Biomedical subjects

Noriko Ueno

Publications and source records attributed to Noriko Ueno.

17 recordsLinked to original sources

PubMed2006

Prediction of peritoneal metastasis in advanced gastric cancer by gene expression profiling of the primary site.

Peritoneal metastasis is the most common cause of tumour progression in advanced gastric cancer. Clinicopathological findings including cytologic examination of peritoneal lavage have been applied to assess the risk of peritoneal metastasis, but are sometimes inadequate for predicting peritoneal metastasis in individuals. Hence, we tried to construct a new prediction system for peritoneal metastasis by using a PCR-based high throughput array with 2304 genes. The prediction system, constructed from the learning set comprised of 30 patients with the most informative 18 genes, classified each case into a 'good signature group' or 'poor signature group'. Then, we confirmed the predictive performance in an additional validation set comprised of 24 patients, and the prediction accuracy for peritoneal metastasis was 75%. Kaplan-Meier analysis with peritoneal metastasis revealed significant difference between these two groups (P=0.0225). By combining our system with conventional clinicopathological factors, we can identify high risk cases for peritoneal metastasis more accurately.

Aged↗

PubMed2006

Direct involvement of the small GTPase Rac in activation of the superoxide-producing NADPH oxidase Nox1.

Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22(phox) at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22(phox): the Noxa1-Noxo1 and Noxo1-p22(phox) interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent alpha-helix of p22phox.

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47phox with p22phox, a subunit of the oxidase catalytic core flavocytochrome b558. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47phox sandwich a short PRR (proline-rich region) of p22phox (amino acids 151-160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47phox SH3 domains results not only in a defective binding to p22phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro152, Pro156 and Arg158 in the p22phox PRR as residues indispensable for the interaction with p47phox. Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47phox with p22phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22phox (amino acids 161-164), adopting an a-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47phox SH3 domains.

Amino Acid Substitution↗

PubMed2006

Regulation of superoxide-producing NADPH oxidases in nonphagocytic cells.

The membrane-integrated protein gp91phox functions as the catalytic center of the superoxide-producing phagocyte NADPH oxidase. Recent studies have identified homologs of gp91phox in nonphagocytic cells, which constitute the NADPH oxidase (Nox) family. Activation of the Nox oxidases leads to production of reactive oxygen species (ROS), thereby participating in a variety of biological events, such as host defense, hormone biosynthesis, and signal transduction. The activity of the Nox enzymes is regulated by various proteins, including the small GTPase Rac; regulatory mechanisms differ dependent on the type of the Nox proteins. For example, an oxidase activator (p47phox or Noxo1) and an oxidase activator (p67phox or Noxa1) are absolutely required for superoxide production by gp91phox and Nox1, but not by Nox3. Rac, albeit probably dispensable to the Nox3 activity, plays an essential role in activation of gp91phox. Thus, functional reconstitution of Nox systems is crucial for the study of Nox regulation. Here we describe a basic method for the reconstitution of Nox systems by expression of oxidase proteins in transfectable cells.

Animals↗

PubMed2005

Coupling between cyclooxygenases and terminal prostanoid synthases.

Biosynthesis of prostanoids is regulated by three sequential enzymatic steps, namely phospholipase A2, cyclooxygenase (COX), and terminal prostanoid synthase. Recent evidence suggests that lineage-specific terminal prostanoid synthases, including prostaglandin (PG) E2, PGD2, PGF2alpha, PGI2, and thromboxane synthases, show distinct functional coupling with upstream COX isozymes, COX-1 and COX-2. This can account, at least in part, for segregated utilization of the two COX isozymes in distinct phases of PG-biosynthetic responses. In terms of their localization and COX preference, terminal prostanoid synthases are classified into three categories: (i) the perinuclear enzymes that prefer COX-2, (ii) the cytosolic enzyme that prefers COX-1, and (iii) the translocating enzyme that utilizes both COXs depending on the stimulus. Additionally, altered supply of arachidonic acid by phospholipase A2s significantly affects the efficiency of COX-terminal prostanoid synthase coupling. In this review, we summarize our recent understanding of the coupling profiles between the two COXs and various terminal prostanoid synthases.

Animals↗

PubMed2005

Prediction of recurrence in advanced gastric cancer patients after curative resection by gene expression profiling.

The prognosis of patients with advanced gastric cancer remains unfavorable. Even after curative resection, 40% of patients with advanced gastric cancer die of recurrence. Conventional clinicopathlogic findings are sometimes inadequate for predicting recurrence in individuals. Hence, we tried to construct a new diagnostic system, which predicts recurrence in patients with advanced gastric cancer after curative resection based on molecular analysis. Gastric cancer progression is a function of multiple genetic events that may affect the expression of large number of genes. We performed gene expression profiling with 2,304 genes in 60 advanced gastric cancer patients who underwent curative resection using a PCR array technique, a high-throughput quantitative RT-PCR technique. The diagnostic system, which was constructed from the learning set comprised of 40 patients with the most informative 29 genes, classified each case into a good-signature or poor-signature group. Then, we confirmed the predictive performance in an additional test set comprised of 20 patients, and the prediction accuracy for recurrence was 75%. Kaplan-Meier analysis revealed significant difference between the good-signature and the poor-signature group (p = 0.0125). Especially in patients with smaller tumor (< or = 5 cm), less developed LN metastasis (N(0,1)), or earlier stage (stages I and II), the prediction accuracy was high (88.9%, 84.6%, or 81.8%, respectively). Our diagnostic system based on systematic analysis of gene expression profiling can predict the recurrence at clinically meaningful level. By combining our system with conventional clinicopathologic factors, we can improve the prediction of recurrence in patients with advanced gastric cancer who underwent curative surgery.

Aged↗

PubMed2005

The NADPH oxidase Nox3 constitutively produces superoxide in a p22phox-dependent manner: its regulation by oxidase organizers and activators.

Nox3, a member of the superoxide-producing NADPH oxidase (Nox) family, participates in otoconia formation in mouse inner ears, which is required for perception of balance and gravity. The activity of other Nox enzymes such as gp91(phox)/Nox2 and Nox1 is known to absolutely require both an organizer protein (p47(phox) or Noxo1) andanactivatorprotein (p67(phox) or Noxa1); for the p47(phox)-dependent activation of these oxidases, treatment of cells with stimulants such as phorbol 12-myristate 13-acetate is also indispensable. Here we show that ectopic expression of Nox3 in various types of cells leads to phorbol 12-myristate 13-acetate-independent constitutive production of a substantial amount of superoxide under the conditions where gp91(phox) and Nox1 fail to generate superoxide, i.e. in the absence of the oxidase organizers and activators. Nox3 likely forms a functional complex with p22(phox); Nox3 physically interacts with and stabilizes p22(phox), and the Nox3-dependent superoxide production is totally dependent on p22(phox). The organizers p47(phox) and Noxo1 are capable of enhancing the superoxide production by Nox3 in the absence of the activators, and the enhancement requires the interaction of the organizers with p22(phox), further indicating a link between Nox3 and p22(phox). The p47(phox)-enhanced Nox3 activity is further facilitated by p67(phox) or Noxa1, whereas the activators cancel the Noxo1-induced enhancement. On the other hand, the small GTPase Rac, essential for the gp91(phox) activity, is likely dispensable to the Nox3 system. Thus Nox3 functions together with p22(phox) as an enzyme constitutively producing superoxide, which can be distinctly regulated by combinatorial use of the organizers and activators.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Prediction of docetaxel response in human breast cancer by gene expression profiling.

PURPOSE: Docetaxel is one of the most effective anticancer drugs available in the treatment of breast cancer. Nearly half of the treated patients, however, do not respond to chemotherapy and suffer from side effects. The ability to reliably predict a patient's response based on tumor gene expression will improve therapeutic decision making and save patients from unnecessary side effects. PATIENTS AND METHODS: A total of 44 breast tumor tissues were sampled by biopsy before treatment with docetaxel, and the response to therapy was clinically evaluated by the degree of reduction in tumor size. Gene expression profiling of the biopsy samples was performed with 2,453 genes using a high-throughput reverse transcriptase polymerase chain reaction technique. Using genes differentially expressed between responders and nonresponders, a diagnostic system based on the weighted-voting algorithm was constructed. RESULTS: This system predicted the clinical response of 26 previously unanalyzed samples with over 80% accuracy, a level promising for clinical applications. Diagnostic profiles in nonresponders were characterized by elevated expression of genes controlling the cellular redox environment (ie, redox genes, such as thioredoxin, glutathione-S-transferase, and peroxiredoxin). Overexpression of these genes protected cultured mammary tumor cells from docetaxel-induced cell death, suggesting that enhancement of the redox system plays a major role in docetaxel resistance. CONCLUSION: These results suggest that the clinical response to docetaxel can be predicted by gene expression patterns in biopsy samples. The results also suggest that one of the molecular mechanisms of the resistance is activation of a group of redox genes.

Adult↗

PubMed2004

Molecular prediction of response to 5-fluorouracil and interferon-alpha combination chemotherapy in advanced hepatocellular carcinoma.

PURPOSE: The prognosis of hepatocellular carcinoma (HCC) is very poor, particularly in patients with tumors that have invaded the major branches of the portal vein. Combination chemotherapy with intra-arterial 5-fluorouracil and subcutaneous interferon-alpha has shown promising results for such advanced HCC, but it is important to develop the ability to accurately predict chemotherapeutic responses. EXPERIMENTAL DESIGN: We analyzed the expression of 3,080 genes using a polymerase chain reaction-based array in 20 HCC patients who were treated with combination chemotherapy after reduction surgery. After unsupervised analyses, a supervised classification method for predicting chemotherapeutic responses was constructed. To minimize the number of predictive genes, we used a random permutation test to select only significant (P < 0.01) genes. A leave-one-out cross-validation confirmed the gene selection. We also prepared an additional 11 cases for validation of predictive performance. RESULTS: Hierarchical clustering analysis and principal component analysis with all 3,080 genes revealed distinct gene expression patterns in responders (those with complete response or partial response) and nonresponders (those with stable disease or progressive disease) to the combination chemotherapy. Using a weighted-voting classification method with either all genes or only significant genes as assessed by permutation testing, the objective responses to treatment were correctly predicted in 17 of 20 cases (accuracy, 85%; positive predictive value, 100%; negative predictive value, 80%). Moreover, patients in the validation dataset could be classified into two distinct prognostic groups using 63 predictive genes. CONCLUSIONS: Molecular analysis of 63 genes can predict the response of patients with advanced HCC and major portal vein tumor thrombi to combination chemotherapy with 5-fluorouracil and interferon-alpha.

Adult↗

PubMed2004

Molecular-based prediction of early recurrence in hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) has a very poor prognosis, due to the high incidence of tumor recurrence. As the current morphological indicators are often insufficient for therapeutic decisions, we sought to identify additional biologic indicators for early recurrence. METHODS: We analyzed gene expression using a PCR-based array of 3,072 genes in 100 HCC patients. Informative genes predicting early intrahepatic recurrence were selected by random permutation testing, and a weighted voting prediction method was constructed. Following estimation of prediction accuracy, a multivariate Cox analysis was performed. RESULTS: By permutation testing, we selected 92 genes demonstrated distinct expression patterns differing significantly between recurrence cases and recurrence-free cases. Our prediction method, using the 20 top-ranked genes, correctly predicted the early intrahepatic recurrence for 29 of 40 cases within the validation group, and the odds ratio was 6.8 (95%CI 1.7-27.5, P = 0.010). The 2-year recurrence rates in the patients with the good signature and those with the poor signature were 29.4 and 73.9%, respectively. Multivariate Cox analysis revealed that molecular-signature was an independent indicator for recurrence (hazard ratio 3.82, 95%CI 1.44-10.10, P = 0.007). CONCLUSIONS: Our molecular-based prediction method using 20 genes is clinically useful to predict early recurrence of HCC.

Aged↗

PubMed2004

Molecular mechanism underlying activation of superoxide-producing NADPH oxidases: roles for their regulatory proteins.

The phagocyte NADPH oxidase is dormant in resting cells but becomes activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants, thereby playing a crucial role in host defence. The catalytic core of this enzyme comprises the two membranous subunits gp91phox/Nox2 and p22phox. The oxidase activation requires the small GTPase Rac and the SH3 domain-containing proteins p47phox and p67phox; they normally exist in the cytoplasm and translocate upon cell stimulation to the membrane. The translocation depends on a stimulus-induced conformational change of p47phox, which leads to the SH3 domain-mediated interaction with p22phox, a binding required for the gp91phox/Nox2-dependent superoxide production. Activation of Nox1, an oxidase that is likely involved in host defence at the colon, requires novel proteins homologous to p47phox and p67phox, designated Noxo1 and Noxa1, respectively. Noxo1 and Noxa1, both expressed abundantly in the colon, are capable of constitutively activating Nox1. The constitutive activation may be due to the property of Noxo1: in contrast with p47phox, Noxo1 seems to normally associate with p22phox, which is required for the Nox1 activation. We will also describe the mechanism underlying regulation of the third oxidase Nox3, which exits in fetal kidney and inner ears.

Animals↗

PubMed2003

Coupling between cyclooxygenases and prostaglandin F(2alpha) synthase. Detection of an inducible, glutathione-activated, membrane-bound prostaglandin F(2alpha)-synthetic activity.

Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF(2alpha) only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF(2alpha) from endogenous AA, even though significant increase in PGF(2alpha) production occurred in cells transfected with COX-2 alone. This poor COX/PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF(2alpha)-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulus-inducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2.

Animals↗

PubMed2003

Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases.

The catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91phox/Nox2, a membrane-integrated protein that forms a heterodimer with p22phox to constitute flavocytochrome b558. The cytochrome becomes activated by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. Here we describe the cloning of human cDNAs for novel proteins homologous to p47phox and p67phox, designated p41nox and p51nox, respectively; the former is encoded by NOXO1 (Nox organizer 1), and the latter is encoded by NOXA1 (Nox activator 1). The novel homologue p41nox interacts with p22phox via the two tandem SH3 domains, as does p47phox. The protein p51nox as well as p67phox can form a complex with p47phox and with p41nox via the C-terminal SH3 domain and binds to GTP-bound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs. These bindings seem to play important roles, since p47phox and p67phox activate the phagocyte oxidase via the same interactions. Indeed, p41nox and p51nox are capable of replacing the corresponding classical homologue in activation of gp91phox. Nox1, a homologue of gp91phox, also can be activated in cells, when it is coexpressed with p41nox and p51nox, with p41nox and p67phox, or with p47phox and p51nox; in the former two cases, Nox1 is partially activated without any stimulants added, suggesting that p41nox is normally in an active state. Thus, the novel homologues p41nox and p51nox likely function together or in combination with a classical one, thereby activating the two Nox family oxidases.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Identification of expressed genes linked to malignancy of human colorectal carcinoma by parametric clustering of quantitative expression data.

BACKGROUND: Individual human carcinomas have distinct biological and clinical properties: gene-expression profiling is expected to unveil the underlying molecular features. Particular interest has been focused on potential diagnostic and therapeutic applications. Solid tumors, such as colorectal carcinoma, present additional obstacles for experimental and data analysis. RESULTS: We analyzed the expression levels of 1,536 genes in 100 colorectal cancer and 11 normal tissues using adaptor-tagged competitive PCR, a high-throughput reverse transcription-PCR technique. A parametric clustering method using the Gaussian mixture model and the Bayes inference revealed three groups of expressed genes. Two contained large numbers of genes. One of these groups correlated well with both the differences between tumor and normal tissues and the presence or absence of distant metastasis, whereas the other correlated only with the tumor/normal difference. The third group comprised a small number of genes. Approximately half showed an identical expression pattern, and cancer tissues were classified into two groups by their expression levels. The high-expression group had strong correlation with distant metastasis, and a poorer survival rate than the low-expression group, indicating possible clinical applications of these genes. In addition to c-yes, a homolog of a viral oncogene, prognostic indicators included genes specific to glial cells, which gives a new link between malignancy and ectopic gene expression. CONCLUSIONS: The malignancy of human colorectal carcinoma is correlated with a unique expression pattern of a specific group of genes, allowing the classification of tumor tissues into two clinically distinct groups.

Cluster Analysis↗

PubMed2003

Molecular features of non-B, non-C hepatocellular carcinoma: a PCR-array gene expression profiling study.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) usually develops following chronic liver inflammation caused by hepatitis C or B virus. Through expression profiling in a rare type of HCC, for which the causes are unknown, we sought to find key genes responsible for each step of hepatocarcinogenesis in the absence of viral influence. METHODS: We used 68 non-B, non-C liver tissues (20 HCC, 17 non-tumor, 31 normal liver) for expression profiling with PCR-array carrying 3072 genes known to be expressed in liver tissues. To select the differentially expressed genes, we performed random permutation testing. A weighted voting classification algorithm was used to confirm the reliability of gene selection. We then compared these genes with the results of previous expression profiling studies. RESULTS: A total of 220 differentially expressed genes were selected by random permutation tests. The classification accuracies using these genes were 91.8, 92.0 and 100.0% by a leave-one-out cross-validation, an additional PCR-array dataset and a Stanford DNA microarray dataset, respectively. By comparing our results with previous reports on virus-infected HCC, four genes (ALB, A2M, ECHS1 and IGFBP3) were commonly selected in some studies. CONCLUSIONS: The 220 differentially expressed genes selected by PCR-array are potentially responsible for hepatocarcinogenesis in the absence of viral influence.

Aged↗

PubMed2002

Comparison of gene expression profiling during postnatal development of mouse dentate gyrus and cerebellum.

Both the dentate gyrus of the hippocampus and the cerebellar cortex consist mainly of granule cells and develop postnatally. The granule cells in both tissues are presumed to be similar. Changes in gene expression were analyzed during the postnatal development of the dentate gyrus. Altogether, expression patterns of 1,937 genes were determined by adaptor-tagged competitive PCR. More than 90% of the genes belong to groups characterized by elevated expression either at earlier or later stages of development. A majority of the genes expressed showed marked changes during the developmental process, but there was little correlation between gene function and expression, unlike that observed during mouse postnatal cerebellar development. Despite anatomical and physiological similarities between these two processes, the gene expression profiles are completely different.

Animals↗

PubMed2002

Molecular classification of primary breast tumors possessing distinct prognostic properties.

The natural progression of breast cancer differs greatly between patients; the precise prediction of this disease course will improve the efficacy of therapeutics. Gene expression profiling may elucidate the undiscovered biological variations between seemingly similar cancers, leading to a new cancer classification system valuable in accurate diagnosis. The expression levels of 2412 genes, derived from 98 cancer samples, were precisely recorded by a high throughput RT-PCR technique, adapter-tagged competitive PCR. Subsequent cluster analysis revealed a molecular profile, correlating with estrogen receptor levels and the presence of lymph node metastases. We analyzed 301 cancer samples for the expression patterns of 21 genes critical in this categorization. The classification of the samples into three major groups was verified utilizing principal component analysis. This molecular classification system correlated significantly with early recurrence, independent of lymph node status. This malignant potential is associated with the expression levels of a group of genes, which comprise a set of candidates potentially useful in diagnostic prediction. These genes and the associated control mechanisms may also be effective therapeutic targets.

Breast Neoplasms↗