研究者詳細 - 時野　隆至

論文 - 時野　隆至

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p53 family members regulate the expression of the apolipoprotein D gene.

Sasaki Y, Negishi H, Koyama R, Anbo N, Ohori K, Idogawa M, Mita H, Toyota M, Imai K, Shinomura Y, Tokino T

The Journal of biological chemistry 284 ( 2 ) 872 - 83 2009年01月 [査読有り] [国際誌]

p73 and p63 are members of the p53 gene family that play an important role in development and homeostasis, mainly by regulating transcription of a variety of genes. We report here that apolipoprotein D (apoD), a member of the lipocalin superfamily of lipid transport proteins, is a direct transcriptional target of the p53 family member genes. We found that the expression of apoD was specifically up-regulated by either TAp73 or TAp63 but not significantly by p53. In addition, apoD transcription is activated in response to cisplatin in a manner dependent on endogenous p73. By using small interference RNA designed to target p73, we demonstrated that silencing endogenous p73 abolishes induction of apoD transcription following cisplatin treatment. We also identified a p73/p63-binding site in the promoter of the apoD gene that is responsive to the p53 family members. The ectopic expression of TAp73 as well as the addition of recombinant human apoD to culture medium induced the osteoblastic differentiation of the human osteosarcoma cell line Saos-2, as assessed by alkaline phosphatase activity. Importantly, apoD knockdown abrogated p73-mediated alkaline phosphatase induction. Moreover, TAp73-mediated apoD expression was able to induce morphological differentiation, as well as expression of neuronal markers, in the human neuroblastoma cell line SH-SY5Y. These results suggest that apoD induction may mediate the activity of p73 in normal development.

DOI PubMed

Prediction of p53 target genes based on integrative analysis of chromatin- immunoprecipitated and sequenced tags,by using Galaxy,a web-based interactive platform for large-scale genome analysis

Mita H, Sasaki Y, Suzuki H, Idogawa M, Kashima L, Anbo N, Akashi H, Tatsumi H, Toyota M, Tokino T

Tumor Research ( Sapporo Medical University ) 43 1 - 23 2008年12月 [査読有り]

Chromatin immunoprecipitation (ChIP) followed by sequencing of immunoprecipitated DNA fragments is the high-throughput method for identifying transcription factor binding sites. In one such method, ChIP-PET, paired-end ditags (PETs) derived from both ends of the immunoprecipitated DNA fragments are sequenced and mapped to the genome. We report here the prediction of p53 target genes by metaanalyzing tags of p53 ChIP-PET and by combining with other genomic annotations, using Galaxy, a web-based platform for large-scale genome analysis. We found 327 of p53 binding sites on the genome of 5-fluorouracil (5-FU)- treated HCT116 colon cancer cells by searching the total 65,509 PETs for PET clusters. The search for p53 target gene, which focused on PET clusters with computationally-predicted p53 binding motif, identified 20 of putative p53 target genes as well as 11 of known p53 targets. Another search for p53 target genes, which focused on PET clusters located within 50-kb flanking regions of transcription start sites of genes, identified 278 of Refseq genes, 79 of noncoding RNAs and 5 of microRNAs as p53 targets which included lots of known validated targets. Our results indicate that sequencing-based ChIP analysis combined with the existing genome annotation is effective method to predict p53 binding loci and target genes, and also show that the Galaxy platform is well-suited for multiple-type analyses and visualization of ChIP data, leading to functional annotation of transcription factor binding sites.

DOI CiNii

DNA methylation and cancer pathways in gastrointestinal tumors.

Suzuki H, Tokino T, Shinomura Y, Imai K, Toyota M

Pharmacogenomics 9 ( 12 ) 1917 - 28 2008年12月 [査読有り] [国際誌]

Cancer is fundamentally a genetic and epigenetic disease that requires the accumulation of genomic alterations that inactivate tumor suppressors and activate proto-oncogenes. In addition to genetic mutation or allelic loss, epigenetic gene silencing associated with DNA methylation is now recognized as an alternative mechanism by which tumor suppressor genes are inactivated. In gastrointestinal cancers, for example, DNA methylation frequently alters the activity in a number of important signaling pathways by silencing expression of genes encoding Wnt antagonists, negative Ras effectors and p53 targets. Indeed, the list of genes aberrantly methylated in cancer is growing, and methylation of a p53 target micoRNA gene has recently been demonstrated. Sites of DNA methylation could be promising markers and targets for risk assessment, early detection and treatment of cancer.

DOI PubMed

LINE-1 hypomethylation is associated with increased CpG island methylation in Helicobacter pylori-related enlarged-fold gastritis.

Yamamoto E, Toyota M, Suzuki H, Kondo Y, Sanomura T, Murayama Y, Ohe-Toyota M, Maruyama R, Nojima M, Ashida M, Fujii K, Sasaki Y, Hayashi N, Mori M, Imai K, Tokino T, Shinomura Y

Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 17 ( 10 ) 2555 - 64 2008年10月 [査読有り] [国際誌]

BACKGROUND: The molecular mechanism by which Helicobacter pylori infection leads to gastric cancer is not fully understood. Similarly, patients with enlarged-fold (EF+) gastritis, one cause of which is H. pylori infection, have an increased risk for gastric cancer, although again molecular mechanism is unclear. In the present study, we analyzed the methylation status of long interspersed nucleotide elements (LINE-1) and three cancer-related genes in a panel of gastric mucosae, with or without EF+ gastritis. METHODS: We used bisulfite pyrosequencing to assess the levels of LINE-1, CDH1, CDH13, and PGP9.5 methylation in 78 gastric mucosa specimens from 48 patients. RESULTS: Levels of LINE-1 methylation were significantly reduced in mucosae from patients with EF+ gastritis. This hypomethylation of LINE-1 was associated with increased methylation of the 5' CpG islands of the genes, which suggests that, in EF+ gastritis, the methylation of the promoter regions of certain genes is accompanied by global demethylation of repetitive sequences. CONCLUSIONS: Our results indicate that genomewide hypomethylation and regional hypermethylation occur in EF+ gastritis and may contribute to the tumorigenesis of diffuse-type gastric cancers.

DOI PubMed

p53による肝癌由来増殖因子(HDGF)の抑制機構(Negative regulation of hepatoma-derived growth factor by p53)

安保直樹, 佐々木泰史, 根岸秀明, 井戸川雅史, 小山良太, 豊田実, 鈴木拓, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 67回 136 - 136 2008年09月

CHFRはNF-kappaBを抑制する(CHFR, a potential tumor suppressor, inhibits the transcriptional activity of NF-kappaB)

鹿島理沙, 見田裕章, 豊田実, 佐々木泰史, 鈴木拓, 井戸川雅史, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 67回 410 - 410 2008年09月

p53ファミリー(p73,p63)の標的遺伝子apolipoprotein Dの同定(p53 family members regulate the expression of the apolipoprotein D gene)

佐々木泰史, 安保直樹, 根岸秀明, 井戸川雅史, 見田裕章, 豊田実, 今井浩三, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 67回 60 - 60 2008年09月

miRNAによるp21誘導抑制能をもつp53発現アデノウイルスベクターによる抗腫瘍効果の増強(Enhancement of antitumor effect by single adenovirus vector expressing p53 and suppressing p21 induction by miRNA)

井戸川雅史, 佐々木泰史, 今井浩三, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 67回 237 - 237 2008年09月

胃癌におけるKRAS遺伝子増幅と細胞内シグナルの活性化(Gene amplification of KRAS and activation of intracellular signaling pathways in gastric cancer)

見田裕章, 豊田実, 青木文夫, 明石浩史, 丸山玲緒, 佐々木泰史, 鈴木拓, 井戸川雅史, 鹿島理沙, サバウ・ソリン, 今井浩三, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 67回 163 - 163 2008年09月

大腸癌におけるmicroRNA-34b/cおよびBTG4のCpGアイランドメチル化によるエピジェネティックな不活化(Epigenetic silencing of microRNA-34b/c and BTG4 is associated with CpG island hypermethylation in colorectal cancer)

鈴木拓, 豊田実, 野島正寛, 佐々木泰史, 時野隆至, 今井浩三, 篠村恭久

日本癌学会総会記事 ( (一社)日本癌学会 ) 67回 71 - 71 2008年09月

口腔扁平上皮癌におけるSFRP遺伝子のエピジェネティックな不活化(Epigenetic inactivation of SFRP genes in oral squamous cell carcinoma)

曽我部陽平, 鈴木拓, 豊田実, 今井崇, 野島正寛, 佐々木泰史, 時野隆至, 平塚博義

日本癌学会総会記事 ( (一社)日本癌学会 ) 67回 174 - 174 2008年09月

DNA hypermethylation contributes to incomplete synthesis of carbohydrate determinants in gastrointestinal cancer.

Kawamura YI, Toyota M, Kawashima R, Hagiwara T, Suzuki H, Imai K, Shinomura Y, Tokino T, Kannagi R, Dohi T

Gastroenterology 135 ( 1 ) 142 - 151 2008年07月 [査読有り] [国際誌]

BACKGROUND & AIMS: It has long been known that malignant transformation is associated with abnormal expression of carbohydrate determinants. The aim of this study was to clarify the cause of cancer-associated abnormal glycosylation in gastrointestinal (GI) cancers. METHODS: We compared the expression levels of "glyco-genes," including glycosyltransferases and glycosidases, in normal GI mucosa and in gastric and colorectal cancer cells. To examine the possibility that DNA hypermethylation contributed to the down-regulation of these genes, we treated GI cancer cells with 5-aza-2'-deoxycytidine (5-aza-dC), an inhibitor of DNA methyltransferase. RESULTS: The silencing of some of these glyco-genes, but not up-regulation of certain molecules, was observed. The Sd(a) carbohydrate was abundantly expressed in the normal GI mucosa, but its expression was significantly decreased in cancer tissues. When human colon and gastric cancer cells were treated with 5-aza-dC, cell surface expression of Sd(a) and the transcription of B4GALNT2, which catalyzes the synthesis of the Sd(a), were induced. The promoter region of the human B4GALNT2 gene was heavily hypermethylated in many of the GI cancer cell lines examined as well as in gastric cancer tissues (39 out of 78 cases). In addition, aberrant methylation of the B4GALNT2 gene was strongly correlated with Epstein-Barr virus-associated gastric carcinomas and occurred coincidentally with hypermethylation of the ST3GAL6 gene. CONCLUSIONS: Epigenetic changes in a group of glycosyltransferases including B4GALNT2 and ST3GAL6 represent a malignant phenotype of gastric cancer caused by silencing of the activity of these enzymes, which action may eventually induce aberrant glycosylation and expression of cancer-associated carbohydrate antigens.

DOI PubMed

Cytoplasmic RASSF2A is a proapoptotic mediator whose expression is epigenetically silenced in gastric cancer.

Maruyama R, Akino K, Toyota M, Suzuki H, Imai T, Ohe-Toyota M, Yamamoto E, Nojima M, Fujikane T, Sasaki Y, Yamashita T, Watanabe Y, Hiratsuka H, Hirata K, Itoh F, Imai K, Shinomura Y, Tokino T

Carcinogenesis 29 ( 7 ) 1312 - 8 2008年07月 [査読有り] [国際誌]

Gastric cancer cells often show altered Ras signaling, though the underlying molecular mechanism is not fully understood. We examined the expression profile of eight ras-association domain family (RASSF) genes plus MST1/2 and found that RASSF2A is the most frequently downregulated in gastric cancer. RASSF2A was completely silenced in 6 of 10 gastric cancer cell lines as a result of promoter methylation, and expression was restored by treating the cells with 5-aza-2'-deoxycytidine. Introduction of RASSF2A into non-expressing cell lines suppressed colony formation and induced apoptosis. These effects were associated with the cytoplasmic localization of RASSF2A and morphological changes to the cells. Complementary DNA microarray analysis revealed that RASSF2A suppresses the expression of inflammatory cytokines, which may in turn suppress angiogenesis and invasion. In primary gastric cancers, aberrant methylation of RASSF2A was detected in 23 of 78 (29.5%) cases, and methylation correlated significantly with an absence of the lymphatic invasion, absence of venous invasion, absence of lymph node metastasis, less advanced stages, Epstein-Barr virus, absence of p53 mutations and the presence of the CpG island methylator phenotype-high. These results suggest that epigenetic inactivation of RASSF2A is required for tumorigenesis in a subset of gastric cancers.

DOI PubMed

Epigenetic silencing of microRNA-34b/c and B-cell translocation gene 4 is associated with CpG island methylation in colorectal cancer.

Toyota M, Suzuki H, Sasaki Y, Maruyama R, Imai K, Shinomura Y, Tokino T

Cancer research 68 ( 11 ) 4123 - 32 2008年06月 [査読有り] [国際誌]

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that, in cancer, expression of some miRNAs cells is silenced in association with CpG island hypermethylation. To identify epigenetically silenced miRNAs in colorectal cancer (CRC), we screened for miRNAs induced in CRC cells by 5-aza-2'-deoxycytidine (DAC) treatment or DNA methyltransferase knockout. We found that miRNA-34b (miR-34b) and miR-34c, two components of the p53 network, are epigenetically silenced in CRC; that this down-regulation of miR-34b/c is associated with hypermethylation of the neighboring CpG island; and that DAC treatment rapidly restores miR-34b/c expression. Methylation of the miR-34b/c CpG island was frequently observed in CRC cell lines (nine of nine, 100%) and in primary CRC tumors (101 of 111, 90%), but not in normal colonic mucosa. Transfection of precursor miR-34b or miR-34c into CRC cells induced dramatic changes in the gene expression profile, and there was significant overlap between the genes down-regulated by miR-34b/c and those down-regulated by DAC. We also found that the miR-34b/c CpG island is a bidirectional promoter which drives expression of both miR-34b/c and B-cell translocation gene 4 (BTG4); that methylation of the CpG island is also associated with transcriptional silencing of BTG4; and that ectopic expression of BTG4 suppresses colony formation by CRC cells. Our results suggest that miR-34b/c and BTG4 are novel tumor suppressors in CRC and that the miR-34b/c CpG island, which bidirectionally regulates miR-34b/c and BTG4, is a frequent target of epigenetic silencing in CRC.

DOI PubMed

Epigenetic inactivation of SFRP genes in oral squamous cell carcinoma.

Sogabe Y, Suzuki H, Toyota M, Ogi K, Imai T, Nojima M, Sasaki Y, Hiratsuka H, Tokino T

International journal of oncology 32 ( 6 ) 1253 - 61 2008年06月 [査読有り] [国際誌]

Although mutations of APC, CTNNB1 (beta-catenin) and AXIN1 are rare in oral squamous cell carcinoma (OSCC), activation of the Wnt signaling pathway is thought to play an important role in oral carcinogenesis. In the present study, we examined the relationship between Wnt signaling and epigenetic alteration of the secreted frizzled-related protein (SFRP) genes in OSCC. We frequently detected loss of membrane localization of beta-catenin and its cytoplasmic or nuclear accumulation in OSCC cell lines, although these cell lines showed no APC or CTNNB1 (beta-catenin) mutations and no methylation of CDH1 (E-cadherin). By contrast, we frequently detected methylation of SFRP1 (7/17, 41%) SFRP2 (16/17, 94%) and SFRP5 (14/17, 82%) in a panel of OSCC cell lines, as well as in specimens of primary tumors collected from 44 OSCC patients (SFRP1, 10/42, 24%; SFRP2, 16/44, 36%; SFRP5, 7/43, 16%). We also observed that OSCC cell lines express various Wnt ligands, and that ectopic expression of SFRPs inhibited cancer cell proliferation. Our results confirm the frequent methylation and silencing of SFRP genes in OSCC, and suggest that their loss of function contributes to activation of Wnt signaling that leads to cell proliferation during oral carcinogenesis.

DOI PubMed

Epigenetic inactivation of RASSF2 in oral squamous cell carcinoma.

Imai T, Toyota M, Suzuki H, Akino K, Ogi K, Sogabe Y, Kashima L, Maruyama R, Nojima M, Mita H, Sasaki Y, Itoh F, Imai K, Shinomura Y, Hiratsuka H, Tokino T

Cancer science 99 ( 5 ) 958 - 66 2008年05月 [査読有り] [国際誌]

Genetic and epigenetic alterations in tumor-suppressor genes play important roles in human neoplasia. Ras signaling is often activated in oral squamous cell carcinoma (OSCC), although Ras mutations are rarely detected in Japanese OSCC patients, and the mechanisms underlying the gene's activation remain unclear. Here, we examined the expression of Ras association family (RASSF) genes in a panel of OSCC cell lines and found that RASSF2 is often downregulated by DNA methylation in OSCC cells. In addition, aberrant methylation of RASSF2 was detected in 12 of 46 (26%) primary OSCC, and 18 (39%) of those OSCC showed methylation of at least one RASSF gene. Ectopic expression of RASSF2 in OSCC cells suppressed cell growth and induced apoptosis. A RASSF2 deletion mutant lacking the Ras-association domain, which was therefore unable to interact with Ras, exhibited less pro-apoptotic activity than the full-length protein, indicating that the pro-apoptotic activity of RASSF2 is related to its association with Ras. Genomic screening of genes regulated by RASSF2 showed that genes involved in immune responses, angiogenesis, and metastasis are suppressed by RASSF2. Our results suggest that epigenetic inactivation of RASSF2 plays an important role in OSCC tumorigenesis, and that RASSF2 may be a useful molecular target for the diagnosis and treatment of OSCC.

DOI PubMed

A requirement for DICER to maintain full promoter CpG island hypermethylation in human cancer cells.

Ting AH, Suzuki H, Cope L, Schuebel KE, Lee BH, Toyota M, Imai K, Shinomura Y, Tokino T, Baylin SB

Cancer research 68 ( 8 ) 2570 - 5 2008年04月 [査読有り] [国際誌]

Promoter hypermethylation is a prevalent phenomenon, found in virtually all cancer types studied thus far, and accounts for tumor suppressor gene silencing in the absence of genetic mutations. The mechanism behind the establishment and maintenance of such aberrant hypermethylation has been under intense study. Here, we have uncovered a link between aberrant gene silencing associated with promoter CpG island DNA methylation and the siRNA/miRNA processing enzyme, DICER, in human cancer cells. By comparing demethylated HCT116 colon cancer cells with HCT116 cells genetically rendered hypomorphic for DICER, we identified a group of epigenetically silenced genes that became reactivated in the absence of functional DICER. This reactivation is associated with a dramatic loss of localized promoter DNA hypermethylation. Thus, intact DICER is required to maintain full promoter DNA hypermethylation of select epigenetically silenced loci in human cancer cells.

DOI PubMed

Histone deacetylase inhibitor FK228 enhances adenovirus-mediated p53 family gene therapy in cancer models.

Sasaki Y, Negishi H, Idogawa M, Suzuki H, Mita H, Toyota M, Shinomura Y, Imai K, Tokino T

Molecular cancer therapeutics 7 ( 4 ) 779 - 87 2008年04月 [査読有り] [国際誌]

Therapeutic replacement of the wild-type p53 gene has been pursued as a potential gene therapy strategy in a variety of cancer types; however, some cancer models are resistant to p53 in vivo and in vitro. Therefore, to improve p53 gene therapy, it is important to overcome the resistance to p53-mediated apoptosis. Histone deacetylase inhibitors are a novel class of chemotherapeutic agents that are able to reverse the malignant phenotype of transformed cells. A natural histone deacetylase inhibitor, FK228, is reported to enhance adenovirus infection due in part to the up-regulation of coxsackievirus adenovirus receptor expression. In this study, preclinical experiments were done to establish a mechanistic rationale for the combination of adenovirus-mediated p53 family gene transfer and FK228 pretreatment in future clinical trials. Pretreatment with FK228 enhanced apoptosis in human cancer cells through enhanced transduction of Ad-p53. FK228 also induced hyperacetylation of the p53 protein and specifically enhanced p53-mediated Noxa expression. Additionally, the combination of FK228 and Ad-p53 induced Bax translocation to the mitochondria. The double knockdown of Bax and Noxa expression by small interfering RNA antagonized the synergistic effect of Ad-p53 and FK228 on apoptosis induction. In human cancer xenograft models, FK228 significantly increased the therapeutic effectiveness of p53 as well as p63 gene therapy. These results provide a strong rationale for combining p53 gene therapy and FK228 pretreatment in cancer therapy.

DOI PubMed

Frequent epigenetic inactivation of Wnt antagonist genes in breast cancer

H. Suzuki, M. Toyota, H. Caraway, E. Gabrielson, T. Ohmura, T. Fujikane, N. Nishikawa, Y. Sogabe, M. Nojima, T. Sonoda, M. Mori, K. Hirata, K. Imai, Y. Shinomura, S. B. Baylin, T. Tokino

BRITISH JOURNAL OF CANCER ( NATURE PUBLISHING GROUP ) 98 ( 6 ) 1147 - 1156 2008年03月 [査読有り]

Although mutation of APC or CTNNB1 ( beta- catenin) is rare in breast cancer, activation of Wnt signalling is nonetheless thought to play an important role in breast tumorigenesis, and epigenetic silencing of Wnt antagonist genes, including the secreted frizzled- related protein ( SFRP) and Dickkopf ( DKK) families, has been observed in various tumours. In breast cancer, frequent methylation and silencing of SFRP1 was recently documented; however, altered expression of other Wnt antagonist genes is largely unknown. In the present study, we found frequent methylation of SFRP family genes in breast cancer cell lines ( SFRP1, 7 out of 11, 64%; SFRP2, 11 out of 11, 100%; SFRP5, 10 out of 11, 91%) and primary breast tumours ( SFRP1, 31 out of 78, 40%; SFRP2, 60 out of 78, 77%; SFRP5, 55 out of 78, 71%). We also observed methylation of DKK1, although less frequently, in cell lines ( 3 out of 11, 27%) and primary tumours ( 15 out of 78, 19%). Breast cancer cell lines express various Wnt ligands, and overexpression of SFRPs inhibited cancer cell growth. In addition, overexpression of a beta- catenin mutant and depletion of SFRP1 using small interfering RNA synergistically upregulated transcriptional activity of T- cell factor/ lymphocyte enhancer factor. Our results confirm the frequent methylation and silencing of Wnt antagonist genes in breast cancer, and suggest that their loss of function contributes to activation of Wnt signalling in breast carcinogenesis.

DOI

Identification of flotillin-2, a major protein on lipid rafts, as a novel target of p53 family members.

Sasaki Y, Oshima Y, Koyama R, Maruyama R, Akashi H, Mita H, Toyota M, Shinomura Y, Imai K, Tokino T

Molecular cancer research : MCR 6 ( 3 ) 395 - 406 2008年03月 [査読有り] [国際誌]

p73 and p63 are members of the p53 gene family and have been shown to play an important role in development and homeostasis mainly by regulating the transcription of a variety of genes. A subset of these genes encodes secreted proteins and receptors that may be involved in the communication between adjacent cells. We report here that flotillin-2, a major hydrophobic protein on biomembrane microdomain lipid rafts, is a direct transcriptional target of the p53 family member genes. It has been suggested that such rafts could play an important role in many cellular processes including signal transduction, membrane trafficking, cytoskeletal organization, and pathogen entry. We found that the expression of flotillin-2 was specifically up-regulated by either TAp73beta or TAp63gamma, but not significantly by p53. In addition, flotillin-2 transcription is activated in response to cisplatin in a manner dependent on endogenous p73. By using small interference RNA designed to target p73, we showed that silencing endogenous p73 abolishes the induction of flotillin-2 transcription following cisplatin treatment. Furthermore, we identified a p73/p63-binding site located upstream of the flotillin-2 gene that is responsive to the p53 family members. This response element is highly conserved between humans and rodents. We also found that ectopic expression of TAp73 as well as TAp63 enhances signal transduction by assessing the interleukin-6-mediated phosphorylation of signal transducers and activators of transcription 3. Thus, in addition to direct transactivation, p53 family member genes enhance a set of cellular processes via lipid rafts.

DOI PubMed

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