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1.
Proc Natl Acad Sci U S A ; 119(31): e2123241119, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35895679

RESUMO

Somatic mutations are accumulated in normal human tissues with aging and exposure to carcinogens. If we can accurately count any passenger mutations in any single DNA molecule, since their quantity is much larger than driver mutations, we can sensitively detect mutation accumulation in polyclonal normal tissues. Duplex sequencing, which tags both DNA strands in one DNA molecule, enables accurate count of such mutations, but requires a very large number of sequencing reads for each single sample of human-genome size. Here, we reduced the genome size to 1/90 using the BamHI restriction enzyme and established a cost-effective pipeline. The enzymatically cleaved and optimal sequencing (EcoSeq) method was able to count somatic mutations in a single DNA molecule with a sensitivity of as low as 3 × 10-8 per base pair (bp), as assessed by measuring artificially prepared mutations. Taking advantages of EcoSeq, we analyzed normal peripheral blood cells of pediatric sarcoma patients who received chemotherapy (n = 10) and those who did not (n = 10). The former had a mutation frequency of 31.2 ± 13.4 × 10-8 per base pair while the latter had 9.0 ± 4.5 × 10-8 per base pair (P < 0.001). The increase in mutation frequency was confirmed by analysis of the same patients before and after chemotherapy, and increased mutation frequencies persisted 46 to 64 mo after chemotherapy, indicating that the mutation accumulation constitutes a risk of secondary leukemia. EcoSeq has the potential to reveal accumulation of somatic mutations and exposure to environmental factors in any DNA samples and will contribute to cancer risk estimation.


Assuntos
Análise Mutacional de DNA , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Taxa de Mutação , Imagem Individual de Molécula , Envelhecimento/genética , Pareamento de Bases , Criança , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação , Imagem Individual de Molécula/métodos
2.
Gut ; 73(2): 255-267, 2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-37751933

RESUMO

OBJECTIVE: The presence of intestinal metaplasia (IM) is a risk factor for gastric cancer. However, it is still controversial whether IM itself is precancerous or paracancerous. Here, we aimed to explore the precancerous nature of IM by analysing epigenetic alterations. DESIGN: Genome-wide DNA methylation analysis was conducted by EPIC BeadArray using IM crypts isolated by Alcian blue staining. Chromatin immunoprecipitation sequencing for H3K27ac and single-cell assay for transposase-accessible chromatin by sequencing were conducted using IM mucosa. NOS2 was induced using Tet-on gene expression system in normal cells. RESULTS: IM crypts had a methylation profile unique from non-IM crypts, showing extensive DNA hypermethylation in promoter CpG islands, including those of tumour-suppressor genes. Also, the IM-specific methylation profile, namely epigenetic footprint, was present in a fraction of gastric cancers with a higher frequency than expected, and suggested to be associated with good overall survival. IM organoids had remarkably high NOS2 expression, and NOS2 induction in normal cells led to accelerated induction of aberrant DNA methylation, namely epigenetic instability, by increasing DNA methyltransferase activity. IM mucosa showed dynamic enhancer reprogramming, including the regions involved in higher NOS2 expression. NOS2 had open chromatin in IM cells but not in gastric cells, and IM cells had frequent closed chromatin of tumour-suppressor genes, indicating their methylation-silencing. NOS2 expression in IM-derived organoids was upregulated by interleukin-17A, a cytokine secreted by extracellular bacterial infection. CONCLUSIONS: IM cells were considered to have a precancerous nature potentially with an increased chance of converting into cancer cells, and an accelerated DNA methylation induction due to abnormal NOS2 expression.


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Lesões Pré-Cancerosas , Neoplasias Gástricas , Humanos , Metilação de DNA , Neoplasias Gástricas/microbiologia , DNA , Cromatina/metabolismo , Metaplasia/genética , Metaplasia/metabolismo , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Mucosa Gástrica/metabolismo , Helicobacter pylori/genética , Infecções por Helicobacter/complicações
3.
Breast Cancer Res Treat ; 201(2): 317-328, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37378696

RESUMO

PURPOSE: HER2-positive breast cancer has a high chance of achieving pathological complete response when HSD17B4, responsible for peroxisomal ß-oxidation of very long-chain fatty acids (VLCFA) and estradiol, is methylation-silenced. Here, we aimed to identify the underlying molecular mechanism. METHODS: Using a HER2-positive breast cancer cell line, BT-474, control and knock-out (KO) clones were obtained. Metabolic characteristics were analyzed using a Seahorse Flux analyzer. RESULTS: HSD17B4 KO suppressed cellular proliferation, and enhanced sensitivity to lapatinib approximately tenfold. The KO led to accumulation of VLCFA and a decrease of polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and arachidonic acid. HSD17B4 KO increased Akt phosphorylation, possibly via decreased DHA, and genes involved in oxidative phosphorylation (OxPhos) and electron transport chain (ETC) were upregulated. Increased mitochondrial ATP production in the KO cells was confirmed by extracellular flux analyzer. Increased OxPhos led to severe dependence of the KO cells on pyruvate from glycolysis. Suppression of glycolysis by lapatinib led to severe delayed suppression of OxPhos in KO cells. CONCLUSION: HSD17B4 KO in BT-474 cells caused a decrease of PUFAs, increased Akt phosphorylation, enhanced glucose dependence of OxPhos, and increased sensitivity to inhibition of HER2, upstream of Akt. This mechanism may be applicable to other HER2-positive glucose-dependent breast cancer cells with HSD17B4 silencing.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Lapatinib/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Metilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Glucose , Linhagem Celular Tumoral , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteína Multifuncional do Peroxissomo-2/genética , Proteína Multifuncional do Peroxissomo-2/metabolismo
4.
Gynecol Oncol ; 167(3): 513-518, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36253303

RESUMO

OBJECTIVE: To establish a quantitative method to evaluate the DNA methylation level of an immediate upstream region of major BRCA1 transcriptional start sites (TSSs), and to investigate whether methylation of the region is a prognostic factor in high-grade serous ovarian cancer patients after neoadjuvant chemotherapy. METHODS: Ninety-two FFPE samples of advanced high-grade serous ovarian cancers after neoadjuvant chemotherapy between 2011 and 2018 were used for mutation and methylation analysis. DNA methylation levels were assessed by pyrosequencing and DNA methylation microarray. An association between methylation level (or a mutation) and progression-free survival was assessed by Kaplan-Meier analysis. RESULT: Major BRCA1 transcripts and CpG sites immediately upstream of their TSSs were identified, and a pyrosequencing method was developed. BRCA1 methylation, BRCA1/2 mutations, and a RAD51C mutation were detected in 17/79 (21.5%), 17/92 (18.5%), and 1/92 (1.1%) high-grade serious ovarian cancer samples. In univariate analysis, BRCA1 methylation and no residual tumor were associated with progression-free survival (BRCA1 methylation: P = 0.025, no residual tumor: P = 0.0026). Multivariate analysis showed that both BRCA1 methylation (P = 0.038, HR = 0.47, 95% CI: 0.21-0.96) and no residual tumor (P = 0.012, HR = 0.49, 95% CI: 0.28-0.85) were significant favorable prognostic factors. CONCLUSION: A quantitative method to estimate the methylation level of the immediate upstream region of major BRCA1 TSSs was established. Methylation of the region of was an independent favorable prognostic factor in high-grade serous ovarian cancer patients.


Assuntos
Metilação de DNA , Neoplasias Ovarianas , Humanos , Feminino , Prognóstico , Sítio de Iniciação de Transcrição , Mutação em Linhagem Germinativa , Neoplasias Ovarianas/patologia , Proteína BRCA1/genética
5.
Gastric Cancer ; 25(2): 336-345, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34557982

RESUMO

BACKGROUND: Prediction of tissue origin of esophagogastric junction (EGJ) adenocarcinomas can be important for therapeutic decision, but no molecular marker is available. Here, we aimed to develop such a marker taking advantage of tissue-specific profiles of DNA methylation. METHODS: DNA methylation profiles of gastric adenocarcinomas (GACs) were obtained by an Infinium HumanMethylation450 BeadChip array, and those of esophageal adenocarcinoma (EACs) were obtained from the TCGA database. DNA from formalin-fixed paraffin-embedded (FFPE) samples was analyzed by bisulfite pyrosequencing. RESULTS: In the screening set, 51 of 145,841 CpG sites in CpG islands were methylated at significantly higher levels in 30 GACs compared to those in 30 EACs. Among them, SLC46A3 and cg09177106 were unmethylated in all the 30 EACs. Predictive powers of these two markers were successfully confirmed in an independent validation set (18 GACs and 18 EACs) (SLC46A3, sensitivity = 77.8%, specificity = 100%; cg09177106, sensitivity = 83.3%, specificity = 94.4%), and could be applied to FFPE samples (37 GACs and 18 EACs) (SLC46A3, P = 0.0001; cg09177106, P = 0.0028). On the other hand, EAC-specific markers informative in the FFPE samples could not be isolated. Using these GAC-specific markers, nine of 46 (19.6%) TCGA EGJ adenocarcinomas were predicted to be GACs. CONCLUSIONS: Two GAC-specific markers, SLC46A3 and cg09177106, had a high specificity for identifying the tissue origin of EGJ adenocarcinoma.


Assuntos
Adenocarcinoma , Neoplasias Esofágicas , Neoplasias Gástricas , Adenocarcinoma/genética , Adenocarcinoma/patologia , Metilação de DNA , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Junção Esofagogástrica/patologia , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
6.
Carcinogenesis ; 42(2): 180-189, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33284950

RESUMO

Cancer-associated fibroblasts (CAFs) tend to have tumor-promoting capacity, and can provide therapeutic targets. Even without cancer cells, CAF phenotypes are stably maintained, and DNA methylation and H3K27me3 changes have been shown to be involved. Here, we searched for a potential therapeutic target in primary CAFs from gastric cancer and a mechanism for its dysregulation. Expression microarray using eight CAFs and seven non-CAFs (NCAFs) revealed that serum amyloid A1 (SAA1), which encodes an acute phase secreted protein, was second most upregulated in CAFs, following IGF2. Conditioned medium (CM) derived from SAA1-overexpressing NCAFs was shown to increase migration of gastric cancer cells compared with that from control NCAFs, and its tumor-promoting effect was comparable to that of CM from CAFs. In addition, increased migration of cancer cells by CM from CAFs was mostly canceled with CM from CAFs with SAA1 knockdown. Chromatin immunoprecipitation (ChIP)-quantitative PCR showed that CAFs had higher levels of H3K27ac, an active enhancer mark, in the promoter and the two far upstream regions of SAA1 than NCAFs. Also, BET bromodomain inhibitors, JQ1 and mivebresib, decreased SAA1 expression and tumor-promoting effects in CAFs, suggesting SAA1 upregulation by enhancer activation in CAFs. Our present data showed that SAA1 is a candidate therapeutic target from gastric CAFs and indicated that increased enhancer acetylation is important for its overexpression.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Proteína Amiloide A Sérica/genética , Neoplasias Gástricas/patologia , Acetilação , Azepinas/farmacologia , Azepinas/uso terapêutico , Fibroblastos Associados a Câncer/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Meios de Cultivo Condicionados/metabolismo , Elementos Facilitadores Genéticos , Gastrectomia , Mucosa Gástrica/citologia , Mucosa Gástrica/patologia , Mucosa Gástrica/cirurgia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Cultura Primária de Células , Piridonas/farmacologia , Piridonas/uso terapêutico , Proteína Amiloide A Sérica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/cirurgia , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Triazóis/uso terapêutico , Regulação para Cima
7.
Br J Cancer ; 125(12): 1647-1656, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34635821

RESUMO

BACKGROUND: The CpG island methylator phenotype of neuroblastoma (NBL) is strongly associated with poor prognosis and can be targeted by 5-aza-2'-deoxycytidine (5-aza-dC). Differentiation therapy is a standard maintenance therapy for high-risk NBLs. However, the in vivo effect of tamibarotene, a synthetic retinoic acid, and the efficacy of its combination with 5-aza-dC have not been studied. Here, we conducted a preclinical study to assess the in vivo tamibarotene effect and the combination. METHODS: Treatment effects were analysed by in vitro cell growth and differentiation state and by in vivo xenograft suppression. Demethylated genes were analysed by DNA methylation microarrays and geneset enrichment. RESULTS: Tamibarotene monotherapy induced neural extension and upregulation of differentiation markers of NBL cells in vitro, and tumour regression without severe side effects in vivo. 5-Aza-dC monotherapy suppressed tumour growth both in vitro and in vivo, and induced demethylation of genes related to nervous system development and function. Pre-treatment with 5-aza-dC in vitro enhanced upregulation of differentiation markers and genes involved in retinoic acid signaling. Pre-treatment with 5-aza-dC in vivo significantly suppressed tumour growth and reduced the variation in tumour sizes. CONCLUSIONS: Epigenetic drug-based differentiation therapy using 5-aza-dC and TBT is a promising strategy for refractory NBLs.


Assuntos
Metilação de DNA/genética , Neuroblastoma/tratamento farmacológico , Retinoides/uso terapêutico , Tretinoína/uso terapêutico , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Neuroblastoma/patologia , Retinoides/farmacologia , Transdução de Sinais , Tretinoína/farmacologia
8.
Genes Cells ; 25(1): 22-32, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31680384

RESUMO

DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Histonas/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Replicação do DNA , Histonas/fisiologia , Humanos , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
9.
BMC Pulm Med ; 21(1): 345, 2021 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-34732194

RESUMO

BACKGROUND: It remains unclear whether methicillin-resistant Staphylococcus aureus (MRSA) pneumonia is associated with higher mortality compared with non-MRSA pneumonia. This study's objective was to compare outcomes including in-hospital mortality and healthcare costs during hospitalisation between patients with MRSA pneumonia and those with non-MRSA pneumonia. METHODS: Using a national inpatient database in Japan, we conducted a 1:4 matched-pair cohort study of inpatients with community-acquired pneumonia from 1 April 2012 to 31 March 2014. In-hospital outcomes (mortality, length of stay and healthcare costs during hospitalisation) were compared between patients with and without MRSA infection. We performed multiple imputation using chained equations followed by multivariable regression analyses fitted with generalised estimating equations to account for clustering within matched pairs. All-cause in-hospital mortality and healthcare costs during hospitalisation were compared for pneumonia patients with and without MRSA infection. RESULTS: Of 450,317 inpatients with community-acquired pneumonia, 3102 patients with MRSA pneumonia were matched with 12,320 patients with non-MRSA pneumonia. The MRSA pneumonia patients had higher mortality, longer hospital stays and higher costs. Multivariable logistic regression analysis revealed that MRSA pneumonia was significantly associated with higher in-hospital mortality compared with non-MRSA pneumonia (adjusted odds ratio = 1.94; 95% confidence interval: 1.72-2.18; p < 0.001). Healthcare costs during hospitalisation were significantly higher for patients with MRSA pneumonia than for those with non-MRSA pneumonia (difference = USD 8502; 95% confidence interval: USD 7959-9045; p < 0.001). CONCLUSIONS: MRSA infection was associated with higher in-hospital mortality and higher healthcare costs during hospitalisation, suggesting that preventing MRSA pneumonia is essential.


Assuntos
Infecções Comunitárias Adquiridas/microbiologia , Infecções Comunitárias Adquiridas/mortalidade , Mortalidade Hospitalar , Pneumonia/microbiologia , Pneumonia/mortalidade , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Infecções Comunitárias Adquiridas/economia , Feminino , Custos de Cuidados de Saúde/estatística & dados numéricos , Humanos , Japão/epidemiologia , Masculino , Staphylococcus aureus Resistente à Meticilina , Pessoa de Meia-Idade , Pneumonia/economia , Pneumonia Estafilocócica , Infecções Estafilocócicas
10.
Proc Natl Acad Sci U S A ; 115(6): 1328-1333, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29358395

RESUMO

Genetic and epigenetic alterations are both involved in carcinogenesis, and their low-level accumulation in normal tissues constitutes cancer risk. However, their relative importance has never been examined, as measurement of low-level mutations has been difficult. Here, we measured low-level accumulations of genetic and epigenetic alterations in normal tissues with low, intermediate, and high cancer risk and analyzed their relative effects on cancer risk in the esophagus and stomach. Accumulation of genetic alterations, estimated as a frequency of rare base substitution mutations, significantly increased according to cancer risk in esophageal mucosae, but not in gastric mucosae. The mutation patterns reflected the exposure to lifestyle risk factors. In contrast, the accumulation of epigenetic alterations, measured as DNA methylation levels of marker genes, significantly increased according to cancer risk in both tissues. Patients with cancer (high-risk individuals) were precisely discriminated from healthy individuals with exposure to risk factors (intermediate-risk individuals) by a combination of alterations in the esophagus (odds ratio, 18.2; 95% confidence interval, 3.69-89.9) and by only epigenetic alterations in the stomach (odds ratio, 7.67; 95% confidence interval, 2.52-23.3). The relative importance of epigenetic alterations upon genetic alterations was 1.04 in the esophagus and 2.31 in the stomach. The differential impacts among tissues will be critically important for effective cancer prevention and precision cancer risk diagnosis.


Assuntos
Carcinoma de Células Escamosas/genética , Epigênese Genética , Neoplasias Esofágicas/genética , Mucosa Gástrica/fisiologia , Neoplasias Gástricas/genética , Biomarcadores Tumorais/genética , Metilação de DNA , Carcinoma de Células Escamosas do Esôfago , Feminino , Estudo de Associação Genômica Ampla , Infecções por Helicobacter/complicações , Humanos , Masculino , Taxa de Mutação , Mutação Puntual , Fatores de Risco , Fator de Transcrição AP-2/genética
11.
Gut ; 69(2): 243-251, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31085554

RESUMO

OBJECTIVE: Cancer-associated fibroblasts (CAFs), a major component of cancer stroma, can confer aggressive properties to cancer cells by secreting multiple factors. Their phenotypes are stably maintained, but the mechanisms are not fully understood. We aimed to show the critical role of epigenetic changes in CAFs in maintaining their tumour-promoting capacity and to show the validity of the epigenomic approach in identifying therapeutic targets from CAFs to starve cancer cells. DESIGN: Twelve pairs of primary gastric CAFs and their corresponding non-CAFs (NCAFs) were established from surgical specimens. Genome-wide DNA methylation and H3K27me3 analyses were conducted by BeadArray 450K and ChIP-on-Chip, respectively. Functions of potential a therapeutic target were analysed by inhibiting it, and prognostic impact was assessed in a database. RESULTS: CAFs had diverse and distinct DNA methylation and H3K27me3 patterns compared with NCAFs. Loss of H3K27me3, but not DNA methylation, in CAFs was enriched for genes involved in stem cell niche, cell growth, tissue development and stromal-epithelial interactions, such as WNT5A, GREM1, NOG and IGF2. Among these, we revealed that WNT5A, which had been considered to be derived from cancer cells, was highly expressed in cancer stromal fibroblasts, and was associated with poor prognosis. Inhibition of secreted WNT5A from CAFs suppressed cancer cell growth and migration. CONCLUSIONS: H3K27me3 plays a crucial role in defining tumour-promoting capacities of CAFs, and multiple stem cell niche factors were secreted from CAFs due to loss of H3K27me3. The validity of the epigenetic approach to uncover therapeutic targets for cancer-starving therapy was demonstrated.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Histona Desmetilases com o Domínio Jumonji/genética , Neoplasias Gástricas/genética , Meios de Cultivo Condicionados , Metilação de DNA , DNA de Neoplasias/genética , Epigenômica/métodos , Ontologia Genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Histona Desmetilases com o Domínio Jumonji/deficiência , Mutação , Nicho de Células-Tronco , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas
12.
Brain ; 142(6): 1675-1689, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31135049

RESUMO

The mechanism by which dopaminergic neurons are selectively affected in Parkinson's disease is not fully understood. In this study, we found a dramatic increase in the expression of catechol-O-methyltransferase (COMT), along with a lower level of DNA methylation, in induced pluripotent stem cell-derived dopaminergic neurons from patients with parkin (PARK2) gene mutations compared to those from healthy controls. In addition, a significant increase in the expression of COMT was found in dopaminergic neurons of isogenic PARK2 induced pluripotent stem cell lines that mimicked loss of function of PARK2 by CRISPR Cas9 technology. In dopamine transporter (DAT)-Cre mice, overexpression of COMT, specifically in dopaminergic neurons of the substantia nigra, produced cataleptic behaviours associated with impaired motor coordination. These findings suggest that upregulation of COMT, likely resulting from DNA hypomethylation, in dopaminergic neurons may contribute to the initial stage of neuronal dysfunction in Parkinson's disease.


Assuntos
Catecol O-Metiltransferase/genética , Neurônios Dopaminérgicos/metabolismo , Doença de Parkinson/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Transgênicos , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Substância Negra/metabolismo
13.
Allergol Int ; 68(1): 101-109, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30197185

RESUMO

BACKGROUND: Bronchial asthma is a chronic airway disease characterized by eosinophilic airway inflammation. Lung fibroblasts activated by IL-13 serve as important sources of chemokines, such as eotaxins, contributing to persistent eosinophilic inflammation. Src-homology 2-containing protein (CISH), belonging to the suppressor of cytokine signaling (SOCS) family, acts as a negative regulator of cytokine induction. The aim of this study was to elucidate the role of CISH in the production of eosinophil chemotactic chemokines in human lung fibroblasts. METHODS: Normal human lung fibroblasts were stimulated by IL-13, and global gene expression profile was assessed by cDNA microarray. Expression changes and downstream of IL-13 signaling were evaluated by quantitative RT-PCR, ELISA or western blotting. Loss- and gain-of-function analyses of CISH were performed by small interfering RNA and vector overexpression, respectively. RESULTS: Ingenuity pathway analysis revealed that IL-13 induced chemokine signaling, including the eotaxin family, while significantly suppressing IFN-α/ß signaling. Among eight SOCS family members, CISH was most strongly induced by IL-13 via phosphorylation of signal transducer and activator of transcription 6 (STAT6). Loss- and gain-of-function studies demonstrated that CISH negatively regulated the expression of CCL26. CONCLUSIONS: These findings suggest that CISH plays a key role in the eosinophilic inflammation associated with bronchial asthma by regulating IL-13-induced CCL26 production. Augmentation of CISH function could be a novel approach for treating eosinophilic inflammation in severe asthma.


Assuntos
Quimiocina CCL26/metabolismo , Fibroblastos/metabolismo , Interleucina-13/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Células Cultivadas , Quimiocina CCL26/genética , Eosinofilia/metabolismo , Humanos , Pulmão , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/genética
14.
Int Arch Allergy Immunol ; 175(1-2): 26-35, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29342461

RESUMO

BACKGROUND: Asthma is a chronic airway inflammatory disease characterized by airway remodeling, in which the bronchial smooth muscle (BSM) cells play an important role. Periostin, a biomarker that reflects Th2-driven inflammatory diseases such as asthma, may play an important role in the asthmatic airway. Although periostin is mainly produced in airway epithelial cells and fibroblasts after interleukin (IL)-13 stimulation, whether BSM cells produce periostin remains unclear. Therefore, we investigated periostin production in BSM cells and the mechanisms involved. METHODS: Human BSM cells were cultured, and the effect of IL-13 stimulation on periostin production was evaluated using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). We evaluated the phosphorylation of signal transducer and activator of transcription factor 6 (STAT6), extracellular signal-regulated kinase (ERK)1/2, and Akt after IL-13 stimulation. Furthermore, using ELISA, we evaluated the influence of several phosphorylation inhibitors on periostin production. RESULTS: Periostin mRNA expression increased in a dose- and time-dependent manner after IL-13 stimulation; periostin production was induced 24 and 48 h after stimulation. IL-13 stimulation induced the phosphorylation of STAT6, ERK1/2, and Akt. IL-13-induced periostin production was attenuated by inhibiting STAT6 phosphorylation and strongly suppressed by inhibiting mitogen-activated protein kinase kinase 1/2 phosphorylation or phosphatidylinositol 3-kinase (PI3K) phosphorylation. CONCLUSIONS: BSM cells produced periostin after IL-13 stimulation, via the JAK/STAT6, ERK1/2, and PI3K/Akt pathways. Understanding the mechanism of periostin production in BSM cells may help to clarify asthma pathogenesis.


Assuntos
Asma/imunologia , Brônquios/imunologia , Moléculas de Adesão Celular/metabolismo , Miócitos de Músculo Liso/fisiologia , Remodelação das Vias Aéreas , Moléculas de Adesão Celular/genética , Células Cultivadas , Humanos , Interleucina-13/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Fator de Transcrição STAT6/metabolismo , Transdução de Sinais , Regulação para Cima
15.
Gastric Cancer ; 21(5): 745-755, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29427040

RESUMO

BACKGROUND: The risk stratification of healthy individuals after Helicobacter pylori eradication is an urgent issue. The assessment of aberrant DNA methylation accumulated in gastric tissues with normal appearance, which can reflect overall epigenomic damage, is a promising strategy. We aimed to establish novel epigenetic cancer risk markers for H. pylori-eradicated individuals. METHODS: Gastric mucosa was collected from eight healthy volunteers without H. pylori infection (G1), 75 healthy individuals with gastric atrophy (G2), and 94 gastric cancer patients (G3) after H. pylori eradication. Genome-wide analysis was conducted using Infinium 450 K and differentially methylated probes were screened using large difference and iEVORA-based methods. Bisulfite pyrosequencing was used for validation. RESULTS: Screening, using 8 G1, 12 G2, and 12 G3 samples, isolated 57 candidates unmethylated in G1 and differentially methylated in G3 compared with G2. Validation for nine candidate markers (FLT3, LINC00643, RPRM, JAM2, ELMO1, BHLHE22, RIMS1, GUSBP5, and ZNF3) in 63 G2 and 82 G3 samples showed that all of them had significantly higher methylation levels in G3 than in G2 (P < 0.0001). Their methylation levels were highly correlated, which indicated that they reflect overall epigenomic damage. The candidates had sufficient performance (AUC: 0.70-0. 80) and high odds ratios (5.43-23.41), some of which were superior to a previous marker, miR-124a-3. The methylation levels of our novel markers were not associated with gastric atrophy, gender, or age. CONCLUSIONS: Novel epigenetic markers for gastric cancer risk optimized for H. pylori-eradicated individuals were established.


Assuntos
Biomarcadores Tumorais/genética , Epigênese Genética , Infecções por Helicobacter/complicações , Neoplasias Gástricas/genética , Adulto , Fatores Etários , Idoso , Atrofia/microbiologia , Metilação de DNA , Feminino , Mucosa Gástrica/patologia , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Infecções por Helicobacter/terapia , Helicobacter pylori/patogenicidade , Humanos , Masculino , Reprodutibilidade dos Testes , Neoplasias Gástricas/microbiologia
16.
Cancer Sci ; 108(3): 316-321, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27992956

RESUMO

Aberrant DNA methylation accumulated in normal tissues, namely methylation burden, is associated with risk of carcinogenesis. The levels of methylation burden are known to be influenced by multiple factors, such as genetic factors and strengths of carcinogenic factors. However, the impact of the degree of exposure to a carcinogenic factor is still unclear. Here, using a Mongolian gerbil model of Helicobacter pylori (H. pylori)-induced gastritis, we aimed to clarify the impact of the degree of exposure on methylation burden in normal gastric tissues. DNA methylation levels of four CpG islands, HE6, SA9, SB5, and SD2, increased by H. pylori infection, depending upon the infection period. After eradication of H. pylori, DNA methylation levels decreased, but tended to be higher in gastric mucosae with a longer infection period. DNA molecules with dense methylation, but not those with sparse methylation, increased depending upon the infection period. DNA methylation levels of one of the four CpG islands, SA9, tended to be higher in gastric mucosae of gerbils infected with H. pylori, even 50 weeks after eradication than in those of non-infected gerbils. These results showed for the first time that the levels of methylation burden in normal tissues are influenced by the degree of exposure to a carcinogenic factor.


Assuntos
Carcinogênese/genética , Metilação de DNA/genética , Mucosa Gástrica/patologia , Gastrite/microbiologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/patologia , Animais , Carcinógenos/toxicidade , Ilhas de CpG/genética , Modelos Animais de Doenças , Mucosa Gástrica/citologia , Mucosa Gástrica/microbiologia , Gerbillinae , Infecções por Helicobacter/microbiologia , Helicobacter pylori
17.
BMC Cancer ; 17(1): 613, 2017 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-28865438

RESUMO

BACKGROUND: The optimal postoperative treatment strategy for small cell lung cancer (SCLC) remains unclear, especially in patients with lymph node metastasis. We aimed to compare the outcomes of patients with SCLC and lymph node metastasis treated with postoperative adjuvant chemotherapy or chemoradiotherapy. METHODS: We retrospectively collected data on patients with postoperative SCLC diagnosed with N1 and N2 lymph node metastasis from the Diagnosis Procedure Combination database in Japan, between July 2010 and March 2015. We extracted data on patient age, sex, comorbidities, and TNM classification at lung surgery; operative procedures, chemotherapy drugs, and radiotherapy during hospitalization; and discharge status. Recurrence-free survival was compared between the chemotherapy and chemoradiotherapy groups using multivariable Cox regression analysis. RESULTS: Median recurrence-free survival was 1146 days (95% confidence interval [CI], 885-1407) in the chemotherapy group (n = 489) and 873 days (95% CI, 464-1282) in the chemoradiotherapy group (n = 75). There was no significant difference between these after adjusting for patient backgrounds (hazard ratio, 1.29; 95% CI, 0.91-1.84). CONCLUSIONS: There was no significant difference in recurrence-free survival between patients with SCLC and N1-2 lymph node metastasis treated with postoperative adjuvant chemotherapy and chemoradiotherapy. Further randomized clinical trials are needed to address this issue.


Assuntos
Quimiorradioterapia , Quimioterapia Adjuvante , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/radioterapia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Feminino , Humanos , Japão , Linfonodos/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Carcinoma de Pequenas Células do Pulmão/epidemiologia , Carcinoma de Pequenas Células do Pulmão/patologia , Resultado do Tratamento
18.
BMC Pulm Med ; 17(1): 108, 2017 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-28778188

RESUMO

BACKGROUND: Asthma exacerbation may require a visit to the emergency room as well as hospitalization and can occasionally be fatal. However, there is limited information about the prognostic factors for asthma exacerbation requiring hospitalization, and no methods are available to predict an inpatient's prognosis. We investigated the clinical features and factors affecting in-hospital mortality of patients with asthma exacerbation and generated a nomogram to predict in-hospital death using a national inpatient database in Japan. METHODS: We retrospectively collected data concerning hospitalization of adult patients with asthma exacerbation between July 2010 and March 2013 using the Japanese Diagnosis Procedure Combination database. We recorded patient characteristics and performed Cox proportional hazards regression analysis to assess the factors associated with all-cause in-hospital mortality. Then, we constructed a nomogram to predict in-hospital death. RESULTS: A total of 19,684 patients with asthma exacerbation were identified; their mean age was 58.8 years (standard deviation, 19.7 years) and median length of hospital stay was 8 days (interquartile range, 5-12 days). Among study patients, 118 died in the hospital (0.6%). Factors associated with higher in-hospital mortality included older age, male sex, reduced level of consciousness, pneumonia, and heart failure. A nomogram was generated to predict the in-hospital death based on the existence of seven variables at admission. The nomogram allowed us to estimate the probability of in-hospital death, and the calibration plot based on these results was well fitted to predict the in-hospital prognosis. CONCLUSION: Our nomogram allows physicians to predict individual risk of in-hospital death in patients with asthma exacerbation.


Assuntos
Asma/mortalidade , Mortalidade Hospitalar , Nomogramas , Adulto , Fatores Etários , Idoso , Asma/epidemiologia , Comorbidade , Transtornos da Consciência/epidemiologia , Bases de Dados Factuais , Progressão da Doença , Feminino , Insuficiência Cardíaca/epidemiologia , Hospitalização , Humanos , Japão/epidemiologia , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Pneumonia/epidemiologia , Prognóstico , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores Sexuais
19.
BMC Pulm Med ; 17(1): 128, 2017 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-28985724

RESUMO

BACKGROUND: Pneumonia is the most common cause of death in patients with dementia, but the outcomes of patients with dementia hospitalized with pneumonia are poorly understood. We sought to illuminate the association between dementia and in-hospital mortality and discharge status in patients hospitalized with pneumonia. METHODS: We used the Diagnosis Procedure Combination database, a national inpatient database in Japan, to identify retrospectively patients aged ≥60 years admitted to hospital with pneumonia during the study period of May 1, 2010 to March 31, 2014. We recorded their sex, age, body mass index, severity of pneumonia and comorbidities (including dementia). The outcomes were in-hospital mortality and discharge home. Multivariable Cox regression analysis was performed to analyze factors influencing discharge home. RESULTS: We identified 470,829 patients hospitalized with pneumonia; 45,031 were recorded as having dementia (9.6%). In-hospital mortality was 13.1% and 13.4% in patients with and without dementia, respectively (P = 0.63). The proportions of patients discharged home were 52.9% and 71.3% in patients with and without dementia, respectively (P < 0.001). The adjusted hazard ratio for discharge home for patients with dementia was 0.68 (95% confidence interval, 0.67-0.69; P < 0.001). CONCLUSIONS: In-hospital mortality from pneumonia did not differ significantly between patients with and without dementia; however, those with dementia were less likely to be discharged home.


Assuntos
Demência/complicações , Tempo de Internação/estatística & dados numéricos , Alta do Paciente/estatística & dados numéricos , Pneumonia/mortalidade , Idoso , Idoso de 80 Anos ou mais , Comorbidade , Feminino , Mortalidade Hospitalar , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Índice de Gravidade de Doença
20.
Gastric Cancer ; 19(2): 361-369, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25678126

RESUMO

BACKGROUND: Tumor samples are unavoidably contaminated with coexisting normal cells. Here, we aimed to establish a DNA methylation marker to estimate the fraction of gastric cancer (GC) cells in any DNA sample by isolating genomic regions specifically methylated in GC cells. METHODS: Genome-wide and gene-specific methylation analyses were conducted with an Infinium HumanMethylation450 BeadChip array and by quantitative methylation-specific PCR, respectively. Purified cancer and noncancer cells were prepared by laser-capture microdissection. TP53 mutation data were obtained from our previous study using next-generation target sequencing. RESULTS: Genome-wide DNA methylation analysis of 12 GC cell lines, 30 GCs, six normal gastric mucosae, one sample of peripheral leukocytes, and four noncancerous gastric mucosae identified OSR2, PPFIA3, and VAV3 as barely methylated in normal cells and highly methylated in cancer cells. Quantitative methylation-specific PCR using 26 independent GCs validated that one or more of them was highly methylated in all of the GCs. Using four pairs of purified cells, we confirmed the three genes were highly methylated (85 % or more) in cancer cells and barely methylated (5 % or less) in noncancer cells. The cancer cell fraction assessed by the panel of the three genes showed good correlation with that assessed by the TP53 mutant allele frequency in 13 GCs (r = 0.77). After correction of the GC cell fraction, unsupervised clustering analysis of the genome-wide DNA methylation profiles yielded clearer clustering. CONCLUSIONS: A DNA methylation marker-namely, the panel of the three genes-is useful to estimate the cancer cell fraction in GCs.


Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Neoplasias Gástricas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Infecções por Helicobacter/genética , Humanos , Mutação , Proteínas Proto-Oncogênicas c-vav/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética
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