect to all biopsy samples analyzed, DNA aneuploidy was found in 32.5% (27/83) of patients, dysplasia in 22.9% (15/83), p53 in 21.7% (18/83) and Ki-ras mutations in 18.1% (15/83) of patients. None of these markers was found in our control group. In 7 out of 10 patients who displayed dysplastic findings during endoscopic surveillance p53 and / or Ki-ras mutations were present in at least one colonoscopy. Statistically significant associations were observed between dysplasia and DNA aneuploidy (P < 0.001), between dysplasia and p53 mutations (P = 0.05) and between dysplasia and p53 and/or Ki-ras mutations (P = 0.002). No significant associations were found between dysplasia and Ki-ras mutations alone. The results for the SSCP analysis showed a much broader variation than those for the flow cytometric analysis. CONCLUSIONS: These results show that screening for p53 and Ki-ras mutations can be a useful adjunct in surveillance of patients with long-standing ulcerative colitis.