Biomedical subjects

Shumpei Ishikawa

Publications and source records attributed to Shumpei Ishikawa.

15 recordsLinked to original sources

PubMed2026

Hox/Meis-dependent gene-regulatory transition underlies cardiopharyngeal neural crest diversification.

Neural crest cells (NCCs) are multipotent migratory cells essential for cardiac development, yet the lineage trajectories and gene regulatory networks underlying their differentiation in the cardiopharyngeal region remain unclear. Here, we integrate single-cell RNA-seq, spatial transcriptomics, and multiomic analyses to construct a comprehensive map of NCC lineages in developing mouse cardiopharyngeal tissues. We identify a transition from Hox-positive pharyngeal NCCs to Hox-negative intracardiac populations associated with the outflow tract cushion, accompanied by a shift in Meis transcription factor binding and gene-regulatory network architecture. By contrast, NCCs forming the aorticopulmonary septum and great vessel smooth muscle retain distinct Hox-codes. A Meis2-Sox9-Scx gene-regulatory network defines a skeletogenic progenitor-like intermediate state that gives rise to coronary artery smooth muscle and semilunar valves. Our findings suggest that the loss of Hox-dependent regional identity enables pharyngeal NCCs to acquire new fates upon entering the cardiac cushion, providing insight into the developmental origins of coronary and valvular calcification.

Journal Article↗

PubMed2006

Global variation in copy number in the human genome.

Copy number variation (CNV) of DNA sequences is functionally significant but has yet to be fully ascertained. We have constructed a first-generation CNV map of the human genome through the study of 270 individuals from four populations with ancestry in Europe, Africa or Asia (the HapMap collection). DNA from these individuals was screened for CNV using two complementary technologies: single-nucleotide polymorphism (SNP) genotyping arrays, and clone-based comparative genomic hybridization. A total of 1,447 copy number variable regions (CNVRs), which can encompass overlapping or adjacent gains or losses, covering 360 megabases (12% of the genome) were identified in these populations. These CNVRs contained hundreds of genes, disease loci, functional elements and segmental duplications. Notably, the CNVRs encompassed more nucleotide content per genome than SNPs, underscoring the importance of CNV in genetic diversity and evolution. The data obtained delineate linkage disequilibrium patterns for many CNVs, and reveal marked variation in copy number among populations. We also demonstrate the utility of this resource for genetic disease studies.

Chromosome Mapping↗

PubMed2006

Genome-wide detection of human copy number variations using high-density DNA oligonucleotide arrays.

Recent reports indicate that copy number variations (CNVs) within the human genome contribute to nucleotide diversity to a larger extent than single nucleotide polymorphisms (SNPs). In addition, the contribution of CNVs to human disease susceptibility may be greater than previously expected, although a complete understanding of the phenotypic consequences of CNVs is incomplete. We have recently reported a comprehensive view of CNVs among 270 HapMap samples using high-density SNP genotyping arrays and BAC array CGH. In this report, we describe a novel algorithm using Affymetrix GeneChip Human Mapping 500K Early Access (500K EA) arrays that identified 1203 CNVs ranging in size from 960 bp to 3.4 Mb. The algorithm consists of three steps: (1) Intensity pre-processing to improve the resolution between pairwise comparisons by directly estimating the allele-specific affinity as well as to reduce signal noise by incorporating probe and target sequence characteristics via an improved version of the Genomic Imbalance Map (GIM) algorithm; (2) CNV extraction using an adapted SW-ARRAY procedure to automatically and robustly detect candidate CNV regions; and (3) copy number inference in which all pairwise comparisons are summarized to more precisely define CNV boundaries and accurately estimate CNV copy number. Independent testing of a subset of CNVs by quantitative PCR and mass spectrometry demonstrated a >90% verification rate. The use of high-resolution oligonucleotide arrays relative to other methods may allow more precise boundary information to be extracted, thereby enabling a more accurate analysis of the relationship between CNVs and other genomic features.

Algorithms↗

PubMed2006

CARAT: a novel method for allelic detection of DNA copy number changes using high density oligonucleotide arrays.

BACKGROUND: DNA copy number alterations are one of the main characteristics of the cancer cell karyotype and can contribute to the complex phenotype of these cells. These alterations can lead to gains in cellular oncogenes as well as losses in tumor suppressor genes and can span small intervals as well as involve entire chromosomes. The ability to accurately detect these changes is central to understanding how they impact the biology of the cell. RESULTS: We describe a novel algorithm called CARAT (Copy Number Analysis with Regression And Tree) that uses probe intensity information to infer copy number in an allele-specific manner from high density DNA oligonuceotide arrays designed to genotype over 100,000 SNPs. Total and allele-specific copy number estimations using CARAT are independently evaluated for a subset of SNPs using quantitative PCR and allelic TaqMan reactions with several human breast cancer cell lines. The sensitivity and specificity of the algorithm are characterized using DNA samples containing differing numbers of X chromosomes as well as a test set of normal individuals. Results from the algorithm show a high degree of agreement with results from independent verification methods. CONCLUSION: Overall, CARAT automatically detects regions with copy number variations and assigns a significance score to each alteration as well as generating allele-specific output. When coupled with SNP genotype calls from the same array, CARAT provides additional detail into the structure of genome wide alterations that can contribute to allelic imbalance.

Algorithms↗

PubMed2006

Chromosomal numerical abnormality profiles of gastrointestinal stromal tumors.

BACKGROUND: Biological variations in and the heterogeneity of gastrointestinal stromal tumors (GISTs) are well known, but chromosomal numerical abnormality (CNA) has not been fully examined especially in this context. The aim of this study is to test CNA as a possible biological predictor of biological behavior of GISTs. METHOD: We applied microwave-assisted FISH protocol to pathological archives of GIST tumors displaying different clinical features to characterize the CNA profile of these tumors. A panel of 18 centromere enumeration probes (CEP) and 24 bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC) probes containing genes like Aurora kinases (AURKs) and other candidate genes involved in human carcinogenesis were used. CNA profiles, histopathological risk categorization and Ki-67 labeling indexes of 23 primary and/or metastatic GIST tumors of 12 subjects (both primary and metastatic in 7 subjects) were compared between primary GIST with and without metastases, and between metastatic and primary portions in 7 individuals. RESULTS: CNA in the primary sites was more extensive in the GISTs with recurrence and metastasis than in those without, especially as to the loss of chromosome 20 and genomic imbalance of AURKA-containing BAC probe on 20q in the cases with metastasis. The consistent loss of one allele of chromosome 14q was also noted. Interestingly, both primary and metastatic tumors in identical individuals had similar CNA profiles. CONCLUSION: The extent of CNA differed between GISTS with and without recurrence or metastasis; thus, FISH analysis of specimens from the primary sites may predict the biological behavior of this tumor.

Adult↗

PubMed2006

Noise reduction from genotyping microarrays using probe level information.

Genomic copy number change is one of the important phenomenon observed in cancer and other genetic disorders. Recently oligonucleotide microarrays have been used to analyze changes in the copy number. Although high density microarrays provide genome wide useful data on copy number, they are often associated with substantial amount of experimental noise that could affect the performance of the analyses. We used the high density oligonucleotide genotyping microarrays in our experiments that uses redundant probe tiling approach for individual SNPs. We found that the noise in the genotyping microarray data is associated with several experimental steps during target preparation and devised an algorithm that takes into account those experimental parameters. Additionally, defective probes that do not hybridize well to the target and therefore could not be modified inherently were detected and omitted automatically by using the algorithm. When we applied the algorithm to actual datasets, we could reduce the noise substantially without compressing the dynamic range. Additionally, combinatorial use of our noise reduction algorithm and conventional breakpoint detection algorithm successfully detected a microamplification of c-myc which was overlooked in the raw data. The algorithm described here is freely available with the software upon request to all non-profit researchers.

Adenocarcinoma↗

PubMed2005

Two subclasses of lung squamous cell carcinoma with different gene expression profiles and prognosis identified by hierarchical clustering and non-negative matrix factorization.

Current clinical and histopathological criteria used to define lung squamous cell carcinomas (SCCs) are insufficient to predict clinical outcome. To make a clinically useful classification by gene expression profiling, we used a 40 386 element cDNA microarray to analyse 48 SCC, nine adenocarcinoma, and 30 normal lung samples. Initial analysis by hierarchical clustering (HC) allowed division of SCCs into two distinct subclasses. An additional independent round of HC induced a similar partition and consensus clustering with the non-negative matrix factorization approach indicated the robustness of this classification. Kaplan-Meier analysis with the log-rank test pointed to a nonsignificant difference in survival (P = 0.071), but the likelihood of survival to 6 years was significantly different between the two groups (40.5 vs 81.8%, P = 0.014, Z-test). Biological process categories characteristic for each subclass were identified statistically and upregulation of cell-proliferation-related genes was evident in the subclass with poor prognosis. In the subclass with better survival, genes involved in differentiated intracellular functions, such as the MAPKKK cascade, ceramide metabolism, or regulation of transcription, were upregulated. This work represents an important step toward the identification of clinically useful classification for lung SCC.

Biomarkers, Tumor↗

PubMed2005

Allelic dosage analysis with genotyping microarrays.

Genomic alternations, including dosage and allelic imbalance, constitute a major basis of neoplastic and other genetic disorders. Using oligonucleotide genotyping microarrays, here we report the development and usage of an algorithm, called genome imbalance map (GIM) algorithm, for allelic as well as total gene dosage analysis. Using the GIM algorithm, global genome imbalance status at over 100,000 loci was simultaneously analyzed with unprecedented accuracy and allelic discrimination.

Algorithms↗

PubMed2005

Overexpression of the aldo-keto reductase family protein AKR1B10 is highly correlated with smokers' non-small cell lung carcinomas.

PURPOSE: Squamous cell carcinoma (SCC) and adenocarcinoma of the lung are currently subject to similar treatment regimens despite distinct differences in histology and epidemiology. The aim of this study is to identify a molecular target with diagnostic and therapeutic values for SCC. EXPERIMENTAL DESIGN: Genes specifically up-regulated in SCC were explored through microarray analysis of 5 SCCs, 5 adenocarcinomas, 10 small cell lung carcinomas, 27 normal tissues, and 40 cancer cell lines. Clinical usefulness of these genes was subsequently examined mainly by immunohistochemical analysis. RESULTS: Seven genes, including aldo-keto reductase family 1, member B10 (AKR1B10), were identified as SCC-specific genes. AKR1B10 was further examined by immunohistochemical analysis of 101 non-small cell lung carcinomas (NSCLC) and its overexpression was observed in 27 of 32 (84.4%) SCCs and 19 of 65 (29.2%) adenocarcinomas. Multiple regression analysis showed that smoking was an independent variable responsible for AKR1B10 overexpression in NSCLCs (P < 0.01) and adenocarcinomas (P < 0.01). AKR1B10 staining was occasionally observed even in squamous metaplasia, a precancerous lesion of SCC. CONCLUSION: AKR1B10 was overexpressed in most cases with SCC, which is closely associated with smoking, and many adenocarcinoma cases of smokers. These results suggest that AKR1B10 is a potential diagnostic marker specific to smokers' NSCLCs and might be involved in tobacco-related carcinogenesis.

Aldehyde Reductase↗

PubMed2005

Differential expression of S100A2 and S100A4 in lung adenocarcinomas: clinicopathological significance, relationship to p53 and identification of their target genes.

Previous studies suggest that some S100 proteins are involved in the progression of certain types of cancer. However, no comprehensive data is currently available on the expression of S100 family genes in lung adenocarcinomas. Oligonucleotide array, quantitative reverse transcription-polymerase chain reaction and western blot analyses of lung adenocarcinoma cell lines and bronchiolar epithelial cells (SAEC and NHBE) revealed that S100A2 and S100A4 were the most strikingly downregulated and upregulated members of the S100 family, respectively. Immunohistochemical analyses of 94 primary lung adenocarcinomas showed that positive S100A2 expression (33/94, 35.1%) was significantly associated with lymphatic invasion (P=0.0233) and positive S100A4 expression (19/94, 20.2%) with vascular invasion (P=0.0454). Interestingly, a strong inverse relationship was found between S100A4 and p53 expression (P=0.0008). Survival analyses showed that S100A4 positivity was associated with poor patient prognosis (P=0.042). S100A2 positivity was not associated with patient survival when the whole patient group was analyzed; however, S100A2 positivity was a favorable prognostic indicator in patients with p53-negative tumors (P=0.0448). Finally, we used oligonucleotide array analyses and identified potential S100A2 and S100A4 target genes involved in cancer progression: S100A2 induced RUNX3 and REPRIMO; S100A4 induced EZRIN, RUNX1 and WISP1; S100A2 repressed EGFR, NFKB2 and RELA2; and S100A4 repressed ANXA10 and IL1RN. Thus, the present study demonstrates involvement of S100A2 and S100A4 in the progression of lung adenocarcinomas and an inverse association between S100A4 and p53 expression, and provides a list of targets regulated by S100A2 and S100A4.

Adenocarcinoma↗

PubMed2004

Selective expression of a subset of neuronal genes in oligodendroglioma with chromosome 1p loss.

Gliomas are classified based mainly on microscopic resemblance to their presumed glial origin such as astrocyte and oligodendrocyte. However, more objective diagnostic criteria are indispensable for the precise treatment of patients. For instance, loss of the short arm of chromosome 1 (1p) in oligodendrogliomas is recognized as an important marker for better response to chemotherapy and longer survival of the patients. To gain insight into their molecular biological background and to identify genes characterizing each subgroup, we investigated gene expression profile of the 4 glioma subsets, oligodendroglioma with and without 1p loss, diffuse astrocytoma and glioblastoma using DNA microarray. Remarkably, most of the genes showing distinctive expression in oligodendroglioma with 1p loss were also highly expressed in normal brain tissues and had neuron-related function, which included MYT1L, INA, RIMS2, SNAP97 and SNCB. Histological analysis also demonstrated that MYT1L, which were abundantly expressed in normal neuron, were certainly present in tumor cells. These results suggest that oligodendroglioma, especially with 1p loss, has more or less neuronal characteristics although oligodendroglioma is thought to originate form glial lineage cell. With further pathological studies, those neuron-related genes might be good diagnostic markers for oligodendroglioma of better prognosis as well.

Astrocytoma↗

PubMed2004

Short report: Lethal malaria in cytosolic phospholipase A2- and phospholipase A2IIA-deficient mice.

Lipid mediators play important roles in the pathogenesis of malaria. Phospholipase A2s are enzymes involved in the production of these mediators, and they function in inflammation. Among them, cytosolic phospholipase A2 (cPLA2) is a key enzyme in the metabolism of arachidonic acid, the first intermediate in the production of lipid mediators. Plasmodium berghei ANKA causes cerebral malaria in CL57B/6 mice, and we recently produced cPLA2-deficient mice with this background. With the expectation of reduced pathogenicity, we performed experimental infection in these mice. Unexpectedly, the infected mice developed cerebral malaria and died at the same time as the control mice, while the parasitemia progressed similarly in both groups. These observations suggest that secretory PLA2s rather than cPLA2 may be involved in the aggravation, although possible compensation by the induction of other enzymes has not been excluded. The present findings are expected to help clarify the involvement of various phospholipase A2s in malaria.

Animals↗

PubMed2003

Functional characterization of rat brain-specific organic anion transporter (Oatp14) at the blood-brain barrier: high affinity transporter for thyroxine.

Oatp14/blood-brain barrier-specific anion transporter 1 (Slc21a14) is a novel member of the organic anion transporting polypeptide (Oatp/OATP) family. Northern blot analysis revealed predominant expression of Oatp14 in the brain, and Western blot analysis revealed its expression in the brain capillary and choroid plexus. Immunohistochemical staining indicated that Oatp14 is expressed in the border of the brain capillary endothelial cells. When expressed in human embryonic kidney 293 cells, Oatp14 transports thyroxine (T4; prothyroid hormone) (Km = 0.18 mum), as well as amphipathic organic anions such as 17beta estradiol-d-17beta-glucuronide (Km = 10 mum), cerivastatin (Km = 1.3 mum), and troglitazone sulfate (Km = 0.76 mum). The uptake of triiodothyronine (T3), an active form produced from T4, was significantly greater in Oatp14-expressed cells than in vector-transfected cells, but the transport activity for T3 was approximately 6-fold lower that for T4. The efflux of T4, preloaded into the cells, from Oatp14-expressed cells was more rapid than that from vector-transfected cells (0.032 versus 0.006 min-1). Therefore, Oatp14 can mediate a bidirectional transport of T4. Sulfobromophthalein, taurocholate, and estrone sulfate were potent inhibitors for Oatp14, whereas digoxin, p-aminohippurate, or leukotriene C4, or organic cations such as tetraetheylammonium or cimetidine had no effect. The expression levels of Oatp14 mRNA and protein were up- and down-regulated under hypo- and hyperthyroid conditions, respectively. Therefore, it may be speculated that Oatp14 plays a role in maintaining the concentration of T4 and, ultimately, T3 in the brain by transporting T4 from the circulating blood to the brain.

Animals↗

PubMed2003

Expression imbalance map: a new visualization method for detection of mRNA expression imbalance regions.

We describe the development of a new visualization method, called the expression imbalance map (EIM), for detecting mRNA expression imbalance regions, reflecting genomic losses and gains at a much higher resolution than conventional technologies such as comparative genomic hybridization (CGH). Simple spatial mapping of the microarray expression profiles on chromosomal location provides little information about genomic structure, because mRNA expression levels do not completely reflect genomic copy number and some microarray probes would be of low quality. The EIM, which does not employ arbitrary selection of thresholds in conjunction with hypergeometric distribution-based algorithm, has a high tolerance of these complex factors. The EIM could detect regionally underexpressed or overexpressed genes (called, here, an expression imbalance region) in lung cancer specimens from their gene expression data of oligonucleotide microarray. Many known as well as potential loci with frequent genomic losses or gains were detected as expression imbalance regions by the EIM. Therefore, the EIM should provide the user with further insight into genomic structure through mRNA expression.

Allelic Imbalance↗

PubMed2003

Glypican-3, overexpressed in hepatocellular carcinoma, modulates FGF2 and BMP-7 signaling.

The Glypican (GPC) family is a prototypical member of the cell-surface heparan sulfate proteoglycans (HSPGs). The HSPGs have been demonstrated to interact with growth factors, act as coreceptors and modulate growth factor activity. Here we show that based on oligonucleotide array analysis, GPC3 was upregulated in hepatocellular carcinoma (HCC). By northern blot analysis, GPC3 mRNA was found to be upregulated in 29 of 52 cases of HCC (55.7%). By Western blot analysis carried out with a monoclonal anti-GPC3 antibody we generated, the GPC3 protein was found to be overexpressed in 6 hepatoma cell lines, HepG2, Hep3B, HT17, HuH6, HuH7 and PLC/PRF/5, as well as 22 tumors (42.3%). To investigate the role of overexpressed GPC3 in liver cancer, we analyzed its effects on cell growth of hepatoblastoma-derived cells. Overexpression of GPC3 modulated cell proliferation by inhibiting fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 7 (BMP-7) activity. An interaction of GPC3 and FGF2 was revealed by co-immunoprecipitation, while GPC3 was found to inhibit BMP-7 signaling through the Smad pathway by reporter gene assay. The modulation of growth factors by GPC3 may help explain its role in liver carcinogenesis. In addition, the ability of HCC cells to express GPC3 at high levels may serve as a new tumor marker for HCC.

Aged↗