研究者詳細 - 佐谷　秀行

論文 - 佐谷　秀行

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Mechanism of hyperploid cell formation induced by microtubule inhibiting drug in glioma cell lines

Tsuiki Hiromasa, Nitta Masayuki, Tada Mitsuhiro, Inagaki Masaki, Ushio Yukitaka, Saya Hideyuki

Oncogene 20 （ 4 ） 420 - 429 2001年01月

ISSN 0950-9232

Checkpoint mechanism plays a crucial role in ensuring genomic integrity during cell cycle. Loss of checkpoint function is known to induce genomic instability and to alter ploidy of dividing cells. In this study, we examined mechanisms of hyperploid formation in glioma cells by treatment with nocodazole, which activates spindle assembly checkpoint by inhibiting microtubule polymerization. By prolonged nocodazole treatment, U251MG human glioma cell, which has a p53 mutation, underwent transient arrest at mitosis, and subsequently exited from mitotic arrest (termed 'mitotic slippage') followed by DNA replication without cytokinesis, resulting in hyperploid formation. Additionally, the heterogeneity in the number of centrosomes per cell increased during the hyperploid formation, suggesting that these hyperploid cells have genomic instability. By employing LN382 glioma cell that has a temperature-sensitive p53 mutation, we found that the activation of p53 prevents hyperploid formation after the prolonged nocodazole treatment. Furthermore, staurosporine, an inhibitor for a broad range of serine/threonine kinases including cdc2, was found to enhance hyperploid formation in U251MG cells by accelerating the induction of mitotic slippage. Interestingly, inhibitors specific for cdc2 kinase prevented the G2 to M transition but did not accelerate mitotic slippage, suggesting that staurosporinesensitive kinases other than cdc2 are required for maintenance of spindle assembly checkpoint. Moreover, the enhancement of hyperploid formation by staurosporine was also blocked by p53-dependent G1 checkpoint. These results suggest that abrogation of G1 checkpoint is a critical factor for formation of hyperploid cells after the mitotic slippage.

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Commentary

Laws E.R. Jr, Saya H., Ushio Y.

Neurologia Medico-Chirurgica 41 （ 5 ） 258 - 259 2001年

ISSN 0470-8105

Identification of the cis-acting region in the NF2 gene promoter as a potential target for mutation and methylation-dependent silencing in schwannoma

Kino Takeshi, Takeshima Hideo, Nakao Mitsuyoshi, Nishi Toru, Yamamoto Keizo, Kimura Takahiro, Saito Yoshiki, Kochi Masato, Kuratsu Jun Ichi, Saya Hideyuki, Ushio Yukitaka

Genes to Cells 6 （ 5 ） 441 - 454 2001年

ISSN 1356-9597

Background: Although mutational inactivation and allelic loss in the NF2 gene appear to be causal events in the majority of vestibular schwannomas, involvement of another potentially important mechanism, transcriptional inactivation, has not been investigated. Results: We cloned and functionally characterized the 5′-flanking region of the human NF2 gene and identified the molecular mechanisms that regulate NF2 expression. Luciferase assay and site-directed mutagenesis demonstrated that a 70-base pair (bp) region (-591 to -522 bp from the translation start site) was essential for the basic expression of the NF2 gene. A gel mobility shift assay indicated recognition by nuclear protein of the unusually long (≈66 bp) sequences in this region. Recognition was inhibited by either mutation of the binding core sequence or by methylation of three CpG sites. Point mutations at these CpG sites significantly decreased promoter activity, suggesting the importance of these sites. In 14 of 23 vestibular schwannomas, these three CpG sites were methylated in a site-specific manner and the methylation status was consistent with the expression of NF2 mRNA. Conclusions: Suppressed expression by aberrant methylation or mutation of the promoter elements could be an alternative mechanism for inactivation of the NF2 gene.

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Analysis of loss of heterozygosity on chromosome 10 in patients with malignant astrocytic tumors

Tada K., Shiraishi S., Kamiryo T., Nakamura H., Hirano H., Kuratsu J. I., Kochi M., Saya H., Ushio Y.

Journal of Neurosurgery 95 （ 4 ） 651 - 659 2001年

ISSN 0022-3085

Object. The most frequent genetic abnormality in human malignant gliomas is loss of heterozygosity (LOH) on chromosome 10. Candidate genes on chromosome 10 that are associated with the prognosis of patients with anaplastic astrocytoma (AA) and glioblastoma (GBM) were evaluated. Methods. The authors used 12 fluorescent microsatellite markers on both arms of chromosome 10 to study LOH in 108 primary astrocytic tumors. The LOH on chromosome 10 was observed in 11 (32%) of 34 AAs and 34 (56%) of 61 GBMS. No LOH was detected in 13 low-grade gliomas. Loss of heterozygosity was not detected in any AA in the seven patients younger than 35 years, but it was discovered in 41% of the patients older than 35 years. The prognostic significance of LOH at each locus was evaluated in 89 patients older than 15 years; 33 (37%) had supratentorial AAs and 56 (63%) had supratentorial GBMs. The Cox proportional hazards model, adjusted for patient age at surgery, the preoperative Karnofsky Performance Scale score, and the extent of surgical resection revealed that LOH on marker D10S209 near the FGFR2 and DMBT1 genes was significantly associated with shorter survival in patients with AA. The LOH on markers D10S215 and D10S541, which contain the PTEN/MMAC1 gene between them, was significantly associated with shorter survival in patients with GBM. Conclusions. In the present study it is found that LOH on chromosome 10 is an age-dependent event for patients with AAs and that LOH on marker D10S209 near the FGFR2 and DMBT1 loci is a significantly unfavorable prognostic factor. It is also reported that LOH on the PTEN/MMAC1 gene is a significantly unfavorable prognostic factor in patients with GBM.

Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides

Nakajima Hiroto, Shimbara Naoki, Shimonishi Yoko, Mimori Tatsuyuki, Niwa Shin Ichiro, Saya Hideyuki

Gene 260 （ 1-2 ） 121 - 131 2000年12月

ISSN 0378-1119

The protein invasin expressed on the cell surface of the pathogenic bacteria Yersinia pseudotuberculosis mediates the entry of this bacterium into cultured mammalian cells. We have developed a system for expression of random peptides on the cell surface of Escherichia coli (E. coli) by creation of a fusion hybrid between a peptide and the invasin protein. The fusion protein constructs consist of part of the outer membrane domain of the invasin protein, six proline spacers, and a decamer of random peptides flanked by cysteine residues (CX₁₀C). Peptides were constitutively expressed on the cell surface in the resulting random decamer peptide library, which we designated as ESPEL (E. coli Surface Peptide Expression Library). The ESPEL was systematically screened for its binding affinity toward human cultured cells. Several bacterial clones were identified whose binding to human cells was mediated by peptides expressed on the bacterial cell surface. Flow cytometric analysis showed that both the identified bacterial clones and these corresponding chemically synthesized peptides bound to human cells specifically. The techniques described provide a new method that uses E. coli random peptide library to select targeting peptides for mammalian cells without any knowledge of the human cellular receptors.

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Molecular analysis of the h-warts/LATS1 gene in human breast cancer.

Morinaga N., Shitara Y., Yanagita Y., Koida T., Kimura M., Asao T., Kimijima I., Takenoshita S., Hirota T., Saya H., Kuwano H.

International Journal of Oncology 17 （ 6 ） 1125 - 1129 2000年12月

ISSN 1019-6439

Loss of heterozygosity (LOH) on chromosome 6q is often observed in breast cancer, suggesting the existence of a putative tumor suppressor. Recently, a human homolog of the Drosophila warts tumor suppressor gene, h-warts/LATS1, was identified and mapped at chromosome 6q24-25.1. Mutation analysis of the h-warts/LATS1 was performed using 25 breast cancer tissues by RT-PCR SSCP analysis. Although LOH of the h-warts/LATS1 was found in one patient, no mutations were found. Two polymorphisms were found, but neither of them caused amino acid substitutions. Further investigations are necessary to elucidate the role of the h-warts/LATS1 gene in the carcinogenesis of breast cancer.

Ras oncoprotein induces CD44 cleavage through phosphoinositide 3-OH kinase and the Rho family of small G proteins

Kawano Yoshiaki, Okamoto Isamu, Murakami Daizo, Itoh Hiroshi, Yoshida Masaki, Ueda Shoichi, Saya Hideyuki

Journal of Biological Chemistry 275 （ 38 ） 29628 - 29635 2000年09月

ISSN 0021-9258

CD44 is a cell surface adhesion molecule for several extracellular matrix components. We previously showed that CD44 expressed in cancer cells is proteolytically cleaved at the ectodomain through membrane-anchored metalloproteases and that CD44 cleavage plays a critical role in cancer cell migration. Therefore, cellular signals that promote the migration and metastatic activity of cancer cells may regulate the CD44 ectodomain cleavage. Here, we demonstrate that the expression of the dominant active mutant of Ha-Ras (Ha-Ras^Val-12) induces redistribution of CD44 to the newly generated membrane ruffling area and CD44 ectodomain cleavage. The migration assay revealed that the CD44 cleavage contributes to the Ha-Ras^Val-12-induced migration of NIH3T3 cells on hyaluronate substrate. Treatment with LY294002, an inhibitor for phosphoinositide 3-OH kinase (PI3K), significantly inhibits Ha-Ras^Val-12-induced CD44 cleavage, whereas that with PD98059, an inhibitor for MEK, does not. The active mutant p110 subunit of PI3K has also been shown to enhance the CD44 cleavage, suggesting that PI3K mediates the Ras-induced CD44 cleavage. Moreover, the expression of dominant negative mutants of Cdc42 and Rac1 inhibits the Ha-Ras^Val-12-induced CD44 cleavage. These results suggest that Ras > PI3K > Cdc42/Rac1 pathway plays an important role in CD44 cleavage and may provide a novel molecular basis to explain how the activated Ras facilitates cancer cell migration.

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Mapping and regulation of the tumor-associated epitope recognized by monoclonal antibody RS-11

Eto Hiroshi, Yoon Sam S., Bode Barrie P., Kamidono Sadao, Makino Keishi, Saya Hideyuki, Nakamura Hideo, Tanabe Kenneth K.

Journal of Biological Chemistry 275 （ 35 ） 27075 - 27083 2000年09月

ISSN 0021-9258

We have previously described a rat monoclonal antibody, RS-11, which recognizes a tumor-associated antigen common to several species. In the present study, we have cloned and characterized the antigen recognized by RS-11. We screened a phage expression library prepared from HeLa cDNA and identified a clone that reacts with RS-11. DNA sequence analysis revealed that this clone contains sequences of keratin 18 (nucleotides 568-1196). We constructed several glutathione S-transferase fusion proteins and synthetic peptides based on this DNA sequence analysis and examined their reactivity with RS-11 to accurately map the RS-11 epitope. We determined that the epitope resides within a region of seven amino acids on the α-helix 2B domain of keratin 18 in which two amino acids (Leu³⁶⁶ and Lys³⁷⁰) are completely conserved among intermediate filaments as well as other keratin members that are immunoreactive with RS-11. These two residues are sequentially discontinuous but spatially adjacent. The RS-11 epitope is constitutively present in human primary cultured hepatocytes; however, its immunoreactivity with RS-11 is up-regulated by malignant transformation or stimulation with either epidermal growth factor or transforming growth factor alpha.

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Mechanism of transcriptional regulation by methyl-CpG binding protein MBD1

Fujita Naoyuki, Shimotake Nobuya, Ohki Izuru, Chiba Tsutomu, Saya Hideyuki, Shirakawa Masahiro, Nakao Mitsuyoshi

Molecular and Cellular Biology 20 （ 14 ） 5107 - 5118 2000年07月

ISSN 0270-7306

MBD1 is a mammalian protein that binds symmetrically-methylated CpG sequences and regulates gene expression in association with DNA methylation. This protein possesses a conserved sequence, named methyl-CpG binding domain (MBD), among a family of methyl-CpG binding proteins that mediate the biological consequences of the methylation. In addition, MBD1 has at least five isoforms due to alternative splicing events, resulting in the presence of CXXC1, CXXC2, and CXXC3 in MBD1 isoforms v1 (MBD1v1) and MBD1v2, and CXXC1 and CXXC2 in MBD1v3 and -v4. In the present study, we have investigated the significance of MBD, CXXC, and the C-terminal transcriptional repression domain (TRD) in MBD1. A bacterially expressed MBD binds efficiently to densely methylated rather than to sparsely methylated DNAs. In both methylation-deficient Drosophila melanogaster SL2 cells and mammalian CHO-K1 cells, MB-D1v1 represses transcription preferentially from both unmethylated and sparsely methylated promoters, while MBD1v3 inhibits densely methylated but not unmethylated promoter activities. The CXXC3 sequence in MBD1v1 is responsible for the ability to bind unmethylated promoter. Furthermore, we have constructed mutant-type MBD1s in which the functionally important residues Arg22, Arg30, Asp32, Tyr34, Arg44, Ser45, and Tyr52 are changed to alanine to investigate the correlation between the structure and function of the MBD in MBD1. Excepting those for Ser45 and Tyr52, none of the recombinant MBD mutants bound to the densely methylated or unmethylated DNAs, and green fluorescent protein-fused MBD1 mutants did not localize properly in the nucleus. All the MBD1v1 and -v3 mutants lost the activity of methylation- dependent gene repression. Based on these findings we have concluded that MBD1 acts as a transcriptional regulator depending on the density of methyl- CpG pairs through the cooperation of MBD, CXXC, and TRD sequences.

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Zyxin, a regulator of actin filament assembly, targets the mitotic apparatus by interacting with h-warts/LATS1 tumor suppressor

Hirota Toru, Morisaki Tetsuro, Nishiyama Yasuyuki, Marumoto Tomotoshi, Tada Kenji, Hara Toshihiro, Masuko Norio, Inagaki Masaki, Hatakeyama Katsuyoshi, Saya Hideyuki

Journal of Cell Biology 149 （ 5 ） 1073 - 1086 2000年05月

ISSN 0021-9525

The mitotic apparatus plays a pivotal role in dividing cells to ensure each daughter cell receives a full set of chromosomes and complement of cytoplasm during mitosis. A human homologue of the Drosophila warts tumor suppressor, h-warts/LATS1, is an evolutionarily conserved serine/threonine kinase and a dynamic component of the mitotic apparatus. We have identified an interaction of h-warts/LATS1 with zyxin, a regulator of actin filament assembly. Zyxin is a component of focal adhesion, however, during mitosis a fraction of cytoplasmic-dispersed zyxin becomes associated with h-warts/LATS1 on the mitotic apparatus. We found that zyxin is phosphorylated specifically during mitosis, most likely by Cdc2 kinase, and that the phosphorylation regulates association with h-warts/LATS1. Furthermore, microinjection of truncated h-warts/LATS1 protein, including the zyxin-binding portion, interfered with localization of zyxin to mitotic apparatus, and the duration of mitosis of these injected cells was significantly longer than that of control cells. These findings suggest that h-warts/LATS1 and zyxin play a crucial role in controlling mitosis progression by forming a regulatory complex on mitotic apparatus.

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Calpain-dependent proteolysis of NF2 protein

Kimura Yoriyoshi, Saya Hideyuki, Nakao Mitsuyoshi

Neuropathology 20 （ 3 ） 153 - 160 2000年

ISSN 0919-6544

The neurofibromatosis type 2 (NF2) protein, known as merlin or schwannomin, is a tumor suppressor, and the NF2 gene has been found to be mutated in the majority of schwannomas and meningiomas, including both sporadically occurring and familial NF2 cases. Although the development of these tumors depends on the loss of merlin, the presence of tumors lacking detectable NF2 mutations suggests different mechanisms for inactivating merlin. Recent studies have demonstrated cleavage of merlin by calpain, a calcium-dependent neutral cysteine protease, and marked activation of the calpain system resulting in the degradation of merlin in these tumors. Increased turnover of merlin by calpain in some schwannomas and meningiomas exemplifies tumorigenesis linked to the calpain-mediated proteolytic pathway.

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Clinical significance of p53 functional loss in squamous cell carcinoma of the oropharynx

Obata Atsushi, Eura Masao, Sasaki Jiichiro, Saya Hideyuki, Chikamatsu Kazuaki, Tada Mitsuhiro, Iggo Richard D., Yumoto Eiji

International Journal of Cancer 86 （ 3 ） 187 - 193 2000年

ISSN 0020-7136

We examined the frequency of p53 mutations in 38 oropharyngeal squamous cell carcinomas (SCC), using both a yeast functional assay and a conventional immunohistochemical staining method (IHC) to detect p53 mutations. We also explored the clinical importance of p53 mutations in oropharyngeal SCC. An accumulation of p53 protein was detected in 17 of the 38 (45%) tumors by IHC, whereas the yeast-based assay detected 6 additional p53 mutations, for a total of 23 tumors (61%) with p53 mutations. The cDNA sequencing analysis revealed that the 6 mutations undetected by IHC consisted of 3 frameshift, 1 nonsense and 2 missense mutations. Thus, the yeast functional assay was more sensitive than conventional IHC for detecting p53 mutations. Subsequently, the relationship between p53 mutations and the clinico-pathological parameters in oropharyngeal SCC was evaluated using the results of the functional assay. Mutation of p53 was not associated with the patient age, sex, tumor stage or degree of tumor cell differentiation. Interestingly, heavy drinking had a significant positive correlation with the p53 mutation, but heavy smoking did not, suggesting that prolonged exposure to alcohol is more related to p53 mutation in oropharyngeal SCC than to tobacco consumption. Radiation sensitivity was examined by comparing tumor size on magnetic resonance images before and after completion of therapy with 45 Gy radiation, in the 18 cases of T2 oropharyngeal SCC that were initially treated by radiotherapy. The results showed that tumors with wild-type p53 decreased in size significantly compared to those with mutant p53. In 33 patients treated with curative intent, the overall survival after the completion of therapy was better in patients with a wild-type p53 tumor than in patients with a mutant p53 tumor. We conclude that p53 mutation is associated with radiation resistance and a decreased probability of survival in oropharyngeal SCC. (C) 2000 Wiley-Liss. Inc.

Comparative analysis of brain proteins from p53-deficient mice by two- dimensional electrophoresis

Araki Norie, Morimasa Tadaomi, Sakai Tomoko, Tokuoh Hiroshi, Yunoue Shunji, Kamo Masaharu, Miyazaki Kenji, Abe Koji, Saya Hideyuki, Tsugita Akira

Electrophoresis 21 （ 9 ） 1880 - 1889 2000年

ISSN 0173-0835

p53 is a tumor suppressor protein that regulates many cellular processes including the cell cycle, DNA repair, and apoptosis. It also serves as a critical regulator of neuronal apoptosis in the central nervous system (CNS). To elucidate the role of p53 in the CNS, brain proteins of p53 knock-out mice (p53-/-) were analyzed by two-dimensional gel electrophoresis (2-DE) and compared with those from p53 wild type (p53+/+) mice. Six types of brain tissue (temporal cortex, cerebellum, hippocampus, striatum, olfactory bulb, and cervical spinal cord) and other control tissues (lung and blood) from 18- week-old non-stress-induced mice were analyzed. The morphology of brains from p53-/- mice appeared to be normal and identical to that of p53+/+ mice, although lungs showed diffuse tumors that may have been caused by p53 deficiency. Comparative 2-D gel analysis showed that, on average, 7 of 886 spots from brain tissue were p53-/- specific, whereas 12 of 1008 spots from lung tissue were p53-/- specific. N-terminal amino acid sequence was determined for p53-/- specific proteins. In all brain tissues from p53-/- mice, a newly identified mouse mitochondrial NADH-ubiquinone oxidoreductase 24 kDa subunit showed decreased expression, and apolipoprotein A1 acidic forms showed increased expression. In addition, brain-type creatine kinase B chain and tubulin β-5 N-terminal fragment were increased in the p53-/- cerebellum, and a new protein in mouse, hydroxyacylglutathione hydrolase (glyoxalase II) was decreased in the temporal cortex of p53-/- mice. The alterations in protein expression identified in this study may imply a p53- related brain function. This is the first proteomic analysis on the p53-/- mouse brain, and further information based on this study will provide new insights into the p53 function in the CNS.

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NE-dlg, a mammalian homolog of Drosophila dlg tumor suppressor, induces growth suppression and impairment of cell adhesion

Hanada Norihisa, Makino Keishi, Koga Hisashi, Morisaki Tetsuro, Kuwahara Hiroaki, Masuko Norio, Tabira Yoichi, Hiraoka Takehisa, Kitamura Nobuo, Kikuchi Akira, Saya Hideyuki

International Journal of Cancer 86 （ 4 ） 480 - 488 2000年

ISSN 0020-7136

Membrane-associated guanylate kinases (MAGUK's) are known to function as scaffolds for forming multiprotein complexes at the synaptic junctions of neuronal cells and at sites of epithelial cell-cell contact. In Drosophila, mutations of the lethal (I)-discs large (dig) gene, which encodes a MAGUK protein, leads to post-synaptic structure defects in neuronal cells and neoplastic overgrowth of epithelial cells. We previously showed that NE-dig (neuronal and endocrine dig), a human homolog of the dig, plays a crucial role in formation of synaptic structure in human neuronal cells. Here we demonstrate that NE-dig, similar to Drosophila dig, is involved in regulation of cell cycle progression and adhesive ability of non-neuronal cells. Overexpression of NE-dlg in proliferating cells including various cancer cell lines induced growth suppression and impairment of cell adhesive ability. Furthermore, NE-dlg overexpression caused the down-regulation of β-catenin in cancer cells regardless of mutations in the APC (adenomatous polyposis coil) gene. The PDZ domains of NE-dlg were found to be essential for the growth suppression, loss of adhesive property and down-regulation of β- catenin. We propose that NE-dlg regulates the cell growth and adhesive ability by controlling the level of β-catenin through an APC-independent pathway. Inactivation of NE-dlg may therefore contribute to development and/or progression of human neoplasms. (C) 2000 Wiley-Liss, Inc.

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Calcium influx triggers the sequential proteolysis of extracellular and cytoplasmic domains of E-cadherin, leading to loss of β-catenin from cell-cell contacts

Ito Kiyoharu, Okamoto Isamu, Araki Norie, Kawano Yoshiaki, Nakao Mitsuyoshi, Fujiyama Shigetoshi, Tomita Kimio, Mimori Tatsuyuki, Saya Hideyuki

Oncogene 18 （ 50 ） 7080 - 7090 1999年11月

ISSN 0950-9232

Cadherins are major cell-cell adhesion molecules in both tumor and normal tissues. Although serum levels of soluble E-cadherin have been shown to be higher in the cancer patients than in healthy volunteers, the detail mechanism regulating release of soluble E-cadherin remains to be elucidated. Here we show that the ectodomain of E-cadherin is proteolytically cleaved from some cancer cells by a membrane-bound metalloprotease to yield soluble form, and the residual membrane-tethered cleavage product is subsequently degraded by intracellular proteolytic pathway. Futhermore, we show that extracellular calcium influx, that is induced by mechanical scraping of cells or ionomycin treatment, enhances the metalloprotease-mediated E-cadherin cleavage and the subsequent degradation of the cytoplasmic domain. Immunocytochemical analysis demonstrates that the sequential proteolysis of E-cadherin triggered by the calcium influx results in translocation of β-catenin from the cell-cell contacts to cytoplasm. Our data suggest that calcium influx-induced proteolysis of E-cadherin not only disrupts the cell-cell adhesion but also activates β-catenin-mediated intracellular signaling pathway, potentially leading to alterations in motility and proliferation activity of cells.

A point mutation of human p53, which was not detected as a mutation by a yeast functional assay, led to apoptosis but not p21(Waf1/Cip1/Sdi1) expression in response to ionizing radiation in a human osteosarcoma cell line, Saos-2

Okaichi Kumio, Wang Li Hong, Sasaki Ji ichiro, Saya Hideyuki, Tada Mitsuhiro, Okumura Yutaka

International Journal of Radiation Oncology Biology Physics 45 （ 4 ） 975 - 980 1999年11月

ISSN 0360-3016

Purpose: The 123A point mutation of p53 showed increased radiosensitivity, whereas other mutations (143A, 175H, and 273H) were not affected. To determine the reason for increased radiosensitivity of the 123A mutation, the response of the transformant of 123A mutation to ionizing radiation (IR) was examined and compared to those of transformants with the wild type p53 or other point mutations (143A, 175H, and 273H).Methods and Materials: Stable transformants with a mutant or wild type p53 made by introducing cDNA into the human osteosarcoma cell line, Saos-2, which lacks an endogenous p53 were used. The transcriptional activity of mutant p53 was examined using a yeast functional assay. The transformants were examined for the accumulation of p53, the induction of p21(Waf1/Cip1/Sdi1) (hereafter referred to as p21), and the other response of p53-responsive genes (MDM2, Bax, and Bcl-2) by Western blotting. Apoptosis was analyzed by detection of DNA fragmentation.Results: The 123A point mutation of p53 was detected as a wild type in the yeast functional assay. The 123A mutant accumulated p53 in response to IR. The 123A mutant did not induce p21, but normally responded to MDM2, Bax, and Bcl-2. The 123A mutant entered apoptosis earlier than the wild type p53 transformant, and induced Fas at earlier in response to IR.Conclusion: The 123A mutant led to apoptosis, but not p21 expression in response to IR. The occurrence of apoptosis, but not induction of p21, corresponded to the radiosensitivity in the transformant. The early occurrence of apoptosis in 123A transformants may depend on the early induction of Fas. Copyright (C) 1999 Elsevier Science Inc.

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The stabilization mechanism of mutant-type p53 by impaired ubiquitination

Nagata Yoichi, Anan Tadashi, Yoshida Tetsuo, Mizukami Tamio, Taya Yoichi, Fujiwara Toshiyoshi, Kato Hirohisa, Saya Hideyuki, Nakao Mitsuyoshi

Oncogene 18 （ 44 ） 6037 - 6049 1999年10月

ISSN 0950-9232

Mutant-type p53 (mt p53) is largely accumulated in cancer cells due to its increased stability. To elucidate the mechanism of mt p53 stabilization, we analysed the turnover of p53 mutated at codon 248 whose alteration is most frequently found in human cancers. Proteasome inhibition induced the accumulation of ubiquitinated mt p53, indicating that the ubiquitinated forms were essentially unstable and degraded by the proteasome. The presence of a small amount of the ubiquitinated mt p53 relative to the abundant non-ubiquitinated form suggested that the mt p53 ubiquitination was a rate-limiting process in the slow turnover. Two phenomena destabilizing mt p53 via the ubiquitin-proteasome degradation were proved to be independent. First, the coexpression of wild-type p53 (wt p53) promoted mt p53 destabilization as feedback regulation. Second, geldanamycin also induced mt p53 destabilization through the dissociation of the protein from hsp90 but not through the restoration of wt p53 function. Neither the mutant-specific conformation nor the N-terminal phosphorylation seemed to contribute directly to the mt p53 stabilization. Further, a two-dimensional gel electrophoresis revealed that most of the post-translationally modified mt p53 was equally subjected to ubiquitination and subsequent proteasomal degradation. These findings are that mt p53 stabilization depends on the ubiquitination due to both the loss of wt p53 function and the hsp90 association.

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A human homolog of Drosophila warts tumor suppressor, h-warts, localized to mitotic apparatus and specifically phosphorylated during mitosis

Nishiyama Yasuyuki, Hirota Toru, Morisaki Tetsuro, Hara Toshihiro, Marumoto Tomotoshi, Iida Shin Ichi, Makino Keishi, Yamamoto Hideyuki, Hiraoka Takehisa, Kitamura Nobuo, Saya Hideyuki

FEBS Letters 459 （ 2 ） 159 - 165 1999年10月

ISSN 0014-5793

We identified a human homolog of Drosophila warts tumor suppressor gene, termed h-warts, which was mapped at chromosome 6q24-25.1. The h-warts protein has a serine/threonine kinase domain and is localized to centrosomes in interphase cells. However, it becomes localized to the mitotic apparatus, including spindle pole bodies, mitotic spindle, and midbody, in a highly dynamic manner during mitosis. Furthermore, h-warts is specifically phosphorylated in cells at mitotic phase, most likely by Cdc2 kinase. These findings suggest that h-warts functions as a component of the mitotic apparatus and is involved in proper progression of mitosis.

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Regulated CD44 cleavage under the control of protein kinase C, calcium influx, and the rho family of small G proteins

Okamoto Isamu, Kawano Yoshiaki, Matsumoto Mitsuhiro, Suga Moritaka, Kaibuchi Kozo, Ando Masayuki, Saya Hideyuki

Journal of Biological Chemistry 274 （ 36 ） 25525 - 25534 1999年09月

ISSN 0021-9258

CD44 is a cell surface receptor for several extracellular matrix components and is implicated in tumor cell invasion and metastasis. Our previous studies have shown that CD44 expressed in cancer cells is proteolytically cleaved at the extracellular domain through membrane- associated metalloproteases and that CD44 cleavage plays a critical role in CD44-mediated tumor cell migration (Okamoto, I., Kawano, Y., Tsuiki, H., Sasaki, J., Nakao, M., Matsumoto, M., Suga, M., Ando, M., Nakajima, M., and Saya, H. (1999) Oncogene 18, 1435-1446). In the present study, we first demonstrate rapid degradation of the membrane-tethered CD44 cleavage product through intracellular proteolytic pathways, and it occurs only after CD44 extracellular cleavage. To address the mechanisms regulating CD44 cleavage at the extracellular domain, we show that 12-O-tetradecanoylphorbol 13-acetate (TPA) and the calcium ionophore ionomycin rapidly enhance metalloprotease- mediated CD44 cleavage in U251MG cells via protein kinase C-dependent and - independent pathways, respectively, suggesting the existence of multiple distinct pathways for regulation of CD44 cleavage. Concomitant with TPA- induced CD44 cleavage, TPA treatment induces redistribution of CD44 and ERM proteins (ezrin, radixin, and moesin) to newly generated membrane ruffling areas. Treatment with lysophosphatidic acid, which is known to activate the Rho-dependent pathway, inhibits TPA-induced CD44 redistribution and CD44 cleavage. Furthermore, overexpression of Rac dominant active mutants results in the redistribution of CD44 to the Rac-induced ruffling areas and the enhancement of CD44 cleavage. These results suggest that the Rho family proteins play a role in regulation of CD44 distribution and cleavage.

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Methylation-mediated transcriptional silencing in euchromatin by methyl- CpG binding protein MBD1 isoforms

Fujita Naoyuki, Takebayashi Shin Ichiro, Okumura Katsuzumi, Kudo Shinichi, Chiba Tsutomu, Saya Hideyuki, Nakao Mitsuyoshi

Molecular and Cellular Biology 19 （ 9 ） 6415 - 6426 1999年09月

ISSN 0270-7306

DNA methylation of promoter-associated CpG islands is involved in the transcriptional repression of vertebrate genes. To investigate the mechanisms underlying gene inactivation by DNA methylation, we characterized a human MBD1 protein, one of the components of MeCP1, which possesses a methyl-CpG binding domain (MBD) and cysteine-rich (CXXC) domains. Four novel MBD1 isoforms (MBD1v1, MBD1v2, MBD1v3, and MBD1v4) were identified by the reverse transcription-PCR method. We found that these transcripts were alternatively spliced in the region of CXXC domains and the C terminus. Green fluorescent protein-fused MBD1 was localized to multiple foci on the human genome, mostly in the euchromatin regions, and particularly concentrated in the pericentromeric region of chromosome 1. Both the MBD sequence and genome methylation were required for proper localization of the MBD1 protein. We further investigated whether MBD1 isoforms are responsible for transcriptional repression of human genes. A bacterially expressed MBD1 protein bound preferentially to methylated DNA fragments containing CpG islands from the tumor suppressor genes p16, VHL, and E-cadherin and from an imprinted SNRPN gene. All MBD1 isoforms inhibited promoter activities of these genes via methylation. Interestingly, MBD1 isoforms v1 and v2 containing three CXXC domains also suppressed unmethylated promoter activities in mammalian cells. These effects were further manifested in Drosophila melanogaster cells, which lack genome methylation. Sp1-activated transcription of methylated p16 and SNRPN promoters was inhibited by all of the MBD1 isoforms, whereas the isoforms v1 and v2 reduced Sp1-activated transcription from unmethylated promoters as well. These findings suggested that the MBD1 isoforms have different roles in methylation-mediated transcriptional silencing in euchromatin.

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