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Takao Sekiya

Publications and source records attributed to Takao Sekiya.

18 recordsLinked to original sources
PubMed2006

Segregation of partly melted DNA molecules.

Segregation of partly melted DNA molecules is a convenient and efficient method to isolate DNA fragments associated with CpG islands. The method stands on the observation that the electrophoretic mobility of partly melted DNA fragments in a denaturing gradient gel is low and that they persist in the gel so long as the remaining helical part is sufficiently resistant to strand dissociation and dissociates slowly. Such features are observed in DNA fragments derived from CpG islands. These DNA fragments are preferentially retained in a denaturing gradient gel after prolonged electric field exposure, permitting the enrichment of DNA fragments derived from CpG islands. The principle and practical application of this method are reviewed.

Base Pairing↗
PubMed2004

Nemo-like kinase suppresses a wide range of transcription factors, including nuclear factor-kappaB.

Nemo-like kinase (NLK) is a serine/threonine kinase that suppresses the transcription activity of the beta-catenin-T-cell factor (TCF) complex through phosphorylation of TCF. Our previous study showed that NLK overexpression induces apoptosis in DLD-1 human colon cancer cells and that apoptosis induction presumably requires a mechanism other than the suppression of beta-catenin-TCF complex. Luciferase reporter gene assay with pNF-kappaB-Luc revealed that NLK could suppress transcription activity of NF-kappaB in a kinase-dependent manner. However, it appeared that transcription co-activators of NF-kappaB, such as CREB binding protein (CBP)/p300, were likely to be the direct targets of NLK, rather than NF-kappaB itself. Luciferase reporter gene analysis of GAL4-CBP fusion proteins revealed that the C-terminal region of CBP was critical for transcription suppression by NLK. In vitro kinase assay showed that NLK could phosphorylate the C-terminal domain of CBP. However, HAT activity was not suppressed by the induction of wild-type NLK in DLD-1 cells. Furthermore, we observed that NLK suppressed the transcription activity of AP-1, Smad, and p53, all of which also utilize CBP as a co-activator. The extent of suppression by NLK was similar among the transcription factors tested (50-60% reduction). Our results suggest that NLK may suppress a wide range of gene expression, possibly through CBP.

CREB-Binding Protein↗
PubMed2004

Segregation of partly melted molecules: isolation of CpG islands by polyacrylamide gel electrophoresis.

The technique of segregation of partly melted molecules (SPM) is a convenient and efficient method to isolate DNA fragments associated with CpG islands. The approach is conceptually simple and uses denaturant gradient gel electrophoresis to separate DNA molecules digested with restriction endonucleases. The SPM methodology has successfully been applied to the identification of genes from anonymous, unsequenced DNA fragments and CpG islands methylated in human cancer. In this article the theoretical background and practical application of the SPM method is reviewed.

CpG Islands↗
PubMed2004

Inhibition of angiogenesis in human glioma cell lines by antisense RNA from the soluble guanylate cyclase genes, GUCY1A3 and GUCY1B3.

Malignant gliomas, most of which show an elevated level of vascular endothelial cell growth factor (VEGF) expression, are well known for their hyper-vascularity. One of the possible inducers of VEGF in tumor cells is nitric oxide (NO), which is synthesized by NO synthase and stimulates soluble guanylate cyclase (GC) in tumor cells. Here, we report that 2 of 9 human glioma cell lines, CCF-STTG1 and U-87MG, overproduced cyclic GMP (cGMP) and showed increased expression of both or either subunits of soluble GC1, GUCY1A3 and GUCY1B3. Transfection of antisense GUCY1A3 or GUCY1B3 into these two glioma cell lines markedly reduced the content of cGMP and expression of VEGF. The angiogenic activity in vitro was subsequently inhibited, which was determined by induction of HUVEC cell growth. Furthermore, subcutaneous tumor formation by U-87MG cells in nude mice was dramatically suppressed to less than 0.05% in volume by transfection of either antisense GUCY1A3 or antisense GUCY1B3, which was accompanied by the significant decrease in vascular index to about 10%. These findings demonstrate that cGMP is an upstream mediator of VEGF expression in glioma cells and that soluble guanylate cyclases could be the target molecules for controlling neo-vascularization in a subset of human malignant gliomas.

Animals↗
PubMed2003

Nemo-like kinase induces apoptosis in DLD-1 human colon cancer cells.

Deregulation of Wnt/beta-catenin signaling is thought to play a critical role in human carcinogenesis. Nemo-like kinase (NLK) is an evolutionarily conserved serine/threonine kinase that suppresses beta-catenin/T-cell factor (TCF) complex transcriptional activity through phosphorylation of TCF. Since NLK may be a tumor suppressor as a negative regulator of Wnt/beta-catenin pathway, we established tetracycline-inducible NLK and its kinase-negative mutant expression in DLD-1 human colon cancer cells to analyze the effect of NLK on cell growth and viability. The induction of wild-type NLK in DLD-1 cells caused suppression of cell growth whereas the kinase-negative mutant did not. Flow cytometry indicated that NLK expression increased the number of apoptotic cells but did not induce obvious cell cycle arrest. Apoptosis induction by wild-type NLK was confirmed using TUNEL assays. Our results suggest that overexpression of NLK may have targets other than TCF for induction of apoptosis in human colon carcinoma cells.

Apoptosis↗
PubMed2003

Specific methylation status of the entire CpG island is not a prerequisite for the formation of an inactive chromatin at the promoter region in cancer cells.

It is well-recognized that DNA methylation causes gene silencing. However, although DNA methylation is closely associated with alterations in chromatin structure, it is not clear whether methylation at a particular locus contributes to the formation of an inactive chromatin structure. In this study, we have investigated the chromatin structure of the CpG island of the human CDH1 gene in cancer cells. The CDH1-CpG island was differentially methylated between silenced cells, however, DNase I hypersensitive site that are present in the expressing cell were commonly lost in silenced cells and transcription factors were excluded from their binding sites. These results demonstrate that formation of an inactive structure was not affected by heterogeneous methylation status of the CpG island.

Carbohydrate Dehydrogenases↗
PubMed2003

Is transvaginal ultrasonography at mid-trimester useful for predicting early spontaneous preterm birth?

Numerous reports have examined the relationship between sonographically determined cervical length and spontaneous preterm birth. Moreover, large screening studies have consistently demonstrated that the shorter the cervical length, the higher the rate of spontaneous preterm delivery. However, the sensitivity and positive predictive value of the cervical length for detecting preterm birth were low. Subsequently, we developed a new sonographic parameter termed "cervical gland area (CGA)". The purpose of this study was to determine whether sonographic cervical findings (shortened cervical length or absence of CGA) at 16 - 19 weeks' gestation could predict spontaneous preterm birth. The absence of CGA as compared to the shortened cervical length showed a higher sensitivity (75.0% vs. 50.0%) and a significantly elevated positive predictive value (54.5% vs. 8.3%) for preterm birth before 32 weeks' gestation. It was concluded that the absence of CGA was a novel and useful sonographic parameter for predicting early spontaneous preterm birth.

Female↗
PubMed2003

Ultrasonographic and clinical appearance of hemorrhagic ovarian cyst diagnosed by transvaginal scan.

We examined clinical and sonographic findings in 112 patients diagnosed as having hemorrhagic ovarian cyst (HOC) who had clinical and transvaginal sonographic follow up. The patients were classified into group A (n=40) with signs and symptoms of acute abdomen and group B (n=72) with no symptoms or mild abdominal pain, and their ultrasonographic and clinical findings were compared. Significant differences were found in mean age, white blood cell (WBC) count, greatest diameter of the mass, shortest diameter of the mass, and size of cross section of the mass. The internal echograms of HOCs were grouped into 4 types: (1) hyperechoic and hypoechoic solid type; (2) reticular or sponge-like type; (3) mixture type of solid and cystic components; and (4) cystic types. In all image types, septum-like or thread-like echoes were seen. Transvaginal sonography (TVS) of type 1, type 2, and type 3 images showed a clear division into hyperechoic and other areas with the passing of time which was finally changed into a cystic pattern and disappeared. HOCs were found more frequently in nulliparous patients (n=79, 70.5%) than in multiparous (n=33, 29.5%). There were many luteal phase (n=86, 76.8%) in comparison with follicular phase (n=13, 11.6%). Thirteen cases were detected during early gestation (n=13, 11.6%). In group A, severe pain reduced or disappeared within 3 h in 37/40 (92.5%) of the patients. Blood flow inside the masses was analyzed in 14 patients by the color Doppler method and showed no significant change. Taken together, this study elucidated the ultrasonographic and clinical characteristics of HOCs, which provide useful information to differentiate HOCs from organic masses and help to avoid unnecessary laparotomy.

Adult↗
PubMed2002

Heterogeneity in the modification and involvement of chromatin components of the CpG island of the silenced human CDH1 gene in cancer cells.

The structural alteration of chromatin has a key role in regulating gene expression. The alteration of chromatin is mediated by modification of its components. Detailed understanding of the relationship between these modifications, notably, methylation of the full-length CpG island, the association of methyl-CpG binding proteins (MBPs), and the acetylation and methylation of histones in gene silencing is vitally important. Currently, however, the manner in which chromatin components, associated with a specific gene, are modified is poorly understood. Here we provide in vivo evidence in cancer cells of the differential association between CpG methylation, MBPs, and histone modification in the entire CpG island of the human E-cadherin (CDH1) gene. Of the cell lines with CDH1 transcriptional repression, the distribution of methyl-CpGs in the CpG island differed markedly. In a cell line with gene silencing, the promoter region was almost methylation-free. Chromatin immunoprecipitation analysis revealed that the acetylation status of histone H4 differed between cell lines. However, deacetylated histone H3 was associated with the CpG island in all silenced cell lines. Binding of MeCP2 was also detected in all silenced cell lines. Additional binding of MBD1 protein was detected in a cell line in which the promoter region was poorly methylated and only histone H3 was deacetylated. Binding of MBD2 protein was detected in all other silenced cell lines. Histone H3 lysine 9 was methylated in all silenced cells, while histone H3 lysine 4 was methylated in some silenced cell lines. These results demonstrate that chromatin components associated with inactive CDH1 chromatin is heterogeneously modified and suggests the presence of multiple pathways for the formation of inactive chromatin.

Acetylation↗
PubMed2002

A comprehensive catalog of CpG islands methylated in human lung adenocarcinomas for the identification of tumor suppressor genes.

CpG island methylation is an important mechanism in gene silencing and is a key epigenetic event in cancer development. As yet, the number and identities of the genes that are inactivated in cancer cells has not been determined. In order to address this issue, we have performed a comprehensive isolation of CpG islands that are methylated in human lung adenocarcinomas. We have isolated approximately 200 CpG islands that are methylated in tumor DNA including those of known tumor-associated genes such as the HOXA5 gene. As the library contains the CpG islands of a number of known tumor suppressor genes it is highly likely that additional, previously unidentified tumor suppressor genes, will be present. On average, 1-2% of CpG islands were methylated specifically in tumors although this figure differed greatly between patients. This study provides an important resource in the search for genes inactivated in tumors and for the investigation of epigenetic dysregulation of gene expression by CpG island methylation.

Adenocarcinoma↗
PubMed2002

Homozygous deletion on the chromosomal region 5q12.3 in human lines of small-cell lung cancers.

Loss of heterozygosity at the polymorphic loci on the long arm of chromosome 5 is observed in about 80% of human small-cell lung cancer (SCLC). Absence of inactivating mutations in the APC gene on 5q14 suggests the involvement of another tumor suppressor gene. We found a homozygous deletion of sequence tagged site sequence G73332on 5q12.3 in 2 of 12 human SCLC cell lines, Lu130 and Lu134. One copy of chromosome 5q was lost in these cell lines, and the remaining allele had a deletion in a more restricted region. A polymerase chain reaction-based analysis of yeast artificial chromosome, bacterial artificial chromosome (BAC), and lambda-phage clones narrowed the region of homozygous deletion to a fragment cloned within one BAC. Sequencing analysis revealed that a DNA fragment of approximately 25 kb was deleted interstitially, probably because of recombination through Alu repetitive sequences in Lu130 and Lu134 cells. This deletion was not detected in normal lymphocyte DNA from 98 unrelated individuals. No candidate genes, however, were detected within this region or in the adjacent 150-kb fragment. The absence of microsatellite instability and the presence of an interstitial deletion as well as gross chromosomal aberration suggest that the genomic integrity of Lu130 and Lu134 cells might possibly be affected by Alu-mediated recombination in addition to chromosomal instability. The identical breakpoints in Lu134 and Lu135 cells as well as the same genotypes at all 33 polymorphic loci examined on various chromosomes strongly suggest that these cell lines share the same genetic materials, at least in part, during the establishment or propagation of cell lines.

Carcinoma, Small Cell↗
PubMed2002

Complete remission of uterine endometrial cancer with multiple lung metastases treated by paclitaxel and carboplatin.

Endometrial cancer is believed to have a better prognosis than cervical cancer. However, this is not necessarily true for cases beyond International Federation of Gynecology and Obstetrics (FIGO) stage III, and advanced endometrial cancer with distant metastases in particular has a poor prognosis. Moreover, there is no established therapy for advanced endometrial cancer. Recently, we treated two patients with endometrial cancer with multiple lung metastases (FIGO stage IVb). Both patients had massive uncontrollable genital bleeding and eventually progressed to anemia. The imminent severe bleeding was considered to be a major reason for exacerbation of their general condition. Therefore, hysterectomy was performed as a counter-measure to improve their general condition. In their postoperative course, the two patients successfully underwent T-J chemotherapy [paclitaxel: 210 m/m2 over 3h; carboplatin: area under the curve (AUC) 5]. Six courses of the regimen were given every 3-4 weeks. Multiple lung shadows in chest X-P and computed tomography (CT) were reduced in number and size after two courses of T-J chemotherapy. The multiple lung metastases either disappeared or just remained as scars after six courses. There has been no evidence of recurrence for 28 months in one patient and 7 months in the other patient.

Anemia↗
PubMed2002

Promoter methylation of TSLC1 and tumor suppression by its gene product in human prostate cancer.

We recently identified TSLC1, a tumor suppressor gene in human lung cancer. Gene silencing by promoter methylation has been observed frequently in adenocarcinoma of the lung, liver, and pancreas. Here, we demonstrate that TSLC1 expression is also absent or markedly reduced in 3 of 4 prostate cancer cell lines. Promoter sequences of TSLC1 were heavily methylated in PPC-1 cells that lacked TSLC1 expression, supporting the idea that promoter methylation is strongly correlated with complete loss of gene expression. Promoter sequences of TSLC1 were also methylated significantly in 7 of 22 (32%) primary prostate cancers. Hypermethylation of the promoter occurred not only in advanced tumors, but also in relatively early-stage tumors. Restoration of TSLC1 expression substantially suppressed tumor formation of PPC-1 cells in nude mice. These findings indicate that alteration of TSLC1 is involved in prostate cancer.

Animals↗
PubMed2002

Detection of the cervical gland area in threatened preterm labor using transvaginal sonography in the assessment of cervical maturation and the outcome of pregnancy.

OBJECTIVES: To detect the cervical gland area in threatened preterm labor, and to determine its detection rate and relationship with cervical maturation and outcome of pregnancy in preterm labor. METHODS: This was a mixed longitudinal and cross-sectional study involving 615 transvaginal scans performed to detect the cervical gland area and measure cervical length in 101 singleton pregnancies with threatened preterm labor. The patients were treated with intravenous administration of ritodrine chloride for regular uterine contractions at 16-35 weeks of gestation. 260 normal singleton pregnancies served as controls. Simultaneously conventional digital examination was used to assess the cervical maturation index. The detection rates of the cervical gland area, measurements of cervical length by sonography, and assessment of the cervical maturation index by digital examination in threatened preterm labor were compared with those of normal singleton pregnancies. In the threatened labor group, the outcome of pregnancy was assessed according to the sonographic absence or presence of the cervical gland area. RESULTS: In the normal pregnancy group, the detection rate of the cervical gland area remained practically constant until the 31st week of pregnancy (97%), but substantially decreased thereafter (70.2% in gestational weeks 32-35). In the threatened preterm labor group, the detection rate of the cervical gland area was constantly lower (44.5%) and the cervical maturation index was higher (4.65 score) than in the normal pregnancy group (83.1% and 1.80 score, respectively). The outcome of pregnancy in the threatened preterm labor group was poorer in the subgroup with the absence of a cervical gland area than in the subgroup with the presence of a cervical gland area (duration of pregnancy 257.0 vs. 271.0 days, birth weight 2,597.2 vs. 2,990.0 g, and admission to delivery interval 38.8 vs. 60.8 days). Highly significant correlations were noted among the detection rates of a cervical gland area and cervical length, cervical maturation index, and outcome of pregnancy. CONCLUSIONS: This study demonstrates for the first time that the sonographic absence of the cervical gland area reflects cervical maturation and could be considered as a predictor of threatened preterm labor and a sign of poor outcome of pregnancy in this condition.

Adolescent↗
PubMed2002

An overview of the analysis of DNA methylation in mammalian genomes.

DNA methylation at position C5 of the pyrimidine ring of cytosine in mammalian genomes has received a great deal of research interest due to its importance in many biological phenomena. It is associated with events such as epigenetic gene silencing and the maintenance of genome integrity. Aberrant DNA methylation, particularly that of chromosomal regions called CpG islands, is an important step in carcinogenesis. In order to elucidate methylation profiling of complex genomes, various methods have been developed. Many of these methods are based on the differential reactivity of cytosine and 5-methylcytosine to various chemicals. The combined use of these chemical reactions and other preexisting methods has enabled the discrimination of cytosine and 5-methylcytosine in complex genomes. The use of proteins that preferentially bind to methylated DNA has also successfully been used to discriminate between methylated and unmethylated sites. The chemical and structural dissection of the in vivo processes of enzymatic methylation and the binding of methyl-CpG binding proteins provides evidence for the complex mechanisms that nature has acquired. In this review we summarize the methods available for the discrimination between cytosine and 5-methylcytosine in complex genomes.

Animals↗