PubMed Health⌕ Search

Biomedical subjects

M Toyota

Publications and source records attributed to M Toyota.

At least 55 records · Page 3Linked to original sources
PubMed2000

Quantitative DNA methylation analysis by fluorescent polymerase chain reaction single-strand conformation polymorphism using an automated DNA sequencer.

A novel DNA methylation assay technique, termed bisulfite single-strand conformation polymorphism (bisulfite-SSCP), is a combination of sodium-bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. After bisulfite treatment followed by PCR amplification, methylated and unmethylated alleles can be simultaneously separated in a nondenaturing gel using an automated DNA sequencer. Using bisulfite-SSCP, methylation of hMLH1 was detected in a quantitative manner. This method is not only simple, quick, accurate, and quantitative, but detailed information about methylation is also available with less work. Methylation analysis of large numbers of samples for multiple loci will be facilitated by bisulfite-SSCP.

Adaptor Proteins, Signal Transducing↗
PubMed2000

DNA methylation and gastrointestinal malignancies: functional consequences and clinical implications.

Evidence now suggests that the transcriptional silencing of selected genes by DNA methylation plays a crucial role in the development and progression of human gastrointestinal malignancies. To date, genes involved in the regulation of the cell cycle, DNA repair, angiogenesis, and apoptosis have all been shown to be inactivated by the hypermethylation of their respective 5' CpG islands. The specific causes and effects of the changes in DNA methylation that occur in cancer remain unknown, however. Nevertheless, recently developed techniques for detecting changes in DNA methylation have dramatically increased the amount of information available on the patterns of methylation that occur as cancers progress. One key process involved in aberrant methylation is related to aging, and because it affects a large number of CpG islands, age-related methylation may be a primary cause of the increased incidence of cancer seen among older individuals. Other patterns of methylation are cancer-specific and are detected only in a subset of tumors exhibiting the CpG island methylator phenotype (CIMP). In this regard, the majority of sporadic colorectal cancers, exhibiting microsatellite instability (MSI) appear to be associated with CIMP, which leads to aberrant methylation of human MutL homologue (hMLH1) and the loss of its expression. We anticipate that as the various components of the molecular machinery involved in aberrant DNA methylation become better understood, they will prove to be useful targets, serving as the basis for the development of new methods for the diagnosis and treatment of gastrointestinal malignancies.

DNA↗
PubMed2000

Epigenetic regulation of androgen receptor gene expression in human prostate cancers.

Epigenetic mechanisms including DNA methylation and histone deacetylation are thought to play important roles in gene transcriptional inactivation. Heterogenous expression of androgen receptor (AR), which appears to be related to variable responses to endocrine therapy in prostate cancer (PCa) may also be due to epigenetic factors. The methylation status of the 5' CpG island of the AR in 3 prostate cancer cell lines and 10 primary and 14 hormone-refractory PCa samples was determined using the bisulfite PCR methods. In DU145, CpG-rich regions of the AR were hypermethylated. By an immunohistochemical analysis, only one PCa sample had no AR expression, the others being heterogenous. Bisulfite sequencing and methylation-specific PCR analysis showed aberrant methylation of AR 5'-regulatory region in 20% of 10 primary and 28% of 14 hormone-refractory PCa samples. To clarify the effect of epigenetic regulation on AR expression, we treated three prostate cancer cell lines with a demethylating agent, 5-aza-2'-deoxycytidine (azaC), and a histone deacetylase inhibitor, Trichostatin A (TSA). In DU145, re-expression of AR mRNA was detected after treatment with azaC and/or TSA. Our results suggest that epigenetic regulations including CpG methylation and histone acetylation may play important roles in the regulation of the AR.

Antimetabolites, Antineoplastic↗
PubMed2000

[Phytochemical study of liverworts Conocephalum conicum and Chiloscyphus polyanthos].

Four hundred samples of Conocephalum conicum, collected in various places have been analyzed by GC-MS. This resulted in the presence of three chemo-types of C. conicum. One of them elaborate (-)-sabinene (type-I) as a major compound. The other two types characteristically contained (+)-bornyl acetate (type-II) and methyl cinnamate (type-III) as major constituent respectively. The structures of three new monoterpenic esters, isolated from the ether extract of C. conicum (type-I and/or II) have been established by chemical and spectral means. They were shown to be monoterpenic esters of (+)-borneol and p-coumaric acid derivatives. The ether extract of the liverwort Chiloscyphus polyanthos afforded sesquiterpenoids which are enantiomeric to those found in another liverwort Lepidozia vitrea. The absolute configurations of the sesquiterpenoids found in the C. polyanthos were determined by spectroscopic evidence, chemical derivatization and/or x-ray crystallographic analysis. The ether extract of the liverwort Porella perrottetiana afforded (-)-alpha-eudesmol, which showed an opposite sign of the optical rotation to that found in higher plants. Present work on the absolute configuration and an optical purity of (-)-alpha-eudesmol strongly suggested that the positive values (e.g. +28.5 degrees) described in many previous papers should be revised. Since the absolute configuration of (-)-alpha-eudesmol was identical to that (+)-beta-eudesmol found in the higher plants, it was apparent that the expression of the positive sign might be revised to (-)-alpha-eudesmol. The optical purity, reconfirmation of the absolute configuration and synthesis of (-)-alpha-eudesmol will be discussed.

Lichens↗
PubMed2000

[Proposal of a new lymph node mapping system for pancreatic head cancer].

The Japanese Pancreatic Society published the 4th edition of the general guidelines for the study of pancreatic cancer (JPS guidelines) in 1993. The current JPS guidelines differ from the UICC classification of lymph nodes. Thirty-five lymph node stations are divided into three groups in the JPS guidelines. Group 1 (n1) corresponds closely with the regional lymph nodes in the UICC classification. Group 2 (n2) and group 3 (n3) are distant metastases in the UICC classification. To clarify practical lymph node mapping, 2,449 resected tubular adenocarcinomas of the pancreas head registered with the JPS were analyzed. Lymph node involvement was observed most frequently in posterior pancreatoduodenal (PPD) and anterior pancreatoduodenal (APD) and then in paraaortic (n2) nodes. Fifty-six patients who achieved 5-year survival were registered, of whom 19 had lymph node involvement. The sites of lymph node involvement were limited to the PPD and/or APD nodes in 14 cases. PPD and APD nodes differ from the other n1 in terms of high incidence of metastasis and fair prognosis. In conclusion, we propose a new practical lymph node mapping system in which PPD and APD nodes are the first station and the other n1 are the second station.

Humans↗
PubMed1999

PPARgamma inhibits the expression of c-MET in human gastric cancer cells through the suppression of Ets.

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) is shown to inhibit the growth of MKN-45 cells, a human gastric cancer cell line, which overexpresses c-Met tyrosine kinase. The aim of the present study was to investigate whether PPARgamma regulates the expression of c-Met. Two days after the activation of PPARgamma by troglitazone, a potent and selective PPARgamma ligand, a dramatic reduction of c-MET transcripts and the c-Met protein in MKN45 cells was observed. The luciferase assay showed that the activation of PPARgamma suppressed -249 to +330 c-MET promoter activity, driven by cotransfection of ETS-1 expression vector. These data demonstrate that PPARgamma activation is capable of suppressing Ets-induced c-MET gene transcription. Thus, it is possible that the growth inhibitory effect of PPARgamma on MKN-45 cells is related to the suppression of c-MET transcription.

Cell Division↗
PubMed1999

Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype.

Aberrant methylation of 5' CpG islands is thought to play an important role in the inactivation of tumor suppressor genes in cancer. In colorectal cancer, a group of tumors is characterized by a hypermethylator phenotype termed CpG island methylator phenotype (CIMP), which includes methylation of such genes as p16 and hMLH1. To study whether CIMP is present in gastric cancer, the methylation status of five newly cloned CpG islands was examined in 56 gastric cancers using bisulfite-PCR. Simultaneous methylation of three loci or more was observed in 23 (41%) of 56 cancers, which suggests that these tumors have the hypermethylator phenotype CIMP. There was a significant concordance between CIMP and the methylation of known genes including p16, and hMLH1; methylation of p16 was detected in 16 (70%) of 23 CIMP+ tumors, 1 (8%) of 12 CIMP intermediate tumors, and 1 (5%) of 21 CIMP- tumors (P<0.0001). Methylation of the hMLH1 gene was detected in three of five tumors that showed microsatellite instability, and all three of the cases were CIMP+. The CIMP phenotype is an early event in gastric cancer, being present in the normal tissue adjacent to cancer in 5 of 56 cases. These results suggest that CIMP may be one of the major pathways that contribute to tumorigenesis in gastric cancers.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Distinct methylation pattern and microsatellite instability in sporadic gastric cancer.

Aberrant 5' CpG island methylation is an alternative mechanism of gene inactivation during the development of cancer as demonstrated for several tumor-suppressor genes. Also, marked relationship of microsatellite instability (MSI) and DNA methylation has been reported in sporadic colorectal cancer, which is a result of epigenetic inactivation of hMLH1 in association of promoter hypermethylation. In the present study, we investigated the 5' CpG island hypermethylation of hMLH1, E-cadherin and p16 in 61 primary gastric cancers (GCs) by using combined bisulfite restriction analysis (COBRA) and methylation-specific PCR (MSP), and their MSI status. Of 61 GCs investigated, 5 (8.1%) tumors presented hMLH1 methylation, 16 (26.2%) and 25 (40.9%) showed E-cadherin and p16 methylation respectively, and 8 (13.1%) presented high-frequency MSI (MSI-H). Of the 8 MSI-H patients, 5 presented hMLH1 methylation, whereas no low-frequency MSI (MSI-L) and microsatellite stable (MSS) cases exhibited hMLH1 methylation (5/8 vs. 0/43, p < 0.00001). Furthermore, these patients also presented E-cadherin and p16 hypermethylation. Our data showed a significant correlation between hMLH1 methylation and MSI in GC, and suggested that a common mechanism of aberrant de novo methylation can be postulated in these cancers.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Inactivation of CACNA1G, a T-type calcium channel gene, by aberrant methylation of its 5' CpG island in human tumors.

Using a newly developed PCR-based technique called methylated CpG island amplification, we have identified several DNA fragments that are aberrantly methylated in a colon cancer cell line. One of the fragments, termed MINT31, mapped to human chromosome 17q21, where frequent loss of heterozygosity is detected in various human tumors. By characterizing the genomic sequence around this area, we identified a gene encoding a T-type calcium channel, CACNA1G, as a target for hypermethylation in human tumors. By reverse transcriptase-PCR we detected expression of CACNA1G in normal colon and bone marrow, but expression was absent in the five tumor cell lines in which methylation was found. After treatment with the methylation inhibitor 5-deoxyazacytidine, the expression of CACNA1G was restored in all five cell lines. Detailed methylation mapping of the 5' CpG island by bisulfite-PCR revealed that methylation of a region 300-800 bp upstream of the translation initiation site closely correlated with the inactivation of CACNA1G. This region contained the transcription start site, as determined by 5' rapid amplification of cDNA ends analysis. Aberrant methylation of CACNA1G was also examined in various human primary tumors and was detected in 17 of 49 (35%) colorectal cancers, 4 of 16 (25%) gastric cancers, and 3 of 23 (13%) acute myelogenous leukemia cases. Inactivation of CACNA1G may play a role in cancer development by modulating calcium signaling, which potentially affects cell proliferation and apoptosis.

Bone Marrow Cells↗
PubMed1999

CpG island methylator phenotype in colorectal cancer.

Aberrant methylation of promoter region CpG islands is associated with transcriptional inactivation of tumor-suppressor genes in neoplasia. To understand global patterns of CpG island methylation in colorectal cancer, we have used a recently developed technique called methylated CpG island amplification to examine 30 newly cloned differentially methylated DNA sequences. Of these 30 clones, 19 (63%) were progressively methylated in an age-dependent manner in normal colon, 7 (23%) were methylated in a cancer-specific manner, and 4 (13%) were methylated only in cell lines. Thus, a majority of CpG islands methylated in colon cancer are also methylated in a subset of normal colonic cells during the process of aging. In contrast, methylation of the cancer-specific clones was found exclusively in a subset of colorectal cancers, which appear to display a CpG island methylator phenotype (CIMP). CIMP+ tumors also have a high incidence of p16 and THBS1 methylation, and they include the majority of sporadic colorectal cancers with microsatellite instability related to hMLH1 methylation. We thus define a pathway in colorectal cancer that appears to be responsible for the majority of sporadic tumors with mismatch repair deficiency.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification.

CpG island methylation has been linked to tumor suppressor gene inactivation in neoplasia and may serve as a useful marker to clone novel cancer-related genes. We have developed a novel PCR-based method, methylated CpG island amplification (MCA), which is useful for both methylation analysis and cloning differentially methylated genes. Using restriction enzymes that have differential sensitivity to 5-methyl-cytosine, followed by adaptor ligation and PCR amplification, methylated CpG rich sequences can be preferentially amplified. In a model experiment using a probe from exon 1 of the p16 gene, signal was detected from MCA products of a colorectal cancer cell line but not in normal colon mucosa. To identify novel CpG islands differentially methylated in colorectal cancer, we have applied MCA coupled with representational difference analysis to the colon cancer cell line Caco2 as a tester and normal colon mucosa as a driver. Using this strategy, we isolated 33 differentially methylated DNA sequences, including fragments identical to several known genes (PAX6, Versican, alpha-tubulin, CSX, OPT, and rRNA gene). The association of hypermethylation of the clones obtained and transcriptional suppression in colorectal cancer was confirmed by examining the Versican gene, which we found to be silenced in methylated cell lines and reactivated by the methylation inhibitor 5-aza-2'-deoxycytidine. We therefore propose that MCA is a useful technique to study methylation and to isolate CpG islands differentially methylated in cancer.

5-Methylcytosine↗
PubMed1999

CpG island methylator phenotypes in aging and cancer.

CpG islands are short stretches of CpG rich regions that are frequently associated with the promoter region of genes. Aberrant methylation of CpG islands is one mechanism of inactivating tumor suppressor genes (TSGs) in neoplasia, and there is growing evidence that altered cytosine methylation play important roles in cancer development. However, the differences in global CpG island methylation patterns between normal and cancer cells remain poorly understood. By examining a large number of loci in a series of cancers, global methylation profiles can be constructed. Such studies revealed that in colorectal cancer, there appears to be two types of methylation that are associated with cancer progression: type A (for age-related) methylation, and type C (for cancer-specific) methylation. Initially, type A methylation arises as a function of age in normal colorectal epithelial cells. By affecting genes that regulate the growth and/or differentiation of these cells, such methylation may result in a predisposition state that precedes tumor formation in the colon. Type C methylation, by contrast, was found exclusively in a subset of cancers, which display a CpG island methylator phenotype (CIMP). CIMP is a novel molecular instability pathway that appears to be responsible for most cases of aberrant TSG methylation in colorectal cancer, and which has important interactions with genetic pathways as well. In fact, CIMP+ tumors account for the majority of sporadic colorectal cancers with microsatellite instability, through methylation of the mismatch repair gene hMLH1. This model whereby age-related methylation increases cell-susceptibility to transformation and cancer-specific methylation results in neoplastic progression in a subset of cases may be applicable to many human neoplasms.

Aging↗
PubMed1999

Induction of apoptosis by gallic acid in lung cancer cells.

The apoptosis-inducing effect of gallic acid (3,4,5-trihydroxybenzoic acid) was investigated in four human lung cancer cell lines, SBC-3 (small cell carcinoma), EBC-1 (squamous cell carcinoma), A549 (adenocarcinoma) and SBC-3/CDDP (cisplatin-resistant subclone of SBC-3). Gallic acid induced apoptosis in a dose-dependent manner as evidenced by analyses of DNA fragmentation, changes in cell morphology and loss of viability. Fifty percent inhibitory concentration (IC50) values of gallic acid on the cell viability of SBC-3, EBC-1 and A549 were around 10, 20 and 60 microg/ml, respectively. The IC50 value for SBC-3/CDDP cells was almost the same as that of SBC-3, suggesting that susceptibility of cells to gallic acid-induced apoptosis is not altered by the acquisition of cisplatin resistance. The apoptotic process was effectively triggered by 30 min exposure to gallic acid. A caspase inhibitor and alpha-tocopherol effectively prevented the gallic acid-induced apoptosis, indicating the involvememt of caspase activation and oxidative processes during the course of apoptosis in gallic acid-treated cancer cells. These findings suggest the possible applicability of gallic acid in lung cancer therapy, especially to circumvent resistance to anti-cancer drugs.

Adenocarcinoma↗
PubMed1998

Generation of polymorphic markers tightly linked to the thymus enlargement loci by phenotype-directed representational difference analysis.

To obtain genetic markers linked to a specific genetic locus, genomic subtraction with a DNA pool of backcross or F2 intercross animals with a specific genotype at the locus is known to be effective. To determine whether the pooling strategy is also effective for isolation of genetic markers linked to a quantitative phenotype that can potentially be controlled by multiple genetic loci, we tested the ability of representational difference analysis (RDA) to isolate genetic markers linked to the thymus enlargement observed in the BUF/Mna (BUF) rat. This is known to be controlled by single major and minor genes, Ten1 and Ten2, on Chromosomes (Chrs) 1 and 13, respectively, both of which have dose effects on the normal WKY/Ncj (WKY) allele. DNA from an inbred WKY rat was used as the tester, and the driver was prepared from a DNA pool of 12 (WKY x BUF)F1 x BUF backcross rats with high thymus ratios (thymus weight/body weight), expected to have dominance of the BUF allele in the responsible loci. By two RDA series with the restriction enzymes BglII and BamHI, respectively, 28 polymorphic markers were isolated, and 8 of them were shown to be linked to Ten1, and one to Ten2. One of the 8 markers linked to Ten1 demonstrated no recombination in 18 rats with high thymus ratios. RDA with a DNA pool based on a quantitative phenotype (phenotype-directed RDA) can thus be considered an efficient approach for direct isolation of polymorphic markers linked to a quantitative trait.

Animals↗
PubMed1998

Cytotoxic 2,3-secoaromadendrane-type sesquiterpenoids from the liverwort Plagiochila ovalifolia.

The ether extract of the Japanese liverwort, Plagiochila ovalifolia Mitt. (Plagiochilaceae) yielded three 2,3-secoaromadendrane-type sesquiterpenoids, plagiochiline-A-15-yl octanoate, 14-hydroxyplagiochiline-A-15-yl 2E,4E-dodecadienoate and 14-hydroxyplagiochiline-A-15-yl 2E,4E,8Z-tetradecatrienoate. The two latter compounds were first isolated from the Plagiochila species. The structures of the new compounds were elucidated by spectroscopy. Plagiochiline-A-15-yl octanoate and 14-hydroxyplagiochiline-A-15-yl 2E,4E-dodecadienoate showed significant cytotoxicity against P-388 murine leukemia tumor cells, both compounds exhibiting an ID50 of 0.05 microgram/ml. Plagiochiline A also showed cytotoxic activity. The ID50 was 3.0 micrograms/ml.

Animals↗
PubMed1998

Linkage mapping of the Bra, Brb and Brg genes for rat protein phosphatase 2A 55 kDa B-regulatory subunit isotypes.

We previously identified the rat Bra, Brb and Brg genes, which encode alpha, beta and gamma isotypes of the 55 kDa B-regulatory subunit of protein phosphatase 2A. Polymerase chain reaction-single strand conformation polymorphism analysis in the present study identified polymorphisms in Bra, Brb and Brg between the ACI and BUF, ZI and TM, and BN and WTC strains, respectively. Linkage analysis using mapping panels composed of F2 or back-crosses of these strains allowed Bra, Brb and Brg to be assigned to chromosomes 15, 18 and 14, respectively. Furthermore, it was revealed that Bra is located close to the Rb1 locus. Using polymorphism in Bra, loss of heterozygosity (LOH) was analyzed for rat mammary tumors induced in (SD x F344) F1 female rats by a food-borne carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, and a typical mammary carcinogen, 7,12-dimethylbenz[a]anthracene. No LOH was detected at the Bra locus.

9,10-Dimethyl-1,2-benzanthracene↗