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Biomedical subjects

Hiroyuki Mano

Publications and source records attributed to Hiroyuki Mano.

At least 37 records · Page 2Linked to original sources
PubMed2005

Retroviral expression screening of oncogenes in pancreatic ductal carcinoma.

Pancreatic ductal carcinoma (PDC) remains one of the most intractable malignancies in humans. In order to clarify the molecular events underlying the carcinogenesis in PDC, we constructed a retroviral cDNA expression library from a PDC cell line, and used it to screen transforming genes in PDC by a focus formation assay with mouse 3T3 fibroblasts. We could obtain a total of 30 transformed cell foci in the screening, and one of the cDNA inserts harvested from such cell clones turned out to encode a wild-type human ARAF1. Unexpectedly, a long terminal repeat-driven overexpression of ARAF1 mRNA was confirmed to induce transformed foci in fibroblasts. The oncogenic potential of ARAF1 was examined by injecting the transformed fibroblasts into athymic nude mice. Importantly, ARAF1 mRNA was highly expressed in pancreatic ductal cell specimens purified from patients with PDC. These results have unveiled the transforming potential of ARAF1 protein, and also suggest that quantity of intracellular ARAF1 may be important in carcinogenesis of various human cancers.

3T3 Cells↗
PubMed2005

Experimental trial for diagnosis of pancreatic ductal carcinoma based on gene expression profiles of pancreatic ductal cells.

Pancreatic ductal carcinoma (PDC) remains one of the most intractable human malignancies, mainly because of the lack of sensitive detection methods. Although gene expression profiling by DNA microarray analysis is a promising tool for the development of such detection systems, a simple comparison of pancreatic tissues may yield misleading data that reflect only differences in cellular composition. To directly compare PDC cells with normal pancreatic ductal cells, we purified MUC1-positive epithelial cells from the pancreatic juices of 25 individuals with a normal pancreas and 24 patients with PDC. The gene expression profiles of these 49 specimens were determined with DNA microarrays containing >44 000 probe sets. Application of both Welch's analysis of variance and effect size-based selection to the expression data resulted in the identification of 21 probe sets corresponding to 20 genes whose expression was highly associated with clinical diagnosis. Furthermore, correspondence analysis and 3-D projection with these probe sets resulted in separation of the transcriptomes of pancreatic ductal cells into distinct but overlapping spaces corresponding to the two clinical classes. To establish an accurate transcriptome-based diagnosis system for PDC, we applied supervised class prediction algorithms to our large data set. With the expression profiles of only five predictor genes, the weighted vote method diagnosed the class of samples with an accuracy of 81.6%. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC.

Carcinoma, Pancreatic Ductal↗
PubMed2004

Schedule-dependent synergism and antagonism between pemetrexed and paclitaxel in human carcinoma cell lines in vitro.

Pemetrexed is a novel multitargeted antifolate with significant clinical activity against a variety of tumors. We studied the schedule-dependent cytotoxic effects of pemetrexed in combination with paclitaxel in vitro to improve our understanding of how this combination might be used clinically. Human lung cancer A549 cells, breast cancer MCF7, ovarian cancer PA1, and colon cancer WiDr cells were exposed to both pemetrexed and paclitaxel in vitro. Cell growth inhibition after 5 days was determined and the effects of drug combinations were analyzed by the isobologram method (Steel and Peckham). Simultaneous exposure to pemetrexed and paclitaxel for 24 h produced antagonistic effects in A549 and PA1 cells, additive/antagonistic effects in MCF7 cells, and additive effects in WiDr cells. Pemetrexed for 24 h followed by paclitaxel for 24 h produced synergistic effects in A549 and MCF7 cells and additive effects in PA1 and WiDr cells, while the reverse sequence produced additive effects in all four cell lines. Cell cycle analysis supported these observations. Our findings suggest that the simultaneous administration of pemetrexed and paclitaxel is suboptimal. The optimal schedule of pemetrexed in combination with paclitaxel is the sequential administration of pemetrexed followed by paclitaxel, and this schedule should be assessed in clinical trials for the treatment of solid tumors.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2004

Cardiac function-related gene expression profiles in human atrial myocytes.

To obtain insights into the molecular pathogenesis of heart failure in humans, we have analyzed the expression profiles of>12,000 genes in a total of 17 human specimens of right atrial myocytes. From this large data set, we here tried to identify gene clusters, expression level of which is correlated precisely with clinical parameter values of cardiac function. We could reveal that cardiac myocytes with normal sinus rhythm were clearly differentiated, in the point of view of gene expression, from those with atrial fibrillation. Further, an expression profile-based prediction of arrhythmia by a newly developed "weighted-distance method" could efficiently diagnose our samples. We could even construct calculation formulae for the values of left ventricular ejection fraction based on the expression level of selected genes. To our best knowledge, this is the first report to indicate that pumping ability of heart can be predicted by any measures of atrium.

Adolescent↗
PubMed2004

The Tyr-kinase inhibitor AG879, that blocks the ETK-PAK1 interaction, suppresses the RAS-induced PAK1 activation and malignant transformation.

AG 879 has been widely used as a Tyr kinase inhibitor specific for ErbB2 and FLK-1, a VEGF receptor. The IC(50) for both ErbB2 and FLK-1 is around 1 microM. AG 879, in combination of PP1 (an inhibitor specific for Src kinase family), suppresses almost completely the growth of RAS-induced sarcomas in nude mice. In this paper we demonstrate that AG 879 even at 10 nM blocks the specific interaction between the Tyr-kinase ETK and PAK1 (a CDC42/ Rac-dependent Ser/Thr kinase) in cell culture. This interaction is essential for both the RAS-induced PAK1 activation and transformation of NIH 3T3 fibroblasts. However, AG 879 at 10 nM does not inhibit either the purified ETK or PAK1 directly in vitro, suggesting that this drug blocks the ETK-PAK1 pathway by targeting a highly sensitive kinase upstream of ETK. Although the Tyr-kinases Src and FAK are known to activate ETK directly, Src is insensitive to AG 879, and FAK is inhibited by 100 nM AG 879, but not by 10 nM AG879. The structure-function relationship analysis of AG 879 derivatives has revealed that both thio and tert-butyl groups of AG 879, but not (thio) amide group, are essential for its biological function (blocking the ETK-PAK1 pathway), suggesting that through the (thio) amide group, AG 879 can be covalently linked to agarose beads to form a bioactive affinity ligand useful for identifying the primary target of this drug.

3T3 Cells↗
PubMed2004

Mutations of BRAF are associated with extensive hMLH1 promoter methylation in sporadic colorectal carcinomas.

Activating mutations of BRAF have been frequently observed in microsatellite unstable (MSI+) colorectal carcinomas (CRCs), in which mutations of BRAF and KRAS are mutually exclusive. Previously, we reported that hypermethylation of hMLH1 might play an important role in the tumorigenesis of right-sided sporadic CRCs with MSI showing less frequency of KRAS/TP53 alteration. Therefore, we have assumed that BRAF mutations might be highly associated with hMLH1 methylation status rather than MSI status. In this study, mutations of BRAF and KRAS and their relationship with MSI and hMLH1 methylation status were examined in 140 resected specimens of CRC. The methylation status was classified into 3 types: full methylation (FM), partial methylation (PM) and nonmethylation (NM). Only FM closely linked to reduced expression of hMLH1 protein. BRAF mutations were found in 16 cases (11%), all leading to the production of BRAF(V599E). As for MSI status, BRAF mutations were found in 43% of MSI+ and 4% of MSI- cases (p < 0.0001). Among the MSI+ individuals, BRAF mutations were more frequent in cases with hMLH1 deficiency (58%) than those with hMSH2 deficiency (0%; p=0.02). Moreover, they were found in 69% of FM, 4% of PM and 4% of NM, revealing a striking difference between FM and the other 2 groups (FM vs. PM or NM; p < 0.0001). These findings suggest that BRAF activation may participate in the carcinogenesis of sporadic CRCs with hMLH1 hypermethylation in the proximal colon, independently of KRAS activation.

Adaptor Proteins, Signal Transducing↗
PubMed2004

DNA microarray analysis of dysplastic morphology associated with acute myeloid leukemia.

OBJECTIVE: Acute myeloid leukemia (AML) develops de novo or secondarily to either myelodysplastic syndrome (MDS) or anticancer treatment (therapy-related leukemia, TRL). Prominent dysplasia of blood cells is apparent in individuals with MDS-related AML as well as in some patients with TRL or even with de novo AML. The clinical entity of AML with multilineage dysplasia (AML-MLD) is likely to be an amalgamation of MDS-related AML and de novo AML-MLD. The aim of this study was to clarify, by the use of high-density oligonucleotide microarrays, whether these subcategories of AML are intrinsically distinct from each other. MATERIALS AND METHODS: The AC133+ hematopoietic stem cell-like fractions were purified from the bone marrow of individuals with de novo AML without dysplasia (n = 15), AML-MLD (n = 11), MDS-related AML (n = 11), or TRL (n = 2), and were subjected to the synthesis of cRNA which was subsequently hybridized to microarray harboring oligonucleotide corresponding to more than 12,000 probe sets. RESULTS: We could identify many genes whose expression was specific to these various subcategories of AML. Furthermore, with the correspondence analysis/three-dimensional projection strategy, we were able to visualize the independent, yet partially overlapping, nature of current AML subcategories on the basis of their transcriptomes. CONCLUSION: Our data indicate the possibility of subclassification of AML based on gene expression profiles of leukemic blasts.

Adolescent↗
PubMed2004

Transcriptional profile of genes induced in human atrial myocardium with pressure overload.

BACKGROUND: The molecular response of human myocardium to mechanical stimuli, particularly the difference between pressure or volume overload cardiac hypertrophy, remains incompletely defined. METHODS: We investigated the transcriptional profile of genes induced in human pressure- or volume-overloaded myocardium with DNA microarray technology. We used right atrial tissue from patients who underwent cardiac surgery. On the basis of pressure data and echocardiographic findings, the patients were divided into three groups: control group (n=3), pressure overload group (mean right atrial pressure of >7 mm Hg, n=3), and volume overload group (moderate or severe tricuspid regurgitation, n=3). Expression profiles of 2139 human genes were investigated with mRNA obtained from the samples. RESULTS: In the pressure overload group, expression of genes of cyclin-dependent kinase inhibitor 1A (CDKI1A, 11.7+/-3.1-fold vs. control), and mitogen-activated protein kinase phosphatase-1 (MKP-1, 26.2+/-2.1-fold) was significantly increased compared with those in control or volume overload group (P<0.05). The specificity of these gene expressions was confirmed by a quantitative "real-time" polymerase chain reaction (PCR) analysis. In addition, mechanical strain induced CDKI1A and MKP-1 protein expressions in neonatal rat cardiac myocytes in an amplitude-dependent manner. In contrast, transcripts of growth factors did not significantly increase. CONCLUSIONS: This study demonstrated that gene expressions of CDKI1A and MKP-1, but not growth factors, are induced in chronic pressure-overloaded myocardium. These findings suggest that suppressors of the cell cycle or cell proliferation may play a critical role in the pathophysiology of pressure overload.

Adolescent↗
PubMed2004

Genistein-induced changes in gene expression in Panc 1 cells at physiological concentrations of genistein.

OBJECTIVES: To investigate the effect of genistein on gene expression in Panc 1 cells using microarray technology. METHODS: Panc 1 cells were treated with 10 micromol/L genistein or DMSO (vehicle control) for 0, 1, 3, 6, or 12 hours. Total RNA from each sample was isolated, and biotin-labeled probes were hybridized to the human genome U133A chip, after which the chip was washed and scanned. Data were analyzed using DMT software (Affymetrix). For genes that showed large changes in expression due to genistein, these changes were confirmed using real-time PCR assays. RESULTS: Two independent microarray experiments showed that genistein significantly changed the expression of 47 genes: up-regulating of egr-1 and IL-8 and down-regulating of EGF-R AKT2, CYP1B1, NELL2, SCD, DNA ligase III, Rad as well as 18s and 28s rRNA and others. These alterations in expression were confirmed using real-time PCR, although the increase in change was not exactly the same in the 2 assays. CONCLUSIONS: Our data suggest the reported apparent ability of genistein to inhibit carcinogenesis may involve a number of pathways. The most obvious target is the EGF-R signaling pathway since the expression of 5 genes related to this pathway was reduced (EGFR, egr-1, AKT2, CYP1B1, and NELL2). Genistein may also act by disabling cancer cell self-protection by inhibiting expression of AKT2, CYP1B1, and DNA ligase III. Furthermore, genistein may inhibit car-cinogenesis by inhibiting expression of SCD. Finally, our data support findings indicating that genistein inhibits rRNA formation, which is an important mechanism by which genistein regulates tumor cell growth.

Adenocarcinoma↗
PubMed2004

High-throughput screening of genome fragments bound to differentially acetylated histones.

Although acetylation-deacetylation of histones contributes to regulation of gene expression, few methods have been available to determine the whole-genome histone acetylation profile in specific cells or tissues. We have now developed a genome-wide screening method, differential chromatin scanning (DCS), to isolate genome fragments embedded in histones subject to differential acetylation. This DCS screening was applied to a human gastric cancer cell line incubated with or without an inhibitor of histone deacetylase (HDAC) activity, resulting in the rapid identification of more than 250 genome fragments. Interestingly, a number of cancer-related genes were revealed to be the targets of HDAC in the cancer cells, including those for tumour protein 73 and cell division cycle 34. Such differential acetylation of histone was also shown to be linked to the regulation of transcriptional activity of the corresponding genes. Among the isolated genome fragments, 94% (32/34) of them were confirmed to be bound to differentially acetylated histones, and the genes corresponding to 78% (7/9) of them exhibited differential transcriptional activity consistent with the level of histone acetylation. With its high fidelity, the DCS method should open a possibility to rapidly compare the genome-wide histone acetylation profiles and to provide novel insights into molecular carcinogenesis.

Acetylation↗
PubMed2004

Stratification of acute myeloid leukemia based on gene expression profiles.

Acute myeloid leukemia (AML) is characterized by clonal growth of immature leukemic blasts and develops either de novo or secondarily to anticancer treatment or to other hematologic disorders. Given that the current classification of AML, which is based on blast karyotype and morphology, is not sufficiently robust to predict the prognosis of each affected individual, new stratification schemes that are of better prognostic value are needed. Global profiling of gene expression in AML blasts has the potential both to identify a small number of genes whose expression is associated with clinical outcome and to provide insight into the molecular pathogenesis of this condition. Emerging genomics tools, especially DNA microarray analysis, have been applied in attempts to isolate new molecular markers for the differential diagnosis of AML and to identify genes that contribute to leukemogenesis. Progress in bioinformatics has also yielded means with which to classify patients according to clinical parameters such as long-term prognosis. The application of such analysis to large sets of gene expression data has begun to provide the basis for a new AML classification that is more powerful with regard to prediction of prognosis.

Biomarkers, Tumor↗
PubMed2003

Reprogramming of human postmitotic neutrophils into macrophages by growth factors.

It is generally recognized that postmitotic neutrophils give rise to polymorphonuclear neutrophils alone. We obtained evidence for a lineage switch of human postmitotic neutrophils into macrophages in culture. When the CD15+CD14- cell population, which predominantly consists of band neutrophils, was cultured with granulocyte macrophage-colony-stimulating factor, tumor necrosis factor-alpha, interferon-gamma, and interleukin-4, and subsequently with macrophage colony-stimulating factor alone, the resultant cells had morphologic, cytochemical, and phenotypic features of macrophages. In contrast to the starting population, they were negative for myeloperoxidase, specific esterase, and lactoferrin, and they up-regulated nonspecific esterase activity and the expression of macrophage colony-stimulating factor receptor, mannose receptor, and HLA-DR. CD15+CD14- cells proceeded to macrophages through the CD15-CD14- cell population. Microarray analysis of gene expression also disclosed the lineage conversion from neutrophils to macrophages. Macrophages derived from CD15+CD14- neutrophils had phagocytic function. Data obtained using 3 different techniques, including Ki-67 staining, bromodeoxyuridine incorporation, and cytoplasmic dye labeling, together with the yield of cells, indicated that the generation of macrophages from CD15+CD14- neutrophils did not result from a contamination of progenitors for macrophages. Our data show that in response to cytokines, postmitotic neutrophils can become macrophages. This may represent another differentiation pathway toward macrophages in human postnatal hematopoiesis.

Cell Differentiation↗
PubMed2003

Mutual regulation of protein-tyrosine phosphatase 20 and protein-tyrosine kinase Tec activities by tyrosine phosphorylation and dephosphorylation.

PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.

Amino Acid Sequence↗
PubMed2003

Proteomic analysis of hematopoietic stem cell-like fractions in leukemic disorders.

DNA microarray analysis has been applied to identify molecular markers of human hematological malignancies. However, the relatively low correlation between the abundance of a given mRNA and that of the encoded protein makes it important to characterize the protein profile directly, or 'proteome,' of malignant cells in addition to the 'transcriptome.' To identify proteins specifically expressed in leukemias, here we isolated AC133(+) hematopoietic stem cell-like fractions from the bone marrow of 13 individuals with various leukemic disorders, and compared their protein profiles by two-dimensional electrophoresis. A total of 11 differentially expressed protein spots corresponding to 10 independent proteins were detected, and peptide fingerprinting combined with mass spectrometry of these proteins revealed them to include NuMA (nuclear protein that associates with the mitotic apparatus), heat shock proteins, and redox regulators. The abundance of NuMA in the leukemic blasts was significantly related to the presence of complex karyotype anomalies. Conditional expression of NuMA in a mouse myeloid cell line resulted in the induction of aneuploidy, cell cycle arrest in G(2)-M phases, and apoptosis. These results demonstrate the potential of proteome analysis with background-matched cell fractions obtained from fresh clinical specimens to provide insight into the mechanism of human leukemogenesis.

Adult↗
PubMed2003

DNA microarray analysis of in vivo progression mechanism of heart failure.

Dahl salt-sensitive rats are genetically hypersensitive to sodium intake. When fed a high sodium diet, they develop systemic hypertension, followed by cardiac hypertrophy and finally heart failure within a few months. Therefore, Dahl rats represent a good model with which to study how heart failure is developed in vivo. By using DNA microarray, we here monitored the transcriptome of >8000 genes in the left ventricular muscles of Dahl rats during the course of cardiovascular damage. Expression of the atrial natriuretic peptide gene was, for instance, induced in myocytes by sodium overload and further enhanced even at the heart failure stage. Interestingly, expression of the gene for the D-binding protein, an apoptotic-related transcriptional factor, became decreased upon the transition to heart failure. To our best knowledge, this is the first report to describe the transcriptome of cardiac myocytes during the disease progression of heart failure.

Animals↗
PubMed2003

Bruton's tyrosine kinase is required for lipopolysaccharide-induced tumor necrosis factor alpha production.

Lipopolysaccharide (LPS), a product of Gram-negative bacteria, is potent mediator of tumor necrosis factor (TNF)alpha production by myeloid/macrophage cells. Inhibitors capable of blocking the signaling events that result in TNF alpha production could provide useful therapeutics for treating septic shock and other inflammatory diseases. Broad spectrum tyrosine inhibitors are known to inhibit TNF alpha production, however, no particular family of tyrosine kinases has been shown to be essential for this process. Here we show that the Bruton's tyrosine kinase (Btk)-deficient mononuclear cells from X-linked agammaglobulinemia patients have impaired LPS-induced TNF alpha production and that LPS rapidly induces Btk kinase activity in normal monocytes. In addition, adenoviral overexpression of Btk in normal human monocytes enhanced TNF alpha production. We examined the role of Btk in TNF alpha production using luciferase reporter adenoviral constructs and have established that overexpression of Btk results in the stabilization of TNF alpha mRNA via the 3' untranslated region. Stimulation with LPS also induced the activation of related tyrosine kinase, Tec, suggesting that the Tec family kinases are important components for LPS-induced TNF alpha production. This study provides the first clear evidence that tyrosine kinases of the Tec family, in particular Btk, are key elements of LPS-induced TNF alpha production and consequently may provide valuable therapeutic targets for intervention in inflammatory conditions.

Adolescent↗
PubMed2003

Antiapoptotic effect of endothelin-1 in rat cardiomyocytes in vitro.

Apoptosis of cardiac myocytes is thought to be a feature of many pathological disorders, including congestive heart failure (CHF) and ischemic heart disease (IHD). Because recent investigations indicate that endothelin-1 (ET-1) plays an important role in CHF and IHD, we investigated the effect of ET-1 on cardiomyocyte apoptosis. The presence of apoptosis in rat cardiomyocytes (H9c2 and neonatal) was evaluated by morphological criteria, electrophoresis of DNA fragments, 4',6'-diamidine-2'-phenylindole staining, and TUNEL analysis. ET-1, but not angiotensin II, prevented apoptosis induced by serum deprivation via ETA receptors in a dose-dependent manner (1 to 100 nmol/L). ET-1 also prevented cytochrome c release from mitochondria to the cytosol. The use of specific pharmacological inhibitors demonstrated that the antiapoptotic effect of ET-1 was mediated through a tyrosine kinase pathway (genistein and AG490) but not through protein kinase C (PKC; calphostin C), mitogen-activated protein kinases (PD98059 and SB203580), or PKA (KT5270) pathways. Adenovirus-mediated gene transfer of kinase-inactive (KI) c-Src reversed the antiapoptotic effect of ET-1. We further investigated whether Bcl-xL, an antiapoptotic molecule, would be upregulated by using a luciferase-based reporter system. ET-1 upregulated Bcl-xL, and this upregulation was inhibited by genistein or AG490 but not by calphostin C. The experiments with KI mutants for various tyrosine kinases revealed that c-Src and Pyk2 (but not JAK1, Jak2, Syk, and Tec) are involved in ET-1-induced upregulation of Bcl-xL expression. These findings suggest that ET-1 prevents apoptosis in cardiac myocytes through the ETA receptor and the subsequent c-Src/Bcl-xL-dependent pathway.

Animals↗