RESUMO
Although microRNAs (miRs) have been implicated in the pathogenesis of various human malignancies, limited information is available regarding mechanisms by which these noncoding RNAs contribute to initiation and progression of tobacco-induced esophageal cancers. In this study, array and quantitative reverse transcriptase-PCR techniques were used to examine miR expression in immortalized esophageal epithelia (IEE) and esophageal adenocarcinoma (EAC) cells cultured in normal media with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly decreased miR-217 expression in these cells. Endogenous levels of miR-217 expression in cultured EAC cells (EACC)/primary EACs were significantly lower than those observed in IEE/ paired normal esophageal tissues. RNA crosslink immunoprecipitation, quantitative reverse transcriptase-PCR (qRT-PCR) and immunoblot experiments demonstrated direct interaction of miR-217 with kallikrein 7 (KLK7), encoding a putative oncogene not previously implicated in EAC. Repression of miR-217 correlated with increased levels of KLK7 in primary EACs, particularly those from smokers. Chromatin and methylated DNA immunoprecipitation experiments demonstrated that CSC-mediated repression of miR-217 coincided with DNMT3b-dependent hypermethylation and decreased occupancy of nuclear factor 1 within the miR-217 genomic locus. Deoxyazacytidine induced miR-217 expression and downregulated KLK7 in EACC; deoxyazacytidine also attenuated CSC-mediated miR-217 repression and upregulation of KLK7 in IEE and EACC. Overexpression of miR-217 significantly decreased, whereas overexpression of KLK7 increased proliferation, invasion and tumorigenicity of EACC. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of EAC via upregulation of KLK7 and suggest that restoration of miR-217 expression may be a novel treatment strategy for these malignancies.
Assuntos
Adenocarcinoma/genética , Carcinogênese/genética , Repressão Epigenética/genética , Neoplasias Esofágicas/genética , MicroRNAs/genética , Nicotiana/efeitos adversos , Fumar/genética , Adenocarcinoma/patologia , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Cromatina/genética , Metilação de DNA/genética , Regulação para Baixo/genética , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Calicreínas/genética , Fatores de Transcrição NFI/genética , Invasividade Neoplásica/genética , Fumaça/efeitos adversos , Regulação para Cima/genéticaRESUMO
A prominent feature of most cancers including Barrett's adenocarcinoma (BAC) is genetic instability, which is associated with development and progression of disease. In this study, we investigated the role of recombinase (hsRAD51), a key component of homologous recombination (HR)/repair, in evolving genomic changes and growth of BAC cells. We show that the expression of RAD51 is elevated in BAC cell lines and tissue specimens, relative to normal cells. HR activity is also elevated and significantly correlates with RAD51 expression in BAC cells. The suppression of RAD51 expression, by short hairpin RNA (shRNA) specifically targeting this gene, significantly prevented BAC cells from acquiring genomic changes to either copy number or heterozygosity (P<0.02) in several independent experiments employing single-nucleotide polymorphism arrays. The reduction in copy-number changes, following shRNA treatment, was confirmed by Comparative Genome Hybridization analyses of the same DNA samples. Moreover, the chromosomal distributions of mutations correlated strongly with frequencies and locations of Alu interspersed repetitive elements on individual chromosomes. We conclude that the hsRAD51 protein level is systematically elevated in BAC, contributes significantly to genomic evolution during serial propagation of these cells and correlates with disease progression. Alu sequences may serve as substrates for elevated HR during cell proliferation in vitro, as they have been reported to do during the evolution of species, and thus may provide additional targets for prevention or treatment of this disease.
Assuntos
Adenocarcinoma/genética , Elementos Alu , Esôfago de Barrett/genética , Neoplasias Esofágicas/genética , Genoma Humano , Rad51 Recombinase/fisiologia , Recombinação Genética , Linhagem Celular Tumoral , Humanos , Perda de Heterozigosidade , MutaçãoRESUMO
Esophageal adenocarcinoma (EA) is characterized by a poor prognosis making the identification of clinically targetable proteins essential for improving patient outcome. We report the involvement of multiple alterations of the MET pathway in EA development and progression. Microarray analysis of Barrett's metaplasia, dysplasia, and EA revealed overexpression of the MET oncogene in EAs but only those with MET gene amplification. STS-amplification mapping revealed that the boundary of the MET amplicon in these EAs is defined by fragile site FRA7G. We also identified an amplicon at 11p13 that resulted in amplification and overexpression of CD44, a gene involved in MET autophosphorylation upon HGF stimulation. Tissue microarrays with phospho-MET-specific antibodies demonstrated a uniformly high abundance of MET activation in primary EA and cells metastatic to lymph nodes but to a lesser extent in a subset of metaplastic and dysplastic Barrett's samples. Increased expression of multiple genes in the MET pathway associated with invasive growth, for example, many MMPs and osteopontin, also was found in EAs. Treatment of EA-derived cell lines with geldanamycin, an inhibitor for tyrosine kinases including MET receptor kinase, reduced cell migration and induced EA cell apoptosis. The data indicate that upregulation of the MET pathway may contribute to the poor outcome of EA patients and that therapeutic agents targeting this pathway may help improve patient survival.
Assuntos
Adenocarcinoma/genética , Sítios Frágeis do Cromossomo , Neoplasias Esofágicas/genética , Amplificação de Genes , Proteínas Proto-Oncogênicas/genética , Receptores de Fatores de Crescimento/genética , Regulação para Cima , Western Blotting , Humanos , Imunoprecipitação , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-met , RNA Mensageiro/genéticaAssuntos
Biomarcadores Tumorais/sangue , Neoplasias/sangue , Fosfoglicerato Quinase/sangue , Anticorpos Monoclonais/metabolismo , Estudos de Coortes , Humanos , Neoplasias/enzimologia , Neoplasias/metabolismo , Neovascularização Patológica , Análise de Sequência com Séries de Oligonucleotídeos , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismoRESUMO
Molecular classification of tumors based on their gene expression profiles promises to significantly refine diagnosis and management of cancer patients. The establishment of organ-specific gene expression patterns represents a crucial first step in the clinical application of the molecular approach. Here, we report on the gene expression profiles of 154 primary adenocarcinomas of the lung, colon, and ovary. Using high-density oligonucleotide arrays with 7129 gene probe sets, comprehensive gene expression profiles of 57 lung, 51 colon, and 46 ovary adenocarcinomas were generated and subjected to principle component analysis and to a cross-validated prediction analysis using nearest neighbor classification. These statistical analyses resulted in the classification of 152 of 154 of the adenocarcinomas in an organ-specific manner and identified genes expressed in a putative tissue-specific manner for each tumor type. Furthermore, two tumors were identified, one in the colon group and another in the ovarian group, that did not conform to their respective organ-specific cohorts. Investigation of these outlier tumors by immunohistochemical profiling revealed the ovarian tumor was consistent with a metastatic adenocarcinoma of colonic origin and the colonic tumor was a pleomorphic mesenchymal tumor, probably a leiomyosarcoma, rather than an epithelial tumor. Our results demonstrate the ability of gene expression profiles to classify tumors and suggest that determination of organ-specific gene expression profiles will play a significant role in a wide variety of clinical settings, including molecular diagnosis and classification.
Assuntos
Adenocarcinoma/genética , Neoplasias do Colo/genética , Perfilação da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Ovarianas/genética , Adenocarcinoma/classificação , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/classificação , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Diagnóstico Diferencial , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Ovarianas/classificação , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologiaRESUMO
The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis as well as leads for therapy. The purpose of this study was to identify proteins that commonly induce a humoral response in lung cancer by using a proteomic approach and to investigate biological processes that may be associated with the development of autoantibodies. Aliquots of solubilized proteins from a lung adenocarcinoma cell line (A549) and from lung tumors were subjected to two-dimensional PAGE, followed by Western blot analysis in which individual sera were tested for primary antibodies. Sera from 54 newly diagnosed patients with lung cancer and 60 patients with other cancers and from 61 noncancer controls were analyzed. Sera from 60% of patients with lung adenocarcinoma and 33% of patients with squamous cell lung carcinoma but none of the noncancer controls exhibited IgG-based reactivity against proteins identified as glycosylated annexins I and/or II. Immunohistochemical analysis showed that annexin I was expressed diffusely in neoplastic cells in lung tumor tissues, whereas annexin II was predominant at the cell surface. Interestingly, IL-6 levels were significantly higher in sera of antibody-positive lung cancer patients compared with antibody-negative patients and controls. We conclude that an immune response manifested by annexins I and II autoantibodies occurs commonly in lung cancer and is associated with high circulating levels of an inflammatory cytokine. The proteomic approach we have implemented has utility for the development of serum-based assays for cancer diagnosis as we report in this paper on the discovery of antiannexins I and/or II in sera from patients with lung cancer.
Assuntos
Anexina A1/imunologia , Anexina A2/imunologia , Anticorpos Antineoplásicos/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Interleucina-6/sangue , Neoplasias Pulmonares/imunologia , Proteínas de Neoplasias/imunologia , Sequência de Aminoácidos , Anexina A1/química , Anexina A1/genética , Anexina A2/química , Anexina A2/genética , Anticorpos Antineoplásicos/sangue , Autoanticorpos/sangue , Autoantígenos/química , Autoantígenos/genética , Western Blotting , Proteína C-Reativa/análise , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma de Células Pequenas/sangue , Carcinoma de Células Pequenas/imunologia , Eletroforese em Gel Bidimensional , Perfilação da Expressão Gênica , Glicosilação , Humanos , Soros Imunes , Interleucina-1/sangue , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Dados de Sequência Molecular , Neoplasias/sangue , Neoplasias/imunologia , Processamento de Proteína Pós-Traducional , Fator de Necrose Tumoral alfa/análiseRESUMO
Common fragile sites have been proposed to play a mechanistic role in chromosome translocations and other rearrangements in cancer cells in vivo based on their behavior in vitro and their co-localization with cancer translocation breakpoints. This hypothesis has been the subject of controversy, because associations have been made at the chromosomal level and because of the large number of both fragile sites and cancer chromosome breakpoints. Tests of this hypothesis at the molecular level are now possible with the cloning of common fragile site loci and the use of fragile site clones in the analysis of rearranged chromosomes. FRA3B, the most frequently seen common fragile site, lies within the large FHIT gene. It is now well established that this region is the site of frequent, large intragenic deletions and aberrant transcripts in a number of tumors and tumor cell lines. In contrast, only one tumor-associated translocation involving the FHIT gene has been reported. We have found translocations in both homologs of chromosome 3 in an early-passage esophageal adenocarcinoma cell line. This cell line showed no normal FHIT transcripts by reverse transcription polymerase chain reaction. Subsequent chromosome analysis showed translocations of the short arms of both homologs of chromosome 3: t(3;16) and t(3;4). The breakpoints of both translocations were shown by fluorescence in situ hybridization and polymerase chain reaction to be in the FHIT gene, at or near the center of the fragile site region. Using rapid amplification of cDNA ends with FHIT primers, a noncoding chimeric transcript resulting from t(3;16) was identified. These data provide direct support for the hypothesis that FRA3B, and likely other common fragile sites, may be "hot spots" for translocations in certain cancers, as they are for deletions, and that such translocations have the potential to form abnormal chimeric transcripts. In addition, the results suggest selection for loss of a functional FHIT gene by the translocation events.
Assuntos
Hidrolases Anidrido Ácido , Adenocarcinoma/genética , Quebra Cromossômica/genética , Fragilidade Cromossômica/genética , Cromossomos Humanos Par 3/genética , Neoplasias Esofágicas/genética , Proteínas de Neoplasias/genética , Proteínas/genética , Translocação Genética/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios Frágeis do Cromossomo , Cromossomos Humanos Par 16/genética , Cromossomos Humanos Par 4/genética , Humanos , Masculino , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
Adenocarcinoma in Barrett's esophagus has been increasing in incidence at a rapid rate for more than two decades. Cyclooxygenase (COX)-2 appears to play an important role in gastrointestinal carcinogenesis, and COX-2 overexpression has been demonstrated both in esophageal adenocarcinomas and in the metaplastic epithelium of Barrett's esophagus. The aim of our study was to determine whether selective inhibition of COX-2 by NS-398 would alter the rates of cell growth and apoptosis in human Barrett's-associated esophageal adenocarcinoma cell lines. COX-1 and COX-2 expression in adenocarcinoma cell lines was determined using reverse transcription-PCR and Western blotting for mRNA and protein, respectively. Esophageal adenocarcinoma cell lines were treated with various concentrations of NS-398 (selective for COX-2 inhibition) and flurbiprofen (selective for COX-1 inhibition). Cell growth was compared in flurbiprofen-treated and untreated tumor cell lines; cell growth and apoptosis were compared in NS-398-treated and untreated tumor cell lines. COX-2 mRNA and protein were detected in two of three cell lines (SEG-1 and FLO); the third cell line, BIC-1, did not express COX-2 mRNA or protein under basal conditions or after stimulation with phorbol 12-myristate 13-acetate. Treatment with COX-1-selective concentrations of flurbiprofen did not affect cell growth in any of the three tumor cell lines. In contrast, treatment with COX-2-selective concentrations of NS-398 significantly suppressed cell growth and increased apoptosis in the cell lines that expressed COX-2 (SEG-1 and FLO), but not in the cell line that did not express COX-2 (BIC-1). We conclude that the administration of a selective inhibitor of COX-2 significantly decreases cell growth and increases apoptosis in Barrett's-associated adenocarcinoma tumor cells that express COX-2. These observations suggest a potential role for selective COX-2 inhibitors in the prevention and treatment of esophageal adenocarcinoma for patients with Barrett's esophagus.
Assuntos
Adenocarcinoma/enzimologia , Apoptose/efeitos dos fármacos , Neoplasias Esofágicas/enzimologia , Isoenzimas/antagonistas & inibidores , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/fisiologia , Esôfago de Barrett/complicações , Esôfago de Barrett/enzimologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/patologia , Flurbiprofeno/farmacologia , Expressão Gênica , Inibidores do Crescimento/farmacologia , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Proteínas de Membrana , Nitrobenzenos/farmacologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato , Sulfonamidas/farmacologia , Células Tumorais CultivadasRESUMO
BACKGROUND: The adenomatous polyposis coli (APC) locus on chromosome 5q21-22 shows frequent loss of heterozygosity (LOH) in esophageal carcinomas. However, the prevalence of truncating mutations in the APC gene in esophageal carcinomas is low. Because hypermethylation of promoter regions is known to affect several other tumor suppressor genes, we investigated whether the APC promoter region is hypermethylated in esophageal cancer patients and whether this abnormality could serve as a prognostic plasma biomarker. METHODS: We assayed DNA from tumor tissue and matched plasma from esophageal cancer patients for hypermethylation of the promoter region of the APC gene. We used the maximal chi-square statistic to identify a discriminatory cutoff value for hypermethylated APC DNA levels in plasma and used bootstrap-like simulations to determine the P: value to test for the strength of this association. This cutoff value was used to generate Kaplan-Meier survival curves. All P values were based on two-sided tests. RESULTS: Hypermethylation of the promoter region of the APC gene occurred in abnormal esophageal tissue in 48 (92%) of 52 patients with esophageal adenocarcinoma, in 16 (50%) of 32 patients with esophageal squamous cell carcinoma, and in 17 (39.5%) of 43 patients with Barrett's metaplasia but not in matching normal esophageal tissues. Hypermethylated APC DNA was observed in the plasma of 13 (25%) of 52 adenocarcinoma patients and in two (6.3%) of 32 squamous carcinoma patients. High plasma levels of methylated APC DNA were statistically significantly associated with reduced patient survival (P =.016). CONCLUSION: The APC promoter region was hypermethylated in tumors of the majority of patients with primary esophageal adenocarcinomas. Levels of hypermethylated APC gene DNA in the plasma may be a useful biomarker of biologically aggressive disease in esophageal adenocarcinoma patients and should be evaluated as a potential biomarker in additional tumor types.
Assuntos
Adenocarcinoma/metabolismo , Polipose Adenomatosa do Colo/genética , Biomarcadores Tumorais/sangue , Cromossomos Humanos Par 5/genética , DNA de Neoplasias/sangue , Neoplasias Esofágicas/metabolismo , Adenocarcinoma/genética , Esôfago de Barrett/metabolismo , Biomarcadores Tumorais/isolamento & purificação , Carcinoma de Células Escamosas/metabolismo , Distribuição de Qui-Quadrado , DNA de Neoplasias/isolamento & purificação , Neoplasias Esofágicas/genética , Mucosa Gástrica/metabolismo , Humanos , Perda de Heterozigosidade , Metilação , Reação em Cadeia da Polimerase/métodos , Lesões Pré-Cancerosas/metabolismo , Prognóstico , Regiões Promotoras Genéticas , Análise de SobrevidaRESUMO
The prolonged use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to exert a chemopreventive effect in esophageal and other gastrointestinal tumors. The precise mechanism by which this occurs, however, is unknown. While the inhibition of COX-2 as a potential explanation for this chemopreventive effect has gained a great deal of support, there also exists evidence supporting the presence of cyclooxygenase-independent pathways through which NSAIDs may exert their effects. In this study, immunohistochemical analysis of 29 Barrett's epithelial samples and 60 esophageal adenocarcinomas demonstrated abundant expression of the COX-2 protein in Barrett's epithelium, but marked heterogeneity of expression in esophageal adenocarcinomas. The three esophageal adenocarcinoma cell lines, Flo-1, Bic-1, and Seg-1, also demonstrated varying expression patterns for COX-1 and COX-2. Indomethacin induced apoptosis in all three cell lines, however, in both a time- and dose-dependent manner. In Flo-1 cells, which expressed almost undetectable levels of COX-1 and COX-2, and in Seg-1, which expressed significant levels of COX-1 and COX-2, indomethacin caused upregulation of the pro-apoptotic protein Bax. The upregulation of Bax was accompanied by the translocation of mitochondrial cytochrome c to the cytoplasm, and activation of caspase 9. Pre-treatment of both cell lines with the specific caspase 9 inhibitor, z-LEHD-FMK, as well as the broad-spectrum caspase inhibitor, z-VAD-FMK, blocked the effect of indomethacin-induced apoptosis. These data demonstrate that induction of apoptosis by indomethacin in esophageal adenocarcinoma cells is associated with the upregulation of Bax expression and mitochondrial cytochrome c translocation, and does not correlate with the expression of COX-2. This may have important implications for identifying new therapeutic targets in this deadly disease.
Assuntos
Adenocarcinoma/patologia , Apoptose/fisiologia , Grupo dos Citocromos c/metabolismo , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Indometacina/farmacologia , Mitocôndrias/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Apoptose/efeitos dos fármacos , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Ciclo-Oxigenase 2 , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Esôfago , Humanos , Isoenzimas/genética , Cinética , Proteínas de Membrana , Mitocôndrias/efeitos dos fármacos , Mucosa/metabolismo , Mucosa/patologia , Transporte Proteico/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína X Associada a bcl-2RESUMO
The incidence of esophageal adenocarcinomas has increased greatly over the past 20 years. The genetic alterations associated with this disease, however, remain largely unknown. We identified recently a novel amplicon at 8p22-23 in esophageal adenocarcinomas using the restriction landmark genomic scanning two-dimensional gel technique. Four known genes within or near this amplicon were initially characterized. The cathepsin B (CTSB) gene was found to be amplified in 13% of esophageal tumors. CTSB was shown previously to be overexpressed without amplification in many other human cancers. An approach termed sequence tagged site-amplification mapping has been implemented in the present study, allowing the 8p22-23 amplicon to be narrowed from 12 cM to a <2-cM minimal amplified area located between markers D8S552 and D8S1759. The CTSB gene maps within this region. To identify other cancer-related candidate genes in this region, a positional candidate gene approach was subsequently applied to characterize this minimal critical region. An expressed sequence tag (EST), which was included in the minimal critical region, demonstrated both amplification and overexpression. This EST and the extended sequence from the EST were determined to be a novel sequence in the 3' untranslated region of the human GATA-4 gene. GATA-4, a member of a zinc finger transcription factor family, was confirmed to be amplified and overexpressed in esophageal adenocarcinomas and was localized within <0.5 kb from CTSB. Furthermore, amplification of 8p22-23 was detected in one of eight gastric cardia adenocarcinomas but was not observed in either human lung adenocarcinomas (n = 39) or in esophageal squamous cell carcinomas (n = 24). The relatively high frequency of the 8p22-23 amplification in esophageal (13.6%) and gastric cardia (12.5%) adenocarcinomas may indicate a specificity of this amplicon for tumors of gastroesophageal origin.
Assuntos
Adenocarcinoma/genética , Cromossomos Humanos Par 8 , Proteínas de Ligação a DNA/genética , Neoplasias Esofágicas/genética , Fatores de Transcrição/genética , Mapeamento Cromossômico , Fator de Transcrição GATA4 , Amplificação de Genes , Humanos , Reação em Cadeia da Polimerase , Dedos de Zinco/genéticaRESUMO
Genomic DNA amplification in tumors is frequently associated with an increased gene copy number of oncogenes or other cancer-related genes. We have used a two-dimensional whole-genome scanning technique to identify gene amplification events in esophageal adenocarcinomas. A multicopy genomic fragment from a tumor two-dimensional gel was cloned, and genomic amplification encompassing this fragment was confirmed by Southern blot analysis. The corresponding DNA sequence was matched by BLAST to a BAC contig, which allowed the use of electronic-PCR to localize this amplicon to 19q12. Sequence tagged site-amplification mapping, an approach recently implemented in our laboratory (Lin, L. et al., Cancer Res., 60: 1341-1347,2000), was used to characterize the amplicon. Genomic DNA from 65 esophageal and 11 gastric cardia adenocarcinomas were investigated for 19q12 amplification using quantitative PCR at 11 sequence tagged site markers neighboring the cloned fragment. The amplicon was narrowed from >8 cM to a minimal critical region spanning <0.8 cM, between D19S919 and D19S882. This region includes the cyclin E gene. Fourteen expressed sequence tags (ESTs) covering the minimal region were then assayed for potential gene overexpression using quantitative reverse transcription-PCR. Seven of the selected ESTs were found to be both amplified and overexpressed. Among these seven ESTs, cyclin E showed the highest frequency of gene amplification and overexpression in the tumors examined, which allowed us to finalize the core-amplified region to <300 kb. These results indicate that cyclin E is the likely target gene selected by the amplification event at 19q12. The fact that cyclin E overexpression was found only in the amplified tumors examined indicates that gene amplification underlies the cyclin E gene overexpression. Our study represents the first extensive analysis of the 19q12 amplicon, and is the first to physically map the core-amplified domain to a region of <300 kb that includes cyclin E. Amplification of 19q12 was found neither in the 28 esophageal squamous cancers nor in the 39 lung adenocarcinomas examined but was observed in 13.8% of esophageal and 9.1% of gastric cardia adenocarcinomas.
Assuntos
Adenocarcinoma/genética , Cromossomos Humanos Par 19 , Ciclina E/genética , Neoplasias Esofágicas/genética , Southern Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Mapeamento Cromossômico , Clonagem Molecular , Mapeamento de Sequências Contíguas , Ciclina E/biossíntese , Eletroforese em Gel Bidimensional , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Esôfago/metabolismo , Esôfago/patologia , Etiquetas de Sequências Expressas , Humanos , Neoplasias Pulmonares/genética , Modelos Genéticos , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologiaRESUMO
We used TaqMan PCR to detect quantitative anomalies of tumor markers in both tumor and serum DNA from esophageal cancer patients. We demonstrated the potential of this methodology by detecting erbB-2 amplifications in a plurality of esophageal tumor samples. These amplifications were corroborated by Southern blots. We then showed the potential of this methodology to detect quantitative anomalies of erbB-2 in serum DNA from individuals with a corresponding amplification in the tumor. The capability of TaqMan PCR to detect abnormalities in serum of esophageal cancer patients creates an opportunity to diagnose esophageal cancer and to monitor the outcome of treatment with a blood test.
Assuntos
Adenocarcinoma/genética , DNA de Neoplasias/sangue , Neoplasias Esofágicas/genética , Genes erbB-2/genética , Adenocarcinoma/sangue , Southern Blotting , Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias Esofágicas/sangue , Amplificação de Genes , Dosagem de Genes , Humanos , Reação em Cadeia da Polimerase , Receptores de Quimiocinas/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Barrett's metaplasia consists of columnar epithelium that replaces the normal esophageal mucosa in patients with chronic gastroesophageal reflux. Because intestinal-type Barrett's metaplasia is the major risk factor for adenocarcinoma development, understanding the mechanisms that predispose the esophageal mucosa to malignant degeneration is clinically important. Glutathione s-transferase (GST)-pi belongs to a class of protective enzymes whose activity has been shown to be much lower in Barrett's metaplasia than in the normal esophagus, where this form of GST is predominant. In the studies described here, using immunocytochemical analysis, we observed higher levels of cytoplasmic GST-pi protein in normal esophageal mucosa than in Barrett's metaplasia. Using northern blot analysis, we also observed lower GST-pi mRNA levels in Barrett's metaplasia than in normal esophagus or adenocarcinomas from the same patients. Using as model systems three Barrett's adenocarcinoma cell lines and short-term organ culture of freshly resected normal esophagus and Barrett's metaplasia, dose-dependent induction of GST-pi mRNA was observed by using butylated hydroxyanisole and dexamethasone. GST-pi mRNA in Barrett's metaplasia was induced up to 2.5-fold with 60 microM butylated hydroxyanisole and nearly fivefold with 320 nM dexamethasone after 24 h. These studies demonstrate the ability to induce protective GST-pi in Barrett's metaplasia and may suggest a mechanism for future chemoprevention studies in patients with this type of epithelium, which is at high risk for malignant degeneration.
Assuntos
Adenocarcinoma/enzimologia , Esôfago de Barrett/enzimologia , Neoplasias Esofágicas/enzimologia , Glutationa Transferase/biossíntese , Isoenzimas/biossíntese , Indução Enzimática , Glutationa S-Transferase pi , Humanos , Técnicas de Cultura de Órgãos , RNA Mensageiro/biossíntese , Células Tumorais CultivadasRESUMO
Villin is an actin-binding cytoskeletal protein required for brush-border formation in the normal small intestinal and renal proximal tubule epithelium. Villin is a marker of cell differentiation in small intestinal and renal cell lineages, and recent studies have shown villin to be highly expressed in 100% of intestinal-type Barrett's metaplasias. This epithelium is the single greatest risk factor for developing esophageal adenocarcinoma and arises when the normal esophageal squamous epithelium is replaced by a small intestine-like columnar epithelium after damage by chronic gastroesophageal reflux. In intestinal-type Barrett's metaplasia, the villin protein exhibits a highly characteristic staining pattern in which strong apical, brush-border staining of columnar epithelial cells is observed. In this study, the ability to identify intestinal metaplastic cells by using this distinct villin staining pattern was examined in endoscopic esophageal brushings from patients with confirmed Barrett's metaplasia. Esophageal brushings from 81% (17 of 21) of patients with Barrett's metaplasia demonstrated individual columnar cells with the characteristic villin staining pattern, whereas all normal esophageal squamous cells, blood cells, and gastric columnar cells were negative for villin expression. Northern blot analysis demonstrated villin mRNA expression in Barrett's metaplasia but not in the normal squamous esophagus or gastric mucosa from the same patients. The combined use of villin immunohistochemical analysis and esophageal brush cytology may provide a simple and effective method of detecting intestinal-type Barrett's metaplasia in patients at higher risk for developing this epithelium, such as those experiencing chronic gastroesophageal reflux symptoms.
Assuntos
Esôfago de Barrett/diagnóstico , Proteínas de Transporte/análise , Esôfago/patologia , Proteínas dos Microfilamentos/análise , Esôfago de Barrett/metabolismo , Biomarcadores , Proteínas de Transporte/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Intestinos/patologia , Masculino , Proteínas dos Microfilamentos/biossíntese , Pessoa de Meia-IdadeRESUMO
The protein expression patterns of normal, metaplastic and malignant oesophageal tissues were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to identify changes associated with Barrett's metaplasia and transformation to oesophageal adenocarcinoma. Heat-shock protein 27 (Hsp27), a small heat-shock protein which is protective against cytotoxic stresses, was abundant in normal oesophagus. However, Hsp27 expression was markedly lower in Barrett's metaplasia and oesophageal adenocarcinomas. This was confirmed by immunohistochemical analysis. Hsp27 protein was most highly expressed in the upper layers of squamous epithelium and exhibited a pattern of expression that corresponded with the degree of squamous maturation. Northern and Southern analysis demonstrated Hsp27 to be regulated at the level of mRNA transcription or abundance. Normal oesophageal tissues were examined for gender differences in Hsp27 expression. Women expressed fourfold higher levels of Hsp27 mRNA, however, this difference was not appreciable in protein expression. Hsp27 protein was inducible by heat shock in Barrett's adenocarcinoma cell lines and an immortalized oesophageal epithelial cell line (HET-1A), but not by oestradiol. These results demonstrate abundant constitutive expression of the stress-response protein Hsp27 in the normal oesophagus, and suggest that low-level expression in Barrett's metaplasia may be one factor which may influence susceptibility to oesophageal adenocarcinoma development.
Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Neoplasias Esofágicas/genética , Esôfago/química , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico/genética , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Esôfago de Barrett/patologia , Transformação Celular Neoplásica/genética , Suscetibilidade a Doenças , Neoplasias Esofágicas/patologia , Esôfago/citologia , Estradiol/farmacologia , Feminino , Proteínas de Choque Térmico/biossíntese , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fatores SexuaisRESUMO
The expression of cytochromes P450 (CYP) in Barrett's esophagus and esophageal squamous mucosa was investigated. Esophagectomy specimens from 23 patients were examined for CYP expression of CYP1A2, CYP3A4, CYP2C9/10, and CYP2E1 by immunohistochemical analysis, and the expression of CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 in these tissues was further confirmed by reverse transcription polymerase chain reaction. Immunohistochemical analysis of esophageal squamous mucosa (n = 12) showed expression of CYP1A2, CYP3A4, CYP2E1, and CYP2C9/10 proteins, but it was noted that cells within the basal proliferative zone did not express CYPs. Immunohistochemical analysis of Barrett's esophagus (n = 13) showed expression of CYP1A2, CYP3A4, CYP2E1, and CYP2C9/10 that was prominent in the basal glandular regions, which are areas containing a high percentage of actively proliferating cells. Immunohistochemical staining for both proliferating cell nuclear antigen and the CYPs further supported the colocalization of CYP expression to areas of active cell proliferation in Barrett's esophagus, whereas in the esophageal squamous epithelium, CYP expression is limited to cells that are not proliferating. RT-PCR with amplification product sequence analysis confirmed CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 mRNA expression in Barrett's esophagus. These data suggest that the potential ability of cells in Barrett's esophagus to both activate carcinogens and proliferate may be important risk factors affecting carcinogenesis in this metaplastic tissue.
Assuntos
Esôfago de Barrett/enzimologia , Sistema Enzimático do Citocromo P-450/biossíntese , Esôfago/enzimologia , Mucosa Intestinal/enzimologia , Lesões Pré-Cancerosas/enzimologia , Western Blotting , Divisão Celular , Citocromo P-450 CYP1A2/biossíntese , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/química , Feminino , Humanos , Imuno-Histoquímica , Masculino , Oxigenases de Função Mista/biossíntese , Antígeno Nuclear de Célula em Proliferação/biossíntese , Isoformas de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The identification of markers that distinguish primary pulmonary adenocarcinomas from pulmonary adenocarcinomas secondary to the digestive tract would be clinically important. Villin, a specific marker in digestive-tract malignancies, was evaluated in 57 pulmonary adenocarcinomas, six samples of proximal bronchial tissue, and five metastatic pulmonary adenocarcinomas (three colon and two esophageal adenocarcinomas) by using immunohistochemical and molecular analyses. Villin was expressed in 31.6% (18 of 57) of the pulmonary adenocarcinomas and showed either a diffuse cytoplasmic pattern (10.5%) or a primary cytoplasmic pattern with minor brush-border staining (21.1%). However, none of those samples demonstrated the primary brush-border staining pattern that was characteristic of all five of the metastatic digestive-tract adenocarcinomas. There was a significant difference in the positive brush-border staining pattern between the primary and metastatic pulmonary adenocarcinomas (P < 0.002). Villin protein was expressed in bronchial epithelial cells, and villin mRNA was detected by reverse transcription-polymerase chain reaction. Northern analysis demonstrated 3.5- and 2.7-kb villin mRNAs in villin protein-positive tumors, but villin mRNA was not detected in non-tumorous lung tissue, indicating the transcriptional upregulation of villin in lung tumors. An additional smaller-sized mRNA (1.8 kb) was observed in six of 10 pulmonary adenocarcinomas and in the bronchoalveolar carcinoma cell line A549. Two small villin mRNAs were cloned from the cell line A549 and were found to represent an alternatively spliced (exon 8-exon 14) 1.85-kb mRNA and a 1.8-kb mRNA that was missing a portion of the 5' region (exon 1-exon 9) of the native villin mRNA. These studies demonstrated that the pattern of villin expression and the presence of altered villin mRNAs may be useful markers for pulmonary adenocarcinomas as well as provide support for the potential origin of villin-expressing tumors from bronchial epithelial cells.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Biomarcadores Tumorais/análise , Proteínas de Transporte/biossíntese , Neoplasias do Colo/metabolismo , Neoplasias Esofágicas/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Proteínas dos Microfilamentos/biossíntese , Metástase Neoplásica/diagnóstico , Proteínas de Neoplasias/biossíntese , Adenocarcinoma/genética , Idoso , Esôfago de Barrett/metabolismo , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Brônquios/metabolismo , Proteínas de Transporte/genética , Diferenciação Celular , Colo/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias Esofágicas/genética , Esôfago/metabolismo , Feminino , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Masculino , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Metástase Neoplásica/genética , Proteínas de Neoplasias/genética , Especificidade de Órgãos , PrognósticoRESUMO
Cathepsin B (CTSB) is overexpressed in tumors of the lung, prostate, colon, breast, and stomach. However, evidence of primary genomic alterations in the CTSB gene during tumor initiation or progression has been lacking. We have found a novel amplicon at 8p22-23 that results in CTSB overexpression in esophageal adenocarcinoma. Amplified genomic NotI-HinfI fragments were identified by two-dimensional DNA electrophoresis. Two amplified fragments (D4 and D5) were cloned and yielded unique sequences. Using bacterial artificial chromosome clones containing either D4 or D5, fluorescent in situ hybridization defined a single region of amplification involving chromosome bands 8p22-23. We investigated the candidate cancer-related gene CTSB, and potential coamplified genes from this region including farnesyl-diphosphate farnesyltransferase (FDFT1), arylamine N-acetyltransferase (NAT-1), lipoprotein lipase (LPL), and an uncharacterized expressed sequence tag (D8S503). Southern blot analysis of 66 esophageal adenocarcinomas demonstrated only CTSB and FDFT1 were consistently amplified in eight (12.1%) of the tumors. Neither NAT-1 nor LPL were amplified. Northern blot analysis showed overexpression of CTSB and FDFT1 mRNA in all six of the amplified esophageal adenocarcinomas analyzed. CTSB mRNA overexpression also was present in two of six nonamplified tumors analyzed. However, FDFT1 mRNA overexpression without amplification was not observed. Western blot analysis confirmed CTSB protein overexpression in tumor specimens with CTSB mRNA overexpression compared with either normal controls or tumors without mRNA overexpression. Abundant extracellular expression of CTSB protein was found in 29 of 40 (72. 5%) of esophageal adenocarcinoma specimens by using immunohistochemical analysis. The finding of an amplicon at 8p22-23 resulting in CTSB gene amplification and overexpression supports an important role for CTSB in esophageal adenocarcinoma and possibly in other tumors.
Assuntos
Adenocarcinoma/enzimologia , Catepsina B/genética , Cromossomos Humanos Par 8 , Neoplasias Esofágicas/enzimologia , Amplificação de Genes , Adenocarcinoma/genética , Sequência de Bases , Clonagem Molecular , Mapeamento de Sequências Contíguas , Primers do DNA , Neoplasias Esofágicas/genética , Humanos , ImmunoblottingRESUMO
The molecular genetic mechanisms underlying esophageal cancer are poorly understood. However, a novel gene that may be involved in esophageal carcinogenesis was recently localized by others to distal 17q by linkage analysis of kindreds with palmoplantar keratoderma and squamous cell carcinoma of the esophagus. To help determine whether a distal 17q gene may also be involved in the pathogenesis of primary Barrett's esophageal and gastric cardia adenocarcinomas, we performed loss of heterozygosity (LOH) analysis of 21 Barrett's and 18 gastric cardia adenocarcinomas at loci spanning 17q: cen-BRCA1-SSTR2-D17S2058-D17S929-D17S722-+ ++D17S937-D17S802-tel. Over 50% of the Barrett's and cardia adenocarcinomas demonstrated loss of an allele at one or more informative distal 17q markers. One common overlapping region of loss involved loci mapped to distal 17q24-proximal 17q25, which tentatively defines a potential chromosomal region distal to BRCA1 involved in the pathogenesis or progression of both types of adenocarcinomas. LOH analysis of DNA from matched microdissected sections of Barrett's metaplasia suggested that loss of D17S2058 in this region may be an early event in the malignant transformation of Barrett's metaplasia. No statistically significant correlations between 17q LOH and tumor stage or patient survival were noted. In summary, LOH mapping of 17q in Barrett's and cardia adenocarcinomas suggests the existence of at least one putative distal 17q tumor suppressor gene involved in the pathogenesis of these tumors.