研究者詳細 - 井戸川　雅史

論文 - 井戸川　雅史

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Mesenchymal Stem Cells Cancel Azoxymethane-Induced Tumor Initiation

Masanao Nasuno, Yoshiaki Arimura, Kanna Nagaishi, Hiroyuki Isshiki, Kei Onodera, Suguru Nakagaki, Shuhei Watanabe, Masashi Idogawa, Kentaro Yamashita, Yasuyoshi Naishiro, Yasushi Adachi, Hiromu Suzuki, Mineko Fujimiya, Kohzoh Imai, Yasuhisa Shinomura

STEM CELLS ( WILEY-BLACKWELL ) 32 ( 4 ) 913 - 925 2014年04月 [査読有り]

The role of mesenchymal stem cells (MSCs) in tumorigenesis remains controversial. Therefore, our goal was to determine whether exogenous MSCs possess intrinsic antineoplastic or proneoplastic properties in azoxymethane (AOM)-induced carcinogenesis. Three in vivo models were studied: an AOM/dextran sulfate sodium colitis-associated carcinoma model, an aberrant crypt foci model, and a model to assess the acute apoptotic response of a genotoxic carcinogen (AARGC). We also performed in vitro coculture experiments. As a result, we found that MSCs partially canceled AOM-induced tumor initiation but not tumor promotion. Moreover, MSCs inhibited the AARGC in colonic epithelial cells because of the removal of O-6-methylguanine (O(6)MeG) adducts through O(6)MeG-DNA methyltransferase activation. Furthermore, MSCs broadly affected the cell-cycle machinery, potentially leading to G1 arrest in vivo. Coculture of IEC-6 rat intestinal cells with MSCs not only arrested the cell cycle at the G1 phase, but also induced apoptosis. The anti-carcinogenetic properties of MSCs in vitro required transforming growth factor (TGF)-beta signaling because such properties were completely abrogated by absorption of TGF-beta under indirect coculture conditions. MSCs inhibited AOM-induced tumor initiation by preventing the initiating cells from sustaining DNA insults and subsequently inducing G1 arrest in the initiated cells that escaped from the AARGC. Furthermore, tumor initiation perturbed by MSCs might potentially dysregulate WNT and TGF-beta-Smad signaling pathways in subsequent tumorigenesis. Obtaining a better understanding of MSC functions in colon carcinogenesis is essential before commencing the broader clinical application of promising MSC-based therapies for cancer-prone patients with inflammatory bowel disease. Stem Cells 2014;32:913-925

DOI PubMed

Conditioned mesenchymal stem cells produce pleiotropic gut trophic factors

Shuhei Watanabe, Yoshiaki Arimura, Kanna Nagaishi, Hiroyuki Isshiki, Kei Onodera, Masanao Nasuno, Kentaro Yamashita, Masashi Idogawa, Yasuyoshi Naishiro, Masaki Murata, Yasushi Adachi, Mineko Fujimiya, Kohzoh Imai, Yasuhisa Shinomura

JOURNAL OF GASTROENTEROLOGY ( SPRINGER JAPAN KK ) 49 ( 2 ) 270 - 282 2014年02月 [査読有り]

Although mounting evidence implicates mesenchymal stem cells (MSCs) in intestinal tissue repair, controversy remains regarding the engraftment, proliferation, and differentiation for repopulating MSCs in recipient tissues. Therefore, we investigated the paracrine and/or endocrine role of MSCs in experimental colitis. We analyzed the therapeutic effects of MSC-conditioned medium (MSC-CM) on dextran sulfate sodium (DSS)- or 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. We investigated the effects of MSC-CM on the epithelial cell viability, mobility, cell cycle, and cytokine production in ex vivo lamina propria/mesenteric lymphocytes, a macrophage cell line, and the mixed lymphocyte reaction. An optimal regimen against colitis was explored. The contents of MSC-CM were analyzed using a WNT signaling pathway polymerase chain reaction array, an inflammatory cytokines antibody array, and liquid chromatography-tandem mass spectrometry analysis. Independent of the systemic administration route, MSC-CM concentrates were effective for the inductive phase of TNBS-induced colitis and for the recovery phase of DSS-induced colitis. Hypoxia appeared to be one of the optimal preconditioning factors assessed by cell motility and viability through activating the PI3K-Akt pathway in rat small intestine epithelial cells, IEC-6. Thus, Hypoxia had profound effects on the contents of MSC-CM, which comprised pleiotropic gut trophic factors involved in each wound healing process, including the anti-inflammatory, proliferative, and tissue remodeling phases. Identification and optimization of potential gut trophic factors in MSC-CM is urgently needed to form the basis for new drug discovery and for optimizing cell-based therapies for inflammatory bowel disease.

DOI PubMed

AKR1B10, a Transcriptional Target of p53, Is Downregulated in Colorectal Cancers Associated with Poor Prognosis

Tomoko Ohashi, Masashi Idogawa, Yasushi Sasaki, Hiromu Suzuki, Takashi Tokino

MOLECULAR CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 11 ( 12 ) 1554 - 1563 2013年12月 [査読有り]

担当区分：筆頭著者 , 責任著者

p53 is one of the most important tumor suppressor genes, and it is frequently inactivated in various cancers. p53 modulates various cellular functions, such as apoptosis and cell-cycle arrest via transcriptional regulation. Recently, p53 has been reported to be involved in a wide range of cellular metabolic pathways, including glycolysis, oxidative phosphorylation, glutaminolysis, and the antioxidant response. To understand the functional mechanism of p53, it is important to find out the direct transcriptional targets of p53. In this study, aldo-keto reductase family 1, member B10 (AKR1B10) was identified as a direct target of the p53 family by cDNA microarray analysis after comparing the mRNA expression of control and H1299 cells that overexpressed with p53 family members. In addition, we found that the expression of AKR1B10 was significantly decreased in colorectal cancers and adenomas as compared with normal colon tissues. Knockdown of AKR1B10 significantly inhibited p53-induced apoptosis in colorectal cancer cells, whereas the overexpression of AKR1B10 enhanced p53-induced apoptosis and inhibited tumor proliferation in vivo. Furthermore, low expression of AKR1B10 in colon cancer patients was correlated with decreased survival and poor prognosis. These results suggest that decreased expression of AKR1B10 could disrupt the tumor suppressive function of p53, which result in decreased survival in colorectal cancer patients. In summary, AKR1B10 may be a novel prognostic predictor and a novel therapeutic target for colorectal cancer. Implications: AKR1B10, a transcriptional target of p53, is also a novel prognostic and therapeutic molecule in colorectal cancer. (C) 2013 AACR.

DOI PubMed

p53によるXPO1の抑制機構とその意義(p53 negatively regulates the expression of the nuclear export protein XPO1, establishing a positive feedback loop)

佐々木泰史, 田村みゆき, 竹田康佑, 井戸川雅史, 丸山玲緒, 鈴木拓, 篠村恭久, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 72回 105 - 105 2013年10月

CLCA2, a target of the p53 family, negatively regulates cancer cell migration and invasion

Yasushi Sasaki, Ryota Koyama, Reo Maruyama, Takehiro Hirano, Miyuki Tamura, Jun Sugisaka, Hiromu Suzuki, Masashi Idogawa, Yasuhisa Shinomura, Takashi Tokino

CANCER BIOLOGY & THERAPY ( LANDES BIOSCIENCE ) 13 ( 14 ) 1512 - 1521 2012年12月 [査読有り]

The tumor suppressor p53 transcriptionally regulates a number of genes that are involved in cell-cycle inhibition, apoptosis and the maintenance of genetic stability. Recent studies suggest that p53 also contributes to the regulation of cell migration and invasion. Here, we show that human chloride channel accessory-2 (CLCA2) is a target gene of the p53 family (p53, p73 and p63). CLCA2 is induced by DNA damage in a p53-dependent manner. The p53 family proteins activate the CLCA2 promoter by binding directly to the conserved consensus p53-binding site present in the CLCA2 promoter. In terms of function, ectopic expression of CLCA2 inhibited cancer cell migration. In contrast, silencing CLCA2 with siRNA stimulated cancer cell migration and invasion. We also found that inactivation of CLCA2 enhanced the expression of focal adhesion kinase (FAK), as well as its promoter activation. A small-molecule FAK inhibitor reduced the effect of CLCA2 siRNA on cell migration and invasion, suggesting that CLCA2 inhibits cancer cell migration and invasion through suppression of the FAK signaling pathway. Furthermore, there was an inverse correlation between CLCA2 and FAK expression in 251 human breast cancer tissues. These results strongly suggest that CLCA2 is involved in the p53 tumor suppressor network and has a significant effect on cell migration and invasion.

DOI PubMed

A novel approach to cancer treatment using structural hybrids of the p53 gene family

Y. Sasaki, Y. Oshima, R. Koyama, M. Tamura, L. Kashima, M. Idogawa, T. Yamashita, M. Toyota, K. Imai, Y. Shinomura, T. Tokino

CANCER GENE THERAPY ( NATURE PUBLISHING GROUP ) 19 ( 11 ) 749 - 756 2012年11月 [査読有り]

The p53 tumor suppressor belongs to a gene family that includes two other structurally and functionally related members: p73 and p63. The regulation of p53 activity differs significantly from that of p73 and p63. To enhance the tumor suppressive activity of p53, we constructed six recombinant adenoviruses that encode hybrid proteins with three functional domains derived from either p53 or TAp63 gamma. The potency of these hybrid molecules in suppressing tumorigenesis was evaluated using in vitro and in vivo models. Of the hybrid molecules tested, one hybrid named p63-53O was the most potent activator of apoptosis in human cancer cells. The p63-53O hybrid is composed of the transcriptional activation domain and DNA-binding domain of TAp63 gamma and the oligomerization domain of p53. The p63-53O hybrid efficiently transactivated p53AIP1. Moreover, silencing of p53AIP1 partially abolished the apoptotic response to p63-53O in human cancer cells. The p53-p63 hybrid molecule is a novel potent anti-proliferative agent for the treatment of cancer.

DOI PubMed

CHFR Protein Regulates Mitotic Checkpoint by Targeting PARP-1 Protein for Ubiquitination and Degradation

Lisa Kashima, Masashi Idogawa, Hiroaki Mita, Miki Shitashige, Tesshi Yamada, Kazuhiro Ogi, Hiromu Suzuki, Minoru Toyota, Hiroyoshi Ariga, Yasushi Sasaki, Takashi Tokino

JOURNAL OF BIOLOGICAL CHEMISTRY ( AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC ) 287 ( 16 ) 12975 - 12984 2012年04月 [査読有り]

The mitotic checkpoint gene CHFR (checkpoint with fork-head-associated (FHA) and RING finger domains) is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase, resulting in the degradation of target proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Here we show poly(ADP-ribose) polymerase 1 (PARP-1) to be a novel CHFR-interacting protein. In CHFR-expressing cells, mitotic stress induced the autoPARylation of PARP-1, resulting in an enhanced interaction between CHFR and PARP-1 and an increase in the polyubiquitination/degradation of PARP-1. The decrease in PARP-1 protein levels promoted cell cycle arrest at prophase, supporting that the cells expressing CHFR were resistant to microtubule inhibitors. In contrast, in CHFR-silenced cells, polyubiquitination was not induced in response to mitotic stress. Thus, PARP-1 protein levels did not decrease, and cells progressed into mitosis under mitotic stress, suggesting that CHFR-silenced cancer cells were sensitized to microtubule inhibitors. Furthermore, we found that cells from Chfr knockout mice and CHFR-silenced primary gastric cancer tissues expressed higher levels of PARP-1 protein, strongly supporting our data that the interaction between CHFR and PARP-1 plays an important role in cell cycle regulation and cancer therapeutic strategies. On the basis of our studies, we demonstrate a significant advantage for use of combinational chemotherapy with PARP inhibitors for cancer cells resistant to microtubule inhibitors.

DOI PubMed

p53 negatively regulates the hepatoma growth factor HDGF.

Sasaki Y, Negishi H, Idogawa M, Yokota I, Koyama R, Kusano M, Suzuki H, Fujita M, Maruyama R, Toyota M, Saito T, Tokino T

Cancer research 71 ( 22 ) 7038 - 47 2011年10月 [査読有り] [国際誌]

Hepatoma-derived growth factor (HDGF) is a secreted heparin-binding growth factor that has been implicated in cancer development and progression. Here, we report that HDGF is a critical target for transcriptional repression by the tumor suppressor p53. Endogenous HDGF expression was decreased in cancer cells with introduction of wild-type p53, which also downregulated HDGF expression after DNA damage. In support of the likelihood that HDGF is a critical driver of cancer cell growth, addition of neutralizing HDGF antibodies to culture media was sufficient to block cell growth, migration, and invasion. Similarly, these effects were elicited by conditioned culture medium from p53-expressing cells, and they could be reversed by the addition of recombinant human HDGF. Interestingly, we found that HDGF was overexpressed also in primary gastric, breast, and lung cancer tissues harboring mutant p53 genes. Mechanistic investigations revealed that p53 repressed HDGF transcription by altering HDAC-dependent chromatin remodeling. Taken together, our results reveal a new pathway in which loss of p53 function contributes to the aggressive pathobiological potential of human cancers by elevating HDGF expression.

DOI PubMed

Identification and characterization of early growth response 2, a zinc-finger transcription factor, as a p53-regulated proapoptotic gene

Ikuko Yokota, Yasushi Sasaki, Lisa Kashima, Masashi Idogawa, Takashi Tokino

INTERNATIONAL JOURNAL OF ONCOLOGY ( SPANDIDOS PUBL LTD ) 37 ( 6 ) 1407 - 1416 2010年12月 [査読有り]

Tumor suppressor p53 is a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress In recent years, many genes have been identified as p53-regulated genes, however, no single target gene has been shown to be required for the apoptotic effect Using microarray analysis, we have identified the transcription factor early growth response 2 (EGR2) as a target of the p53 family, specifically p53, p63 and p73 EGR2 expression was up-regulated by DNA damage-induced p53 activity, as well as by overexpression of p53 family genes Furthermore, we identified a responsive element to p53, TAp63, and TAp73 within the EGR2 gene This response element is highly conserved between human and rodents We also found that overexpression of EGR2 induced apoptosis when combined with anticancer agents Conversely, inactivation of EGR2 attenuated p53-mediated apoptosis The results presented here suggest that EGR2 is a direct transcriptional target of p53 family that can in part mediate the p53-dependent apoptotic pathway

DOI

p53 family network and human cancer

Sasaki Y, Idogawa M, Yokota I, Kashima L, Tokino T

Tumor Research 45 33 - 44 2010年12月 [査読有り]

CHFR, a potential tumor suppressor, downregulates interleukin-8 through the inhibition of NF-κB

L. Kashima, M. Toyota, M. Toyota, H. Mita, H. Suzuki, H. Suzuki, M. Idogawa, K. Ogi, Y. Sasaki, T. Tokino

Oncogene ( NATURE PUBLISHING GROUP ) 28 ( 29 ) 2643 - 2653 2009年07月 [査読有り]

The mitotic checkpoint gene CHFR (checkpoint with forkhead and ring finger domains) is silenced in various human cancers by promoter hypermethylation, suggesting that CHFR is a tumor suppressor. Here, we show that CHFR functions as a negative regulator of the nuclear factor-κB (NF-κB) pathway. Expression of CHFR inhibited NF-κB reporter activity, whereas knockdown of CHFR activated reporter activity. These activities are independent of its RING finger domain. Furthermore, we found that CHFR physically interacts with p65 in cells. Electrophoretic mobility shift assays (EMSAs) and ELISA-based NF-κB-binding assays showed that CHFR negatively regulated transcriptional activity of p65. In addition, our data show that interleukin (IL)-8 is significantly downregulated by CHFR, and that the migration of human endothelial cells is suppressed in culture medium conditioned from CHFR-expressing cancer cells. Using a xenograft model, we show that neovascularization is suppressed by adenovirus-mediated transfer of CHFR. These results indicate that expression of CHFR markedly reduces the expression of IL-8 through the inhibition of NF-κB. As the NF-κB signaling pathway plays a critical role in the development and progression of cancer, our findings show the functional relationship between epigenetic alteration and inflammation/angiogenesis in human cancer cells, thereby showing several potential targets for therapeutic intervention. © 2009 Macmillan Publishers Limited All rights reserved.

DOI PubMed

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

Hiroaki Mita, Minoru Toyota, Fumio Aoki, Hirofumi Akashi, Reo Maruyama, Yasushi Sasaki, Hiromu Suzuki, Masashi Idogawa, Lisa Kashima, Kazuyoshi Yanagihara, Masahiro Fujita, Masao Hosokawa, Masanobu Kusano, Sorin Vasile Sabau, Haruyuki Tatsumi, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

BMC CANCER ( BIOMED CENTRAL LTD ) 9 2009年06月 [査読有り]

Background: Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. Methods: DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. Results: DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8-18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8-50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity. Conclusion: Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified KRAS as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of KRAS amplification and overexpression.

DOI PubMed

A Single Recombinant Adenovirus Expressing p53 and p21-targeting Artificial microRNAs Efficiently Induces Apoptosis in Human Cancer Cells

Masashi Idogawa, Yasushi Sasaki, Hiromu Suzuki, Hiroaki Mita, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

CLINICAL CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 15 ( 11 ) 3725 - 3732 2009年06月 [査読有り]

担当区分：筆頭著者

Purpose: Gene transfer involving p53 is viewed as a potentially effective cancer therapy, but does not result in a good therapeutic response in all human cancers. The activation of p53 induces either cell cycle arrest or apoptosis. Cell cycle arrest in response to p53 activation is mediated primarily through the induction of the cyclin-dependent kinase inhibitor p21. Because p21 also has an inhibitory effect on p53-mediated apoptosis, the suppression of p53-induced p21 expression would be expected to result in the preferential induction of apoptosis. However, p21 also has tumor-suppressive properties. In this study, we developed an adenovirus vector that expresses p53 and suppresses p21 simultaneously to enhance p53-mediated apoptosis. Experimental Design: We constructed a replication-deficient recombinant adenovirus (Ad-p53/miR-p21) that enabled cocistronic expression of the p53 protein and artificial microRNAs that targeted p21, and examined the therapeutic effectiveness of this vector in vitro and in vivo. Results: The levels of p21 were significantly attenuated following infection with Ad-p53/miR-p21. In colorectal and hepatocellular carcinoma cells, infection with Ad-p53/miR-p21 augmented apoptosis as compared with an adenovirus that expressed p53 alone (Ad-p53/miR-control). Ad-p53/miR-p21 also significantly increased the chemosensitivity of cancer cells to adriamycin (doxorubicin). In a xenograft tumor model in nude mice, tumor volume was significantly decreased following the direct injection of Ad-p53/miR-p21 into the tumor, as compared with the injection of Ad-p53/miR-control. Conclusion: These results suggest that adenovirus-mediated transduction of p53 and p21-specific microRNAs may be useful for gene therapy of human cancers.

DOI PubMed

p53により発現抑制される分泌性増殖因子HDGFの同定胃癌組織における発現解析

佐々木泰史, 井戸川雅史, 鈴木拓, 豊田実, 今井浩三, 篠村恭久

日本消化器病学会雑誌 ( (一財)日本消化器病学会 ) 106 ( 臨増総会 ) A234 - A234 2009年03月

p53 Family Members Regulate the Expression of the Apolipoprotein D Gene

Yasushi Sasaki, Hideaki Negishi, Ryota Koyama, Naoki Anbo, Kanae Ohori, Masashi Idogawa, Hiroaki Mita, Minoru Toyota, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

JOURNAL OF BIOLOGICAL CHEMISTRY ( AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC ) 284 ( 2 ) 872 - 883 2009年01月 [査読有り]

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 apolipoproteinD( 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

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

Yasushi Sasaki, Hideaki Negishi, Masashi Idogawa, Hiromu Suzuki, Hiroaki Mita, Minoru Toyota, Yasuhisa Shinornura, Kohzoh Imai, Takashi Tokino

MOLECULAR CANCER THERAPEUTICS ( AMER ASSOC CANCER RESEARCH ) 7 ( 4 ) 779 - 787 2008年04月 [査読有り]

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

Genomic Mapping of 12p Amplifications in Gastric Cancer Reveal a 0.5-Mb Target Region Including KRAS Locus

Mita, Hiroaki, Aoki, Fumio, Akashi, Hirofumi, Maruyama, Reo, Sasaki, Yasushi, Suzuki, Hiromu, Idogawa, Masashi, Kashima, Lisa, Toyota, Minoru, Imai, Kohzoh, Shinomura, Yasuhisa, Tokino, Takashi

Tumor Biology 29 83 - 83 2008年 [査読有り]

Frequent epigenetic inactivation of DICKKOPF family genes in human gastrointestinal tumors

Hironobu Sato, Hiromu Suzuki, Minoru Toyota, Masanori Nojima, Reo Maruyama, Shigeru Sasaki, Hideyasu Takagi, Yohei Sogabe, Yasushi Sasaki, Masashi Idogawa, Tomoko Sonoda, Mitsuru Mori, Kohzoh Imai, Takashi Tokino, Yasuhisa Shinomura

CARCINOGENESIS ( OXFORD UNIV PRESS ) 28 ( 12 ) 2459 - 2466 2007年12月 [査読有り]

Activation of Wnt signaling has been implicated in tumorigenesis, and epigenetic silencing of Wnt antagonist genes has been detected in various cancers. In the present study, we examined the expression and methylation of DICKKOPF (DKK) family genes in gastrointestinal cancer cell lines. We found that all known DKK genes were frequently silenced in colorectal cancer (CRC) cells (DKK1, 3/9, 33%; DKK2, 8/9, 89%; DKK3, 5/9, 56% and DKK4, 5/9, 56%), but not in normal colon mucosa. DKK1, -2 and -3 have 5' CpG islands, and show an inverse relation between expression and methylation. DKK methylation also was frequently observed in gastric cancer (GC) cell lines (DKK1, 6/16, 38%; DKK2, 15/16, 94% and DKK3, 10/16, 63%), but was seen less frequently in hepatocellular carcinoma and pancreatic cancer cell lines. DKKs also were frequently methylated in primary CRCs (DKK1, 7/58, 12%; DKK2, 45/58, 78% and DKK3, 12/58, 21%) and GCs (DKK1, 15/31, 48%; DKK2, 26/31, 84% and DKK3, 12/31, 39%). Against a background of CTNNB1 or APC mutations, Dickkopfs (Dkks) were less effective inhibitors of Wnt signaling than secreted frizzled-related proteins, though over-expression of Dkks suppressed colony formation of CRC cells with such mutations. Our results demonstrate that DKKs are frequent targets of epigenetic silencing in gastrointestinal tumors, and that loss of DKKs may facilitate tumorigenesis through beta-catenin/T-cell factor-independent mechanisms.

DOI PubMed

アデノウイルス介在性p53ファミリー遺伝子療法効果のヒストンデアセチラーゼ阻害剤FK228による増大(The Histone Deacetylase Inhibitor FK228 Increases the Efficacy of Adenovirus-mediated p53 Family Gene Therapy)

佐々木泰史, 根岸秀明, 井戸川雅史, 鈴木拓, 見田裕章, 豊田実, 丸山玲緒, 明石浩史, 篠村恭久, 今井浩三, 時野隆至

日本癌学会総会記事 ( (一社)日本癌学会 ) 66回 129 - 129 2007年08月

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