RESUMO
Chronic inflammation contributes to the development of various forms of cancer. The polyamine catabolic enzyme spermine oxidase (SMOX) is induced in chronic inflammatory conditions, including Helicobacter pylori-associated gastritis, where its production of hydrogen peroxide contributes to DNA damage and subsequent tumorigenesis. MicroRNA expression levels are also altered in inflammatory conditions; specifically, the tumor suppressor miR-124 becomes silenced by DNA methylation. We sought to determine if this repression of miR-124 is associated with elevated SMOX activity and concluded that miR-124 is indeed a negative regulator of SMOX. In gastric adenocarcinoma cells harboring highly methylated and silenced mir-124 gene loci, 5-azacytidine treatment allowed miR-124 re-expression and decreased SMOX expression. Overexpression of an exogenous miR-124-3p mimic repressed SMOX mRNA and protein expression as well as H2O2 production by >50% within 24 h. Reporter assays indicated that direct interaction of miR-124 with the 3'-untranslated region of SMOX mRNA contributes to this negative regulation. Importantly, overexpression of miR-124 before infection with H. pylori prevented the induction of SMOX believed to contribute to inflammation-associated tumorigenesis. Compelling human in vivo data from H. pylori-positive gastritis tissues indicated that the mir-124 gene loci are more heavily methylated in a Colombian population characterized by elevated SMOX expression and a high risk for gastric cancer. Furthermore, the degree of mir-124 methylation significantly correlated with SMOX expression throughout the population. These results indicate a protective role for miR-124 through the inhibition of SMOX-mediated DNA damage in the etiology of H. pylori-associated gastric cancer.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica , Inativação Gênica , MicroRNAs/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Regiões 3' não Traduzidas , Biópsia , Metilação de DNA , Regulação para Baixo , Gastrite/etiologia , Gastrite/patologia , Infecções por Helicobacter/complicações , Helicobacter pylori , Humanos , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/patologia , Poliamina OxidaseRESUMO
Helicobacter pylori infection causes gastric cancer, the third leading cause of cancer death worldwide. More than half of the world's population is infected, making universal eradication impractical. Clinical trials suggest that antibiotic treatment only reduces gastric cancer risk in patients with non-atrophic gastritis (NAG), and is ineffective once preneoplastic lesions of multifocal atrophic gastritis (MAG) and intestinal metaplasia (IM) have occurred. Therefore, additional strategies for risk stratification and chemoprevention of gastric cancer are needed. We have implicated polyamines, generated by the rate-limiting enzyme ornithine decarboxylase (ODC), in gastric carcinogenesis. During H. pylori infection, the enzyme spermine oxidase (SMOX) is induced, which generates hydrogen peroxide from the catabolism of the polyamine spermine. Herein, we assessed the role of SMOX in the increased gastric cancer risk in Colombia associated with the Andean mountain region when compared with the low-risk region on the Pacific coast. When cocultured with gastric epithelial cells, clinical strains of H. pylori from the high-risk region induced more SMOX expression and oxidative DNA damage, and less apoptosis than low-risk strains. These findings were not attributable to differences in the cytotoxin-associated gene A oncoprotein. Gastric tissues from subjects from the high-risk region exhibited greater levels of SMOX and oxidative DNA damage by immunohistochemistry and flow cytometry, and this occurred in NAG, MAG and IM. In Mongolian gerbils, a prototype colonizing strain from the high-risk region induced more SMOX, DNA damage, dysplasia and adenocarcinoma than a colonizing strain from the low-risk region. Treatment of gerbils with either α-difluoromethylornithine, an inhibitor of ODC, or MDL 72527 (N(1),N(4)-Di(buta-2,3-dien-1-yl)butane-1,4-diamine dihydrochloride), an inhibitor of SMOX, reduced gastric dysplasia and carcinoma, as well as apoptosis-resistant cells with DNA damage. These data indicate that aberrant activation of polyamine-driven oxidative stress is a marker of gastric cancer risk and a target for chemoprevention.
Assuntos
Adenocarcinoma , Infecções por Helicobacter/complicações , Helicobacter pylori/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/fisiologia , Neoplasias Gástricas , Adenocarcinoma/epidemiologia , Adenocarcinoma/genética , Adenocarcinoma/microbiologia , Adulto , Animais , Células Cultivadas , Colômbia/epidemiologia , Dano ao DNA/genética , Indução Enzimática , Gerbillinae , Infecções por Helicobacter/genética , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/genética , Fatores de Risco , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Poliamina OxidaseRESUMO
The C-terminus of the Helicobacter pylori CagA protein is polymorphic, bearing different EPIYA sequences (EPIYA-A, B, C or D), and one or more CagA multimerization (CM) motifs. The number of EPIYA-C motifs is associated with precancerous lesions and gastric cancer (GC). The relationship between EPIYA, CM motifs and gastric lesions was examined in H. pylori-infected Colombian patients from areas of high and low risk for GC. Genomic DNA was extracted from H. pylori strains cultured from gastric biopsies from 80 adults with dyspeptic symptoms. Sixty-seven (83.8%) of 80 strains were cagA positive. The 3' region of cagA was sequenced, and EPIYA and CM motifs were identified. CagA proteins contained one (64.2%), two (34.3%) or three EPIYA-C motifs (1.5%), all with Western type CagA-specific sequences. Strains with one EPIYA-C motif were associated with less severe gastric lesions (non-atrophic and multifocal atrophic gastritis), whereas strains with multiple EPIYA-C motifs were associated with more severe lesions (intestinal metaplasia and dysplasia) (p <0.001). In 54 strains, the CM motifs were identical to those common in Western strains. Thirteen strains from the low-risk area contained two different CM motifs: one of Western type located within the EPIYA-C segment and another following the EPIYA-C segment and resembling the CM motif found in East Asian strains. These strains induced significantly shorter projections in AGS cells and an attenuated reduction in levels of CagA upon immunodepletion of SHP-2 than strains possessing Western/Western motifs. This novel finding may partially explain the difference in GC incidence in these populations.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Infecções por Helicobacter/complicações , Helicobacter pylori/genética , Lesões Pré-Cancerosas/microbiologia , Neoplasias Gástricas/microbiologia , Adulto , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Colômbia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Polimorfismo Genético , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
AIMS: To define regions of loss on the distal portion of chromosome 12q in gastric adenocarcinoma. METHODS: Microsatellite analysis on chromosome 12 was performed on 19 human gastric cancer cell lines using 77 markers, 71 of which were within or distal to 12q21; some portions of this region showed extended regions of homozygosity (ERHs) in 10 of 19 gastric cancer cell lines. In addition, microdissected tumour cells from 76 primary gastric adenocarcinomas were examined using 13 markers of interest implicated by the cell line data; 70% of these showed allelic imbalance (AI) at one or more markers in or distal to 12q21. RESULTS: Mapping ERHs in the cell lines and sites of AI in the tumours identified three regions that contain putative tumour suppressor genes: region A is located within 2.8 Mb between markers D12S1667 and D12S88; region B, within 1.9 Mb between markers D12S1607 and D12S78; and region C, in 0.74 Mb between markers D12S342 and D12S324. Fluorescence in situ hybridisation (FISH) analysis in two cell lines confirmed that two of the ERHs reflected deletions, not amplifications, of D12S81 in region A and D12S340 in region C. FISH analysis of marker D12S1075 within an ERH containing region B in one cell line showed neither amplification nor deletion. AI on 12q was not associated with prognosis, but was associated with ethnicity of the patient. CONCLUSIONS: These results identify regions on chromosome 12 that appear to contain tumour suppressor genes important in the development of gastric cancer.
Assuntos
Adenocarcinoma/genética , Desequilíbrio Alélico , Cromossomos Humanos Par 12/genética , Neoplasias Gástricas/genética , Adenocarcinoma/etnologia , Adenocarcinoma/patologia , Genes Supressores de Tumor , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente/métodos , Repetições de Microssatélites , Reação em Cadeia da Polimerase/métodos , Neoplasias Gástricas/etnologia , Neoplasias Gástricas/patologia , Células Tumorais CultivadasRESUMO
AIMS: It has been shown previously (by immunohistochemistry) that gastric adenocarcinomas harbouring Epstein-Barr virus (EBV) frequently lose p16 protein. This study aimed to examine the mechanisms of inactivation of the CDKN2A gene and correlate the results with clinicopathological features. METHODS: Methylation specific polymerase chain reaction was used to detect CDKN2A promoter methylation in gastric adenocarcinomas from American patients. In addition, immunohistochemistry was used to detect the loss of the p16 protein and in situ hybridisation was used to detect the presence of EBV. The tumours were also analysed for the presence of microsatellite instability. RESULTS: Eleven (10%) of 107 tumours harboured EBV in the malignant cells. In gastric cancers without EBV, 32% exhibited CDKN2A promoter methylation and 26% had p16 protein loss. In contrast, 91% of the tumours containing EBV had CDKN2A promoter methylation (p = 0.0003) and 90% showed p16 protein loss (p = 0.0001). The presence of EBV was also associated with male sex (p = 0.03) and was more common in tumours from Texas Hispanics than from non-Hispanic whites or African-Americans (p = 0.01). EBV was not associated with microsatellite instability, histological subtype, stage, or grade of the tumour, or age or survival time of the patient. CONCLUSIONS: The presence of EBV in gastric adenocarcinomas is strongly associated with CDKN2A inactivation by promoter methylation. In addition, these findings suggest that there are ethnic differences in tumour virology and pathogenesis.
Assuntos
Adenocarcinoma/virologia , Infecções por Vírus Epstein-Barr/complicações , Genes p16 , Herpesvirus Humano 4/isolamento & purificação , Neoplasias Gástricas/virologia , Adenocarcinoma/etnologia , Adenocarcinoma/genética , Negro ou Afro-Americano , Idoso , Idoso de 80 Anos ou mais , Metilação de DNA , DNA de Neoplasias/genética , Feminino , Inativação Gênica , Hispânico ou Latino , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Neoplasias Gástricas/etnologia , Neoplasias Gástricas/genética , Estados Unidos/epidemiologiaRESUMO
The CDKN2A gene encodes a cyclin-dependent kinase inhibitor, p16, which promotes cell cycle arrest. Methylation of the promoter region of the gene transcriptionally inactivates the gene. We have analyzed the methylation status of the promoter region of the CDKN2A gene in gastric adenocarcinomas using methylation-specific polymerase chain reaction. We also examined the tumors by immunohistochemistry for p16 protein. Of 114 gastric adenocarcinomas analyzed by immunohistochemistry, 34 cases (30%) were negative for p16 protein. Twenty-four of these 34 cases (71%) had methylation of the promoter region of the CDKN2A gene. Methylation of the promoter was strongly associated with loss of p16 protein by immunohistochemistry (P <0.0001). Neither stage, grade, anatomic site, or histologic subtype of the tumor nor age, gender, ethnic origin, or survival time of the patient were significantly different between the groups characterized by tumors with and without methylation. CDKN2A promoter methylation was not significantly associated with microsatellite instability.
Assuntos
Adenocarcinoma/genética , Metilação de DNA , Genes p16 , Regiões Promotoras Genéticas , Neoplasias Gástricas/genética , Adenocarcinoma/química , Adenocarcinoma/mortalidade , Idoso , Inibidor p16 de Quinase Dependente de Ciclina/análise , DNA de Neoplasias/análise , DNA de Neoplasias/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Reação em Cadeia da Polimerase , Neoplasias Gástricas/química , Neoplasias Gástricas/mortalidade , Taxa de SobrevidaRESUMO
We examined 169 cases of gastric adenocarcinoma for microsatellite instability (MSI), using a panel of 8 microsatellite markers. Of these cases, 142 were from the United States, a country of relatively low risk for gastric cancer. Comparing microdissected tumors to normal cells from the same patient, we classified tumors as being microsatellite-stable (MSS) or having a low frequency of MSI (MSI-L, up to 30% of markers different in the tumor) or a high frequency of MSI (MSI-H, 30% or more of markers different). Among our American cases, we identified 26 (18.2%) showing MSI-H and 15 (10.6%) showing MSI-L. Twenty cases were from Korean patients, and they showed no significant differences in proportions of MSI-H and MSI-L from the American cases. MSI-H tumors in the American patients were characterized by elevated frequencies of band shifts in repeat sequences of the BAX (50%), transforming growth factor-beta receptor type II (TGFbetaRII, 68.9%), beta(2)-microglobulin (21.4%) and E2F4 (51.7%) genes. Alterations in E2F4 in MSI-H tumors were always integral multiples of 3 nucleotides lost or gained, which would not cause a frameshift mutation, and within the range of normal polymorphisms for this sequence. North American patients (n = 127) with MSI-H and MSI-L tumors had a longer median survival of 541 days and 587 days, respectively, compared to 265 days for patients with MSS tumors (p = 0.027). This survival difference may result from a significantly greater tendency for metastases in the MSS group (p = 0.031).
Assuntos
Repetições de Microssatélites , Proteínas Proto-Oncogênicas c-bcl-2 , Neoplasias Gástricas/genética , Humanos , Metástase Neoplásica , Prognóstico , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genética , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/patologia , Taxa de Sobrevida , Proteína X Associada a bcl-2 , Microglobulina beta-2/genéticaRESUMO
Like most cancers, prostate cancer (CaP) is believed to be the result of the accumulation of genetic alterations within cells. Previous studies have implicated numerous chromosomal regions with elevated rates of allelic imbalance (AI), using mostly primary CaPs with an unknown disease outcome. These regions of AI are proposed sites for tumor suppressor genes. One of the regions previously implicated as coding for at least one tumor suppressor gene is the long arm of chromosome 18 (18q). To confirm this observation, as well as to narrow the critical region for this putative tumor suppressor, we analyzed 32 metastatic CaP specimens for AI on chromosome 18q. Thirty-one of these 32 specimens (96.8%) exhibited AI at one or more loci on chromosome 18q. Our analysis using 17 polymorphic markers revealed statistically significant AI on chromosome 18q at 3 markers, D18S35, D18S64 and D18S461. Using these markers as a guide, we have been able to identify 2 distinct minimum regions of AI on 18q. The first region is between the genetic markers D18S1119 and D18S64. The second region lies more distal on the long arm of the chromosome and is between the genetic markers D18S848 and D18S58. To determine if 18q loss is a late event in the progression of CaP, we also examined prostatic intraepithelial neoplasia (PIN) and primary prostate tumors from 17 patients for AI with a subset of 18q markers. We found significantly higher AI in the metastatic samples. Our results are consistent with 18q losses occurring late in CaP progression.
Assuntos
Cromossomos Humanos Par 18 , Genes Supressores de Tumor , Perda de Heterozigosidade , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Carcinoma in Situ/genética , Mapeamento Cromossômico , Marcadores Genéticos , Humanos , Masculino , Repetições de Microssatélites , Metástase Neoplásica , Polimorfismo GenéticoRESUMO
Gastric adenocarcinomas (n = 125) were analyzed by immunohistochemistry for the presence of p16, the CDKN2A gene product. This protein was lost in 31 of 125 cases (25%), and loss was associated with location of the tumor in the body of the stomach (P = .001). Loss of p16 was also associated with the presence of Epstein-Barr virus (EBV) in tumor cells as determined by in situ hybridization (P = .022). This effect may relate to anatomic site, because EBV-associated tumors originate more frequently in the body of the stomach. When p16 status was evaluated for ethnic origin of the patient (non-Hispanic white, Hispanic, or black), a strong trend (P = .057) was found for African-American patients to have fewer p16-negative tumors than other patients. This also may relate to anatomic location, because fewer tumors from black patients arose in the body of the stomach (P = .022). No significant associations were detected between p16 status and histological subtype (intestinal v diffuse), the presence of microsatellite instability, grade or stage of the tumor, or age, gender, or survival of the patient. In conclusion, p16 loss is quite common in gastric adenocarcinoma, and such loss is more common in EBV-infected tumors arising in the body of the stomach.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/virologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Herpesvirus Humano 4/isolamento & purificação , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/virologia , Adenocarcinoma/patologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , RNA Viral/metabolismo , Estômago/patologia , Neoplasias Gástricas/patologiaRESUMO
The p16/MTS1 gene is altered by deletion, mutation, or hypermethylation in a wide variety of human cancers. As a result of deficient p16 protein, these cancers lack a critical mechanism for halting G1/S cell cycle progression. In the current study, 59 cases of nasopharyngeal carcinoma were evaluated for expression of the p16 tumor suppressor protein by immunohistochemical analysis of paraffin-embedded tissue. There was no detectable p16 in 38/59 cases (64%), which implies a very high rate of p16 inactivation in this type of cancer. On the other hand, the retinoblastoma gene product, which also regulates the G1 to S phase transition of the cell cycle, was consistently expressed in nasopharyngeal carcinomas by immunohistochemical analysis. These results implicate p16 inactivation but not Rb alteration in the stepwise progression of nasopharyngeal carcinogenesis.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Proteína do Retinoblastoma/metabolismo , Herpesvirus Humano 4/isolamento & purificação , Humanos , Imuno-Histoquímica , Hibridização In Situ , RNA Viral/metabolismo , Estudos Retrospectivos , Proteína Supressora de Tumor p53/metabolismoRESUMO
OBJECTIVE: To characterize the expression of alpha subunits of integrin adhesion molecules in peritoneal tissue in vivo and in vitro. METHOD: Peritoneum from the anterior abdominal wall (n = 22) and the serosa of the posterior uterus (n = 11) was obtained from women of reproductive age without endometriosis who were undergoing surgery for benign conditions. Immunohistochemical studies were performed on serial sections of peritoneum from the anterior abdominal wall, the uterine serosa, mesothelial monolayer cultures, and peritoneum explants from the abdominal wall using monoclonal antibodies to alpha subunits of integrin adhesion molecules. Electron microscopy was performed to localize these adhesion molecules in the mesothelium. RESULTS: The mesothelial expression of alpha integrin subunits was identical in the anterior peritoneum and uterine serosa. In vivo the mesothelium strongly expressed alpha 2 and alpha 3 and variably expressed alpha 6. In the monolayer cultures there was moderate/strong staining for alpha 2, alpha 3, and alpha 5; there was minimal expression of alpha v. In the explants there was moderate/strong expression of alpha 2, alpha 3, alpha 5, and alpha v; alpha 6 was variably expressed. The ultrastructure of the mesothelium was unique in the anterior peritoneum, uterine serosa, and the monolayer cultures. The integrin subunits were distributed throughout the cytoplasm, were expressed in the plasma membrane, and were present on the surface (i.e., towards the peritoneal cavity) of the mesothelium. CONCLUSION: Integrins are expressed by the mesothelium of the peritoneum. The mesothelium expression of integrins in vivo differs from that of the mesothelium integrin expression in monolayer culture and explant culture.
Assuntos
Integrinas/análise , Peritônio/química , Útero/química , Antígenos CD/análise , Células Cultivadas , Epitélio/química , Epitélio/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica , Integrina alfa1 , Integrina alfa2 , Integrina alfa3 , Integrina alfa4 , Integrina alfa5 , Integrina alfa6 , Integrina alfaV , Microscopia ImunoeletrônicaRESUMO
Eighty-three cases of nasopharyngeal carcinoma were evaluated for the presence of Epstein-Barr virus (EBV) infection in tumor cells by in situ hybridization to EBER1 transcripts, and for p53 expression by immunostains using the D07 antibody which detects native and mutant forms of the p53 protein. A highly significant association was found between EBV infection and p53 overexpression (P = .0004), with 77% of cases coexpressing both markers. This newly discovered association suggests that EBV is not an innocent bystander with respect to p53 accumulation. One possible mediator of the interaction between EBV and p53, viral BZLF1, was not colocalized with p53 in these tumors, suggesting that BZLF1 is not the factor responsible for p53 accumulation. From an epidemiological standpoint, this series of cancers represents an international cohort in which cases from an endemic part of the world (Hong Kong) were examined alongside cases from the United States, where the disease is 50-fold less prevalent. The cancers from Hong Kong tended to be less differentiated and more frequently associated with EBV, suggesting that biological differences might underlie epidemiological variations in tumor prevalence. Finally, we examined 18 potential premalignant lesions of the surface epithelium of the nasopharynx. Although our numbers are small, our data suggest that p53 accumulation might precede EBV infection in the transition from metaplasia to carcinoma in situ. Further studies are needed to dissect the stepwise progression of nasopharyngeal carcinogenesis.
Assuntos
Carcinoma in Situ/metabolismo , Carcinoma de Células Escamosas/metabolismo , Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 4/isolamento & purificação , Neoplasias Nasofaríngeas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Infecções Tumorais por Vírus/metabolismo , Proteínas Virais , Carcinoma in Situ/patologia , Carcinoma in Situ/virologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Proteínas de Ligação a DNA/metabolismo , Epitélio/patologia , Epitélio/virologia , Infecções por Herpesviridae/patologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/virologia , Nasofaringe/patologia , Nasofaringe/virologia , RNA Viral/metabolismo , Transativadores/metabolismo , Infecções Tumorais por Vírus/patologiaRESUMO
We evaluated the effect of six different histologic stains on the productivity of PCR amplification of DNA isolated from paraffin-embedded tissue samples. The tissue was collected from glass slides by microdissection techniques, whereby tiny portions of tissue are visually identified through a microscope and selectively resected for subsequent DNA extraction and PCR amplification. We found that the success of PCR amplification depended on the type of histologic stain that was used to facilitate microscopic visualization of the undeparaffinized tissue section. The best results were obtained with methyl green and nuclear fast red, while Wright's stain yielded less PCR product. Two other stains, Evans blue and light-green SF yellowish (also known as the counterstain for geomori methenamine silver stain) yielded sufficient PCR products; however, their staining characteristics did not afford satisfactory visualization of nuclear chromatin to discriminate between benign and malignant cells. Our most significant finding was that a commonly used histologic stain, hematoxylin, failed to produce DNA templates that could be consistently amplified by PCR. In conclusion, it is prudent to avoid hematoxylin stains when preparing tissues as starting material for PCR. Among the remaining five stains that were evaluated, the best choice depends on the differential staining characteristics of the cells to be dissected.
Assuntos
Corantes , Reação em Cadeia da Polimerase/métodos , Dissecação , Humanos , Parafina , Inclusão do TecidoRESUMO
Helicobacter pylori is a spiral, gram-negative bacterium which causes chronic gastritis and plays a critical role in peptic ulcer disease, gastric carcinoma, and gastric lymphoma. H. pylori expresses significant urease activity which is an essential virulence factor. Since a significant fraction of urease activity is located on the surface of the bacterium, the urease molecule is a logical choice as an antigen for a vaccine; currently recombinant urease apoenzyme is being tested as a vaccine in phase II clinical trials. We have recently demonstrated that urease and HspB (a homolog of the GroEL heat shock protein) become associated with the surface of H. pylori in vitro in a novel manner: these cytoplasmic proteins are released by bacterial autolysis and become adsorbed to the surface of intact bacteria, reflecting the unique characteristics of the outer membrane. To determine if similar mechanisms are operative in vivo, we determined the ultrastructural locations of urease and HspB within bacteria present in human gastric biopsies. Our results demonstrate that both urease and HspB are located within the cytoplasm of all bacteria examined in human gastric biopsies. Interestingly, a significant proportion of the bacteria examined also possessed variable amounts of surface-associated urease and HspB antigen (from 5 to 50% of the total antigenic material), indicating that in vivo, H. pylori has surface characteristics which enable it to adsorb cytoplasmic proteins. This is consistent with our altruistic autolysis model in which H. pylori uses genetically programmed bacterial autolysis to release urease and other cytoplasmic proteins which are subsequently adsorbed onto the surface of neighboring viable bacteria. These observations have important implications regarding pathogenesis and development of vaccines for H. pylori.
Assuntos
Proteínas de Bactérias/análise , Mucosa Gástrica/metabolismo , Proteínas de Choque Térmico/análise , Helicobacter pylori/enzimologia , Urease/análise , Biópsia , Humanos , Microscopia Imunoeletrônica , Estômago/microbiologia , Estômago/patologia , Estômago/ultraestruturaRESUMO
Ninety-five cases of adenocarcinoma of the stomach were evaluated for the presence of Epstein-Barr virus (EBV) using a sensitive in situ hybridization assay targeting Epstein-Barr virus-encoded RNA 1 (EBER1) transcripts. EBER1 was detected in 11 of 95 (12%) of cases. When present, the virus was localized to malignant epithelial cells and to dysplastic gastric epithelium, but was not seen in normal-appearing gastric epithelium or intestinal metaplasia. The EBV DNA was monoclonal in all three cases tested by Southern blot analysis of the EBV terminal repeat fragment. These findings suggest that the virus was present before malignant transformation. The presence of EBV was strongly associated with increased numbers of tumor-infiltrating T lymphocytes; however, EBV was not associated with prolonged survival. Neither p53 nor bcl-2 were consistently detected in the EBV-associated tumors. Specifically, 6 of 11 EBV-positive carcinomas had accumulation of p53 protein by immunohistochemical analysis, which was similar to the prevalence of p53 accumulation in EBV-negative specimens and suggests that EBV infection does not substitute for p53 mutations during tumorigenesis. The bcl-2 oncoprotein was expressed in a third of the carcinoma specimens tested, but bcl-2 expression did not correlate with the presence of EBV or with expression of EBV latent membrane protein 1. In conclusion, EBV infection appears to precede malignant transformation in a significant fraction of gastric carcinomas, but neither bcl-2 expression nor p53 accumulation appear to be consistently associated with the presence of the virus.
Assuntos
Adenocarcinoma/virologia , Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 4 , Proteínas Proto-Oncogênicas/biossíntese , Neoplasias Gástricas/virologia , Proteína Supressora de Tumor p53/biossíntese , Infecções Tumorais por Vírus/metabolismo , Proteínas Virais , Adenocarcinoma/etnologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Southern Blotting , Proteínas de Ligação a DNA/análise , Feminino , Infecções por Herpesviridae/etnologia , Infecções por Herpesviridae/patologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/isolamento & purificação , Hispânico ou Latino , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2 , Estudos Retrospectivos , Neoplasias Gástricas/etnologia , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Transativadores/análise , Infecções Tumorais por Vírus/etnologia , Infecções Tumorais por Vírus/patologia , Proteínas da Matriz Viral/análiseRESUMO
In order to detect regions of DNA containing tumor suppressor genes involved in the development of gastric cancer, we performed an allelotype study on 78 gastric adenocarcinomas from a population composed largely of Texan Hispanics and Anglos, two ethnic groups that have a ratio of incidence rates of gastric cancer of approximately 2:1. In total, 42 microsatellite markers were employed, which detected at least one site per arm of each autosome in the human genome. These included several markers linked to known tumor suppressor genes (TP53, APC, DCC, RB1, and BRCA1). Sites showing quantitative allelic imbalance (AI) greater than 30% were located on 3p (36%), 11q (31%), 12q (38%), 13q (33%), 17p near TP53 (74%), and 17q near BRCAI (32%). Among the 22% of cases showing microsatellite instability (MI), a subset (4 of 17) showed instability at 59% or more of sites tested. No ethnic bias was detected in cases showing MI or in cases with AI at sites with rates of AI above 30%. Tumors of the intestinal subtype were significantly more likely than diffuse tumors to show AI at DI3S170 (P = 0.01). A deletion map of chromosome arm 3p was prepared for tumors with AI at D3S1478. These data indicate that a tumor suppressor gene on chromosome arm 3p is involved in the development of a subset of gastric cancers.
Assuntos
Adenocarcinoma/genética , Alelos , Deleção Cromossômica , Cromossomos Humanos Par 3/genética , Neoplasias Gástricas/genética , Genes Supressores de Tumor/genética , Marcadores Genéticos , Humanos , Repetições de MicrossatélitesRESUMO
PURPOSE: We report a case of adrenal cortical carcinoma in an infant, which was incidentally discovered by renal sonography after a urinary tract infection. The previous death of a sibling after rhabdomyosarcoma in infancy prompted a search for a heritable p53 tumor suppressor gene mutation in this family. PATIENTS AND METHODS: Starting with frozen adrenal carcinoma tissue, polymerase chain reaction (PCR) amplification followed by direct sequencing of exons 4-8 of p53 was used to search for a mutation. When a mutation was identified in exon 6 of the tumor p53 sequence, PCR amplification and direct sequencing of exon 6 alone was then performed on DNA from peripheral blood lymphocytes (PBLs) of all immediate family members to determine whether a germline mutation was present. A different set of primers was used by a second laboratory at our institution to independently confirm the presence of the mutation in the adrenal carcinoma and in paraffin-embedded rhabdomyosarcoma tissue of the deceased sibling. RESULTS: A C-to-T transition was identified at a CpG site in codon 196 resulting in a change from arginine to a stop codon (CGA to TGA). The identical mutation, present as the sole p53 allele in the tumor DNA samples and in the heterozygous state with wild type p53 allele in DNA from PBLs (germline), was found in the adrenal carcinoma, the rhabdomyosarcoma, and the PBLs of the tumor-bearing child and her healthy father and 5-year-old brother. This nonsense mutation of p53 has never before been reported in the germline. The extended pedigree showed only one known additional cancer. CONCLUSIONS: A novel germline p53 mutation was identified by investigation of a sibling pair with cancers associated with the Li-Fraumeni syndrome in a family with an otherwise negative history for cancer. The implications of this case for identification of carriers of p53 germline mutations and their clinical management are discussed.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Genes p53/genética , Mutação em Linhagem Germinativa/genética , Sequência de Bases , Feminino , Humanos , Lactente , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da PolimeraseRESUMO
Gastric cancer is more than twice as common in Hispanics as in Anglos in Texas, while colorectal cancer is almost twice as common in Anglos as Hispanics. To test the hypothesis that mutations in the p53 tumour suppressor gene are involved in these differences, we examined 131 gastric and 138 colorectal cancers from Hispanic and Anglo patients from South Texas and Mexico using immunohistochemistry (IHC) as a screening assay for p53 mutations. The fraction of p53 positive cases was not significantly different in gastric cancers from Hispanics compared to Anglos (43% versus 61%, respectively, p = 0.13) or in colorectal cancer (57% versus 58%, respectively, p = 1.0), suggesting that p53 mutations are not involved in causing the different incidences of these cancers in these populations. In addition, the types of p53 mutations arising in gastric tumours from Hispanic patients were consistent with those reported in gastric tumours in other populations. Sequencing of mutations in five gastric cancers revealed two G:C to A:T transitions, two A:T to G:C transitions and one complex deletion. In contrast with findings in studies in other tumour types, neither stage nor survival was associated with p53 positive staining by IHC in either gastric or colorectal tumours in this study. Positive p53 immunostaining was associated with the diffuse histological subtype in gastric carcinoma (p = 0.05) and high histological grade in colorectal carcinoma (p = 0.04).
Assuntos
Neoplasias Colorretais/genética , Genes p53 , Hispânico ou Latino/genética , Mutação , Neoplasias Gástricas/genética , Sequência de Bases , Códon , Neoplasias Colorretais/química , Neoplasias Colorretais/etnologia , Éxons , Feminino , Humanos , Imuno-Histoquímica , Masculino , México , Dados de Sequência Molecular , Neoplasias Gástricas/química , Neoplasias Gástricas/etnologia , Texas , Proteína Supressora de Tumor p53/análise , Proteína Supressora de Tumor p53/metabolismoRESUMO
Four different isotypes of beta-tubulin are known to be expressed in mammalian brain. Monoclonal antibodies against beta II, beta III, and beta IV were used to characterize the beta-tubulin isotypes in two ciliated bovine tissues: non-motile sensory cilia of retinal rod cells and motile cilia of tracheal epithelium. Retinal rod outer segment (ROS) connecting cilia and cytoskeletons were purified by density gradient centrifugation. This preparation contained more than 20 major protein components, as shown by dodecyl sulfate polyacrylamide gel electrophoresis. Electroblots were used to quantitate the relative amounts of beta II, beta III, and beta IV. The connecting cilium and cytoskeleton of the rod outer segment has less type III beta-tubulin than brain and more type IV. The ratio of beta IV to beta II in the ROS is nearly a factor of 8 larger than in brain. Electron microscopic immunocytochemistry showed extensive labeling of cilia by anti-type IV in thin sections of retinas and trachea, and also in purified ROS cilia and cytoskeletons. Labeling of cilia by anti-beta II was also observed, although in the purified ROS cilia and cytoskeleton, the anti-beta II labeling was primarily on amorphous non-ciliary material. The results suggest that both motile and non-motile cilia are enriched in the type IV beta-tubulin subunit.
Assuntos
Cílios/química , Tubulina (Proteína)/análise , Sequência de Aminoácidos , Animais , Encéfalo/ultraestrutura , Química Encefálica , Bovinos , Citoesqueleto/química , Epitélio/química , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Segmento Externo da Célula Bastonete/química , Segmento Externo da Célula Bastonete/ultraestrutura , Traqueia/química , Traqueia/ultraestrutura , Tubulina (Proteína)/química , Tubulina (Proteína)/fisiologiaRESUMO
Thirteen mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa (ADRP) have been produced by transfection of cloned cDNA into tissue culture cells. Three mutants [class I: Phe-45----Leu, Gln-344----termination (deletion of C-terminal positions 344-348), and Pro-347----Leu] resemble wild-type rhodopsin in yield, regenerability with 11-cis-retinal, and plasma membrane localization. Ten mutants [class II: Thr-17----Met, Pro-23----His, Thr-58----Arg, Val-87----Asp, Gly-89----Asp, Gly-106----Trp, Arg-135----Leu, Arg-135----Trp, Tyr-178----Cys, and Asp-190----Gly] accumulate to significantly lower levels, regenerate with 11-cis-retinal variably or not at all, and are transported inefficiently to the plasma membrane, remaining primarily in the endoplasmic reticulum. These data suggest that there are at least two distinct biochemical defects associated with different rhodopsin mutants in ADRP.