Biomedical subjects

Naohiko Seki

Publications and source records attributed to Naohiko Seki.

At least 37 records · Page 2Linked to original sources

PubMed2005

Increased infectivity of adenovirus type 5 bearing type 11 or type 35 fibers to human esophageal and oral carcinoma cells.

Esophageal and oral carcinomas are relatively resistant to adenovirus serotype 5 (Ad5)-mediated gene transfer, primarily because expression of the cellular receptors for Ad5, the coxsackievirus and adenovirus receptor, is often downregulated in these types of tumor. The types of Ad in which the receptor expression is not suppressed in tumors are therefore better vectors for gene transfer into tumors. CD46, a cellular receptor for Ad subtype B2, such as Ad11 and Ad35, is well expressed in a number of esophageal and oral tumor cells. Since the infectivity of Ad to target cells is mainly influenced by the interaction between their fibers and the cellular receptors, we examined the infectivity of chimeric Ad5, whose fiber structure was substituted with that of type 11 or 35 (Ad5/11 or Ad5/35), to 6 human oral and 11 esophagus carcinoma cells. We found that the chimeric Ad, in particular Ad5/35, infected more effectively than Ad5 in all the tumors tested. However, the efficacy of Ad5/35- and Ad5/11-mediated transduction was not correlated with the expression level of CD46 or CD80/86, a cellular receptor of the Ad subtype B1, in the target cells. These data suggest that the Ad subtype B2 are suitable vectors of gene transfer for human squamous cell carcinomas of the upper gastrointestinal tract, and that the infectivity of the Ad subtype B2 can possibly be regulated by other receptors besides CD46.

Adenoviridae↗

PubMed2005

Identification of candidate radioresistant genes in human squamous cell carcinoma cells through gene expression analysis using DNA microarrays.

Radiation therapy is currently the standard adjuvant approach for oral squamous cell carcinoma (OSCC) patients. Individual OSCCs display a wide range of radiosensitivity (RS). To identify genes associated with radioresistance (RR) of OSCC and establish a useful method of predicting radio-therapeutic effectiveness, we examined the gene expression patterns of OSCC cell lines that exhibited different responses to ionizing radiation (IR) by clonogenic survival assay using an in-house cDNA microarray consisting of 2,201 human genes and quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR). Microarray analysis showed overexpression of 7 genes in the radioresistant cell line, HSC2, and 2 genes in the radiosensitive cell line, HSC3. The changes in expression levels in 7 of 9 genes (Cytokeratin18, DTNBP1, ASNA1, Tcp20, Cyclophilin F, KIAA0218, and HBp17) were confirmed with QRT-PCR. Of these, the genetic alterations of Tcp20, whose expression was remarkably elevated in radioresistant HSC2 cells after IR, were investigated. The escalation of X-ray doses resulted in an enhanced Tcp20 expression level in HSC2 cells compared to radiosensitive HSC3 cells (P<0.05, Mann-Whitney U test). These results suggest that the identified genes, which include Tcp20, may play an important role in conferring RR to OSCC, and could also be useful in identifying cases of OSCC with more radioresistance.

Carcinoma, Squamous Cell↗

PubMed2004

Gene expression profiles in liver regeneration with oval cell induction.

The liver has the unique ability to regenerate even in adulthood. While mature hepatocytes can proliferate by themselves, stem cells also play a critical role in liver regeneration and oval cells are considered to be the progeny of activated hepatic stem cells. We herein investigated the gene expression profiles in the conditions inducing oval cells, using microarray analysis. Two approaches were used to induce oval cells. In the first, animals were treated with a combination of an N-2-acetylaminofluorene (AAF)-containing diet and partial hepatectomy (PHx). In the second, animals were supplied with chow containing a 1:1 mixture of choline-deficient and normal diets, as well as 0.075% ethionine in drinking water. Using in-house cDNA microarrays consisting of 2304 cDNA clones from the mouse liver, 69 and 89 genes, respectively, were found to be up-regulated in these two models. Six genes, i.e., those for insulin-like growth factor binding protein-1, CYP4a14, carnitine octanoyltransferase, osteopontin, and two expressed sequence tags (ESTs) were up-regulated in these models but not in ordinary model with PHx alone. They might be specifically activated in the induction of oval cells, and help to clarify the nature of stem cell stimulation that occurs during liver regeneration.

2-Acetylaminofluorene↗

PubMed2004

Smad4-independent regulation of p21/WAF1 by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, the Smad4-null pancreatic cancer cell line BxPC-3 was transfected with either the Smad4 expression vector or the empty vector and incubated in the presence or absence of TGF-beta. The cells were analysed using a cDNA microarray, which included 2280 named genes to screen for target genes regulated by TGF-beta in either a Smad4-dependent or -independent manner. The microarray and subsequent quantitative RT-PCR analysis demonstrated that the Smad4-independent and -dependent signaling pathways driven by TGF-beta upregulated only one of the 2280 genes, respectively, suggesting that Smad4-independent signaling downstream of TGF-beta might be as widespread as Smad4-dependent signaling. In this study, we demonstrated that the cyclin-dependent kinase inhibitor p21/WAF1, which has been considered the major effector of the Smad-dependent growth inhibitory signal of TGF-beta, is upregulated in a Smad4-independent manner. The upregulation occurs through Smad2/3-dependent transcriptional activation of the p21/WAF1 promoter region. These results suggest a novel mechanism of gene regulation, that is, a novel signal mediator other than Smad4.

Active Transport, Cell Nucleus↗

PubMed2004

Identification of genes up-regulated by histone deacetylase inhibition with cDNA microarray and exploration of epigenetic alterations on hepatoma cells.

BACKGROUND/AIMS: Epigenetics is the key factor in the regulation of gene expression. We conducted cDNA microarray analysis to screen for genes induced by histone deacetylase (HDAC) inhibition and examined epigenetic alterations. METHODS: Microarray analysis was performed in six hepatoma cell lines and primary hepatocytes treated with trichostatin A (TSA). mRNA expression of several genes was examined by reverse transcription-polymerase chain reaction in TSA-treated cells and hepatoma samples. Acetylated histones and methylation status in 5'CpG islands was assessed by chromatin immunoprecipitation (ChIP) assay and bisulfite genomic sequencing, respectively. RESULTS: Fifty-seven genes showed greater than 2-fold change after TSA treatment in multiple cell lines. Among them, four genes including p21(WAF1) exhibited substantial induction (greater than 5-fold changes). Decreased mRNA levels of these genes in hepatoma tissues were observed in more than half of patients. ChIP assay, in general, demonstrated a good correlation between mRNA expression and histone acetylation, but only a limited correlation with the methylated DNA in the promoter region. CONCLUSIONS: We identified 57 up-regulated genes by TSA treatment in hepatoma cells and some of them appeared to be cancer-related genes in hepatomas. The alterations in acetylated histones are likely closely associated with gene expression.

Acetylation↗

PubMed2004

Gene expression of periostin in the early stage of fracture healing detected by cDNA microarray analysis.

To comprehensively evaluate gene expression in the early stage of fracture healing, we used a cDNA microarray with 2304 cDNA clones derived from an oligo-capped mouse embryo library. Closed mid-diaphyseal fractures were created in mouse tibiae and expression profiles were analyzed 3 days after fracture. Six genes were up-regulated in comparison to those in unfractured bones and these included three genes previously identified but never shown to be present in fractures, periostin, calumenin, and FHL-1. Cloning of these genes has been completed but their expression pattern and function during fracture healing and bone formation remain to be elucidated. Up-regulation of the six genes was reconfirmed by semi-quantitative RT-PCR analysis. Spatial and temporal expression of one of the newly identified fracture-induced genes, periostin, was analyzed using in situ hybridization, because it displayed the highest up-regulation ratio. A signal for periostin was detected in undifferentiated mesenchymal cells and immature preosteoblastic cells in the periosteal tissues between days 3 and 14 after fracture. Northern analysis showed that periostin gene expression rapidly increased by day 3, reached a peak on day 7, and declined by day 14. These findings suggest that periostin is a specific marker for preosteoblasts and may play an important role in periosteal callus formation during the early stage of fracture healing.

Animals↗

PubMed2004

An mRNA amplification procedure with directional cDNA cloning and strand-specific cRNA synthesis for comprehensive gene expression analysis.

We developed an integrated system suitable for comprehensive gene expression studies including construction and analysis of cDNA microarrays starting from a small amount of mRNA. We amplified total mRNA first as cDNA mixtures by polymerase chain reaction and then as strand-specific cRNA mixtures by in vitro transcription. These amplified cDNA and cRNA enabled determination of mRNA levels by hybridization analyses such as Southern, Northern, reverse-Northern macroarray, and cDNA microarray analyses, as well as construction of the cDNA library with a unidirectional cDNA insert. By using strand-specific cRNA derived from rat primary-cultured hepatocytes, we detected putative antisense transcripts for the metallothionein gene. cDNA microarray analysis for genes regulated by glucocorticoids and glucagon in the hepatocytes revealed that a number of genes involved in signal transduction and transcriptional regulation were up- or down-regulated. The present system is widely applicable to gene expression analysis with limited amounts of RNA samples.

Animals↗

PubMed2004

Cloning of cDNA encoding a regeneration-associated muscle protease whose expression is attenuated in cell lines derived from Duchenne muscular dystrophy patients.

In the dystrophin-mutant mdx mouse, an animal model for Duchenne muscular dystrophy (DMD), damaged skeletal muscles are efficiently regenerated and thus the animals thrive. The phenotypic differences between DMD patients and the mdx mice suggest the existence of factors that modulate the muscle wasting in the mdx mice. To identify these factors, we searched for mRNAs affected by the mdx mutation by using cDNA microarrays with newly established skeletal muscle cell lines from mdx and normal mice. We found that in the mdx muscle cell line, 12 genes, including L-arginine:glycine amidinotransferase and thymosin beta4, are up-regulated, whereas 7 genes, including selenoprotein P and a novel regeneration-associated muscle protease (RAMP), are down-regulated. Northern blot analysis and in situ hybridization revealed that RAMP mRNA is predominantly expressed in normal skeletal muscle and brain, and its production is enhanced in the regenerating area of injured skeletal muscle in mice. RAMP expression was much lower in individual muscle cell lines derived from biopsies of six DMD patients compared to a normal muscle cell line. These results suggest that RAMP may play a role in the regeneration of skeletal muscle and that its down-regulation could be involved in the progression of DMD in humans.

Amino Acid Sequence↗

PubMed2004

S100A11 gene identified by in-house cDNA microarray as an accurate predictor of lymph node metastases of gastric cancer.

Gastric cancer is one of the most common malignancies in the world, and in Asian countries its incidence and mortality rates are very high. Worldwide, Japan ranks first in the incidence of this type of cancer for both sexes. To shed light on the mechanisms underlying the development and/or progression of gastric cancer, we compared the expression profiles in gastric cancer cells obtained from surgical dissection of 20 gastric adenocarcinoma specimens with those in the corresponding non-cancerous mucosa, by cDNA microarray analysis. In total, 8,000 cDNA clones were randomly picked up and their 5'-end nucleotide sequences were determined. On the basis of sequence information, 4,608 independent clones were selected and used to produce the cDNA microarray. We identified 26 genes that were commonly up-regulated and 44 genes that were commonly down-regulated in cancerous tissues. To validate the cDNA microarray analysis, real-time PCR was performed. We found that gene S100A11 expression was associated with the development of lymph node metastases. S100A11 gene expression was clearly up-regulated in specimens from patients with lymph node metastases relative to those from patients without lymph node metastases. S100A11 gene expression status was useful to distinguish gastric cancers with lymph node metastases from those without lymph node metastasis. This genome-wide information contributes to an improved understanding of molecular changes during the development of gastric cancers. It may also help clinicians predict the development of lymph node metastases and assist researchers in identifying novel therapeutic targets for patients with gastric cancer.

Adenocarcinoma↗

PubMed2003

Antiapoptotic regulation by hepatitis C virus core protein through up-regulation of inhibitor of caspase-activated DNase.

The hepatitis C virus (HCV) core protein is considered to influence multiple cellular processes. We developed a human hepatoblastoma HepG2-derived inducible cell line, Hep191, which allows tightly regulated expression of the core protein at relatively low but physiological levels under control of the ecdysone-regulated promoter. By transcriptional profiling, we identified differentially expressed genes, some of which are involved in cell growth or apoptosis such as inhibitor of caspase-activated DNase (ICAD), defender against cell death 1, tumor necrosis factor (TNF) receptor 1, and cytochrome c oxidase subunit VIII. Furthermore, we found that core protein expression increases a steady-state level of ICAD protein, possibly through enhancing its promoter activity, and inhibits caspase-3 activity induced by anti-Fas antibody. Since Fas- or TNF-mediated DNA fragmentation is suppressed in the core-induced Hep191 cells, these findings suggest that expression of HCV core at physiological levels confers blocking activity of caspase-activated DNase and consequently inhibiting apoptotic cell death.

Apoptosis↗

PubMed2003

Gene expression profiling reveals the mechanism and pathophysiology of mouse liver regeneration.

Comprehensive analysis of the changes in gene expression during liver regeneration was carried out by using an in-house microarray composed of 2,304 distinct mouse liver cDNA clones. Mice were subjected to partial two-thirds hepatectomy, and changes in mRNA levels were monitored up to 48 h. Of the 2,304 genes analyzed, 496 genes showed expression levels measurable at all time points after the partial hepatectomy. 317 genes were up- or down-regulated 2-fold or more at least at one time point during liver regeneration and were classified into eight clusters based on their expression patterns. With a more stringent cut-off value of +/-2 S.D., 68 genes were listed and were classified into five clusters. In these two analyses with different clustering criteria, functionally categorized genes showed similar cluster distributions. Genes involved in protein synthesis and posttranslational processing were significantly enriched in the cluster characterized by rapid gene activation and subsequent persistence. This suggests the importance of modulating the efficiency of protein supply and/or altering the composition of protein population from the early phase of hepatocyte proliferation. Genes for two major liver functions, i.e. plasma protein secretion and intermediate metabolism were enriched in distinct clusters exhibiting the features of gradual gene activation and sustained repression, respectively. Therefore, these genes are differentially regulated during the regeneration, possibly leading to changes in the flow of amino acids and energy from enzyme proteins to plasma proteins in their synthesis. Thus, clustering analysis of expression patterns of functionally classified genes gave insights into mechanism and pathophysiology of liver regeneration.

Animals↗

PubMed2003

Two-peaked synchronization in day/night expression rhythms of the fibrinogen gene cluster in the mouse liver.

Genes expressed with day/night rhythms in the mouse liver were searched for by microarray analysis using an in-house array harboring mouse liver cDNAs. The rhythmic expression with a single peak and trough level was confirmed by RNA blot analysis for 3beta-Hsd and Gabarapl1 genes exhibiting a peak in the light phase and Spot14, Hspa8, Hspa5, and Hsp84-1 genes showing a peak in the dark phase. On the other hand, mRNA levels for all of the three fibrinogen subunits, Aalpha, Bbeta and gamma, exhibited two peaks each in the light and dark phases in a synchronized manner. This two-peaked rhythmic pattern of fibrinogen genes as well as the single peak-trough pattern of other genes was diminished or almost completely lost in the liver of Clock mutant mice, suggesting that the two-peaked expression is also under the control of oscillation-generating genes. In constant darkness, the first peak of the expression rhythm of fibrinogen genes was almost intact, but the second peak disappeared. Therefore, although the first peak in the subjective day is a component of the innate circadian rhythm, the second peak seems to require light stimuli. Fasting in constant darkness caused shifts of time phases of the circadian rhythms. Protein levels of the fibrinogen subunits in whole blood also exhibited circadian rhythms. In the mouse and human loci of the fibrinogen gene cluster, a number of sequence elements resembling circadian transcription factor-binding sites were found. The fibrinogen gene locus provides a unique system for the study of two-peaked day/night rhythms of gene expression in a synchronized form.

Animals↗

PubMed2003

Differential cellular gene expression induced by hepatitis B and C viruses.

Hepatitis B virus (HBV) is a hepatotropic virus that causes acute and chronic hepatocellular injury and hepatocellular carcinoma. To clarify how HBV proteins regulate host cellular gene expression, we used our in-house cDNA microarray and HepG2.2.15 cells, which are derived from HepG2 cells and produce all HBV proteins. Of 2304 genes investigated, several genes were differentially expressed in HepG2.2.15 cells compared with HepG2 cells. These genes included insulin-like growth factor II and alpha-fetoprotein, consistent with previous reports. Furthermore, we previously performed similar microarray analyses to clarify the effects of hepatitis C virus (HCV) proteins on host cells, using a HepG2-derivative cell line, which produces all HCV proteins. Using these two microarray results, we compared the differences in cellular gene expression induced by HBV and HCV proteins. The expression of the majority of genes investigated differed only slightly between HBV and HCV protein-producing cells. However, HBV and HCV proteins clearly regulated several genes in a reciprocal manner. Combined, these microarray results shed new light on the effects of HBV proteins on cellular gene expression and on the differences in the pathogenic activities of these two hepatitis viruses.

Carcinoma, Hepatocellular↗

PubMed2003

Comparing gene expression profiles in human liver, gastric, and pancreatic tissues using full-length-enriched cDNA libraries.

In the post-genome-sequencing era, full-length cDNA-sequence resources are extremely useful for functional analyses of genes. In addition, comprehensive gene profiling of human tissues at the mRNA level is also useful in understanding the molecular mechanisms of tissue-specific functions and disease pathogenesis. In this study, to obtain a wide variety of full-length cDNA clones derived from digestive tissues, numerous expressed sequence tags were generated from libraries enriched with full-length cDNAs. In total, 13575 sequences were obtained from three cDNA libraries, which were constructed from tissues and cell lines of human liver, stomach, and pancreas. The integration of overlapping clones categorized the sequences into 5936 clusters (1666, 2746, and 2222 clusters in the liver, stomach, and pancreas, respectively). Of these, 1138 clones were scored as full-length cDNAs. Surprisingly, the redundant clones from all three tissues were assembled to show that only 101 genes (1.7% of the assembled 5936 genes) were shared. These results suggest that functional differences between tissues are probably related to their divergent gene expression profiles, and form a basis for understanding the molecular mechanisms underlying tissue-specific pathogenesis that are expressed in different organs. In addition, the full-length cDNAs obtained in this study should prove useful for future functional analyses of the genes expressed in digestive tissues.

Journal Article↗

PubMed2003

Influence of hepatitis B virus genotypes on the progression of chronic type B liver disease.

To investigate the hepatitis B virus (HBV) genotype-related differences in the progression of liver disease, 585 patients with chronic HBV infection including 258 with histologically verified chronic liver disease (CLD) and 74 with hepatocellular carcinoma (HCC) were examined. The mean ages of both patients with advanced fibrosis (F3 or F4) and with HCC were significantly older in genotype B than in genotype C patients (P =.018, P =.024, respectively). Both the hepatitis B e antigen (HBeAg) negativity rate at biopsy and the cumulative HBe seroconversion rate in patients with CLD were significantly higher in genotype B patients than genotype C patients (P <.01, P =.022, respectively). Multivariate analysis revealed that genotype B, presence of precore mutation, high ALT levels, and severe histologic activity were independent factors for HBe seroconversion. Among all the biopsy-proven CLD patients, the ratio of patients with advanced fibrosis in genotype B was significantly lower than that in genotype C (4/30 vs. 74/224, respectively; P =.034). This difference was more remarkable in younger patients (< or =45 years; 1/25 vs. 47/180, respectively; P =.020), and there was no difference in older patients (>45 years). The distribution of each genotype between CLD and HCC was very similar (B and C: 11.2% and 87.0% vs. 10.8% and 89.2%, respectively). In conclusion, our results suggest that, although the patients with genotype B experience earlier HBe seroconversion, slower progression of liver fibrosis, and slower development of HCC, the life-long risk of progression to advanced fibrosis and development of HCC may not differ among genotypes B- and C-related chronic liver disease.

Adult↗

PubMed2003

Relevance network between chemosensitivity and transcriptome in human hepatoma cells.

Generally, hepatoma is not a chemosensitive tumor, and the mechanism of resistance to anticancer drugs is not fully elucidated. We aimed to comprehensively evaluate the relationship between chemosensitivity and gene expression profile in human hepatoma cells, by using microarray analysis, and analyze the data by constructing relevance networks. In eight hepatoma cell lines (HLE, HLF, Huh7, Hep3B, PLC/PRF/5, SK-Hep1, Huh6, and HepG2), the baseline expression levels of 2300 genes were measured by cDNA microarray. The concentrations of eight anticancer drugs (nimustine, mitomycin C, cisplatin, carboplatin, doxorubicin, epirubicin, mitoxantrone, and 5-fluorouracil) needed for 50% growth inhibition were examined and used as a measure of chemosensitivity. These data were combined and comprehensive pair-wise correlations between gene expression levels and the 50% growth inhibition values were calculated. Significant correlations with significance were used to construct networks of similarity. Fifty-two relations, including 42 genes, were selected. Among them, nearly 20% were various types of transporters, and most of them negatively correlated with chemosensitivity. Transporter associated with antigen processing 1 was associated with resistance to mitoxantrone, consistent with previous reports. Other transporters were not reported previously to associate with chemosensitivity. Resistance to doxorubicin and its analogue, epirubicin, were positively correlated with topoisomerase II beta expression, whereas it negatively correlated with expression of carboxypeptidases A3 and Z. Response to nimustine was associated with expression of superoxide dismutase 2. Relevance networks identified several negative correlations between gene expression and resistance, which were missed by hierarchical clustering. Our results suggested the necessity of systematically evaluating the transporting systems that may play a major role in resistance in hepatoma. This may provide useful information to modify anticancer drug action in hepatoma.

Antineoplastic Agents↗

PubMed2003

Liver chip and gene shaving.

A comprehensive profile of genes expressed at the mRNA level in various human tissues is considered to be important for understanding the molecular mechanisms of the tissue-specific function and the pathogenesis of related diseases. Here, the gene expression profiling in three human digestive tissues, liver, stomach, and pancreas, was catalogued by generating a large number of expressed sequence tags, and clarified how quantitatively the gene expressions are different. After assembling the redundant clones among three tissues, the results showed that only 1.7% among the assembled genes was expressed commonly in the investigated tissues. These results suggest that the significant functional divergences in different tissues must be related to the divergence of the gene expression profiles. Recently, microarray technologies are widely used. Considering the results that different genes express in different tissues, however, it is important to spot the cDNAs derived from the same tissues or cells examined to acquire information efficiently. For the study of digestive diseases, we constructed an in-house microarray by using the cDNA sets derived from the digestive tissues (liver and gastric chip). In addition, because the amount of information acquired by the microarray analyses is huge, the power of bioinformatics for unifying the obtained data is indispensable. Some examples of the strategies for handling the microarray data obtained by our in-house microarrays are shown in this article.

Gene Expression Profiling↗

PubMed2003

In-house cDNA microarray analysis of gene expression profiles involved in SCC cell lines.

To analyze gene expression in oral cancer, we produced a specialized in-house cDNA microarray. The cDNA library was constructed from surgical specimens of oral squamous cell carcinoma (SCC) using an oligo-capping method. cDNA clones (n=4,800) were randomly selected and their 5'-end nucleotide sequences were determined. Overlapping clones were excluded, and 1,423 independent clones were selected and used for microarray production. Compared to the public nucleotide sequence database, 61% of our cDNA clones were full-length. By correlating expression patterns across SCC cell lines, we identified 53 genes (7 up-regulated and 46 down-regulated) that are differentially expressed in SCC cell lines compared to normal mucosa. Semi-quantitative RT-PCR analysis confirmed these findings and validity of our cDNA microarray. Using specimens from SCC patients, we investigated the expression status of the IL-1ra gene, which showed the down-regulated gene by microarray analysis. Gene expression clearly fell in the SCC specimens relative to their references, which indicated that our in-house cDNA microarray system rapidly identified and characterized candidate biomarkers for clinical use.

Carcinoma, Squamous Cell↗