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Takeshi Sawada,
Eiichiro Yamamoto,
Hiromu Suzuki,
Masanori Nojima,
Reo Maruyama,
Yoshihiro Shioi,
Risaburo Akasaka,
Seiko Kamimae, Taku Harada,
Masami Ashida,
Masahiro Kai,
Yasushi Adachi,
Hiroyuki Yamamoto,
Kohzoh Imai,
Minoru Toyota,
Fumio Itoh,
Tamotsu Sugai
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ABSTRACT: Colorectal cancers (CRCs) exhibit multiple genetic alterations, including allelic imbalances (copy number alterations, CNAs) at various chromosomal loci. In addition to genetic aberrations, DNA methylation also plays important roles in the development of CRC. To better understand the clinical relevance of these genetic and epigenetic abnormalities in CRC, we performed an integrative analysis of copy number changes on a genome-wide scale and assessed mutations of TP53, KRAS, BRAF, and PIK3CA and DNA methylation of six marker genes in single glands isolated from 39 primary tumors. Array-based comparative genomic hybridization (array-CGH) analysis revealed that genomic losses commonly occurred at 3q26.1, 4q13.2, 6q21.32, 7q34, 8p12-23.3, 15qcen and 18, while gains were commonly found at 1q21.3-23.1, 7p22.3-q34, 13q12.11-14.11, and 20. The total numbers and lengths of the CNAs were significantly associated with the aberrant DNA methylation and Dukes' stages. Moreover, hierarchical clustering analysis of the array-CGH data suggested that tumors could be categorized into four subgroups. Tumors with frequent DNA methylation were most strongly enriched in subgroups with infrequent CNAs. Importantly, Dukes' D tumors were enriched in the subgroup showing the greatest genomic losses, whereas Dukes' C tumors were enriched in the subgroup with the greatest genomic gains. Our data suggest an inverse relationship between chromosomal instability and aberrant methylation and a positive association between genomic losses and distant metastasis and between genomic gains and lymph node metastasis in CRC. Therefore, DNA copy number profiles may be predictive of the metastatic behavior of CRCs. © 2012 Wiley Periodicals, Inc.
Genes Chromosomes and Cancer 10/2012; · 3.31 Impact Factor
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Eiichiro Yamamoto,
Hiromu Suzuki,
Hiro-O Yamano,
Reo Maruyama,
Masanori Nojima,
Seiko Kamimae,
Takeshi Sawada,
Masami Ashida,
Kenjiro Yoshikawa,
Tomoaki Kimura, [......], Taku Harada,
Ryo Suzuki,
Akiko Sato,
Masahiro Kai,
Yasushi Sasaki,
Takashi Tokino,
Tamotsu Sugai,
Kohzoh Imai,
Yasuhisa Shinomura,
Minoru Toyota
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ABSTRACT: The concept of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) is widely accepted, although the timing of its occurrence and its interaction with other genetic defects are not fully understood. Our aim in this study was to unravel the molecular development of CIMP cancers by dissecting their genetic and epigenetic signatures in precancerous and malignant colorectal lesions. We characterized the methylation profile and BRAF/KRAS mutation status in 368 colorectal tissue samples, including precancerous and malignant lesions. In addition, genome-wide copy number aberrations, methylation profiles, and mutations of BRAF, KRAS, TP53, and PIK3CA pathway genes were examined in 84 colorectal lesions. Genome-wide methylation analysis of CpG islands and selected marker genes revealed that CRC precursor lesions are in three methylation subgroups: CIMP-high, CIMP-low, and CIMP-negative. Interestingly, a subset of CIMP-positive malignant lesions exhibited frequent copy number gains on chromosomes 7 and 19 and genetic defects in the AKT/PIK3CA pathway genes. Analysis of mixed lesions containing both precancerous and malignant components revealed that most aberrant methylation is acquired at the precursor stage, whereas copy number aberrations are acquired during the progression from precursor to malignant lesion. Our integrative genomic and epigenetic analysis suggests early onset of CIMP during CRC development and indicates a previously unknown CRC development pathway in which epigenetic instability associates with genomic alterations.
American Journal Of Pathology 09/2012; 181(5):1847-61. · 4.89 Impact Factor
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Hiroyuki Takamaru,
Eiichiro Yamamoto,
Hiromu Suzuki,
Masanori Nojima,
Reo Maruyama,
Hiro-O Yamano,
Kenjiro Yoshikawa,
Tomoaki Kimura, Taku Harada,
Masami Ashida,
Ryo Suzuki,
Hiroyuki Yamamoto,
Masahiro Kai,
Takashi Tokino,
Tamotsu Sugai,
Kohzoh Imai,
Minoru Toyota,
Yasuhisa Shinomura
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ABSTRACT: Aberrant DNA methylation is implicated in the epigenetic field defect seen in gastric cancer. Our aim in this study was to identify predictive biomarkers by screening for DNA methylation in noncancerous background gastric mucosa from patients with gastric cancer. Using methylated-CpG island amplification coupled with CpG island microarray (MCAM) analysis, we identified 224 genes that were methylated in the noncancerous gastric mucosa of patients with gastric cancer. Among them, RASGRF1 methylation was significantly elevated in gastric mucosa from patients with either intestinal or diffuse type gastric cancer, as compared with mucosa from healthy individuals (8.3% vs. 22.4%, P < 0.001; 8.3% vs. 19.4%, P < 0.001). RASGRF1 methylation was independent of mucosal atrophy and could be used to distinguish both serum pepsinogen test-positive [sensitivity, 70.0%; specificity, 86.7%; area under the receiver operator characteristic (ROC) curve, AUC, 0.763] and -negative patients with gastric cancer (sensitivity, 72.2%; specificity, 87.0%; AUC, 0.844) from healthy individuals. Ectopic expression of RASGRF1 suppressed colony formation and Matrigel invasion by gastric cancer cells, suggesting it may be involved in gastric tumorigenesis. Collectively, our data suggest that RASGRF1 methylation is significantly involved in an epigenetic field defect in the stomach, and that it could be a useful biomarker to identify individuals at high risk for gastric cancer. Cancer Prev Res; 5(10); 1203-12. ©2012 AACR.
Cancer Prevention Research 09/2012; 5(10):1203-12. · 4.91 Impact Factor
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ABSTRACT: A 39-year-old patient with Crohn's disease (CD) was referred to our hospital for maintenance treatment of CD. He was diagnosed as having CD of the small and large intestines at 32 years old. He underwent partial resection of the ileum at 35 years old because of ileal perforation. He had received enteral nutritional supplement (1200 kcal/d) and metronidazole preparation (500 mg/d), and was in remission Crohn's disease activity index 73. We performed a routine gastroduodenal endoscopic examination, which revealed the representative endoscopic findings of gastroduodenal lesions in CD, namely, bamboo-joint-like appearance of the gastric body and cardia and a notched sign in the duodenum. These findings were clearly observed by using indigo carmine dye spraying. In our patient, typical gastroduodenal findings were observed even in the remission stage, suggesting that these findings would contribute to the early diagnosis of CD not only in the active stage but also during remission.
World journal of gastrointestinal endoscopy. 03/2012; 4(3):96-8.
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Tomoaki Kimura,
Eiichiro Yamamoto,
Hiro-O Yamano,
Hiromu Suzuki,
Seiko Kamimae,
Masanori Nojima,
Takeshi Sawada,
Masami Ashida,
Kenjiro Yoshikawa,
Ryo Takagi,
Ryusuke Kato, Taku Harada,
Ryo Suzuki,
Reo Maruyama,
Masahiro Kai,
Kohzoh Imai,
Yasuhisa Shinomura,
Tamotsu Sugai,
Minoru Toyota
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ABSTRACT: Sessile serrated adenomas (SSAs) are known to be precursors of sporadic colorectal cancers (CRCs) with microsatellite instability (MSI), and to be tightly associated with BRAF mutation and the CpG island methylator phenotype (CIMP). Consequently, colonoscopic identification of SSAs has important implications for preventing CRCs, but accurate endoscopic diagnosis is often difficult. Our aim was to clarify which endoscopic findings are specific to SSAs.
The morphological, histological and molecular features of 261 specimens from 226 colorectal tumors were analyzed. Surface microstructures were analyzed using magnifying endoscopy. Mutation in BRAF and KRAS was examined by pyrosequencing. Methylation of p16, IGFBP7, MLH1 and MINT1, -2, -12 and -31 was analyzed using bisulfite pyrosequencing.
Through retrospective analysis of a training set (n=145), we identified a novel surface microstructure, the Type II open-shape pit pattern (Type II-O), which was specific to SSAs with BRAF mutation and CIMP. Subsequent prospective analysis of an independent validation set (n=116) confirmed that the Type II-O pattern is highly predictive of SSAs (sensitivity, 65.5%; specificity, 97.3%). BRAF mutation and CIMP occurred with significant frequency in Type II-O-positive serrated lesions. Progression of SSAs to more advanced lesions was associated with further accumulation of aberrant DNA methylation and additional morphological changes, including the Type III, IV and V pit patterns.
Our results suggest the Type II-O pit pattern is a useful hallmark of the premalignant stage of CRCs with MSI and CIMP, which could serve to improve the efficacy of colonoscopic surveillance.
The American Journal of Gastroenterology 03/2012; 107(3):460-9. · 7.28 Impact Factor
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Seiko Kamimae,
Eiichiro Yamamoto,
Hiro-o Yamano,
Masanori Nojima,
Hiromu Suzuki,
Masami Ashida,
Tomo Hatahira,
Akiko Sato,
Tomoaki Kimura,
Kenjiro Yoshikawa, [......],
Hiroyuki Takamaru,
Reo Maruyama,
Masahiro Kai,
Morie Nishiwaki,
Tamotsu Sugai,
Yasushi Sasaki,
Takashi Tokino,
Yasuhisa Shinomura,
Kohzoh Imai,
Minoru Toyota
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ABSTRACT: Although conventional colonoscopy is considered the gold standard for detecting colorectal tumors, accurate staging is often difficult because advanced histology may be present in small colorectal lesions. We collected DNA present in mucosal wash fluid from patients undergoing colonoscopy and then assessed the methylation levels of four genes frequently methylated in colorectal cancers to detect invasive tumors. We found that methylation levels in wash fluid were significantly higher in patients with invasive than those with noninvasive tumors. Cytologic and K-ras mutation analyses suggested that mucosal wash fluid from invasive tumors contained greater numbers of tumor cells than wash fluid from noninvasive tumors. Among the four genes, levels of mir-34b/c methylation had the greatest correlation with the invasion and showed the largest area under the receiver operating characteristic curve (AUC = 0.796). Using cutoff points of mir-34b/c methylation determined by efficiency considerations, the sensitivity/specificity were 0.861/0.657 for the 13.0% (high sensitivity) and 0.765/0.833 for the 17.8% (well-balanced) cutoffs. In the validation test set, the AUC was also very high (0.915), the sensitivity/specificity were 0.870/0.875 for 13.0% and 0.565/0.958 for 17.8%. Using the diagnostic tree constructed by an objective algorithm, the diagnostic accuracy of the invasiveness of colorectal cancer was 91.3% for the training set and 85.1% for the test set. Our results suggest that analysis of the methylation of DNA in mucosal wash fluid may be a good molecular marker for predicting the invasiveness of colorectal tumors.
Cancer Prevention Research 05/2011; 4(5):674-83. · 4.91 Impact Factor
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ABSTRACT: Inhibitory effects of reduced glutathione (GSH) on serum enzymes including alanine aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK) were investigated in the hypoglycemic rabbits. Hypoglycemia lasting for 60 min was induced by intravenous injection of insulin (10U/kg) and then recovered by intravenous glucose injection. Serum levels of ALT, AST, LDH and CK increased significantly (p<0.05) at 6h after the induction of hypoglycemia. Plasma GSH, oxidized glutathione (GSSG) and total glutathione (TGSH) began to increase significantly (p<0.05) at 1h after the insulin injection, and GSSG/TGSH ratio rose significantly (p<0.05) at 6h after the induction of hypoglycemia. GSSG contents and GSSG/TGSH ratio in quadriceps significantly increased during hypoglycemia. Administration of GSH significantly decreased plasma GSSG levels, GSSG/TGSH ratio (p<0.05) and suppressed the rise of serum enzymes induced by hypoglycemia. These results suggest that GSH administration may play a preventive role for increases of serum enzymes by experimental hypoglycemia.
Diabetes Research and Clinical Practice 09/2007; 77(3):357-62. · 2.75 Impact Factor
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ABSTRACT: Basic helix-loop-helix (bHLH) transcription factors are known as key regulators for mesenchymal differentiation. The present study showed that overexpression of Twist-1, a bHLH transcription factor, suppresses bone morphogenetic protein (BMP)-induced osteoblast differentiation, and downregulation of endogenous Twist-1 enhances BMP signaling. Maximal inhibition of BMP signaling was observed when Twist-1 was bound to E47, which markedly enhanced the stability of Twist-1. Co-immunoprecipitation assays revealed that Twist-1 formed a complex with Smad4 and histone deacetylase (HDAC) 1 in MC3T3-E1 cells stably expressing Twist-1. With trichostatin, an HDAC inhibitor, osteogenic factors such as alkaline phosphatase, Runx2 and osteopontin increased. Those results suggested that Twist-1 inhibited BMP signaling by recruiting HDAC1 to Smad4. Furthermore, the inhibitory effects of Twist-1 on BMP signaling were overcome by Id1 through induction of Twist-1 degradation. These findings suggest that Twist-1 can act as an inhibitor of BMP signaling, and Id1 can regulate BMP signaling through a positive feedback loop repressing Twist-1 function. These two molecules may therefore regulate differentiation of mesenchymal cells into progeny such as osteoblasts by controlling BMP signaling.
Journal of Cell Science 05/2007; 120(Pt 8):1350-7. · 6.11 Impact Factor
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ABSTRACT: Isolation of effective therapeutic genes is critical for the advancement of gene therapy for various diseases, including vascular diseases and cancers. The goal of the present study was to screen a human cDNA library, using a hemagglutinating virus of Japan envelope (HVJ-E) vector, to isolate candidate genes with potent therapeutic potential. The advantages of a high-throughput functional screening system based on the HVJ-E vector include (1) rapid preparation of the vector containing the DNA library, (2) effective fusion-mediated transfer of the plasmids to various cells with minimal toxicity, and (3) easy cloning of candidate genes by transformation of Escherichia coli. These advantages resulted in a lower probability of damage to isolated clones and in minimization of the time needed to screen for candidate genes. Screening of a human heart library for candidate genes to regulate endothelial cell growth identified three growth-stimulating genes, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and c-fos promoter activity, the products of which were more potent than vascular endothelial growth factor. Similarly, two growth-inhibiting genes were identified, the effects of which were similar to angiostatin. Overall, this novel system will help advance our understanding of cell biology and promote the utility of human gene therapy.
Human Gene Therapy 05/2006; 17(4):470-5. · 4.22 Impact Factor
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ABSTRACT: 1. We investigated whether chronic suppression of the renin-angiotensin system, which is known to be associated with reductions in microvascular density and vasodilator responsiveness of skeletal muscle, could affect exercise capacity in normotensive rats. 2. Rats were placed on normal rat chow, normal rat chow with captopril (100 mg/kg per day) or a high-salt diet (HS; 4%) for 4 weeks. Following these interventions, rats with indwelling carotid artery catheters were submitted to stepwise increasing exercise on a motor treadmill at a speed of 10, 20 and 30 m/min for 4 min while blood lactate was measured. 3. Blood lactate after exercise at a speed of 20 m/min was significantly higher and the duration during which rats were able to run at a speed of 30 m/min was significantly shorter in captopril-treated rats and rats fed an HS diet compared with control rats. 4. We conclude that chronic treatment with captopril or HS diet could reduce the exercise capacity in inactive normotensive rats, probably through chronic inhibition of the renin-angiotensin system.
Clinical and Experimental Pharmacology and Physiology 05/2004; 31(4):197-201. · 1.85 Impact Factor
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ABSTRACT: The relative contributions of scapulothoracic and glenohumeral motion at different rates of shoulder motion were studied through adduction to abduction in the scapular plane. Nineteen shoulders of 10 healthy individuals (all men, 24-30 years of age) were analyzed using an image intensifier and a high-resolution digital video system. High- and low-speed motion consisted of 2 and 4 seconds per one cycle, respectively, from abduction to adduction in the scapular plane. Glenohumeral and scapulothoracic ratios were fixed at low speed and these results agree with the finding of other researchers. Ratios at high speed were not fixed and differed significantly from those at low speed. Ratios were high at the beginning of abduction or adduction and at angles beyond 40 degrees abduction, then decreased according to the arm movement. Glenohumeral motion at high speed was more dominant at the beginning of abduction or adduction beyond the setting phase, then became less dominant according to the arm movement, compared with the motion at low speed.
Clinical Orthopaedics and Related Research 09/2002; · 2.53 Impact Factor
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[show abstract]
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ABSTRACT: The relative contributions of scapulothoracic and glenohumeral motion at different rates of shoulder motion were studied through adduction to abduction in the scapular plane. Nineteen shoulders of 10 healthy individuals (all men, 24–30 years of age) were analyzed using an image intensifier and a high-resolution digital video system. High- and low-speed motion consisted of 2 and 4 seconds per one cycle, respectively, from abduction to adduction in the scapular plane. Glenohumeral and scapulothoracic ratios were fixed at low speed and these results agree with the finding of other researchers. Ratios at high speed were not fixed and differed significantly from those at low speed. Ratios were high at the beginning of abduction or adduction and at angles beyond 40° abduction, then decreased according to the arm movement. Glenohumeral motion at high speed was more dominant at the beginning of abduction or adduction beyond the setting phase, then became less dominant according to the arm movement, compared with the motion at low speed.
Clinical Orthopaedics and Related Research 07/2002; 401:119-124. · 2.53 Impact Factor
