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T Ushijima

Publications and source records attributed to T Ushijima.

At least 19 recordsLinked to original sources
PubMed2006

Genome-wide profiling of promoter methylation in human.

DNA methylation in the promoter region of a gene is associated with a loss of that gene's expression and plays an important role in gene silencing. The inactivation of tumor-suppressor genes by aberrant methylation in the promoter region is well recognized in carcinogenesis. However, there has been little study in this area when it comes to genome-wide profiling of the promoter methylation. Here, we developed a genome-wide profiling method called Microarray-based Integrated Analysis of Methylation by Isoschizomers to analyse the DNA methylation of promoter regions of 8091 human genes. With this method, resistance to both the methylation-sensitive restriction enzyme HpaII and the methylation-insensitive isoschizomer MspI was compared between samples by using a microarray with promoter regions of the 8091 genes. The reliability of the difference in HpaII resistance was judged using the difference in MspI resistance. We demonstrated the utility of this method by finding epigenetic mutations in cancer. Aberrant hypermethylation is known to inactivate tumour suppressor genes. Using this method, we found that frequency of the aberrant promoter hypermethylation in cancer is higher than previously hypothesized. Aberrant hypomethylation is known to induce activation of oncogenes in cancer. Genome-wide analysis of hypomethylated promoter sequences in cancer demonstrated low CG/GC ratio of these sequences, suggesting that CpG-poor genes are sensitive to demethylation activity in cancer.

Adenocarcinoma↗
PubMed2006

Decreased fidelity in replicating DNA methylation patterns in cancer cells leads to dense methylation of a CpG island.

Cancer cells that have a large number of aberrantly methylated CpG islands (CGIs) are known to have CpG island methylator phenotype (CIMP), and decreased fidelity in replicating methylation patters has been analyzed as an underlying mechanism. First we developed a method to analyze the number of errors in replicating CpG methylation patterns in a defined period. A single cell was expanded into 106 cells, and the number of errors during the culture was measured by counting the deviation from the original methylation patterns. It was shown that methylated status of a CpG site was more stably inherited than unmethylated status, suggesting that the genome is constantly exposed to de novo methylation. Promoter CGIs showed higher fidelities than CGIs outside promoter regions. We then analyzed error rates in two gastric cancer cell lines without CIMP and two with CIMP for five promoter CGIs. Two CIMP(-) cell lines showed error rates smaller than 1.0x10(-3) errors per site per generation (99.90%-100% fidelity) for all the five CGIs. In contrast, AGS cells showed significantly elevated error rates, mainly due to increased de novo methylation, in three CGIs (1.6- to 3.2-fold), and KATOIII cells showed a significantly elevated error rate in one CGI (2.2-fold). Presence of densely methylated DNA molecules was observed only in KATOIII and AGS. These data demonstrated that some cancer cells have decreased fidelity in replicating CpG methylation patterns that underlie CIMP.

Breast↗
PubMed2005

Sparse and wavy hair: a new model for hypoplasia of hair follicle and mammary glands on rat chromosome 17.

Mutant animals in the skin and hair have been used to identify important genes in biomedical research. We describe a new mutant rat, sparse and wavy hair (swh), that spontaneously arose in a colony of inbred WTC rats. The mutant phenotype was characterized by sparse and wavy hair, which was most prominent at age 3-4 weeks, and was inherited in an autosomal recessive manner. The swh/swh rats showed impaired gain of body weight, and their hair follicles were reduced both in number and size, associated with hypoplasia of the sebaceous glands and the subcutaneous fat tissue. Female swh/swh rats were unable to suckle their offspring. Their mammary glands were hypoplastic, and differentiation of mammary epithelial and myoepithelial cells was impaired. Linkage analysis of 579 backcross rats localized the swh locus to a .35-cM region between D17Rat131 and D17Rat50 in the distal end of rat Chr 17. The swh locus spanned the 3.7-Mb genomic region where 24 genes have been mapped and corresponded to the centromere region of the mouse Chr 2 or the region of the human Chr 10p11.1-p14. None of the genes or loci described in mouse or human hair and skin diseases mapped to these regions. These findings suggest that the rat swh is a novel mutation associated with impaired development of the skin appendages, such as hair follicles, sebaceous glands, and mammary glands, and will provide an experimental model to clarify a gene and mechanisms for development of skin appendages.

Animals↗
PubMed2005

Sox2 expression in human stomach adenocarcinomas with gastric and gastric-and-intestinal-mixed phenotypes.

AIMS: Other than ectopic expression of intestinal transcription factors, Cdx1 and Cdx2, the molecular mechanisms underlying gastric and intestinal phenotypes of human stomach adenocarcinomas have yet to be clarified in detail. We have reported that Sox2, an HMG-box gastric transcription factor, is expressed in normal gastric mucosa and down-regulated in intestinal metaplasia. METHODS AND RESULTS: We analysed mRNA levels of Sox2 and other differentiation markers in 50 surgically resected stomach adenocarcinomas, immunohistochemically classified into gastric (G), gastric-and-intestinal (GI)-mixed, solely intestinal (I), and null (N) types. Sox2 was found to be transcribed in G and GI-mixed type adenocarcinomas in accordance with MUC5AC and MUC6 expression, while Cdx1 and Cdx2 were up-regulated in GI-mixed and I types along with the expression of MUC2 and villin. In the N type, both gastric and intestinal transcription factors were suppressed. Immunohistochemistry confirmed expression of Sox2 in MUC5AC+ lesions and Cdx2 localization together with MUC2. A stomach adenocarcinoma cell line, KATOIII, demonstrated both MUC5AC and Sox2, although MUC5AC mRNA was not detected in the Sox2+ AGS cell line. CONCLUSIONS: Sox2 may play an important role in maintaining a gastric phenotype in stomach cancers as well as in normal tissue, in cooperation with other cofactor(s).

Adenocarcinoma↗
PubMed2002

Down-regulation of Lsm1 is involved in human prostate cancer progression.

Elucidation of genetic alterations is an approach to understanding the underlying molecular mechanisms of progression of human prostate cancers. We have searched for genes differentially expressed in advanced prostate cancers using cDNA-representational difference analysis, and thereby isolated the Lsm1 as one of down-regulated gene. An Lsm1 expression vector was transfected into PC3 cells, normally featuring down-regulated Lsm1, and four transfectants were established. No differences in morphology or cell proliferation were evident in comparison with parent PC3 or PC3/mock-transfectants. In contrast, significant suppression of invasive potential or metastatic ability of Lsm1 transfectants was observed in the Matrigel chemoinvasion assay and in nude mice, respectively. With human prostate cancers, almost all of informative prostatectomised cases without neoadjuvant therapy showed allelic retention in the Lsm1 region, whereas refractory cancers frequently showed allelic loss in this region. No critical gene mutations were found in open reading frame of Lsm1 in prostate cancers examined by PCR-SSCP analysis, including localised and refractory cancers. These results suggest that Lsm1 is deeply involved in prostate cancer progression through its down-regulation, independent of any gene mutation.

Animals↗
PubMed2001

Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis.

Aberrantly hypermethylated genes in human lung cancers were searched for by a genome scanning technique, methylation-sensitive-representational difference analysis (MS-RDA). A total of 59 DNA fragments were isolated as those methylated more heavily in either/both of two lung squamous cell carcinoma cell lines, EBC-1 and LK-2, than in a primary culture of normal human bronchial epithelium, NHBE. Thirty-four DNA fragments, whose hypermethylation was confirmed in primary squamous cell carcinomas, were sequenced. By database searches, 17 of them were shown to be located within 2 kb of putative CpG islands, and five of the 17 DNA fragments had transcribed regions of known genes in their vicinities. By RT-PCR of the five genes in the carcinoma cell lines and NHBE, decreased expression of HTR1B (5-hydroxytryptamine receptor 1B) and EDN1 (endothelin-1) was observed. Sequencing after bisulfite modification showed that the CpG island in the promoter region of HTR1B was hypermethylated, while that of EDN1 was not. Demethylation and re-expression of HTR1B were observed after treatment of LK-2 cells with 5-aza-2'-deoxycytidine. In primary lung cancers, decreased mRNA expression of HTR1B was observed in 11 of 20 cases, and that of EDN1 was in 16 of 20 cases. Immunohistochemical analysis of endothelin-1 confirmed that its immunoreactivity was reduced in squamous cell carcinoma cells compared with that in normal bronchial epithelial cells. Considering that endothelin-1 induces apoptosis in melanoma cells and that silencing of endothelin receptor B is observed in prostate cancers, its reduced expression was speculated to confer a growth advantage to lung cancer cells. MS-RDA was shown to isolate DNA fragments that are hypermethylated and silenced, such as HTR1B, and those whose expressions are altered and the methylation statuses outside the promoter region are altered, such as EDN1.

Aged↗
PubMed2001

The presence of single nucleotide instability in human breast cancer cell lines.

The presence of single nucleotide instability, an increase of spontaneous point mutation rates (MR: number of mutations per cell division) without microsatellite instability, was demonstrated previously in two rat mammary carcinoma cell lines. In this study, spontaneous point MRs were analyzed in human breast cancer cell lines by the fluctuation test using the hypoxanthine-guanine phosphoribosyltransferase (hprt) marker gene. MRs obtained for six breast cancer cell lines, MCF-7, ZR-75-1, T-47D, MDA-MB-231, MDA-MB-468, and BT-474, all of which were proficient in G/T mismatch binding and reported to be negative for microsatellite instability, were 7.6, 4.6, 6.3, 2.2, 5.6, and 19 x 10(-7) mutations/hprt/cell division. Those in normal human mammary epithelial cells and in a colon cancer cell line with proficient mismatch repair, SW480, were 1.6 and 1.4 x 10(-7) mutations/hprt/cell division, respectively. These findings showed that single nucleotide instability was also present in five of the six human breast cancer cell lines and strongly indicates it has important roles in human and rat mammary carcinogenesis.

Base Pair Mismatch↗
PubMed2001

Linkage disequilibrium pattern in the L-myc gene in Italian and Japanese non-small-cell lung-cancer patients.

Italian and Japanese non-small-cell lung-cancer patients were genotyped for an intragenic L-myc EcoRI restriction site polymorphism previously reported to be associated with lung-tumor prognosis in Asian populations but not in Caucasians. Screening of the L-myc sequence in Italian samples allowed identification of 2 additional 3'-UTR SNPs, located 2.3-3.0 kb from the EcoRI polymorphism, but no coding polymorphism was found. No significant association was found between any of the 3 SNPs and lung-tumor prognosis in Italian patients, consistent with the reported difference between Caucasian and Asian populations. Moreover, the newly discovered polymorphisms in the Italian group were not present in Japanese patients. Significant LD between EcoRI and the 2 other SNPs was detected in the Italian population, whereas no significant LD between the 2 3'-UTR markers was detected despite their close proximity (0.7 kb). Thus, the disparate conclusions about the role of L-myc polymorphism in tumor prognosis among different populations may rest in population-specific LD between the functional gene and the L-myc polymorphism.

Adenocarcinoma↗
PubMed2001

LacI transgenic animal study: relationships among DNA-adduct levels, mutant frequencies and cancer incidences.

In the processes of carcinogenesis caused by genotoxic carcinogens, DNA-adduct formation and resultant genetic changes are crucially important. In this report, the relationship between DNA-adduct levels and mutant frequencies (MFs), DNA-adduct levels and cancer incidences, and MFs and cancer incidences induced by heterocyclic amines (HCAs), to which humans are exposed on daily basis were investigated. There was no direct correlation between adduct levels and MFs detected after feeding Big Blue mice with 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), in a comparison among various organs. Further, there was no direct correlation between DNA-adduct levels and cancer incidences, in a comparison among various organs of F344 rats. Since DNA-adducts are fixed as mutations after cell proliferation, and mutations in cancer-related genes result in cancer development, it is expected that MFs directly correlate with cancer incidences. However, there was no direct correlation between MFs and cancer incidences. Possible mechanisms involved in the discordance between DNA damage markers and cancer incidences are discussed.

Amines↗
PubMed2001

Single nucleotide instability without microsatellite instability in rat mammary carcinomas.

Mutation frequencies (MnFs) of the lacI transgene and mutation rates (MRs) of the endogenous hprt gene were analyzed in two mammary carcinoma cell lines that we established from mammary carcinomas that had been induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in female lacI-transgenic rats. Using the lacI transgene, corrected MnF, which is the number of independent lacI mutations that occurred while 102 cells expanded into 10(7) cells and which reflect the dynamic increase of point mutations, was measured. The corrected MnFs in the two mammary carcinoma cell lines (59 x 10(-6) and 72 x 10(-6) mutations) were significantly higher than that in the primary culture of normal mammary epithelium (4.7 x 10(-6)). MRs of the hprt gene in the two mammary carcinoma cell lines (8.2 x 10(-7) and 11 x 10(-7) mutations/hprt/cell division) were also higher than the same control (1.4 x 10(-7)). A:T to C:G transversion was observed at significantly higher frequencies in the two cell lines (6 of 24 and 6 of 25 for lacI; 10 of 67 and 19 of 92 for hprt) than in the control (0 of 6 for lacI; 0 of 4 for hprt). Taking advantage of the lacI transgene, high frequencies of A:T to C:G transversion (6 of 38 and 8 of 33, respectively) was also confirmed in the primary carcinomas of the two cell lines, which indicated the presence of a common abnormality in the cell lines and in the primary carcinomas. Both the established cell lines and their primary carcinomas were negative for microsatellite instability, which is known to be caused mainly by mismatch repair insufficiency and to increase point mutations, and for p53 mutations. These findings showed that the two cell lines, and possibly their primary carcinomas, had increases in the MRs of point mutations attributable to a mechanism(s) different from mismatch repair insufficiency, and we would suggest that such a state be designated as single nucleotide instability (SNI).

Animals↗
PubMed2001

Attractin/mahogany/zitter plays a critical role in myelination of the central nervous system.

The rat zitter (zi) mutation induces hypomyelination and vacuolation in the central nervous system (CNS), which result in early-onset tremor and progressive flaccid paresis. By positional cloning, we found a marked decrease in Attractin (Atrn) mRNA in the brain of the zi/zi rat and identified zi as an 8-bp deletion at a splice donor site of Atrn. Atrn has been known to play multiple roles in regulating physiological processes that are involved in monocyte-T cell interaction, agouti-related hair pigmentation, and control of energy homeostasis. Rat Atrn gene encoded two isoforms, a secreted and a membrane form, as a result of alternative splicing. The zi mutation at the Atrn locus darkened coat color when introduced into agouti rats, as also described in mahogany (mg) mice, carrying the homozygous mutation at the Atrn locus. Transgenic rescue experiments showed that the membrane-type Atrn complemented both neurological alteration and abnormal pigmentation in zi/zi rats, but that the secreted-type Atrn complemented neither mutant phenotype. Furthermore, we discovered that mg mice exhibited hypomyelination and vacuolation in the CNS associated with body tremor. We conclude from these results that the membrane Atrn has a critical role in normal myelination in the CNS and would provide insights into the physiology of myelination as well as the etiology of myelin diseases.

Agouti Signaling Protein↗
PubMed2001

[Peri-operative management in patients with thoracic aortic aneurysm complicated with cardiac tamponade].

We retrospectively summarized the peri-operative hemodynamic status and respiratory function in patients with thoracic aneurysms complicated with cardiac tamponade (Tamponade Group; n = 6) in comparison with patients complicated with hemorrhagic pericardial effusion without symptom of cardiac tamponade (Effusion Group; n = 10). There were no difference with regard to preoperative heart rate, pericardial effusion quantity, frequency of emergent operation and operation time. Preoperative hemodynamic status was worse in Tamponade Group than in Effusion Group. The cardiac indices of 12 h, 24 h, 48 h after operation in Tamponade Group were 2.60 +/- 0.60, 2.29 +/- 0.42, 1.95 +/- 0.33 l/min/m2 respectively, those were not different in Effusion Group. The differences between O2 tension in alveolar and arterial blood O2 tension (A-aDO2) and oxygenation indices (OI) of 12 h and 24 h after operation were significantly worse in Tamponade Group than in Effusion Group, but those of 48 h were not different. The operative mortality was 50% in Tamponade Group, and none in Effusion Group. It is very important to maintain hemodynamic status before operation, to perform complete repair (graft replacement) as early as possible and to care intensively after operation for thoracic aortic aneurysm complicated with cardiac tamponade.

Adolescent↗
PubMed2000

Interpretation of mutational spectra from different genes: analyses of PhIP-induced mutational specificity in the lacI and cII transgenes from colon of Big Blue rats.

The cII assay provides an alternative choice to the lacI transgene for mutational studies involving Big Blue(R) transgenic mice and rats, or permits the evaluation of mutational responses in both genes. Here, we compare the mutational response of the cII gene from colon of Big Blue(R) F344 rats treated with a dietary mutagen and animal carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), to those previously determined in the lacI transgene from colon of the same group of animals. A cursory inspection of PhIP-induced mutational spectra (MS) in cII and lacI suggests that the two transgenes respond differently to PhIP-induced mutation. However, a more thorough analysis of the MS in the two transgenes, including consideration of the number of mutational target sequences in each gene and nearest neighbor analyses of mutated nucleotides, indicates that PhIP-induced mutational specificity is similar in both genes. The evaluation of PhIP-induced mutational responses in these two transgenes serves as a model for intergenic mutational analyses.

Animals↗
PubMed2000

Acquired loss of p53 induces blastic transformation in p210(bcr/abl)-expressing hematopoietic cells: a transgenic study for blast crisis of human CML.

Chronic myelogenous leukemia (CML) begins with an indolent chronic phase but inevitably progresses to a fatal blast crisis. Although the Philadelphia chromosome, which generates p210(bcr/abl), is a unique chromosomal abnormality in the chronic phase, additional chromosomal abnormalities are frequently detected in the blast crisis, suggesting that superimposed genetic events are responsible for disease progression. To investigate whether loss of p53 plays a role in the evolution of CML, we crossmated p210(bcr/abl)-transgenic (BCR/ABL(tg/-)) mice with p53-heterozygous (p53(+/-)) mice and generated p210(bcr/abl)-transgenic, p53-heterozygous (BCR/ABL(tg/-)p53(+/-)) mice, in which a somatic alteration in the residual normal p53 allele directly abrogates p53 function. The BCR/ABL(tg/-)p53(+/-) mice died in a short period compared with their wild-type (BCR/ABL(-/-)p53(+/+)), p53 heterozygous (BCR/ABL(-/-)p53(+/-)), and p210(bcr/abl) transgenic (BCR/ABL(tg/-)p53(+/+)) litter mates. They had rapid proliferation of blast cells, which was preceded by subclinical or clinical signs of a myeloproliferative disorder resembling human CML. The blast cells were clonal in origin and expressed p210(bcr/abl) with an increased kinase activity. Interestingly, the residual normal p53 allele was frequently and preferentially lost in the tumor tissues, implying that a certain mechanism facilitating the loss of p53 allele exists in p210(bcr/abl)-expressing hematopoietic cells. Our study presents in vivo evidence that acquired loss of p53 contributes to the blastic transformation of p210(bcr/abl)-expressing hematopoietic cells and provides insights into the molecular mechanism for blast crisis of human CML. (Blood. 2000;95:1144-1150)

Animals↗
PubMed2000

Chromosomal mapping of genes controlling development, histological grade, depth of invasion, and size of rat stomach carcinomas.

Rat stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are widely used as a model of differentiated-type human stomach cancers. ACI/N (ACT) rats are susceptible and BUF/Nac (BUF) rats are resistant to MNNG-induced stomach carcinogenesis, and the presence of an autosomal gene with a dominant BUF allele has been suggested. In this study, we performed a carcinogenicity test by giving MNNG in drinking water to 117 male ACI x (ACIxBUF)F1 backcross rats. Each of 100 effective rats was diagnosed for its "carcinoma development" and when it was bearing stomach carcinoma(s), for histological grade, depth of invasion, and size and number of tumors. Carcinoma development was diagnosed based both on the age of the rat and on the presence of stomach carcinoma(s). Linkage analysis was performed with the genotypes of 161 loci, covering 1637 cM of the rat genome. Contrary to our original expectations, the most influential gene was the one on chromosome (chr.) 15, Gastric cancer susceptibility gene 1 (Gcs1), which confers susceptibility to stomach carcinogenesis (LOD, 3.8) with a dominant BUF allele by promoting conversion from adenomas to carcinomas. Two resistance genes on chr. 4 and chr. 3, Gastric cancer resistance gene 1 (Gcr1) and Gcr2, were shown to confer dominant resistance (LOD, 2.7 and 2.6, respectively). Gcs1, Gcr1, and Gcr2 exerted additive effects on the development of stomach carcinomas. A gene on chr. 16, Gcr3, was indicated to reduce the depth of invasion (LOD, 2.2) and sizes of tumors (LOD, 1.9). No linkage was obtained using the number of tumors. These findings show that the coordinate effect of a susceptibility gene, Gcs1, and two resistance genes, Gcr1 and Gcr2, is responsible for the development of MNNG-induced stomach carcinomas and that Gcr3 is responsible for the growth of a stomach carcinoma, reflected in the depth of invasion and in the tumor size.

Animals↗