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Minoru Toyota

Publications and source records attributed to Minoru Toyota.

At least 19 recordsLinked to original sources
PubMed2007

Identification of DFNA5 as a target of epigenetic inactivation in gastric cancer.

Epigenetic gene inactivation plays a key role in the development of various types of cancer. Using methylated CpG island amplification coupled with representational difference analysis to identify genes inactivated by DNA methylation in gastric cancer, we identified seven DNA fragments corresponding to the 5' CpG islands of the affected genes. One of the clones recovered was identical to the 5' flanking region of DFNA5, a gene previously shown to be associated with deafness and induced by DNA damage. Further analysis revealed that DFNA5 is expressed in normal tissues but is down-regulated in gastric cancer cell lines due to methylation of the region around its transcription start site. Treating gastric cancer cells that lacked DFNA5 expression with a methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the gene's expression. Methylation of DFNA5 was detected in 50% of primary gastric tumors, and was correlated with positivity for Epstein-Barr virus and the absence of metastasis. Moreover, introduction of exogenous DFNA5 into silenced cells suppressed colony formation. Taken together, these data suggest that the silencing of DFNA5 occurs frequently in gastric cancer and may play a key role in development and progression of the disease.

Aged↗
PubMed2006

Comparative genome analysis identifies the vitamin D receptor gene as a direct target of p53-mediated transcriptional activation.

p53 is the most frequently mutated tumor suppressor gene in human neoplasia and encodes a transcriptional coactivator. Identification of p53 target genes is therefore key to understanding the role of p53 in tumorigenesis. To identify novel p53 target genes, we first used a comparative genomics approach to identify p53 binding sequences conserved in the human and mouse genome. We hypothesized that potential p53 binding sequences that are conserved are more likely to be functional. Using stringent filtering procedures, 32 genes were newly identified as putative p53 targets, and their responsiveness to p53 in human cancer cells was confirmed by reverse transcription-PCR and real-time PCR. Among them, we focused on the vitamin D receptor (VDR) gene because vitamin D3 has recently been used for chemoprevention of human tumors. VDR is induced by p53 as well as several other p53 family members, and analysis of chromatin immunoprecipitation showed that p53 protein binds to conserved intronic sequences of the VDR gene in vivo. Introduction of VDR into cells resulted in induction of several genes known to be p53 targets and suppression of colorectal cancer cell growth. In addition, p53 induced VDR target genes in a vitamin D3-dependent manner. Our in silico approach is a powerful method for identification of functional p53 binding sites and p53 target genes that are conserved among humans and other organisms and for further understanding the function of p53 in tumorigenesis.

Animals↗
PubMed2006

Genetic, epigenetic, and clinicopathologic features of gastric carcinomas with the CpG island methylator phenotype and an association with Epstein-Barr virus.

BACKGROUND: The CpG island methylator phenotype (CIMP), which is characterized by simultaneous methylation of the CpG islands of multiple genes, has been recognized as one of the important mechanisms in gastrointestinal carcinogenesis. METHODS: Methylation of the 5 methylated-in-tumors (MINT) loci and 12 tumor-related genes in 78 primary gastric carcinomas was examined using combined bisulfite-restriction analysis. Epstein-Barr virus (EBV)-associated gastric tumors were detected using real-time polymerase chain reaction analysis followed by an evaluation of the correlations between CIMP status, EBV-association, and genetic alteration of p53 and K-ras. The authors compared the clinicopathologic features of gastric carcinomas that had high CIMP methylation (CIMP-H) with tumors that had low CIMP methylation (CIMP-L) or negative CIMP methylation (CIMP-N). RESULTS: The methylation profiles of 12 genes showed nonrandom methylation, supporting the presence of CIMP in gastric carcinoma. No p53 mutations were detected among CIMP-H tumors, and no EBV association was detected in tumors that showed mutation of p53 and K-ras. In a multiple logistic regression model with CIMP-H as the dependent variable, proximal location (P = .011), diffuse type (P = .019), and less advanced pathologic TNM status (P = .043) contributed significantly to CIMP-H. Patients who had CIMP-N gastric tumors had a significantly worse survival than patients who had CIMP-H tumors (P = .004) or CIMP-L tumors (P = .012). EBV-associated tumors were associated strongly with CIMP-H, hypermethylation of tumor-related genes, and no p53 or K-ras mutation. CONCLUSIONS: CIMP status appeared to be associated with distinct genetic, epigenetic, and clinicopathologic features in gastric carcinomas. The finding that gastric carcinomas arose through different molecular pathways may affect not only tumor characteristics but also patient prognosis.

Aged↗
PubMed2006

Knowledge of epigenetic influence for prostate cancer therapy.

Prostate cancer is one of the most prevalent cancers in men in many countries, increasing in frequency with age through the most advanced years. The standard treatment for newly diagnosed metastatic tumors is androgen ablation. However, advanced prostate cancer nevertheless often develops in many cases. Although hormonal manipulation and chemotherapy have uncertain value for advanced lesions, especially androgen-independent, recent studies of docetaxel-based chemotherapy in men with androgen-independent prostate cancer have shown a survival benefit. Intensive investigations have shown that aberrant epigenetic features. including aberrant DNA methylation, make an important contribution to carcinogenesis as well as genetic alterations. Hypermethylation of CpG islands in promoter regions can lead to silencing of tumor-suppressor genes, while hypomethylation of the genome leads to instability. This review attempts to provide up-to-date information regarding the significance of epigenetics for human prostate cancer, with aberrations offering dues to therapy and possibly also providing targets for anticancer drugs.

Animals↗
PubMed2006

Activation of the ribosomal protein L13 gene in human gastrointestinal cancer.

Although ribosomal proteins are major components of ribosomes, recent data have shown them to have extraribosomal functions apart from ribosome and protein biosynthesis. In our earlier study, we showed that ribosomal protein L13 mRNA was up-regulated in response to DNA damage in hamster cells. We report here that L13 expression is up-regulated in human gastrointestinal cancers. We also examined the biological role of L13 on human cancer cells. Knocking down L13 expression using small interfering RNA (siRNA) resulted in drastic attenuation of cancer cell growth with significant G1 and G2/M arrest of the cell cycle. Moreover, L13 siRNA significantly enhanced the cellular sensitivity to certain DNA damaging agents and, concordantly, L13-overexpressing cells demonstrated greater chemoresistance compared to parent cells, suggesting an inverse correlation between L13 expression and chemosensitivity. By using semiquantitative RT-PCR, we analyzed expression of L13 in freshly resected cancer tissue of the stomach, colorectum and liver. Up-regulation of L13 mRNA expression was observed in 10 (28%) of 36 gastric, 19 (41%) of 46 colorectal and 5 (20%) of 25 liver cancer tissue samples compared to adjacent normal tissue samples. We also found that increased expression of the L13 gene correlated with clinical staging in gastric cancers. The results of this study suggest that L13 plays an essential role in the progression of some gastrointestinal malignancies.

Animals↗
PubMed2005

Screening for genomic fragments that are methylated specifically in colorectal carcinoma with a methylated MLH1 promoter.

A subset of colorectal carcinomas (CRCs) is associated with microsatellite instability (MSI) of the genome. Although extensive methylation of CpG islands within the promoter regions of DNA mismatch repair genes such as MLH1 is thought to play a central role in tumorigenesis for MSI-positive sporadic CRCs, it has been obscure whether such aberrant epigenetic regulation occurs more widely and affects other cancer-related genes in vivo. Here, by using methylated CpG island amplification coupled with representational difference analysis (MCA-RDA), we screened genomic fragments that are selectively methylated in CRCs positive for MLH1 methylation, resulting in the identification of hundreds of CpG islands containing genomic fragments. Methylation status of such CpG islands was verified for 28 genomic clones in 8 CRC specimens positive for MLH1 methylation and the corresponding paired normal colon tissue as well as in 8 CRC specimens negative for methylation. Many of the CpG islands were preferentially methylated in the MLH1 methylation-positive CRC specimens, although methylation of some of them was more widespread. These data provide insights into the complex regulation of the methylation status of CpG islands in CRCs positive for MSI and MLH1 methylation.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Small interfering RNA-induced CHFR silencing sensitizes oral squamous cell cancer cells to microtubule inhibitors.

Alterations in the function of cell cycle checkpoints are frequently detected in oral squamous cell carcinomas (OSCCs), and are often associated with the sensitivity of the cancer cells to chemotherapeutic drugs. Recently, a mitotic checkpoint gene, Chfr, was shown to be inactivated by promoter methylation and point mutations in various human tumors. Here we show that the absence of its product, CHFR, is associated with mitotic checkpoint dysfunction, and that cancer cells lacking CHFR are sensitive to microtubule inhibitors. Checkpoint impairment appears to be caused by a prophase defect in this case, as OSCC cells lacking CHFR showed phosphorylation of histone H3 on Ser10 and translocation of cyclin B1 to the nucleus. When CHFR-deficient OSCC cells were treated with a microtubule inhibitor (docetaxel or paclitaxel), significant numbers of apoptotic cells were observed. Moreover, disruption of CHFR using small interfering RNA (siRNA) impaired the mitotic checkpoint, thereby reducing the ability of OSCC cells to arrest at G2/M phase and making them more sensitive to microtubule inhibitors. Our results suggest that CHFR could be a useful molecular target for chemotherapy.

Adult↗
PubMed2005

Aberrant methylation and silencing of the BNIP3 gene in colorectal and gastric cancer.

BNIP3 protein is a proapoptotic member of the Bcl-2 family that is expressed in hypoxic regions of tumors. To examine its role in the progression of gastrointestinal cancer, we examined the expression and DNA methylation status of BNIP3 gene in a panel of colorectal and gastric cancer cell lines. BNIP3 was not expressed in 14 of the 24 cell lines tested, and its absence was not caused by gene mutation or by altered expression of hypoxia inducible factor-1, a key transcription factor that regulates BNIP3 expression. On the other hand, methylation of the 5' CpG island of BNIP3 was closely correlated with silencing the gene. Moreover, treating methylated cells with the methyltransferase inhibitor 5-aza-2'-deoxycytidine restored hypoxia-induced expression of BNIP3 mRNA and protein, which in turn led to cell death. Aberrant methylation of BNIP3 was also detected in 66% of primary colorectal and 49% of primary gastric cancers, but not in normal tissue samples collected from areas adjacent to the tumors. Apparently, epigenetic alteration of BNIP3 is a frequent and cancer-specific event, which suggests that inactivation of BNIP3 likely plays a key role in the progression of some gastrointestinal cancers and that it may be a useful molecular target for therapy.

Acetylation↗
PubMed2005

Upregulation of BNIP3 by 5-aza-2'-deoxycytidine sensitizes pancreatic cancer cells to hypoxia-mediated cell death.

BACKGROUND: Pancreatic cancer cells often show resistance to hypoxia-mediated apoptosis, but the molecular mechanism underlying that resistance remains unknown. The purpose of the present study, therefore, was to examine the role of epigenetic gene alteration in the resistance to hypoxia-mediated apoptosis among pancreatic cancer cells. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression of five genes associated with hypoxia-mediated apoptosis (PUMA, Caspase-8 [CASP8], APAF-1, BNIP3, and BNIP3L) in a panel of pancreatic cancer cell lines. Protein expression was examined by Western blot analysis, using lysates from cells incubated under normoxic or hypoxic conditions. The methylation status of the genes was determined using bisulfite-PCR and sequencing. The percentages of cells that were apoptotic were determined using flow cytometry. RESULTS: Under normoxic conditions, the expression of the BNIP3 gene varied among the 12 pancreatic cancer cell lines tested, with 50% of them showing no BNIP3 expression at all, whereas expression of the other four genes was readily detected in all 12 cell lines. DNA methylation of BNIP3's CpG island in the region around the transcription start site of the gene was closely associated with its silencing. The expression of BNIP3 was restored by the methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC), as was the hypoxia-mediated pancreatic cancer cell death. CONCLUSIONS: BNIP3 expression is silenced in some pancreatic cancer cells by the methylation of its CpG island. Demethylation of BNIP3, using a methyltransferase inhibitor, restores the gene's expression and induces hypoxia-mediated cell death. BNIP3 may thus be a useful target for new therapies aimed at treating pancreatic cancer.

Base Sequence↗
PubMed2005

The Ras effector RASSF2 is a novel tumor-suppressor gene in human colorectal cancer.

BACKGROUND & AIMS: Activation of Ras signaling is a hallmark of colorectal cancer (CRC), but the roles of negative regulators of Ras are not fully understood. Our aim was to address that question by surveying genetic and epigenetic alterations of Ras-Ras effector genes in CRC cells. METHODS: The expression and methylation status of 6 RASSF family genes were examined using RT-PCR and bisulfite PCR in CRC cell lines and in primary CRCs and colorectal adenomas. Colony formation assays and flow cytometry were used to assess the tumor suppressor activities of RASSF1 and RASSF2. Immunofluorescence microscopy was used to determine the effect of altered RASSF2 expression on cell morphology. Mutations of K- ras , BRAF, and p53 were identified using single-strand conformation analysis and direct sequencing. RESULTS: Aberrant methylation and histone deacetylation of RASSF2 was associated with the gene's silencing in CRC. The activities of RASSF2, which were distinct from those of RASSF1, included induction of morphologic changes and apoptosis; moreover, its ability to prevent cell transformation suggests that RASSF2 acts as a tumor suppressor in CRC. Primary CRCs that showed K- ras /BRAF mutations also frequently showed RASSF2 methylation, and inactivation of RASSF2 enhanced K- ras -induced oncogenic transformation. RASSF2 methylation was also frequently identified in colorectal adenomas. CONCLUSIONS: RASSF2 is a novel tumor suppressor gene that regulates Ras signaling and plays a pivotal role in the early stages of colorectal tumorigenesis.

Acetylation↗
PubMed2005

CIMP, at last.

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Adaptor Proteins, Signal Transducing↗
PubMed2005

Epigenetic changes in solid and hematopoietic tumors.

There are three connected molecular mechanisms of epigenetic cellular memory in mammalian cells: DNA methylation, histone modifications, and RNA interference. The first two have now been firmly linked to neoplastic transformation. Hypermethylation of CpG-rich promoters triggers local histone code modifications resulting in a cellular camouflage mechanism that sequesters gene promoters away from transcription factors and results in stable silencing. This normally restricted mechanism is ubiquitously used in cancer to silence hundreds of genes, among which some critically contribute to the neoplastic phenotype. Virtually every pathway important to cancer formation is affected by this process. Methylation profiling of human cancers reveals tissue-specific epigenetic signatures, as well as tumor-specific signatures, reflecting in particular the presence of epigenetic instability in a subset of cancers affected by the CpG island methylator phenotype. Generally, methylation patterns can be traced to a tissue-specific, proliferation-dependent accumulation of aberrant promoter methylation in aging tissues, a process that can be accelerated by chronic inflammation and less well-defined mechanisms including, possibly, diet and genetic predisposition. The epigenetic machinery can also be altered in cancer by specific lesions in epigenetic effector genes, or by aberrant recruitment of these genes by mutant transcription factors and coactivators. Epigenetic patterns are proving clinically useful in human oncology via risk assessment, early detection, and prognostic classification. Pharmacologic manipulation of these patterns-epigenetic therapy-is also poised to change the way we treat cancer in the clinic.

Alleles↗
PubMed2005

Mucin expression profile is related to biological and clinical characteristics of intraductal papillary-mucinous tumors of the pancreas.

OBJECTIVES: Biologic and clinical characteristics of intraductal papillary-mucinous tumors of the pancreas (IPMTs) were studied in reference to immunohistochemical mucin (MUC1, MUC2, and MUC5AC) expression. METHODS: Histologic grade, immunohistochemical ki-67 and p53 expression, and findings in imaging tests of 21 IPMTs (9 carcinomas, 6 borderline tumors, and 6 adenomas) were examined according to the mucin expression profile. RESULTS: IPMTs were divided into groups: M1 group (MUC1+, n = 4), M2 group (MUC2 + MUC1-, n = 12), and M5 group (MUC5AC + MUC1-MUC2-, n = 5). The M2 group was subdivided into M2s (strongly positive) and M2w (weakly positive) groups. The rates of carcinoma in the M1, M2s, M2w, and M5 groups were 100%, 40%, 0%, and 0%, respectively. The Ki-67 labeling indexes were significantly higher in the M1 and M2s groups. p53 staining was positive in 50% and 40% of the IPMTs in the M1 and M2s groups, respectively, but in none of the IPMT in the M2w and M5 groups. Morphologic changes in imaging tests during the observation periods were most remarkable in the M1 group. CONCLUSIONS: Our results suggest that MUC1 is related to malignant character but MUC5AC alone is related to benign character in IPMTs and that malignant potential of IPMTs expressing MUC2 depends on the degree of MUC2 expression.

Adenocarcinoma, Mucinous↗
PubMed2005

[SFRP, a family of new colorectal tumor suppressor candidate genes].

Aberrant Wnt pathway signaling is an early progression event in up to 90% of colorectal cancers (CRC). It occurs mainly through mutations of APC or beta-catenin. We have identified frequent promoter hypermethylation and gene silencing of the secreted frizzled-related protein (SFRP) genes in CRC. SFRPs possess a domain similar to one in the Wnt receptor Frizzled proteins and can inhibit Wnt receptor binding to downregulate pathway signaling in development. We next found that restoration of SFRP function in CRC cells attenuates Wnt signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in CRC progression and may, thus, provide constitutive Wnt signaling which is required to complement downstream mutations in the evolution of CRC.

Colorectal Neoplasms↗
PubMed2005

The role of T-fimbrin in the response to DNA damage: silencing of T-fimbrin by small interfering RNA sensitizes human liver cancer cells to DNA-damaging agents.

Fimbrins (also known as plastins) are actin-binding proteins of the cortical cytoskeleton present in all cells and conserved from yeast to mammals. We previously reported that the up-regulation of T-fimbrin, a fimbrin isoform, in association with G2 arrest following DNA damage and that a lack of T-fimbrin expression shortens the radiation-induced G2 arrest in Chinese hamster ovarian cells. In this study, we further investigated the role of T-fimbrin in DNA-damage response using a panel of human liver cancer cell lines and small interfering RNA technology. T-fimbrin was differentially expressed in human liver cancer cell lines. Colony formation assays revealed that cell lines lacking T-fimbrin expression were highly sensitive to DNA damage compared to cell lines that express T-fimbrin. Using siRNA designed to target T-fimbrin, we demonstrated that silencing endogenous T-fimbrin causes a marked increase in the cellular sensitivity to VP-16 and UV irradiation. Moreover, T-fimbrin deletion abrogated UV-mediated cell cycle checkpoint, and consequently led to increased apoptotic cell death in resistant cells. These findings suggest that the status of T-fimbrin expression may be a useful molecular marker for predicting the responsiveness of cancer cells to treatment with chemotherapeutic drugs.

Actins↗
PubMed2004

Identification of differentially methylated CpG islands in prostate cancer.

Epigenetic change such as DNA methylation is one important mechanism for regulating gene expression as genetic change, such as mutation or loss of heterozygosity. Methylation of cancer-related genes has been shown to play an important role in carcinogenesis and tumor progression. Using methylated CpG island amplification (MCA)/representational difference analysis (RDA), we identified four CpG islands in neurotrophin tyrosine kinase receptor type 2 (NTRK2), Protocadherine Flamingo1 and MFPC (Methylated Fragments in Prostate Cancer) 7 and 8. Bisulfite sequencing revealed that 2 regions of NTRK2 as well as MFPC7 and MFPC8 were aberrantly methylated in prostate cancer cell lines, and COBRA showed that 48 (76.24%), 37 (58.7%) and 14 (22.2%) of 63 prostate cancer tissues were methylated, respectively, for these sites. On the other hand, none of 13 benign prostate samples were methylated, except for 1 (7.7%) with NTRK2. For NTRK2, mRNA expression was negative in prostate cancer cell lines (LNCaP and DU145) but was recovered on a methyltransferase inhibitor (5-Aza-CdR) treatment. The role of NTRK2 within NTRK remains unclear. Our results suggest that these 3 hypermethylated DNA fragments also may be markers of prostate cancer detection.

Animals↗