Showing papers by "Hiroyuki Mano published in 2004"

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

Abstract: 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.

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

Abstract: 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.

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

Abstract: 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.

DNA microarray analysis of natural killer cell-type lymphoproliferative disease of granular lymphocytes with purified CD3-CD56+ fractions.

Abstract: Natural killer (NK) cell-type lymphoproliferative disease of granular lymphocytes (LDGL) is characterized by the outgrowth of CD3(-)CD16/56(+) NK cells, and can be further subdivided into two distinct categories: aggressive NK cell leukemia (ANKL) and chronic NK lymphocytosis (CNKL). To gain insights into the pathophysiology of NK cell-type LDGL, we here purified CD3(-)CD56(+) fractions from healthy individuals (n=9) and those with CNKL (n=9) or ANKL (n=1), and compared the expression profiles of >12 000 genes. A total of 15 'LDGL-associated genes' were identified, and a correspondence analysis on such genes could clearly indicate that LDGL samples share a 'molecular signature' distinct from that of normal NK cells. With a newly invented class prediction algorithm, 'weighted distance method', all 19 samples received a clinically matched diagnosis, and, furthermore, a detailed cross-validation trial for the prediction of normal or CNKL status could achieve a high accuracy (77.8%). By applying another statistical approach, we could extract other sets of genes, expression of which was specific to either normal or LDGL NK cells. Together with sophisticated statistical methods, gene expression profiling of a background-matched NK cell fraction thus provides us a wealth of information for the LDGL condition.

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

Abstract: 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.

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

Abstract: 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

Stratification of acute myeloid leukemia based on gene expression profiles.

Abstract: 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.

Primary cell preparation of human renal tubular cells for transcriptome analysis.

Abstract: We initiated a toxicogenomics project using Affymetrix GeneChip® HG-U133A and HG-U133B arrays harboring 45,000 probe sets representing more than 39,000 transcripts to analyze gene expression in primary cultures of human cells after exposure to chemicals that cause tissue toxicity. In order to assess the quality of the samples studied, we prepared primary human renal cortical cell cultures from surgically resected human kidney and evaluated the origin of the cells and the effects of cryopreservation. We analyzed the primary cultures using GeneChip and compared their expression patterns with those in the Novartis Research Foundation (GNF) Gene Expression Database. The comparison with the GNF database revealed that the gene expression pattern of the cultured cells was compatible with kidney cells, indicating that we had purified human renal cortical cells. Due to the purification procedure, the primary cultured cells could be a mixture of renal components; however, we identified the major population as renal...

The Tyr-Kinase Inhibitor AG879, That Blocks the ETK-PAK1 Interaction, Suppresses the RAS-Induced PAK1 Activation and Malignant

Abstract: AG 879 has been widely used as a Tyr kinase inhibitor specific for ErbB2 and FLK-1, a VEGF receptor. The IC50 for both ErbB2 and FLK-1 is around 1 ∝M. 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.