Details of a Researcher - TOKINO Takashi

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Induction of cellular genes is mediated by the Bel1 transactivator in foamy virus-infected human cells

A Wagner, A Doerks, M Aboud, A Alonso, T Tokino, RM Flugel, M Lochelt

JOURNAL OF VIROLOGY ( AMER SOC MICROBIOLOGY ) 74 ( 10 ) 4441 - 4447 2000.05

To gain insight into human foamy virus (HFV; also called spumaretrovirus)-induced alterations of cellular genes, the expression profiles of defined genes in HFV-infected primary human cells were analyzed by cDNA array assays. Several distinct cellular genes activated by HFV infection were identified; the identities of the cellular genes were confirmed by RNA blot analyses. Compared with mock-infected controls, the concentrations of cellular Kip2, Egr-1, COUP-TF1, insulin-like growth factor II (IGF-II), and EphB3 mRNAs were significantly increased in HFV-infected cells and showed a gene-specific and time-dependent induction. Immunoblot analyses with antibodies against some of the cellular gene products revealed increased levels of the corresponding proteins. To investigate mechanisms of HFV-induced alterations in cellular gene expression, the capacity of known HFV genes to increase expression of defined cellular genes was analyzed by transient expression experiments. Plasmids that encode the HFV Bell transcriptional transactivator were necessary and sufficient to strongly increase expression of p57Kip2, IGF-II, and EphB3 genes in 293T cells. Potential mechanisms and consequences of activation of cellular genes during HFV infection and Bell transactivation of the Kip2 gene are discussed.

DOI PubMed CiNii

Nora, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis

E Oda, R Ohki, H Murasawa, J Nemoto, T Shibue, T Yamashita, T Tokino, T Taniguchi, N Tanaka

SCIENCE ( AMER ASSOC ADVANCEMENT SCIENCE ) 288 ( 5468 ) 1053 - 1058 2000.05

A critical function of tumor suppressor p53 is the induction of apoptosis in cells exposed to noxious stresses. We report a previously unidentified pro-apoptotic gene, Nora. Expression of Nora induction in primary mouse cells exposed to x-ray irradiation was dependent on p53, Nora encodes a Bcl-2 homology 3 (BH3)-only member of the Bcl-2 family of proteins; this member contains the BH3 region but not other BH domains. When ectopically expressed, Nora underwent BH3 motif-dependent Localization to mitochondria and interacted with anti-apoptotic Bcl-2 family members, resulting in the activation of caspase-9. We also demonstrate that blocking the endogenous Nora induction results in the suppression of apoptosis. Nora may thus represent a mediator of p53-dependent apoptosis.

DOI PubMed CiNii

Isolation and characterization of a novel TP53-inducible gene, TP53TG5, which suppresses growth and shows cell cycle-dependent transition of expression

S Isaka, Y Takei, T Tokino, K Koyama, Y Miyoshi, M Suzuki, E Takahashi, C Azuma, Y Murata, Y Nakamura

GENES CHROMOSOMES & CANCER ( WILEY-LISS ) 27 ( 4 ) 345 - 352 2000.04

Through a strategy of direct cloning of TP53-binding DNA sequences from human genome DNA, we have identified a novel TP53-target gene, termed TP53TG5 (TP53-target gene 5). This gene, localized to chromosome band 20q13.1 by fluorescence in situ hybridization, encodes a 290-amino-acid peptide with no significant homology with any known proteins in the public database. A colony-formation assay using human glioblastoma cell line T98G, which lacks wild-type TP53 and expresses no endogenous TP53TG5, revealed a growth-suppressive effect of the TP53TG5 gene product. Furthermore, immunohistochemical studies, following transfection of T98G with plasmid designed to express green fluorescent protein-fused TP53TG5, revealed cell cycle-dependent intracellular localization of this protein. Our results suggest that functional studies of TP53TG5 may provide new insights into the complex physiological activities of TP53. (C) 2000 Wiley-Liss, Inc.

DOI

Mutation of the SRC gene in endometrial carcinoma

M Sugimura, K Kobayashi, S Sagae, Y Nishioka, S Ishioka, K Terasawa, T Tokino, R Kudo

JAPANESE JOURNAL OF CANCER RESEARCH ( BUSINESS CENTER ACADEMIC SOCIETIES JAPAN ) 91 ( 4 ) 395 - 398 2000.04

Recently, an activating mutation of the SRC gene has been implicated in about one-tenth of advanced colon cancers. The SRC 531 mutation results in truncation of SRC directly C-terminal to the regulatory Tyr 530 and appears to activate the Tyr 530, To investigate whether mutation of SRC plays an important role in the development and progression of gynecological tumors, we performed mutational analysis of the entire coding region of SRC in 70 ovarian carcinomas, 68 endometrial carcinomas and 3 endometrial stromal sarcomas by means of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by nucleotide sequencing and restriction fragment Length polymorphism (RFLP) analysis. We found one truncated mutation at codon 531 (Gln to Stop) in an endometrial carcinoma. However, we found no mutation of this gene in ovarian carcinoma or endometrial stromal sarcoma. Our results suggest that mutation of SRC may be implicated in a small proportion of endometrial carcinomas.

AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1

S Satoh, Y Daigo, Y Furukawa, T Kato, N Miwa, T Nishiwaki, T Kawasoe, H Ishiguro, M Fujita, T Tokino, Y Sasaki, S Imaoka, M Murata, T Shimano, Y Yamaoka, Y Nakamura

NATURE GENETICS ( NATURE AMERICA INC ) 24 ( 3 ) 245 - 250 2000.03

The Wnt signalling pathway is essential for development and organogenesis(1-3). Wnt signalling stabilizes beta-catenin, which accumulates in the cytoplasm, binds to T-cell factor (TCF; also known as lymphocyte enhancer-binding factor, LEF) and then upregulates downstream genes(4-6). Mutations in CTNNB1 (encoding beta-catenin) or ape (adenomatous polyposis coli) have been reported in human neoplasms including colon cancers and hepatocellular carcinomas(7-13) (HCCs), Because HCCs tend to show accumulation of beta-catenin more often than mutations in CTNNB1, we looked for mutations in AXIN1, encoding a key factor for Wnt signalling, in 6 HCC cell lines and 100 primary HCCs. Among the 4 cell lines and 87 HCCs in which we did not detect CTNNB1 mutations, we identified AXIN1 mutations in 3 cell lines and 6 mutations in 5 of the primary HCCs. In cell lines containing mutations in either gene, we observed increased DNA binding of TCF associated with beta-catenin in nuclei. Adenovirus mediated gene transfer of wild-type AXIN1 induced apoptosis in hepatocellular and colorectal cancer cells that had accumulated beta-catenin as a consequence of either APC, CTNNB1 or AXIN1 mutation, suggesting that axin may be an effective therapeutic molecule for suppressing growth of hepatocellular and colorectal cancers.

DOI PubMed CiNii

Adenovirus-mediated transfer of p53-related genes induces apoptosis of human cancer cells

S Ishida, T Yamashita, U Nakaya, T Tokino

JAPANESE JOURNAL OF CANCER RESEARCH ( BUSINESS CENTER ACADEMIC SOCIETIES JAPAN ) 91 ( 2 ) 174 - 180 2000.02

Two p53-related genes, p73 and p51, were recently identified as structural homologues of the p53 tumor suppressor gene, suggesting that the roles of these tno genes may be similar to those of p53, including growth suppression and induction of apoptosis, Here we show that introduction of p73 or p51 cDNAs into cultured human cancer cells suppressed colony formation in the presence of G418, We then examined the ability of various isoforms of p73 and p51 to activate transcription of a reporter gene. This assay showed that p73 beta and p51A activated transcription through a consensus p53 binding sequence, while p73 alpha and p51B isoforms minimally transactivated the p53 reporter gene. To characterize further the biological functions of the p53-related genes, we constructed recombinant adenoviruses containing the p73 and p51 cDNAs. Ad-p73 beta and Ad-pS1A induced endogenous p21 gene expression more effectively than Ad-p73 alpha and Ad-p51B, respectively. To evaluate the mode of cell death induced by p53-related genes, Ad-p73 and Ad-D51 were used to infect human cancer cells. Infection of Ad-p73 beta, Ad-pS1A or Ad-p51B resulted in DNA fragmentation in a subset of cancer cell lines more efficiently than did infection of Ad-p53. We then examined the combined effect of each p53-related gene and the EIA oncogene in the induction of apoptosis, The EIA oncogene cooperated with p51 as well as p53 to induce apoptosis, while p73 resulted in a weak induction of apoptosis by EIA. Overall, apoptosis induction by p51B and p73 alpha isoforms may he due to mechanisms other than transcriptional activation of p53-target genes. Our results suggest that p53-related genes are both similar to and different from p53 in their pathways leading to growth suppression.

The role of p53-target genes in human cancer

Takashi Tokino, Y Nakamura

CRITICAL REVIEWS IN ONCOLOGY HEMATOLOGY ( ELSEVIER SCIENCE INC ) 33 ( 1 ) 1 - 6 2000.01

Book review, literature introduction, etc.

The tumor suppressor gene p53 is mutated in a large proportion of human cancers. In some cellular conditions like DNA damage, the p53 gene is induced and its gene product is posttranscriptionally activated. p53 works as a transcriptional activator and induces the expression of its downstream target genes. This review will explain why expression of the normal p53 gene leads to tumor growth suppression. The p53 has several biological effects involving cell-cycle arrest, DNA replication and repair, proliferation, apoptosis, angiogenesis inhibition, and cellular stress response. These effects of the p53 result mainly from the activation of expression of a large number of p53-target genes. Here we have focused on the biological functions of the transcriptional targets of p53. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

DOI

p53類似遺伝子p73,p51の抗腫よう効果についての検討

佐々木泰史, 森本一郎, 中谷宇一郎, 石田勢津子, 今井浩三, 鬼原史, 田中敏博, 中村祐輔, 時野隆至

Japanese Journal of Cancer Research 91 ( Supplement (Sept) ) 2000

J-GLOBAL

Ewing肉腫特異的なEWS-ETS融合転写因子の標的遺伝子

吉田幸一, 佐々木泰史, 梅沢明弘, 秦順一, 鬼原史, 田中敏博, 中村祐輔, 時野隆至

Japanese Journal of Cancer Research 91 ( Supplement (Sept) ) 2000

J-GLOBAL

頭頚部癌におけるβ-catenin遺伝子の蛋白発現とmutation,増殖因子受容体の発現

小田島哲世, 佐々木泰史, 山口晃, 田中信幸, 池田健, 佐藤昌明, 時野隆至

Japanese Journal of Cancer Research 91 ( Supplement (Sept) ) 2000

J-GLOBAL

p53ノックアストマウスを用いたp53標的遺伝子の同定

森本一郎, 佐々木泰史, 石田勢津子, 中谷宇一郎, 山下利春, 伊東文生, 今井浩三, 時野隆至

Japanese Journal of Cancer Research 91 ( Supplement (Sept) ) 2000

J-GLOBAL

Ewing肉腫/PNETに特異的EWS-ETS融合転写因子の標的遺伝子

吉田幸一, 佐々木泰史, 梅沢明弘, 秦順一, 鬼原史, 田中敏博, 中村祐輔, 時野隆至

DNAチップ技術研究会プログラム・抄録集 1st 2000

J-GLOBAL

ヒト大腸癌由来細胞株におけるp53類似遺伝子p73,p51によるアポトーシス誘導と抗癌剤との併用効果

石田勢津子, 佐々木泰史, 森本一郎, 中谷宇一郎, 山下利春, 今井浩三, 時野隆至

Japanese Journal of Cancer Research 91 ( Supplement (Sept) ) 2000

J-GLOBAL

Localization of membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 at tight junctions of epithelial cells

N Ide, Y Hata, H Nishioka, K Hirao, Yao, I, M Deguchi, A Mizoguchi, H Nishimori, T Tokino, Y Nakamura, Y Takai

ONCOGENE ( STOCKTON PRESS ) 18 ( 54 ) 7810 - 7815 1999.12

Membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 and Synapse-associated protein (SAP) 97/human Discs-large tumor suppressor gene (hDLG) are ubiquitous isoforms of synaptic scaffolding molecule (S-SCAM) and Postsynaptic density(PSD)-95/SAP90, both of which are implicated in the structures of synapses, respectively. SAP97/hDLG is localized at epithelial junctions and may function as a scaffolding protein, but the subcellular localization or the function of MAGI-1/BAP1 has not been clarified. In intestinal epithelial cells, MAGI-1/BAP1 was localized at tight junctions, whereas SAP97/hDLG was localized diffusely at cell-cell junctions. In Madine Darby canine kidney (MDCK) cells, MAGI-1/BAP1 was colocalized with ZO-1, whereas SAP97/hDLG was colocalized with E-cadherin. In MDCK cells, dominant active and negative mutants of Rad small G protein changed the amounts of SAP97/hDLG at cell-cell junctions, but not that of MAGI-1/BAP1. When MDCK cells were switched to a low Ca2+ medium, E-cadherin disappeared from the plasma membrane, and cells were dissociated. The phorbol 12-myristate 13-acetate-treatment after the low Ca2+ switch induced a tight junction-like structure. MAGI-1/BAP1 was recruited with ZO-1 to this structure, but SAP97/hDLG or E-cadherin was not. These findings suggest that MAGI-1/BAP1 is a component of tight junctions of epithelial cells, and that its role is different from that of SAP97/hDLG.

DOI PubMed CiNii

Induction of transformation and p53-dependent apoptosis by adenovirus type 5 E4orf6/7 cDNA

S Yamano, T Tokino, M Yasuda, M Kaneuchi, M Takahashi, Y Niitsu, K Fujinaga, T Yamashita

JOURNAL OF VIROLOGY ( AMER SOC MICROBIOLOGY ) 73 ( 12 ) 10095 - 10103 1999.12

Adenovirus (Ad) E4orf6/7, one of the early gene products of human Ads, forms a stable complex with the cellular transcription factor E2F to activate transcription from the Ad E2 promoter. E2F cDNAs have growth-promoting and apoptosis-inducing activities when overexpressed in cells. We cloned Ad5 E40rf6/7 cDNA in both simian virus 40- and human cytomegalovirus-based expression vectors to examine its transforming and apoptotic activities. The cloned E4orf6/7 collaborated with a retinoblastoma protein (RB)-nonbinding and therefore E2F-nonreleasing mutant of Ad5 ELA (dl922/947) to morphologically transform primary rat cells, suggesting that E2F is an important cellular protein functioning downstream of E1A for transformation. In a G418 colony formation assay, E4orf6/7 was shown to suppress growth of untransformed rat cells. Moreover, a recombinant Ad expressing Ad5 E4orf6/7 induced apoptosis in rat cells when coinfected with wild-type p53-expressing Ad. Mutational analysis of E4orf6/7 revealed that both of the domains required for growth inhibition and transformation by E4orf6/7 lay in the C-terminal region, which is essential for transactivation from the upstream sequence of an E2a promoter containing E2F-binding sites. However, the smallest mutant of E4orf6/7, encoding the C-terminal 59 amino acids, failed to complement the RE-nonbinding dl/922/947 mutant despite showing growth inhibition and E2F transactivation activities. Thus, it is suggested that a subregion of E40rf6/7 which is required for growth inhibition and transformation in collaboration with dl922/947 overlaps the transactivation domain of E4orf6/7.

Retrovirus vectors designed for efficient transduction of cytotoxic or cytostatic genes

M Ui, M Takada, T Arai, K Matsumoto, K Yamada, T Nakahata, T Nishiwaki, Y Furukawa, Takashi Tokino, Y Nakamura, H Iba

GENE THERAPY ( STOCKTON PRESS ) 6 ( 10 ) 1670 - 1678 1999.10

It is difficult to establish stable packaging cell lines producing retrovirus vectors for the expression of anti-oncogenes with cytotoxic or cytostatic potential, because these genes would also affect the growth of the packaging cell lines. To overcome this problem, we designed a transcriptional unit pBabeLPL for vector RNA production, in which the transcription of the exogenous genes is completely suppressed by the presence of a preceding insertion containing the puromycin resistance gene (puro) and a poly(A) addition signal. This insertion is flanked by a tandem pair of loxP, and is designed to be excised after the introduction of Cre recombinase, when transcription of the exogenous gene will be started from the 5'-LTR. The transcriptional unit carrying LacZ or p53 as the exogenous gene was introduced into a previously constructed prepackaging cell lines PtG-S2, in which the expression of VSV-G is also designed to be initiated by the introduction of Cre recombinase, while the gag-pol gene is expressed continuously. After the introduction of Cre recombinase by an adenovirus vector, LacZ- or p53- expressing VSV-G-pseudotyped retrovirus vectors with the designed structure were produced at high virus titers. The p53 virus was shown to be able to transduce p53 into the entire population of several human cancer cell lines and to induce their growth arrest at the GI phase, indicating that this vector-producing system will be advantageous for human gene therapy.

DOI

細胞傷害性や細胞増殖抑制活性を持つ遺伝子発現用レトロウイルスベクターの設計と開発

宇井基泰, 新井徹, 中畑龍俊, 西脇忠, 古川洋一, 時野隆至, 中村祐輔, 伊庭英夫

日本癌学会総会記事 ( 日本癌学会 ) 58回 319 - 319 1999.08

Mutational analysis of STK11 gene in ovarian carcinomas

Y Nishioka, K Kobayashi, S Sagae, M Sugimura, S Ishioka, M Nagata, K Terasawa, T Tokino, R Kudo

JAPANESE JOURNAL OF CANCER RESEARCH ( ELSEVIER SCI IRELAND LTD ) 90 ( 6 ) 629 - 632 1999.06

Recently STK11, the causative gene of Peutz-Jeghers syndrome (PJS) was identified on chromosome 19p13.3. PJS is often accompanied by several malignancies, including breast tumor, adenoma malignum of the uterine cervix, and ovarian tumor. To investigate the involvement of STK11 gene in the development of ovarian carcinomas, we analyzed 30 ovarian carcinomas for loss of heterozygosity (LOH) and STK11 gene mutations. We found one missense mutation (codon 281, Pro to Leu) with heterozygous and somatic status. This mutation occurred at codon 281, which lies within the mutational hot spot (codon 279-281) of STK11 gene previously reported in PJS. We also detected LOH in 2 (11%) of 19 informative ovarian carcinomas. Our results suggest that mutations of the STK11 gene may play a limited role in the development of ovarian carcinomas.

Mutational analysis of beta-catenin gene in Japanese ovarian carcinomas: Frequent mutations in endometrioid carcinomas

S Sagae, K Kobayashi, Y Nishioka, M Sugimura, S Ishioka, M Nagata, K Terasawa, T Tokino, R Kudo

JAPANESE JOURNAL OF CANCER RESEARCH ( ELSEVIER SCI IRELAND LTD ) 90 ( 5 ) 510 - 515 1999.05

To investigate the contribution of the beta-catenin gene to the development of ovarian carcinomas, mutational analysis of exon 3 of the beta-catenin gene was conducted. We analyzed 61 primary ovarian carcinomas, consisting of 49 non-endometrioid-type and 12 endometrioid-type tumors, for genetic alteration of the beta-catenin gene, Five carcinomas showed beta-catenin mutations (S37C, T41I, T41A), including 4 (33%) of 12 endometrioid-type tumors and 1 (14%) of 7 mucinous-type tumors. All of these mutations altered at the serine/threonine residues that are potential sites of GSK3-beta phosphorylation, We detected no carcinomas with interstitial deletion involving exon 3 of beta-catenin, Furthermore, we immunohistochemically studied 27 of the 61 ovarian carcinomas. Both nuclear and cytoplasmic beta-catenin expressions were demonstrated in 3 of the 27 ovarian carcinomas for which tissue samples were available for examination. All 4 cases exhibited mutations in exon 3 of beta-catenin, including a mucinous carcinoma. Our results suggested that beta-catenin gene mutation at potential GSK3-beta phosphorylation sites results in accumulation of beta-catenin protein within the cells and its translocation to nuclei. Accumulated beta-catenin protein may be involved in the development of endometrioid-type ovarian carcinomas, and some mucinous-type ovarian carcinomas.

Recombinant adenovirus expressing wild-type p53 is antiangiogenic: A proposed mechanism for bystander effect

M Nishizaki, T Fujiwara, T Tanida, A Hizuta, H Nishimori, Takashi Tokino, Y Nakamura, M Bouvet, JA Roth, N Tanaka

CLINICAL CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 5 ( 5 ) 1015 - 1023 1999.05

Angiogenesis is required for the growth and progression of malignancies, Recent studies have demonstrated that genetic alterations may accompany acquisition of the angiogenic phenotype, The tumor suppressor gene p53 is most frequently mutated in human cancers and is also known to be a transcriptional regulator of a variety of genes. Here, we investigated the antiangiogenic effect of the wild-type p53 (wt-p53) gene transfer on a human non-small cell lung cancer cell line. Mutant p53-expressing H226Br non-small cell lung cancer cells were transduced with the wt-p53 gene using a recombinant adenoviral vector (Ad5CMVp53) and applied to semiquantitative reverse transcription-PCRs for the detection of altered mRNA expression of angiogenic and/or antiangiogenic factors. In vivo neovascularization assay of Ad5CMVp53-infected cells was then performed using a membrane-diffusion chamber system s.c. transplanted in nu/nu mice. We also evaluated the effect of Ad5CMVp53-infected H226Br cells on nontransduced tumor cells in vivo by s.c. inoculating mixture of cells into nu/nu mice. Ad5CMVp53 infection markedly inhibited the expression of an angiogenic factor, vascular endothelial growth factor, and increased the expression of a novel antiangiogenic factor, brain-specific angiogenesis inhibitor 1, resulting in reduced neovascularization in vivo. Mixing experiments showed that tumor cells transduced with the wt-p53 gene inhibited the in vivo tumor growth of adjacent nontransduced cells. Our data suggest that a recombinant adenovirus expressing the wt-p53 gene is antiangiogenic, which may explain, in part, the mechanism of the bystander effect induced by the wt-p53 gene transfer on adjacent tumor cells.

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Misc - TOKINO Takashi