論文 - 時野　隆至

IGFBP7 is a p53-responsive gene specifically silenced in colorectal cancer with CpG island methylator phenotype.

Suzuki H, Igarashi S, Nojima M, Maruyama R, Yamamoto E, Kai M, Akashi H, Watanabe Y, Yamamoto H, Sasaki Y, Itoh F, Imai K, Sugai T, Shen L, Issa JP, Shinomura Y, Tokino T, Toyota M

Carcinogenesis   31 ( 3 ) 342 - 9  2010年03月  [査読有り]  [国際誌]

　概要を見る

A subset of colorectal cancers (CRCs) show simultaneous methylation of multiple genes; these tumors have the CpG island methylator phenotype (CIMP). CRCs with CIMP show a specific pattern of genetic alterations, including a high frequency of BRAF mutations and a low frequency of p53 mutations. We therefore hypothesized that genes inactivated by DNA methylation are involved in the BRAF- and p53-signaling pathways. Among those, we examined the epigenetic inactivation of insulin-like growth factor-binding protein 7 (IGFBP7) expression in CRCs. We found that in CRC cell lines, the silencing of IGFBP7 expression was correlated with high levels of DNA methylation and low levels of histone H3K4 methylation. Luciferase and chromatin immunoprecipitation assays in unmethylated cells revealed that p53 induces expression of IGFBP7 upon binding to a p53 response element within intron 1 of the gene. Treating methylated CRC cell lines with 5-aza-2'-deoxycytidine restored p53-induced IGFBP7 expression. Levels of IGFBP7 methylation were also significantly higher in primary CRC specimens than in normal colonic tissue (P < 0.001). Methylation of IGFBP7 was correlated with BRAF mutations, an absence of p53 mutations and the presence of CIMP. Thus, epigenetic inactivation of IGFBP7 appears to play a key role in tumorigenesis of CRCs with CIMP by enabling escape from p53-induced senescence.

DOI PubMed

N-Propionyl-Cysteaminylphenol-Magnetite Conjugate (NPrCAP/M) Is a Nanoparticle for the Targeted Growth Suppression of Melanoma Cells

Makito Sato, Toshiharu Yamashita, Masae Ohkura, Yasue Osai, Akiko Sato, Tomoaki Takada, Hidenobu Matsusaka, Ichiro Ono, Yasuaki Tamura, Noriyuki Sato, Yasushi Sasaki, Akira Ito, Hiroyuki Honda, Kazumasa Wakamatsu, Shosuke Ito, Kowichi Jimbow

JOURNAL OF INVESTIGATIVE DERMATOLOGY ( NATURE PUBLISHING GROUP )  129 ( 9 ) 2233 - 2241  2009年09月  [査読有り]

　概要を見る

A magnetite nanoparticle, NPrCAP/M, was produced for intracellular hyperthermia treatment of melanoma by conjugating N-propionyl-cysteaminylphenol (NPrCAP) with magnetite and used for the study of selective targeting and degradation of melanoma cells. NPrCAP/M, like NPrCAP, was integrated as a substrate in the oxidative reaction by mushroom tyrosinase. Melanoma, but not non-melanoma, cells incorporated larger amounts of iron than magnetite from NPrCAP/M. When mice bearing a B16F1 melanoma and a lymphoma on opposite flanks were given NPrCAP/M, iron was observed only in B16F1 melanoma cells and iron particles (NPrCAP/M) were identified within late-stage melanosomes by electron microscopy. When cells were treated with NPrCAP/M or magnetite and heated to 43 degrees C by an external alternating magnetic field (AMF), melanoma cells were degraded 1.7- to 5.4-fold more significantly by NPrCAP/M than by magnetite. Growth of transplanted B16 melanoma was suppressed effectively by NPrCAP/M-mediated hyperthermia, suggesting a clinical application of NPrCAP/M to lesional therapy for melanoma. Finally, melanoma cells treated with NPrCAP/M plus AMF showed little sub-G1 fraction and no caspase 3 activation, suggesting that the NPrCAP/M-mediated hyperthermia induced non-apoptotic cell death. These results suggest that NPrCAP/M may be useful in targeted therapy for melanoma by inducing non-apoptotic cell death after appropriate heating by the AMF.

DOI

アデノウイルスを用いたp53蛋白とp21発現抑制人工miRNAの共発現による癌治療効果の改善(A single adenovirus expressing p53 protein and p21-targeting artificial microRNAs improves therapeutic outcome in cancer)

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 )  68回   492 - 492  2009年08月

潜在的腫瘍抑制因子CHFRはNFκB抑制を介してインターロイキン-8を下方制御する(CHFR, a potential tumor suppressor, downregulates interleukin-8 via inhibition of NF-kappaB)

鹿島 理沙, 豊田 実, 見田 裕章, 鈴木 拓, 井戸川 雅史, 荻 和弘, 佐々木 泰史, 時野 隆至

日本癌学会総会記事 ( (一社)日本癌学会 )  68回   155 - 155  2009年08月

p53誘導性膜貫通蛋白質hCLCA2は癌細胞移動/接着を制御する(hCLCA2, a p53 inducible transmembrane protein regulates cancer cell migration and adhesion)

佐々木 泰史, 安保 直樹, 鹿島 理沙, 井戸川 雅史, 鈴木 拓, 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 )  68回   140 - 140  2009年08月

p53ファミリーの標的遺伝子ICAM2の同定と機能解析(Identification and characterization of the intercellular adhesion molecule-2 gene as a novel p53 family target)

石田 勢津子, 安保 直樹, 鹿島 理沙, 井戸川 雅史, 佐々木 泰史, 時野 隆至

日本癌学会総会記事 ( (一社)日本癌学会 )  68回   141 - 141  2009年08月

p53ファミリーの標的遺伝子EGR2の同定と機能解析(Identification and characterization of early growth response gene-2 as a p53-regulated proapoptotic gene)

横田 育子, 佐々木 泰史, 安保 直樹, 鹿島 理沙, 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 )  68回   141 - 141  2009年08月

p53応答配列のゲノム網羅的in silico解析によるp53誘導性non-coding RNAの同定(Genome wide in silico analysis of p53 response elements identified non-coding RNA regulated by p53)

高丸 博之, 丸山 玲緒, 豊田 実, 鈴木 拓, 佐々木 泰史, 野島 正寛, 明石 浩史, 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 )  68回   141 - 141  2009年08月

DNAメチル化の網羅的解析によって解明された、多発性骨髄腫におけるRASD1遺伝子不活化とデキサメサゾン耐性の相関(Genomic Screening by DNA Methylation Revealed an Association between RASD 1 Inactivation and Dex Resistance in MM)

野島 正寛, 丸山 玲緒, 安井 寛, 鈴木 拓, 佐々木 泰史, 麻奥 英毅, 酒井 基, 石田 禎夫, 森 満, 今井 浩三, 時野 隆至, 豊田 実, 篠村 恭久

日本癌学会総会記事 ( (一社)日本癌学会 )  68回   224 - 224  2009年08月

Genomic screening for genes silenced by DNA methylation revealed an association between RASD1 inactivation and dexamethasone resistance in multiple myeloma.

Nojima M, Maruyama R, Yasui H, Suzuki H, Maruyama Y, Tarasawa I, Sasaki Y, Asaoku H, Sakai H, Hayashi T, Mori M, Imai K, Tokino T, Ishida T, Toyota M, Shinomura Y

Clinical cancer research : an official journal of the American Association for Cancer Research   15 ( 13 ) 4356 - 64  2009年07月  [査読有り]  [国際誌]

　概要を見る

PURPOSE: Epigenetic changes such as DNA methylation play a key role in the development and progression of multiple myeloma. Our aim in the present study was to use genomic screening to identify genes targeted for epigenetic inactivation in multiple myeloma and assess their role in the development of resistance to dexamethasone. EXPERIMENTAL DESIGN: Gene expression was examined using microarray screening, reverse transcription-PCR, and real-time quantitative PCR. DNA methylation was examined using bisulfite PCR, bisulfite sequencing, and bisulfite pyrosequencing in 14 multiple myeloma cell lines, 87 multiple myeloma specimens, and 12 control bone marrow samples. WST-8 assays were used to assess cell viability after treatment with 5-aza-2'-deoxycytidine and/or dexamethasone. RESULTS: Microarray analysis was done to screen for genes up-regulated by 5-aza-2'-deoxycytidine. In RPMI8226 cells, 128 genes were up-regulated, whereas 83 genes were up-regulated in KMS12PE cells. Methylation of 22 genes with CpG islands in their 5' regions, including RASD1, was confirmed. Methylation of RASD1 was associated with its inactivation, which correlated with resistance to dexamethasone. Treating multiple myeloma cells with 5-aza-2'-deoxycytidine restored sensitivity to dexamethasone. Methylation of RASD1 was also detected in a subset of primary multiple myeloma specimens, and the levels of methylation were increased after repeated antitumor treatments. Gene signature analysis revealed various genes to be synergistically induced by treatment with a combination of 5-aza-2'-deoxycytidine plus dexamethasone. CONCLUSION: Our findings indicate that epigenetic inactivation of genes, including RASD1, plays a key role in the development of dexamethasone resistance in multiple myeloma. Moreover, they show the utility of demethylation therapy in cases of advanced multiple myeloma.

DOI PubMed

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

　概要を見る

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.

Mita H, Toyota M, Aoki F, Akashi H, Maruyama R, Sasaki Y, Suzuki H, Idogawa M, Kashima L, Yanagihara K, Fujita M, Hosokawa M, Kusano M, Sabau SV, Tatsumi H, Imai K, Shinomura Y, Tokino T

BMC cancer   9   198 - 198  2009年06月  [査読有り]  [国際誌]

　概要を見る

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

Overexpression of Aurora A by loss of CHFR gene expression increases the growth and survival of HTLV-1-infected T cells through enhanced NF-kappaB activity.

Tomita M, Toyota M, Ishikawa C, Nakazato T, Okudaira T, Matsuda T, Uchihara JN, Taira N, Ohshiro K, Senba M, Tanaka Y, Ohshima K, Saya H, Tokino T, Mori N

International journal of cancer   124 ( 11 ) 2607 - 15  2009年06月  [査読有り]  [国際誌]

　概要を見る

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia (ATL). Aurora A, a mitotic checkpoint protein, is overexpressed in human cancer cells. The cell cycle-dependent turnover of Aurora A is regulated by E3 ubiquitin ligases such as checkpoint with fork head-associated and ring finger (CHFR). Here, we found overexpression of Aurora A protein in HTLV-1-infected T-cell lines and primary ATL cells. The expression of CHFR mRNA was reduced in these cells by abnormal methylation of CHFR promoter region. Knockdown of Aurora A using small interfering RNA suppressed the growth of HTLV-1-infected T-cell line. Transfection of Aurora A expression plasmid enhanced Tax-induced nuclear factor-kappaB (NF-kappaB) reporter activity. Transfection of CHFR expression plasmid into an HTLV-1-infected T-cell line reduced cell growth, Aurora A protein level and constitutive NF-kappaB reporter activity. Aurora kinase inhibitor suppressed the growth and survival of HTLV-1-infected T-cell lines and primary ATL cells. It also reduced constitutive NF-kappaB activity in an HTLV-1-infected T-cell line by reducing IkappaB kinase beta phosphorylation and the expression of antiapoptotic protein survivin. Our results suggested that loss of CHFR expression resulted to accumulation of Aurora A, which increased NF-kappaB activity. These findings highlight the critical role of Aurora A in HTLV-1-infected T cells, making this molecule a potentially suitable target for future therapies for ATL.

DOI PubMed

A single recombinant adenovirus expressing p53 and p21-targeting artificial microRNAs efficiently induces apoptosis in human cancer cells.

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

Clinical cancer research : an official journal of the American Association for Cancer Research   15 ( 11 ) 3725 - 32  2009年06月  [査読有り]  [国際誌]

　概要を見る

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

A novel biomarker for cervical cancer.

Tokino T

Cancer biology & therapy   8 ( 12 ) 1154 - 5  2009年06月  [査読有り]  [国際誌]

PubMed

miRNAによるp21発現誘導抑制能を付加したp53発現ベクターによる癌治療効果の増強

井戸川 雅史, 佐々木 泰史, 篠村 恭久, 今井 浩三, 時野 隆至

Drug Delivery System ( 日本DDS学会 )  24 ( 3 ) 332 - 332  2009年06月

ヒストン脱アセチル化酵素阻害剤の併用によるp53遺伝子治療の作用増強

佐々木 泰史, 今井 浩三, 篠村 恭久, 時野 隆至

Drug Delivery System ( 日本DDS学会 )  24 ( 3 ) 319 - 319  2009年06月

Cancer epigenomics: implications of DNA methylation in personalized cancer therapy.

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

Cancer science   100 ( 5 ) 787 - 91  2009年05月  [査読有り]  [国際誌]

　概要を見る

Genetic alterations in cancer can provide information for predicting a tumor's sensitivity to chemotherapeutic drugs. But although such information is certainly useful, the relatively low frequency of mutations seen in many cancers limits the utility of pharmacogenomics in large numbers of cancer patients, necessitating consideration of other approaches. Epigenetic changes such as DNA methylation are a hallmark of human cancers. Methylation of genes involved in DNA repair and maintaining genome integrity (e.g. MGMT, hMLH1, WRN, and FANCF), and cell-cycle checkpoint genes (e.g. CHFR and 14-3-3 sigma, CDK10, and p73), all reportedly influence the sensitivity to chemotherapeutic drugs, suggesting that DNA methylation could serve as a molecular marker for predicting the responsiveness of tumors to chemotherapy. However, the comprehensive study of pharmacoepigenomics awaits the advent of genome-wide analysis of DNA methylation using microarrays and next-generation sequencers.

DOI PubMed

p53 family members regulate the expression of the apolipoprotein D gene

Yasushi Sasaki, Naoki Anbo, Masashi Idogawa, Hiroaki Mita, Minoru Toyota, Kohzoh Imai, Yasuhisa Shinomura, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH )  69  2009年05月  [査読有り]

A single recombinant adenovirus expressing p53 and p21-targeting artificial microRNAs efficiently induces apoptosis in human cancer cells.

Masashi Idogawa, Yasushi Sasaki, Kohzoh Mai, Yasuhisa Shinomura, Takashi Tokino

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH )  69  2009年05月  [査読有り]

DOI