RESUMEN
Ease of imaging and abundance of genetic tools make the zebrafish an attractive model host to understand host-pathogen interactions. However, basic knowledge regarding the identity of genes involved in antiviral immune responses is still lagging in this species. We conducted a microarray analysis of the larval zebrafish response to two models of RNA virus infections with very different outcomes. Chikungunya virus (CHIKV) induces a rapid and protective IFN response. Infection with infectious hematopoietic necrosis virus is lethal and is associated with a delayed and inefficient IFN response. A typical signature of IFN-stimulated genes (ISGs) was observed with both viruses, but was stronger for CHIKV. We further compared the zebrafish and human ISG repertoires and made a genomic and phylogenic characterization of the main gene families. We describe a core set of well-induced ISGs conserved across vertebrates, as well as multigenic families diversified independently in each taxon. The conservation of ISGs involved in antiviral signaling indicates conservation of the main feedback loops in these pathways. Whole-mount in situ hybridization of selected transcripts in infected larvae revealed a typical pattern of expression for ISGs in the liver, gut, and blood vessels with both viruses. We further show that some inflammatory genes were additionally induced through IFN-independent pathways by infectious hematopoietic necrosis virus and not by CHIKV. This study provides a useful reference set for the analysis of host-virus interactions in zebrafish and highlights the differences between protective and nonprotective antiviral innate responses.
Asunto(s)
Infecciones por Alphavirus/genética , Inmunidad Innata/genética , Interferones/genética , Infecciones por Rhabdoviridae/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Infecciones por Alphavirus/inmunología , Animales , Fiebre Chikungunya , Regulación de la Expresión Génica , Humanos , Inmunidad Innata/inmunología , Hibridación in Situ , Virus de la Necrosis Hematopoyética Infecciosa/inmunología , Interferones/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones por Rhabdoviridae/inmunología , Pez Cebra/inmunología , Pez Cebra/virología , Proteínas de Pez Cebra/inmunologíaRESUMEN
About 40% of osteosarcoma patients die of metastases. Novel strategies to improve treatment of metastatic patients require a better understanding of the processes involved, like angiogenesis, migration, and the immune response. However, the rarity of osteosarcoma and its heterogeneity make this neoplasm difficult to study. Recently we reported malignant transformation of mouse mesenchymal stem cells (MSCs) which formed osteosarcoma upon transplantation into mice. Here we studied these cells in zebrafish embryos and found that transformed MSCs induced angiogenesis and migrated through the bodies of the embryos, but this was never observed with non-transformed normal MSCs (progenitors of the transformed MSCs). Whole genome expression analysis of both the cells and the host showed that angiogenesis and migration-related genes matrix metalloproteinase 19 (Mmp-19) and erythroblastosis virus E26 oncogene homologue 1 (Ets-1) were overexpressed in transformed MSCs compared to normal MSCs. Investigating the host response, embryos injected with transformed MSCs showed decreased expression of immune response-related genes, especially major histocompatibility complex class 1 (mhc1ze), as compared to embryos injected with normal MSCs. These findings contribute to the identification of genetic events involved in angiogenesis, migration, and host response providing targets as well as an appropriate model for high-throughput drug screens.
Asunto(s)
Neoplasias Óseas/enzimología , Movimiento Celular , Metaloproteinasas de la Matriz Secretadas/metabolismo , Neovascularización Patológica/enzimología , Osteosarcoma/enzimología , Proteína Proto-Oncogénica c-ets-1/metabolismo , Escape del Tumor , Pez Cebra , Animales , Animales Modificados Genéticamente , Neoplasias Óseas/irrigación sanguínea , Neoplasias Óseas/genética , Neoplasias Óseas/inmunología , Neoplasias Óseas/patología , Carbocianinas/metabolismo , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Células Cultivadas , Colorantes Fluorescentes/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Metaloproteinasas de la Matriz Secretadas/genética , Trasplante de Células Madre Mesenquimatosas , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Invasividad Neoplásica , Neovascularización Patológica/genética , Neovascularización Patológica/inmunología , Neovascularización Patológica/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteosarcoma/irrigación sanguínea , Osteosarcoma/genética , Osteosarcoma/inmunología , Osteosarcoma/secundario , Proteína Proto-Oncogénica c-ets-1/genética , Factores de Tiempo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proteína Fluorescente RojaRESUMEN
Fragile X syndrome is the most common cause of inherited mental retardation and the second most common cause of mental impairment after trisomy 21. It occurs because of a failure to express the fragile X mental retardation protein. The most common molecular basis for the disease is the abnormal expansion of the number of CGG repeats in the fragile X mental retardation 1 gene (FMR1). Based on the number of repeats, it is possible to distinguish four types of alleles: normal (5 to 44 repeats), intermediate (45 to 54), premutation (55 to 200), and full mutation (>200). Today, the diagnosis of fragile X syndrome is performed through a combination of PCR to identify fewer than 100 repeats and of Southern blot analysis to identify longer alleles and the methylation status of the FMR1 promoter. We have developed a methylation-specific multiplex ligation-dependent probe amplification assay to analyze male fragile X syndrome cases with long repeat tracts that are not amplifiable by PCR. This inexpensive, rapid and robust technique provides not only a clear distinction between male pre- and full-mutation FMR1 alleles, but also permits the identification of genomic deletions, a less frequent cause of fragile X syndrome.
Asunto(s)
Alelos , Análisis Mutacional de ADN/métodos , Sondas de ADN , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Mutación , Técnicas de Amplificación de Ácido Nucleico , Metilación de ADN , Humanos , Masculino , Repeticiones de Trinucleótidos/genéticaRESUMEN
Gastric cancer is thought to result from a combination of environmental factors and the accumulation of specific genetic alterations due to increasing genetic instability, and consequently affects mainly older patients. Less than 10% of patients present with the disease before 45 years of age (early onset gastric carcinoma) and these patients are believed to develop gastric carcinomas with a molecular genetic profile differing from that of sporadic carcinomas occurring at a later age. In young patients, the role of genetics is presumably greater than in older patients, with less of an impact from environmental carcinogens. As a result, hereditary gastric cancers and early onset gastric cancers can provide vital information about molecular genetic pathways in sporadic cancers and may aid in the unraveling of gastric carcinogenesis. This review focuses on the molecular genetics of gastric cancer and also focuses on early onset gastric cancers as well as familial gastric cancers such as hereditary diffuse gastric cancer. An overview of the various pathways of importance in gastric cancer, as discovered through in-vitro, primary cancer and mouse model studies, is presented and the clinical importance of CDH1 mutations is discussed.
Asunto(s)
Transformación Celular Neoplásica/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Edad de Inicio , Animales , Cadherinas/genética , Cadherinas/metabolismo , Transformación Celular Neoplásica/patología , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Síndromes Neoplásicos Hereditarios/genética , Síndromes Neoplásicos Hereditarios/patologíaRESUMEN
We report the molecular characterization of 8 primary gastric carcinomas, corresponding xenografts, and 2 novel gastric carcinoma cell lines. We compared the tumors and cell lines, with respect to histology, immunohistochemistry, copy number, and hypermethylation of up to 38 genes using methylation-specific multiplex ligation-dependent probe amplification, and TP53 and CDH1 mutation analysis where relevant. The primary tumors and xenografts were histologically comparable and shared expression of 11 of 14 immunohistochemical markers (E-cadherin, beta-catenin, COX-2, p53, p16, TFF1, cyclin E, MLH1, SMAD4, p27, KLK3, CASR, CHFR, and DAPK1). Gains of CASR, DAPK1, and KLK3--not yet described in gastric cancer--were present in the primary tumors, xenografts, and cell lines. The most prominent losses occurred at CDKN2A (p16), CDKN2B (p15), CDKN1B (p27/KIP1), and ATM. Except for ATM, these losses were found only in the cell line or xenograft, suggesting an association with tumor progression. However, examination of p16 and p27 in 174 gastric cancers using tissue microarrays revealed no significant correlation with tumor stage or lymph node status. Further losses and hypermethylation were detected for MLH1, CHFR, RASSF1, and ESR, and were also seen in primary tumors. Loss of CHFR expression correlated significantly with the diffuse phenotype. Interestingly, we found the highest rate of methylation in primary tumors which gave rise to cell lines. In addition, both cell lines harbored mutations in CDH1, encoding E-cadherin. Xenografts and gastric cancer cell lines remain an invaluable research tool in the uncovering of the multistep progression of cancer. The frequent gains, losses, and hypermethylation reported in this study indicate that the involved genes or chromosomal regions may be relevant to gastric carcinogenesis.
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Adenocarcinoma/genética , Biomarcadores de Tumor/análisis , Línea Celular Tumoral , Neoplasias Experimentales/genética , Neoplasias Gástricas/genética , Trasplante Heterólogo , Anciano , Anciano de 80 o más Años , Animales , Islas de CpG , Metilación de ADN , Epigénesis Genética , Humanos , Inmunohistoquímica , Ratones , Persona de Mediana Edad , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Matrices TisularesRESUMEN
Recent studies claim a critical role for RUNX3 in gastric epithelial homeostasis. However, conflicting results exist regarding RUNX3 expression in the stomach and its potential role as a tumour-suppressor gene (TSG) in gastric carcinogenesis. Our aim was to evaluate the role of RUNX3 in early-onset gastric carcinomas (EOGCs). We analysed 41 EOGCs for RUNX3 aberrations using loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) analyses. LOH of markers flanking RUNX3 was relatively common, indicating that loss of the gene may play a role in gastric carcinogenesis. However, FISH analysis of selected cases and a panel of 14 gastric carcinoma-derived cell lines showed widespread presence of multiple copies of centromere 1. While RUNX3 copy numbers were generally equal to or fewer than those of centromere 1, at least two copies were present in almost all cells analysed. Accordingly, a subpopulation of tumour cells in 12/37 cases showed RUNX3 protein expression. However, expression was not detected in the adjacent nontumorous mucosa of any case. Together, these observations indicate that chromosome 1 aberrations occur frequently in EOGCs and are reflected in the LOH and IHC patterns found. Our findings refute a role for RUNX3 as a TSG in EOGCs.
Asunto(s)
Carcinoma/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/fisiología , Genes Supresores de Tumor , Neoplasias Gástricas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma/patología , Línea Celular Tumoral , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Humanos , Hibridación Fluorescente in Situ , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Neoplasias Gástricas/patología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/fisiologíaRESUMEN
BACKGROUND: Colorectal cancers arising in patients with familial adenomatous polyposis (FAP) can be largely prevented by polyp surveillance and prophylactic colectomy. As a result, duodenal adenocarcinoma has become a leading cause of death in patients with FAP. Cyclooxygenase 2 (COX-2) inhibition is effective against colorectal polyposis in FAP, but is less effective in treating duodenal polyps. We compared the expression of COX-2 in duodenal and colorectal adenomas from patients with FAP and from patients with sporadic neoplasms and correlated expression to a COX-2 promoter polymorphism (-765G/-->C) that is reported to influence COX-2 expression. METHODS: The study population included 36 FAP patients with colonic adenomas, 22 FAP patients with duodenal adenomas, 22 patients with sporadic duodenal adenomas, and 17 patients with sporadic duodenal adenocarcinoma. Neoplastic and corresponding normal tissue COX-2 expressions were determined using immunohistochemistry on tissue microarrays. The prevalence and ethnic distribution of a polymorphism in the COX-2 promoter that influences COX-2 expression (-765G --> C) were determined in DNA from 274 individuals by real-time quantitative PCR. RESULTS: Among patients with FAP, histologically normal duodenal mucosa showed higher COX-2 expression than normal colonic mucosa (P < 0.02), and duodenal adenomas had higher COX-2 expression than colonic adenomas (P = 0.01). In addition, the normal duodenum of patients with FAP showed higher COX-2 expression than the normal duodenal mucosa of patients with sporadic adenomas (P < 0.05). COX-2 expression was significantly higher in the normal-appearing (P < 0.01) mucosa of patients with FAP carrying the -765GG genotype compared with those carrying the -765GC or -765CC genotypes. The -765C genotype was more common in African Americans than in Caucasians (52% versus 33%, P < 0.01). CONCLUSIONS: High COX-2 expression in the normal and adenomatous duodenal mucosa of patients with FAP may explain the poorer response of these neoplasms to chemoprevention with COX-2 inhibitors.
Asunto(s)
Adenoma/patología , Poliposis Adenomatosa del Colon/patología , Neoplasias del Colon/patología , Neoplasias Duodenales/patología , Polimorfismo de Nucleótido Simple , Prostaglandina-Endoperóxido Sintasas/genética , Adenoma/enzimología , Adenoma/genética , Poliposis Adenomatosa del Colon/enzimología , Poliposis Adenomatosa del Colon/genética , Adulto , Anciano , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Ciclooxigenasa 2 , Análisis Mutacional de ADN , Neoplasias Duodenales/enzimología , Neoplasias Duodenales/genética , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genotipo , Humanos , Inmunohistoquímica , Masculino , Proteínas de la Membrana , Persona de Mediana Edad , Regiones Promotoras Genéticas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismoRESUMEN
BACKGROUND: Both Helicobacter pylori genotype and host genetic polymorphisms play a role in determining the clinical consequences of H. pylori infection. We investigated whether there are any combinations of bacterial and host genotypes that are particularly associated with the occurrence of gastric carcinoma. METHODS: Genotypic variations in virulence-associated genes of H. pylori vacA (s and m regions) and cagA were determined in 221 subjects with chronic gastritis and 222 patients with gastric carcinoma by polymerase chain reaction (PCR)-line probe assay. Polymorphisms in the human interleukin 1 beta (IL-1B) gene (IL-1B-511*C or IL-1B-511*T) and in the IL-1 receptor antagonist gene (IL-1RN intron 2 variable number of tandem repeats) were evaluated by PCR and single-strand conformation polymorphism analysis. All statistical tests were two-sided. RESULTS: Infection with vacAs1-, vacAm1-, and cagA-positive strains of H. pylori was associated with an increased risk for gastric carcinoma, with odds ratios (ORs) of 17 (95% confidence interval [CI] = 7.8 to 38), 6.7 (95% CI = 3.6 to 12), and 15 (95% CI = 7.4 to 29), respectively. IL-1B-511*T carriers (IL-1B-511*T/*T or IL-1B-511*T/*C) homozygous for the short allele of IL-1RN (IL-1RN*2/*2) had an increased gastric carcinoma risk (OR = 3.3, 95% CI = 1.3 to 8.2). For each combination of bacterial/host genotype, the odds of having gastric carcinoma were greatest in those with both bacterial and host high-risk genotypes: vacAs1/IL-1B-511*T carrier (OR = 87, 95% CI = 11 to 679), vacAm1/IL-1B-511*T carrier (OR = 7.4, 95% CI = 3.2 to 17), cagA-positive/IL-1B-511*T carrier (OR = 25, 95% CI = 8.2 to 77), vacAs1/IL-1RN*2/*2 (OR = 32, 95% CI = 7.8 to 134), vacAm1/IL-1RN*2/*2 (OR = 8.8, 95% CI = 2.2 to 35), and cagA-positive/IL-1RN*2/*2 (OR = 23, 95% CI = 7.0 to 72). CONCLUSION: Combined bacterial/host genotyping may provide an important tool in defining disease risk and targeting H. pylori eradication to high-risk individuals.
Asunto(s)
Antígenos Bacterianos , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Heterocigoto , Homocigoto , Interleucina-1/genética , Polimorfismo Genético , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiología , Adulto , Proteínas Bacterianas/genética , Carcinoma/genética , Carcinoma/microbiología , Enfermedad Crónica , Intervalos de Confianza , Femenino , Gastritis/genética , Gastritis/microbiología , Predisposición Genética a la Enfermedad , Infecciones por Helicobacter/genética , Humanos , Intestinos/microbiología , Intestinos/patología , Masculino , Metaplasia/microbiología , Persona de Mediana Edad , Repeticiones de Minisatélite , Oportunidad Relativa , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Riesgo , Neoplasias Gástricas/patologíaRESUMEN
Collision tumors are thought to arise from the accidental meeting of two independent tumors. Here we present five gastroesophageal junction tumors consisting of two collision tumors and three composite tumors (characterized by two divergent lineages originating from the same neoplastic clonal proliferation), as diagnosed on histology. In an attempt to prove this distinction at a genetic level, we performed TP53 sequence analysis and p53 immunohistochemistry. In addition, loss of heterozygosity (LOH) analysis using 10 microsatellite markers was carried out. An identical TP53 mutation and a similar pattern of retention and LOH were found in both neoplastic components of the presumed collision tumors, suggesting that both components are derived from a single precursor cell that undergoes divergent differentiation in the evolution of the tumor. In the composite group, 1 case had a genetic basis for the possible diagnosis of a collision tumor, with a TP53 mutation in the adenocarcinoma component only, and a different pattern of retention and loss of heterozygosity. These findings imply that it is not possible to recognize true collision tumors from immunohistologic appearance alone and suggest that the long-standing histologic criteria for the diagnosis of these neoplasms have no molecular basis.
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Adenocarcinoma/genética , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Unión Esofagogástrica , Neoplasias Gástricas/genética , Adenocarcinoma/patología , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/patología , Genes p53/genética , Humanos , Pérdida de Heterocigocidad/genética , Repeticiones de Microsatélite/genética , Mutación/genética , Análisis de Secuencia de ADN/métodos , Neoplasias Gástricas/patologíaRESUMEN
One-third of the world population is infected with Mycobacterium tuberculosis and multi-drug resistant strains are rapidly evolving. The noticeable absence of a whole organism high-throughput screening system for studying the progression of tuberculosis is fast becoming the bottleneck in tuberculosis research. We successfully developed such a system using the zebrafish Mycobacterium marinum infection model, which is a well-characterized model for tuberculosis progression with biomedical significance, mimicking hallmarks of human tuberculosis pathology. Importantly, we demonstrate the suitability of our system to directly study M. tuberculosis, showing for the first time that the human pathogen can propagate in this vertebrate model, resulting in similar early disease symptoms to those observed upon M. marinum infection. Our system is capable of screening for disease progression via robotic yolk injection of early embryos and visual flow screening of late-stage larvae. We also show that this system can reliably recapitulate the standard caudal vein injection method with a throughput level of 2,000 embryos per hour. We additionally demonstrate the possibility of studying signal transduction leading to disease progression using reverse genetics at high-throughput levels. Importantly, we use reference compounds to validate our system in the testing of molecules that prevent tuberculosis progression, making it highly suited for investigating novel anti-tuberculosis compounds in vivo.
Asunto(s)
Biomarcadores/análisis , Ensayos Analíticos de Alto Rendimiento/métodos , Tuberculosis/diagnóstico , Animales , Antituberculosos/aislamiento & purificación , Antituberculosos/uso terapéutico , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Embrión no Mamífero , Humanos , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/patología , Mycobacterium marinum/fisiología , Pronóstico , Tuberculosis/tratamiento farmacológico , Tuberculosis/patología , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo , Pez Cebra/fisiologíaRESUMEN
We report on a fragile X mosaic male full mutation/normal allele detected by PCR and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). This combined analysis provides a diagnostic approach for fragile X syndrome (FXS). The method assesses the presence of expansion (full mutation), the CpG methylation status and could determine copy number changes (large deletions/duplications) along the FMR1 and FMR2 (fragile X mental retardation) genes. The method avoids detection of premutations, which makes it applicable for newborn screening. It can also be used in clarification of mosaic cases. The PCR results in our patient showed one normal allele; three repeats larger than his mother's one. The MS-MLPA showed hypermethylated full mutation pattern in the proband. Both results are compatible with FXS mosaic case full mutation/normal allele. The patient demonstrates atypical mild clinical manifestation of the disease, which correlates to the presence of a normal size allele in the patient's cells.
RESUMEN
Mutations in the breast cancer susceptibility gene 2 (BRCA2) are commonly found in familial pancreatic cancer. Recently, EMSY (11q13.5) has been described as a BRCA2 interacting protein capable of binding and inactivating the protein domain encoded by exon 3 of the BRCA2 gene. Amplification of EMSY occurs in 13% of sporadic breast cancers and is directly linked to increased expression. Here we investigate the amplification status of this new potential oncogene in 59 sporadic pancreatic cancers using fluorescence in situ hybridization (FISH) and tissue microarray (TMA). Real-time quantitative RT-PCR was performed on 20 pancreatic cancer cell lines and overexpression was calculated using the delta-delta-Ct-method. Amplification of EMSY was found in 8/59 cases (13.6%). 9/20 (45%) cell line samples showed overexpression of EMSY. In conclusion, sporadic pancreatic cancer shows amplification of EMSY at prevalence similar to that found in other cancers.
RESUMEN
Diffuse-type gastric carcinomas (GCs) are often difficult to characterize because of contamination of tumor samples by surrounding normal tissue. As such, information regarding chromosomal aberrations in this subtype of GCs is limited. In this study, we used representational difference analysis to pinpoint genomic amplifications occurring in diffuse-type GCs. We found nine differential products from two novel regions of amplification in two tumors: one product mapped to 19p13.1 and eight mapped to a 1.8-Mb region in chromosomal segment 11p12-13. These amplifications were confirmed using Southern blot analysis and occurred in 3/16 and 6/15 diffuse-type GCs, respectively. CD44, a well characterized cellular adhesion molecule involved in several human malignancies, is encoded by a gene located within 200 kb of the 11p12-13 amplification fragments. We confirmed that overexpression of isoform CD44v6 was correlated with amplification at 11p12-13 in 11/12 diffuse-type GCs. Since diffuse-type GCs occur more frequently in early-onset gastric carcinomas (EOGCs, presented at 45 years of age or younger) than in "conventional" GCs, and the tumors carrying the original amplifications were EOGCs, we investigated overexpression of CD44v6 in 107 EOGCs and 88 conventional GCs using tissue microarrays. We found frequent CD44v6 overexpression in both tumor groups (76% and 57% respectively) and, interestingly, significantly more cases with overexpression of CD44v6 in EOGCs than in conventional GCs (P = 0.005), irrespective of histology. These findings provide further evidence for both the relevance of CD44 in GC and for distinct molecular characteristics of EOGCs when compared with those of GCs occurring at a later age.
Asunto(s)
Adenocarcinoma/genética , Cromosomas Humanos Par 11/genética , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica/fisiología , Glicoproteínas/genética , Receptores de Hialuranos/genética , Neoplasias Gástricas/genética , Adenocarcinoma/metabolismo , Edad de Inicio , Humanos , Persona de Mediana Edad , Neoplasias Gástricas/metabolismoRESUMEN
Many studies examine the molecular genetics of gastric cancer, but few look at young patients in particular and there is no comparison of molecular expression between early-onset gastric cancer (< or = 45 years old) and conventional gastric cancers. Expression of cycloxygenase-2 (COX-2) is elevated in gastric adenocarcinomas compared to non-neoplastic mucosa, and in light of studies showing reduced risk of gastric cancer in nonsteroidal anti-inflammatory drug users, we have chosen to investigate the expression of COX-2 and related molecules in 113 early-onset gastric cancers and compare it with 91 conventional gastric cancers, using tissue microarrays. These markers include molecules known to be important in conventional gastric carcinogenesis, such as E-Cadherin, p53, COX-2, Trefoil Factor-1 (TFF1), beta-catenin, p16 and c-myc; as well as molecules not yet described as being important in gastric cancer, such as the transcription factor c-jun, the COX-2 mRNA stabilizer HuR, and C/EBP-beta, a transcription factor for COX-2. All markers showed a statistically significant difference between early-onset gastric cancers and conventional gastric cancers, using a chi2 test. In particular, early-onset gastric cancers displayed a COX-2 Low, TFF1-expressing phenotype, whereas COX-2 overexpression and loss of TFF1 was found in conventional cancers, and this difference between early-onset gastric cancers and conventional cancers remained statistically significant when adjusted for location and histology (P<0.0001 and P = 0.002 respectively). We found that COX-2 overexpression correlates significantly with loss of TFF1 (P = 0.001), overexpression of C/EBP-beta (P<0.001) and cytoplasmic HuR (P = 0.016). COX-2 was significantly associated with p53 positivity (P = 0.003). Abnormalities in E-Cadherin correlated significantly with diffuse phenotype, whereas high expression of COX-2, loss of TFF1 and overexpression of C/EBP-beta correlated with the intestinal phenotype. Our results provide further evidence that early-onset gastric cancer exhibits a distinctive expression profile that may have practical implications.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Gástricas/patología , Adulto , Edad de Inicio , Antígenos de Superficie/análisis , Proteína beta Potenciadora de Unión a CCAAT/análisis , Cadherinas/análisis , Distribución de Chi-Cuadrado , Ciclooxigenasa 2/análisis , Proteínas ELAV , Proteína 1 Similar a ELAV , Humanos , Inmunohistoquímica , Proteínas de la Membrana/análisis , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-myb/análisis , Proteínas de Unión al ARN/análisis , Neoplasias Gástricas/epidemiología , Neoplasias Gástricas/metabolismo , Factor Trefoil-1 , Proteína p53 Supresora de Tumor/análisis , Proteínas Supresoras de Tumor/análisis , beta Catenina/análisisRESUMEN
The non-neoplastic pancreatic parenchyma adjacent to infiltrating ductal adenocarcinoma demonstrates inflammation, fibrosis, acinar cell loss and small duct-like metaplasia of acinar cells. Similar morphologic changes are also observed in the setting of chronic pancreatitis. In addition, peritumoral acini have been shown to have alterations in gene expression even in the absence of morphological changes. To better understand the pancreatic acinar responses to infiltrating pancreatic ductal adenocarcinoma, we characterized gene expression patterns of pancreatic acinar tissue adjacent to infiltrating pancreatic ductal adenocarcinomas and compared them to gene expression patterns of acinar tissue affected by chronic pancreatitis as well as to those of normal pancreatic acini. Fresh-frozen pancreatic acinar tissue was microdissected from nine patients (three with pancreatic cancer, three with chronic pancreatitis, three with normal pancreata) using laser capture microdissection, and extracted RNA from each microdissection was subjected to two rounds of linear amplification and hybridized to oligonucleotide microarrays. Gene expression patterns were confirmed using quantitative RT-PCR and/or immunohistochemistry. A total of 20 genes was found to be overexpressed in peritumoral acinar tissue compared to normal acinar tissue and to acini affected by chronic pancreatitis. These 20 genes included pancreatitis-associated protein (HIP/PAP), a gene known to be overexpressed in acini adjacent to infiltrating pancreatic cancer, and the gene cartilage glycoprotein-39 (HC gp-39 or TKL-40). Serum HC gp-39 protein levels were significantly higher in patients with pancreatic cancer and in those with chronic pancreatitis than in controls without pancreatic disease. There was no significant difference in the levels of serum HC gp-39 in patients with pancreatic cancer and those with chronic pancreatitis. Our results demonstrate some of the molecular alterations in acinar cells that occur in response to adjacent infiltrating pancreatic ductal adenocarcinoma and reveal that such alterations can provide a rich source of markers of pancreatic cancer.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Perfilación de la Expresión Génica , Páncreas/patología , Neoplasias Pancreáticas/patología , Adipoquinas , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proteína 1 Similar a Quitinasa-3 , Glicoproteínas/sangre , Glicoproteínas/genética , Humanos , Inmunohistoquímica , Rayos Láser , Lectinas , Microdisección/métodos , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Páncreas/química , Páncreas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Asociadas a Pancreatitis , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
It has been advanced that the trefoil factor (TFF) 1 gene is a candidate tumor-suppressor gene and may be involved in the development and/or progression of human gastric cancer. We aimed to clarify the putative role of TFF1 in gastric carcinogenesis. Ninety gastric carcinomas and eight gastric carcinoma-derived cell lines were screened for TFF1 mutations; subsets of the primary tumors and of the cell lines were subjected to loss of heterozygosity (LOH), immunohistochemistry, and promoter methylation analyses. TFF1 mutations were not detected in any of 90 gastric carcinomas. Eight (28%) of 28 informative cases displayed LOH at the TFF1 locus and absence of TFF1 staining by immunohistochemistry. These results indicate a frequent loss of TFF1 expression in gastric carcinomas through a mutation-independent mechanism. Extensive TFF1 promoter methylation was observed in nonexpressing gastric carcinoma-derived cell lines and tissues. Expressing cell lines, as well as normal gastric mucosa, presented little or no methylation of the promoter. Gastric carcinoma DNA presented de novo methylation of the promoter. These results point to the involvement of promoter methylation in the shutting down of TFF1. We conclude that TFF1 point mutations seem to be a rare event in gastric carcinogenesis. The loss of expression of TFF1 in a proportion of gastric carcinomas may be explained by LOH and methylation of the TFF1 promoter region. Our results further support the role of TFF1 inactivation in gastric carcinogenesis, in agreement with the results obtained in the Tff1-knockout mice model.
Asunto(s)
Metilación de ADN , Sustancias de Crecimiento/genética , Pérdida de Heterocigocidad , Mutación , Péptidos/genética , Regiones Promotoras Genéticas , Proteínas , Neoplasias Gástricas/genética , Eliminación de Gen , Humanos , Reacción en Cadena de la Polimerasa/métodos , Neoplasias Gástricas/patología , Neoplasias Gástricas/cirugía , Factor Trefoil-1 , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
Gastric cancer is thought to result from a combination of environmental factors and accumulation of specific genetic alterations, and consequently mainly affects older patients (>50 years of age). Fewer than 10% of patients present with the disease before 45 years of age and these young patients are thought to develop carcinomas with a different molecular genetic profile from that of sporadic carcinomas occurring at a later age. Forty early-onset gastric carcinoma resection specimens were characterized for microsatellite instability (MSI) and loss of heterozygosity status using 22 polymorphic microsatellite markers. Twenty-four biopsies were additionally evaluated for the presence of MSI. No MSI was observed in any of the cases analysed. Losses were infrequent, but were most common for the D1S234 (26.1%) and D1S1676 (17.4%) markers, flanking the RUNX3 gene; for the p53ALU (23.1%) and TP53 (15.4%) markers, near the TP53 gene; and for the D16S2624 (17.2%) marker, near the E-cadherin (CDH1) gene. All cases with loss of CDH1, as well as 6/7 cases with loss of TP53, displayed aberrant staining of the corresponding proteins, pointing to a functional role for these proteins in early-onset gastric carcinogenesis. No germline CDH1, TP53 or RUNX3 mutations were detected in any of the cases analysed. No correlation was observed between non-functional E-cadherin and the histological type of the tumours analysed. Finally, Epstein-Barr virus was not detected in any of the cases analysed. On the basis of these results, early-onset gastric carcinomas appear to have characteristics distinct from gastric carcinomas occurring at a later age.
Asunto(s)
Adenocarcinoma/genética , Pérdida de Heterocigocidad , Neoplasias Gástricas/genética , Adenocarcinoma/metabolismo , Adolescente , Adulto , Edad de Inicio , Cadherinas/metabolismo , ADN de Neoplasias/genética , Femenino , Humanos , Masculino , Repeticiones de Microsatélite , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Neoplasias Gástricas/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND & AIMS: Pro-inflammatory polymorphisms within the genes interleukin (IL)-1B and IL-1RN are associated with risk for gastric carcinoma (GC) in Helicobacter pylori-infected individuals. We aimed to determine the association between variation of the tumor necrosis factor (TNF)-alpha gene and the risk for chronic atrophic gastritis (CAG) and GC. We also investigated the extent to which the combined effect of proinflammatory genetic polymorphisms (IL-1B, IL-1RN, and TNF-alpha), and the combined effect of TNF-alpha and bacterial genotypes each influence such a risk. METHODS: In a case-control study including 306 controls, 221 individuals with chronic gastritis, and 287 GC patients, the TNF-alpha-308 and IL-1B-511 bi-allelic polymorphisms, the IL-1RN variable number of tandem repeats (VNTR), and the H. pylori genes vacA (s and m regions) and cagA were genotyped. RESULTS: We found that carriers of the TNF-alpha-308*A allele are at increased risk for GC development with an odds ratio (OR) of 1.9 (95% confidence interval [CI], 1.3-2.7). For both CAG and GC, the odds of developing disease increased with the number of high-risk genotypes. Individuals carrying high-risk genotypes at the 3 loci are at increased risk for CAG and GC with an OR of 5.8 (95% CI, 1.1-31.0) and 9.7 (95% CI, 2.6-36.0), respectively. The risk for GC was not affected significantly by the combination of bacterial and TNF-alpha-308 genotypes. CONCLUSIONS: These findings show that a proinflammatory polymorphism in the TNF-alpha gene is associated with increased risk for GC, and that it is possible to define a specific genetic profile associated with highest risk for CAG and GC.