Biomedical subjects

Seiichi Ishida

Publications and source records attributed to Seiichi Ishida.

At least 19 recordsLinked to original sources

PubMed2006

Gene expression property of high-density three-dimensional tissue of HepG2 cells formed in radial-flow bioreactor.

In our previous study, we examined three-dimensional culture using 5-ml radial-flow bioreactor (RFB) and showed that genes encoding cell cycle related proteins were suppressed in a stable phase. In this study, we analyzed the gene expression profiles of RFB-cultivated HepG2 cells and found that vascular endothelial growth factor (VEGF) production was strongly induced in the stable phase compared with the growth phase or static two-dimensional culture. When human umbilical vein endothelial cells (HUVECs) were grown under the conditioned medium of the stable phase, it was found that the formation of new blood vessels was induced in the angiogenesis model. DNA microarray analysis showed that the expression levels of both genes related to cell cycle arrest and which are known as tumor markers have increased in the stable phase. This result suggests that HepG2 cells in the stable phase maintain an active tumor phenotype. In addition, the expression of genes induced in the hypoxic condition was also induced in the stable phase. When the culture was carried out under a higher dissolved oxygen (DO) concentration, VEGF production did not decrease significantly and the new blood-vessel-forming ability of the conditioned medium was not suppressed. This suggests that the induction of VEGF production in a stable phase is not affected by DO during the tested level. These results suggest that the RFB cell culture system may be used to assess tumor progression mechanism under three-dimensional condition in vitro.

Biomarkers, Tumor↗

PubMed2005

Thyroid hormone targets matrix Gla protein gene associated with vascular smooth muscle calcification.

Thyroid hormones have marked cardiovascular effects in vivo. However, their direct effects on vascular smooth muscle cells have been unclear. Because thyroid hormones play critical roles in bone remodeling, we hypothesized that they are also associated with vascular smooth muscle calcification, one of the pathological features of vascular sclerosis. To test this hypothesis, we examined the effects of 3',3,5-triiodo-L-thyronine (T3) on the expression of calcification-associated genes in rat aortic smooth muscle cells (RAOSMCs). Quantitative RT-PCRs revealed that a physiological concentration of T3 (15 pmol/L free T3) increased mRNA level of matrix Gla protein (MGP), which acts as a potent inhibitor of vascular calcification in vivo, by 3-fold in RAOSMCs, as well as in cultured human coronary artery smooth muscle cells. In RAOSMCs transiently transfected with a luciferase reporter gene driven by the MGP promoter, T3 significantly stimulated luciferase activity. In addition, RNA interference against thyroid hormone receptor-alpha gene diminished the effect of T3 on MGP expression. Aortic smooth muscle tissues from methimazole-induced hypothyroid rats (400 mg/L drinking water; 4 weeks) also showed a 68% decrease in the MGP mRNA level, as well as a 33% increase in calcium content compared with that from the control euthyroid animals, whereas hyperthyroidism (0.2 mg T3/kg IP; 10 days) upregulated MGP mRNA by 4.5-fold and reduced calcium content by 11%. Our findings suggest that a physiological concentration of thyroid hormone directly facilitates MGP gene expression in smooth muscle cells via thyroid hormone nuclear receptors, leading to prevention of vascular calcification in vivo.

Animals↗

PubMed2005

Polymorphic tandem repeat sequences of the thymidylate synthase gene correlates with cellular-based sensitivity to fluoropyrimidine antitumor agents.

PURPOSE: Thymidylate synthase (TS) is one of the target molecules for the antitumor effects of fluoropyrimidine drugs. The cellular thymidylate synthase level is one of the determining factors for the antitumor activity of fluoropyrimidines. TYMS, which encodes TS, has been reported to possess 28-bp tandem repeat sequences in its 5'-untranslated region, the number of which varies. In addition, single nucleotide polymorphisms have also been shown in a triple repeat sequence. In this study, correlation between the polymorphic tandem repeat sequences of the TYMS gene and the antitumor activities of 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FUdR) were investigated with 30 established human cell lines derived from solid tumors. METHODS: A reporter assay system was developed in order to compare the ability of the transactivation mediated by the double (2R) and triple (c- or g-type, 3Rc or 3Rg, respectively) repeat sequences using a human colon cancer cell line, DLD-1. The 50% inhibitory concentration (IC(50)) of cell growth by 5-FU and FUdR was measured with 30 different established cell lines of human solid tumors. Genotypes based on the number of the 28-bp TYMS tandem repeat for the above cell lines were determined by electrophoretical analysis of PCR products containing the repeat sequences and nucleotide sequencing. RESULTS: The reporter activity mediated by the 3Rg sequence was significantly higher than that by the 2R and 3Rc sequences. Activities mediated by the 2R and 3Rc sequences were comparable. According to the reporter assay, 2R and 3Rc were judged as low TS expression alleles and 3Rg as a high TS expression allele. On the basis of IC(50) values, cells possessing the 2R/2R and 2R/3R repeat of TYMS were significantly more sensitive to FUdR than those with the 3R/3R repeat. Cells possessing 3Rg/3Rg (a high TS expression genotype) were significantly less sensitive to FUdR than cells with 2R/2R, 2R/3Rc, and 3Rc/3Rc (low TS expression genotypes). CONCLUSIONS: Our results of the reporter assays using 2R, 3Rc, and 3Rg repeat sequences prompted us to classify 3Rg as a high TS expression allele, and 2R and 3Rc as low TS expression alleles. The cells with low TS expression alleles were shown to exhibit significantly higher FUdR sensitivity than the cells with high TS expression alleles for the first time. These results were consistent with numerous previous in vitro and in vivo findings that tumors showing high TS expression were less sensitive to fluoropyrimidines. These results support the idea that genotyping the tandem repeat sequences of TYMS in the 5'-untranslated region is useful for individualized therapy involving fluoropyrimidine antitumor drugs.

Antimetabolites, Antineoplastic↗

PubMed2005

Functional analysis of six human aryl hydrocarbon receptor variants in a Japanese population.

Aryl hydrocarbon receptor (AhR) is an important transcriptional regulator involved in the induction of CYP1A1, CYP1A2, CYP1B1, UGT1A1, and UGT1A6. In this study, functional properties of four novel naturally occurring human AhR variants (K401R, N487D, I514T, and K17T/R554K) were examined along with the single variants K17T and R554K. The luciferase reporter assay using the CYP1A1 promoter reporter in HeLa cells treated with beta-naphthoflavone or 3-methylcholanthrene, which are known as typical agonists for AhR, showed that reporter activities of the K401R and N487D variants were reduced to 40 to 58% of those of wild-type (WT) but not of the other variants. Similarly, the K401R and N487D variants also reduced the omeprazole-induced reporter activities to approximately 56 and 74% of those of the WT, respectively. The reduced activities of the two variants were probably caused by the reduced protein expression levels, since the protein levels of the K401R and N487D variants were approximately 52 and 47% of the WT, respectively, without any changes in their mRNA levels. The reduced protein levels were recovered by treatment with a proteasome inhibitor MG132 [N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal], suggesting that the reduced protein levels were caused by the accelerated proteasomal degradation by a proteasome. Together, the current data demonstrate that the K401R and N487D variants reduce their apparent transcriptional activities, both ligand-induced and omeprazole-induced activation, probably through reduced protein expression. Thus, these two variants may influence drug metabolism through reduced induction of CYP1A1 and other target enzymes.

Genes, Reporter↗

PubMed2005

Cytoprotection against oxidative stress-induced damage of astrocytes by extracellular ATP via P2Y1 receptors.

Oxidative stress is the main cause of neuronal damage in traumatic brain injury, hypoxia/reperfusion injury, and neurodegenerative disorders. Although extracellular nucleosides, especially adenosine, are well known to protect against neuronal damage in such pathological conditions, the effects of these nucleosides or nucleotides on glial cell damage remain largely unknown. We report that ATP but not adenosine protects against the cell death of cultured astrocytes induced by hydrogen peroxide (H2O2). ATP ameliorated the H2O2-induced decrease in cell viability of astrocytes in an incubation time- and concentration-dependent fashion. Protection by ATP was inhibited by P2 receptor antagonists and was mimicked by P2Y1 receptor agonists but not by adenosine. The expressions of P2Y1 mRNAs and functional P2Y1 receptors in astrocytes were confirmed. Thus, ATP, acting on P2Y1 receptors in astrocytes, showed a protective action against H2O2. The astrocytic protection by the P2Y1 receptor agonist 2-methylthio-ADP was inhibited by an intracellular Ca2+ chelator and a blocker of phospholipase C, indicating the involvement of intracellular signals mediated by Gq/11-coupled P2Y1 receptors. The ATP-induced protection was inhibited by cycloheximide, a protein synthesis inhibitor, and it took more than 12 h for the onset of the protective action. In the DNA microarray analysis, ATP induced a dramatic upregulation of various oxidoreductase genes. Taken together, ATP acts on P2Y1 receptors coupled to Gq/11, resulting in the upregulation of oxidoreductase genes, leading to the protection of astrocytes against H2O2.

Adenosine Triphosphate↗

PubMed2005

Gene expression profiling of dexamethasone-treated RBL-2H3 cells: induction of anti-inflammatory molecules.

Glucocorticoids are well known for their anti-inflammatory effect through the regulation of gene expression in many types of immune cells, including mast cells. However, the genes that are involved in suppression of mast cell-mediated inflammation by glucocorticoids have not been fully identified. Therefore, we examined the dexamethasone (Dex)-responsive genes in RBL-2H3 mast cells using a high-density oligonucleotide microarray technique. Gene expression profiling revealed that the antigen-induced up-regulation of pro-inflammatory factors, including monocyte chemoattractant protein-1, was markedly inhibited by 100 nM Dex. On the other hand, Dex treatment itself caused the substantial up-regulation of many genes, including phenylethanolamine-N-methyl transferase (PNMT) and cytokine-inducible SH2-containing protein (CISH), in the mast cells. The expression of these two genes significantly increased 6 h after Dex exposure and lasted for more than 24 h. Considering that PNMT is the rate-determining enzyme in epinephrine synthesis and that CISH is a suppressor of cytokine signaling, these Dex-responsive genes may be potential anti-inflammatory factors. Thus, gene expression profiling suggested that Dex might exert its anti-inflammatory effect through two pathways in mast cells: the suppression and induction of potentially pro- and anti-inflammatory factors, respectively.

Animals↗

PubMed2005

Three-dimensional high-density culture of HepG2 cells in a 5-ml radial-flow bioreactor for construction of artificial liver.

A three-dimensional high-density cell culture is essential for the construction of an artificial tissue. Many researchers have reported that three-dimensional cell culture enhances cell function. The use of a radial-flow bioreactor (RFB) has enabled the cultivation of cells at high density for constructing a three-dimensional tissue. In this study, we have developed a novel, small RFB, which has a bed volume of 5 ml and is equipped with a porous support as an immobilized scaffold; its performance was tested using the hepatoblastoma cell line, HepG2. Among the other supports tested here, hydroxyl apatite was selected from the viewpoint of its ability to support good cell growth at high density with uniform distribution in a bioreactor. The HepG2 cells grew well in the scaffold under a sufficient supply of nutrients by radial flow and were used to construct a three-dimensional tissue in the scaffold. The concentration of the cells cultivated in this 5-ml RFB reached 10(8) cells/ml and the glucose consumption rate was almost similar to that obtained when using a 30-ml RFB, which has already been reported previously. This high glucose consumption continued over 7 d after the growth phase. Furthermore, albumin production was maintained in the stable phase. Gene expression profiles of cells obtained from long-term cultures in the 5-ml RFB were analyzed. It was found that the expressions of genes encoding the cell cycle-related proteins, cyclins, and cell cycle division 2 (cdc2) were suppressed in the stable phase. In addition, the number of cells incorporating 5'-bromo-2'-deoxyuridine (BrdU) in the stable phase markedly decreased compared with that in the growth phase. These results indicated that the majority of cells in the stable phase remain in the G0/G1 phase. Furthermore, this implies that the three-dimensional tissue constructed in the 5-ml RFB showed the high function similar to a normal liver in the human body. Therefore, the 5-ml RFB was considered as a useful tool and a substitute method for animal experiments.

Bioreactors↗

PubMed2005

New approaches to mechanism analysis for drug discovery using DNA microarray data combined with KeyMolnet.

We have developed a comprehensive information platform, named KeyMolnet, for drug discovery and life science research in the post-genome era. Using KeyMolnet, we show new approaches to research into the biological mechanism in DNA microarray analysis. Thanks to the DNA microarray technology, it is now possible to obtain very large quantities of gene expression data at a time. However, it is still difficult to extract meaningful information from such large quantities of data and to analyze the relationship between gene expression data and biological function. We therefore developed an advanced tool that can generate molecular networks upon demand, and beyond signaling "cross-talks," can connect them to physiological phenomena and medical and drug information. Here we show the methods of mechanism analysis using the DNA microarray data and KeyMolnet, as well as the possible mechanism of inducing apoptosis in the human promyelocytic leukemia cell line, HL-60, treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), using the time series of gene expression data from DNA microarray experiments. KeyMolnet enables practical approaches to research into biological mechanisms, which in turn contribute to new discoveries in the medical, pharmaceutical and life sciences.

Apoptosis↗

PubMed2004

Differential modulation of PI3-kinase/Akt pathway during all-trans retinoic acid- and Am80-induced HL-60 cell differentiation revealed by DNA microarray analysis.

All-trans retinoic acid (ATRA) and Am80 are natural and synthetic derivatives of Vitamin A and have been used in the fields of oncology and dermatology for years. Their action was considered to be achieved mainly through binding to nuclear hormone receptors, retinoic acid receptors (RARs), although they have been observed to have different biological effects. For example, the two compounds have similar effects on differentiation but different effects on proliferation in human promyelocytic leukemia cell line HL-60 cells. To elucidate the genes responsible for this and other differences, we attempted for the first time to determine the genes whose expressions were differentially modulated during the time course of HL-60 cell differentiation by ATRA and Am80 treatment up to 72h utilizing DNA microarray and clustering analyses. As a result, the expressions of 204 genes were found to be modulated differentially by ATRA and Am80. Among them, we focused on two components of the PI3-kinase/Akt signal transduction pathway, phosphoinositide-3-kinase, beta-catalytic subunit and ribosomal protein S6 kinase polypeptide 1, which are related to the regulation of cell proliferation and apoptosis. Their expressions were specifically suppressed by ATRA, which coincided with the suppressive effects of ATRA on the HL-60 cell proliferation. Moreover, PI3-kinase inhibitors suppressed the proliferation of Am80-treated cells to the same extent as ATRA did. These results indicated that these gene products play a role in HL-60 cell growth suppression during the late stage of differentiation. The complete data and a list of the genes are available at .

Antineoplastic Agents↗

PubMed2004

Improvement in reliability of probabilistic test of significant differences in GeneChip experiments.

A probabilistic test (FUMI theory) for GeneChip experiments has been proposed for selecting the genes which show significant differences in the gene expression levels between a single pair of treatment and control. This paper describes that the reliability of the judgment by the FUMI theory can be enhanced, when the selected genes are referred to biomolecular-functional networks of a commercial database. The genes judged as being differently expressed are grouped into a cluster in the biomolecular networks. It is also demonstrated that false positive genes have a trend in the networks to be isolated from each other, and also away from the clustered genes, since the false positive genes are randomly selected.

Algorithms↗

PubMed2003

Distinct gene expression phenotypes of cells lacking Rb and Rb family members.

The study of tumor suppressor gene function has been aided by the creation of discrete gene alterations in the mouse. One such example can be seen in the study of tumor suppressor gene function in general and the retinoblastoma (Rb) tumor suppressor in particular. Because the phenotype of a cell is a direct reflection of the gene activity within that cell, a comprehensive analysis of changes in gene activity resulting from the loss of Rb function has the potential to greatly enhance our understanding of Rb biology. We have used DNA microarray analysis to identify gene expression profiles in wild-type and Rb-null mouse embryo fibroblasts, as well as cells lacking other Rb family members, as an approach to developing a more complete understanding of Rb function. In so doing, we have identified gene expression phenotypes that characterize the loss of Rb function, that distinguish a Rb-null cell from a wild-type cell as well as a p107/p130-null cell, and that identify gene regulatory pathways unique to these events. Importantly, the Rb gene expression patterns can identify murine tumors that result from Rb loss of function. We suggest that this is an approach to the eventual understanding of gene regulatory pathways that define a phenotypic state, including those events that lead to tumor development.

Animals↗

PubMed2003

Gene expression phenotypic models that predict the activity of oncogenic pathways.

High-density DNA microarrays measure expression of large numbers of genes in one assay. The ability to find underlying structure in complex gene expression data sets and rigorously test association of that structure with biological conditions is essential to developing multi-faceted views of the gene activity that defines cellular phenotype. We sought to connect features of gene expression data with biological hypotheses by integrating 'metagene' patterns from DNA microarray experiments in the characterization and prediction of oncogenic phenotypes. We applied these techniques to the analysis of regulatory pathways controlled by the genes HRAS (Harvey rat sarcoma viral oncogene homolog), MYC (myelocytomatosis viral oncogene homolog) and E2F1, E2F2 and E2F3 (encoding E2F transcription factors 1, 2 and 3, respectively). The phenotypic models accurately predict the activity of these pathways in the context of normal cell proliferation. Moreover, the metagene models trained with gene expression patterns evoked by ectopic production of Myc or Ras proteins in primary tissue culture cells properly predict the activity of in vivo tumor models that result from deregulation of the MYC or HRAS pathways. We conclude that these gene expression phenotypes have the potential to characterize the complex genetic alterations that typify the neoplastic state, whether in vitro or in vivo, in a way that truly reflects the complexity of the regulatory pathways that are affected.

Animals↗

PubMed2003

Glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan (CPT-11), by human UGT1A1 variants, G71R, P229Q, and Y486D.

7-Ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of antitumor agent irinotecan (CPT-11), is conjugated and detoxified to SN-38-glucuronide by UDP-glucuronosyltransferase (UGT) 1A1. Genetic polymorphisms in UGT1A1 are thought to contribute to severe diarrhea and/or leukopenia caused by CPT-11. In this regard, it has been reported that polymorphisms in the promoter region could affect the CPT-11 pharmacokinetics and interindividual variation of toxicity. However, little information is available on the influence of UGT1A1 polymorphisms in the coding region on the SN-38 glucuronidation activity. In the present study, wild-type (WT) and three variant (G71R, P229Q, and Y486D) cDNAs of human UGT1A1s were transiently expressed in COS-1 cells, and the kinetic parameters of these UGT1A1s were determined for SN-38 glucuronidation. A partially reduced UGT1A1 protein expression was observed in COS-1 cells for G71R and Y486D. WT UGT1A1 catalyzed SN-38 glucuronidation with an apparent K(m) value of 11.5 microM, whereas those of G71R, P229Q, and Y486D were 14.0, 18.0, and 63.5 microM, respectively. The SN-38 glucuronidation efficiency ratio (V(max)/K(m)) normalized for the level of expression was 1.4, 0.66 (47% of WT), 0.73 (52%), and 0.07 (5%) microl/min/mg of protein for WT, G71R, P229Q, and Y486D, respectively. Thus, the SN-38 glucuronidation activity of Y486D was drastically reduced, whereas the reduction in the G71R and P229Q activities was fractional. The decreased SN-38 glucuronidation efficiency ratio of G71R and P229Q could be critical in combination with other polymorphisms in the UGT1A1 gene.

Amino Acid Substitution↗

PubMed2003

Test of significant differences with a priori probability in microarray experiments.

A traditional method for comparing two expression levels of genes in microarray experiments is the two-sample t-test. Because of the difficulty in using a large number of microarrays, an alternative method is required which can provide a reliable judgment of the comparison from a small number of replicates, even from a single pair of control and treatment. We present a method for detecting the changes in the gene expression levels under two different conditions in microarray experiments. Our method targets a single experiment for each condition, while retaining the statistical advantages of the t-test. The new proposals are: 1) standard deviation (SD) estimates of the expression levels which are an indicator for significant differences are given a priori as a function of the expression levels; 2) the limit of detection (LOD) for the expression levels is used to eliminate the majority of genes expressed at extremely low levels. The a priori SD estimates are obtained from six replicates under a fixed condition and are shown to be the approximate, but proper description of the expression uncertainty covering diverse conditions (e.g., different samples (human and rat) and different DNA chips). The LOD is defined as three times blank SD according to the IUPAC recommendation. A cell line (HL60) which will undergo macrophage differentiation on treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) is taken as an example. Our method is compared with the t-test for the data on duplicate TPA experiments and the former alone is evaluated with the data on a single TPA experiment. The errors from sample preparation and instrumental analysis are discussed.

Animals↗

PubMed2003

Clinically potential subclasses of retinoid synergists revealed by gene expression profiling.

Retinoids have chemopreventive and therapeutic potency in oncology and dermatology, although their application is restricted by many undesirable side effects. For the development of more effective and less toxic retinoids, gene expression analyses using DNA microarrays have the potential to supplement conventional screening methods, which are based on the changes in cell morphology and/or function. In this study, we applied the class prediction algorithm, which was used in the molecular phenotyping of tumors, for the classification of synthetic retinoids (Am80 and Tp80) and retinoid synergists (HX630, TZ335, and PA024) as all-trans retinoic acid-like, 9-cis retinoic acid-like, and control-like classes. By analyzing the effects of all-trans retinoic acid and 9-cis retinoic acid on the gene expressions in a human promyelocytic leukemia cell line, HL60, we successfully selected 50 marker genes whose expression pattern could distinguish these classes. Moreover, the classification revealed the existence of two subclasses among the retinoid synergists used with Am80. Close inspection of the DNA microarray analyses indicated that these two subclasses had different effects on the apoptosis of HL60 cells, and this was confirmed by in vivo experiments. These results indicate that the retinoidal activity of Am80, which has already been used in clinical trials, could be modulated differently by the two classes of retinoid synergists. Thus, these two subclasses of retinoid synergists have the potency to widen the usage of Am80. Our analyses demonstrated that the gene expression profiling could provide important information for developing useful retinoid synergists by compensating conventional screening methods.

Alitretinoin↗

PubMed2002

Characterization of human CYP1A1/1A2 induction by DNA microarray and alpha-naphthoflavone.

DNA microarrays and real time PCR were used to analyze the mechanism of gene induction by CYP1A1 inducers, beta-naphthoflavone, and omeprazole, in the human hepatocellular carcinoma HepG2 cells. Reproducible and significant inductions were observed in a limited number of genes including CYP1A1 and CYP1A2. Genes induced by omeprazole included several protein tyrosine kinase targets. This result confirmed that omeprazole could modulate gene expressions through protein tyrosine kinase-mediated pathway. Induction ratios were considerably different from CYP1A1 and CYP1A2 (>10-fold) to other induced genes (<5-fold). alpha-Naphthoflavone, which is known as an antagonist to 2,3,7,8-tetrachlorodibenzo-p-dioxin, inhibited the inductions of heme oxygenase 1, glutamate-cysteine ligase (modifier unit), and thioredoxin reductase by beta-naphthoflavone but not those of CYP1A1 and CYP1A2. It unexpectedly enhanced the beta-naphthoflavone-mediated CYP1A1 and CYP1A2 induction. These results suggest that the CYP1A1 and CYP1A2 genes, which share their 5(') enhancer regions, are regulated differently from the other genes.

Antigens, Differentiation↗

PubMed2002

Substitution of arginine for cysteine 643 of the glucocorticoid receptor reduces its steroid-binding affinity and transcriptional activity.

To investigate the mechanism for glucocorticoid resistance in leukemic cells, we sequenced the coding region of glucocorticoid receptor (GR) gene in ten Japanese leukemic cells. We identified a novel heterozygous mutation (C643R) in the ligand-binding domain in P30/OHK cells. Western blot analysis for COS-7 cells transfected with the wild-type or C643R mutant GR plasmid revealed similar protein expression levels. In the ligand-binding assay, the dissociation constant of the C643R GR was six-fold higher than that of the wild-type GR. Moreover, the C643R GR showed no transcriptional activity in the luciferase reporter assay.

Amino Acid Substitution↗

PubMed2002

Polymorphisms in the ABCC2 (cMOAT/MRP2) gene found in 72 established cell lines derived from Japanese individuals: an association between single nucleotide polymorphisms in the 5'-untranslated region and exon 28.

We found nucleotide variability in the 5'-upstream region and exonic sequences of a gene-encoding canalicular multispecific organic anion transporter/multidrug resistance-associated protein 2 (cMOAT/MRP2) by polymerase chain reaction-based sequencing using genomic DNA from 72 established cell lines derived from 72 Japanese individuals. Four single nucleotide polymorphisms (SNPs) were found in the 5'-untranslational region and 21 in the exonic regions. Of them, 14 were nonsynonymous SNPs. One deletion of seven consecutive adenines resulting in a frameshift variant was also found. Four SNPs, c-24t, g1249a (V417I), c2366t (S789F), and c3972t (I1324I), were the same as those recently reported. A strong association was found between c-24t (5'-untranslated region) and c3972t (exon 28), with the promoter activity of the former worth being compared.

5' Untranslated Regions↗