Tomoaki Wada

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 BRAFV599E. 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.

Epigenetic silencing of AXIN2 in colorectal carcinoma with microsatellite instability.

Mutation or epigenetic silencing of mismatch repair genes, such as MLH1 and MSH2, results in microsatellite instability (MSI) in the genome of a subset of colorectal carcinomas (CRCs). However, little is yet known of genes that directly contribute to tumor formation in such cancers. To characterize MSI-dependent changes in gene expression, we have now compared transcriptomes between fresh CRC specimens positive or negative for MSI (n=10 for each) with the use of high-density oligonucleotide microarrays harboring >44 000 probe sets. Correspondence analysis of the expression patterns of isolated MSI-associated genes revealed that the transcriptome of MSI+ CRCs is clearly distinct from that of MSI− CRCs. Such MSI-associated genes included that for AXIN2, an important component of the WNT signaling pathway. AXIN2 was silenced, apparently as a result of extensive methylation of its promoter region, specifically in MSI+ CRC specimens. Forced expression of AXIN2, either by treatment with 5′-azacytidine or by transfection with AXIN2 cDNA, resulted in rapid cell death in an MSI+ CRC cell line. These data indicate that epigenetic silencing of AXIN2 is specifically associated with carcinogenesis in MSI+ CRCs.

DNA microarray analysis of stage progression mechanism in myelodysplastic syndrome

Summary. Myelodysplastic syndrome (MDS) is a clonal disorder of haematopoietic stem cells. Despite the high incidence of MDS in the elderly, effective treatment of individuals in its advanced stages is problematic. DNA microarray analysis is a potentially informative approach to the development of new treatments for MDS. However, a simple comparison of ‘transcriptomes’ of bone marrow mononuclear cells among individuals at distinct stages of MDS would result in the identification of genes whose expression differences only reflect differences in the proportion of MDS blasts within bone marrow. Such a ‘population shift’ effect has now been avoided by purification of haematopoietic stem‐like cells that are positive for the cell surface marker AC133 from the bone marrow of healthy volunteers and 30 patients at various stages of MDS. Microarray analysis with the AC133+ cells from these individuals resulted in the identification of sets of genes with expression that was specific to either indolent or advanced stages of MDS. The former group of genes included that for PIASy, which catalyses protein modification with the ubiquitin‐like molecule SUMO. Induction of PIASy expression in a mouse myeloid cell line induced apoptosis. A loss of PIASy expression may therefore contribute directly to the growth of MDS blasts and stage progression.

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 G2–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.

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.

Genome-Wide Screening for Target Regions of Histone Deacetylases in Cardiomyocytes

The acetylation status of core histones in cardiomyocytes has been linked to the development of cardiac hypertrophy and heart failure. Little is known, however, of the genes affected by abnormal histone acetylation in such pathological conditions. We recently developed a genome-wide screening method, differential chromatin scanning (DCS), to isolate genomic fragments associated with histones subject to differential acetylation. We have now applied DCS to H9C2 rat embryonic cardiomyocytes incubated with or without trichostatin A (TSA), a specific inhibitor of histone deacetylase (HDAC) activity. About 200 genomic fragments were readily isolated by DCS on the basis of the preferential acetylation of associated histones in TSA-treated cells. Quantitation of the amount of DNA in chromatin immunoprecipitates prepared with antibodies to acetylated histone H3 revealed that 37 of 38 randomly chosen DCS clones were preferentially precipitated from the TSA-treated cells, thus verifying the high fidelity of DCS. Epigenetic regulation of DCS clones was further confirmed in cells treated with sodium butyrate, another HDAC inhibitor, as well as in cardiac myocytes isolated from neonatal rats. The mRNA level of 9 (39%) of 23 genes corresponding to DCS clones changed in parallel with the level of histone acetylation in H9C2 cells. Furthermore, a physiological hypertrophic stimulus, cardiotrophin-1, affected the acetylation level of histones associated with genomic regions corresponding to certain DCS clones. Our data thus establish a genome-wide profile of HDAC targets in cardiomyocytes, which should provide a basis for further investigations into the role of epigenetic modification in cardiac disorders.

Screening of genes specifically activated in the pancreatic juice ductal cells from the patients with pancreatic ductal carcinoma.

Pancreatic ductal carcinoma (PDC) is one of the most intractable human malignancies. Surgical resection of PDC at curable stages is hampered by a lack of sensitive and reliable detection methods. Given that DNA microarray analysis allows the expression of thousands of genes to be monitored simultaneously, it offers a potentially suitable approach to the identification of molecular markers for the clinical diagnosis of PDC. However, a simple comparison between the transcriptomes of normal and cancerous pancreatic tissue is likely to yield misleading pseudopositive data that reflect mainly the different cellular compositions of the specimens. Indeed, a microarray comparison of normal and cancerous tissue identified the INSULIN gene as one of the genes whose expression was most specific to normal tissue. To eliminate such a “population‐shift” effect, the pancreatic ductal epithelial cells were purified by MUC1‐based affinity chromatography from pancreatic juice isolated from both healthy individuals and PDC patients. Analysis of these background‐matched samples with DNA microarrays representing 3456 human genes resulted in the identification of candidate genes for PDC‐specific markers, including those for AC133 and carcinoembryonic antigen‐related cell adhesion molecule 7 (CEACAM7). Specific expression of these genes in the ductal cells of the patients with PDC was confirmed by quantitative real‐time polymerase chain reaction analysis. Microarray analysis with purified pancreatic ductal cells has thus provided a basis for the development of a sensitive method for the detection of PDC that relies on pancreatic juice, which is routinely obtained in the clinical setting. (Cancer Sci 2003; 94: 263–270)

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 μmol/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.

Evidence for activation of Amh gene expression by steroidogenic factor 1

The anti-Müllerian hormone gene (Amh) is responsible for regression in males of the Müllerian ducts. The molecular mechanism of regulation of chicken Amh expression is poorly understood. To investigate the regulation of chicken Amh expression, we have cloned Amh cDNAs from quail and duck as well as the promoter regions of the gene from chicken, quail, and duck. The expression patterns of Amh during embryonic development in these three species were found to be similar, suggesting that the regulatory mechanisms of Amh expression are conserved. The sequence of the proximal promoter of Amh contains a putative binding site for steroidogenic factor 1 (SF1), the protein product of which can up-regulate Amh in mammals. We showed here that SF1 is able to activate the chicken Amh promoter and binds to its putative SF1 binding site. These results suggest that SF1 plays a role in regulation of Amh expression in avian species.