Details of a Researcher - Saya, Hideyuki

Papers - Saya, Hideyuki

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Tissue culture methods to study neurological disorders

Watabe Kazuhiko, Sakamoto Tsuyoshi, Kawazoe Yoko, Michikawa Makoto, Miyamoto Katsuichi, Yamamura Takashi, Saya Hideyuki, Araki Norie

Neuropathology 23 ( 1 ) 68 - 78 2003.03

ISSN 0919-6544

Previously, the authors have established spontaneously immortalized cell lines from long-term cultures of normal adult mouse Schwann cells. Establishment of such Schwann cell lines derived from murine disease models may greatly facilitate studies of the cellular mechanisms of their peripheral nervous system lesions in the relevant diseases. Recently, the authors have established immortalized Schwann cell lines derived from Niemann-Pick disease type C mice (NPC; spm/spm) and globoid cell leukodystrophy mice (twitcher). In the present study, long-term cultures were maintained of Schwann cells derived from dorsal root ganglia and consecutive peripheral nerves of another NPC mouse (npc^nih/npc^nih, npc^nih/+), myelin P0 protein-deficient mice (P0-/-, P0+/-) with their wild-type littermates (P0+/+), and neurofibromatosis type 1 gene (NF1)-deficient mice (Nf1^Fcr/+) for 8-10 months, and immortalized cell lines from all these animals established spontaneously. These cell lines had spindle-shaped Schwann cell morphology and distinct Schwann cell phenotypes and retained genomic and biochemical abnormalities, sufficiently representing the in vivo pathological features of the mutant mice. These immortalized Schwann cell lines can be useful in studies of nervous system lesions in these mutant mice and relevant human disorders.

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Caspase activation in neuronal and glial apoptosis following spinal cord injury in mice

Kuratsu Junichi, Kurisu Kaoro, Laws Edward R., Saya Hideyuki

Neurologia Medico-Chirurgica 43 ( 1 ) 29 - 30 2003.01

ISSN 0470-8105

The aryl hydrocarbon receptor nuclear transporter is modulated by the SUMO-1 conjugation system

Tojo Masahide, Matsuzaki Kazuhito, Minami Takeshi, Honda Yoshiomi, Yasuda Hideyo, Chiba Tsutomu, Saya Hideyuki, Fujii-Kuriyama Yoshiaki, Nakao Mitsuyoshi

Journal of Biological Chemistry 277 ( 48 ) 46576 - 46585 2002.11

ISSN 0021-9258

The aryl hydrocarbon receptor nuclear transporter (ARNT) is a member of the basic helix-loop-helix/PAS (Per-ARNT-Sim) family of transcription factors, which are important for cell regulation in response to environmental conditions. ARNT is an indispensable partner of the aryl hydrocarbon receptor (AHR) or hypoxia-inducible factor-1α. This protein is also able to form homodimers such as ARNT/ARNT. However, the molecular mechanism that regulates the transcriptional activity of ARNT remains to be elucidated. Here, we report that ARNT is modified by SUMO-1 chiefly at Lys²⁴⁵ within the PAS domain of this protein, both in vivo and in vitro. Substitution of the target lysine with alanine enhanced the transcriptional potential of ARNT per se. Furthermore, green fluorescent protein-fused ARNT tended to form nuclear foci in ∼20% of the transfected cells, and the foci partly colocalized with PML nuclear bodies. PML, one of the well known substrates for sumoylation, was found to augment the transcriptional activities of ARNT. ARNT bound AHR or PML, whereas the sumoylated form of ARNT associated with AHR, but not with PML, resulting in a reduced effect of PML, on transactivation by ARNT. Our data suggest that the sumoylation of ARNT modulates its transcriptional role through affecting the ability of ARNT to interact with cooperative molecules such as PML. This exemplifies a crucial role of protein sumoylation in modulating protein-protein interactions.

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Roles of aurora-A kinase in mitotic entry and G2 checkpoint in mammalian cells

Marumoto Tomotoshi, Hirota Toru, Morisaki Tetsuro, Kunitoku Naoko, Zhang Dongwei, Ichikawa Yasuko, Sasayama Takashi, Kuninaka Shinji, Mimori Tatsuyuki, Tamaki Norihiko, Kimura Masashi, Okano Yukio, Saya Hideyuki

Genes to Cells 7 ( 11 ) 1173 - 1182 2002.11

ISSN 1356-9597

Background: Various mitotic events are controlled by Cdc2-cyclin B and other mitotic kinases. Aurora/ Ipl1-related mitotic kinases were proved to play key roles in mitotic progression in diverse lower organisms. Aurora-A is a mammalian counterpart of aurora/Ipl1-related kinases and is thought to be a potential oncogene. However, the regulation of aurora-A activation and the commitment of aurora-A in the progression of G2-M phase are largely unknown in mammalian cells. Results: We demonstrated that aurora-A is activated depending on the activation of Cdc2-cyclin B in mammalian cells. Since Cdc2-cyclin B does not directly phosphorylate aurora-A, indirect pathways such as the inhibition of PP1 by Cdc2-cyclin B may act for the activation of aurora-A kinase. Microinjection of anti-aurora-A antibodies into HeLa cells at late G2 phase caused a significant delay in mitotic entry. Furthermore, aurora-A activation at G2-M transition was inhibited by DNA damage, and the over-expression of aurora-A induced the abrogation of the DNA damage-induced G2 checkpoint. Conclusions: Aurora-A is activated downstream of Cdc2-cyclin B and plays crucial roles in proper mitotic entry and G2 checkpoint control. Dysregulation of aurora-A induces abnormal G2-M transition in mammalian cells and may lead to chromosome instability, which results in the development and progression of malignant tumours.

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WARTS tumor suppressor is phosphorylated by Cdc2/cyclin B at spindle poles during mitosis

Morisaki Tetsuro, Hirota Toru, Iida Shin ichi, Marumoto Tomotoshi, Hara Toshihiro, Nishiyama Yasuyuki, Kawasuzi Michio, Hiraoka Takehisa, Mimori Tatsuyuki, Araki Norie, Izawa Ichiro, Inagaki Masaki, Saya Hideyuki

FEBS Letters 529 ( 2-3 ) 319 - 324 2002.10

ISSN 0014-5793

Identification of physiological substrates for Cdc2/cyclin B is crucial for understanding the functional link between mitotic events and Cdc2/cyclin B activation. A human homologue of the Drosophila warts tumor suppressor, termed WARTS, is a serine/threonine kinase and a dynamic component of the mitotic apparatus. We have found that Cdc2/cyclin B forms a complex with a fraction of WARTS in the centrosome and phosphorylates the Ser613 site of WARTS during mitosis. Immunocytochemical analysis has shown that the S613-phosphorylated WARTS appears in the spindle poles at prometaphase and disappears at telophase. Our findings suggest that Cdc/cyclin B regulates functions of WARTS on the mitotic apparatus.

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Proteomic analysis of human brain identifies α-enolase as a novel autoantigen in Hashimoto's encephalopathy

Ochi Hirofumi, Horiuchi Izumi, Araki Norie, Toda Tosifusa, Araki Tomohiro, Sato Kaori, Murai Hiroyuki, Osoegawa Manabu, Yamada Takeshi, Okamura Ken, Ogino Tomoaki, Mizumoto Kiyohisa, Yamashita Hirohumi, Saya Hideyuki, Kira Jun ichi

FEBS Letters 528 ( 1-3 ) 197 - 202 2002.09

ISSN 0014-5793

Hashimoto's encephalopathy (HE) is a rare autoimmune disease associated with Hashimoto's thyroiditis (HT). To identify the HE-related autoantigens, we developed a human brain proteome map using two-dimensional electrophoresis and applied it to the immuno-screening of brain proteins that react with autoantibodies in HE patients. After sequential MALDI-TOF-MASS analysis, immuno-positive spots of 48 kDa (pI 7.3-7.8) detected from HE patient sera were identified as a novel autoimmuno-antigen, α-enolase, harboring several modifications. Specific high reactivities against human α-enolase were significant in HE patients with excellent corticosteroid sensitivity, whereas the patients with fair or poor sensitivity to the corticosteroid treatment showed less reactivities than cut-off level. Although a few HT patients showed faint reactions to α-enolase, 95% of HT patients, patients with other neurological disorders, and healthy subjects tested were all negative. These results suggest that the detection of anti-α-enolase antibody is useful for defining HE-related pathology, and this proteomic strategy is a powerful method for identifying autoantigens of various central nervous system diseases with unknown autoimmune etiologies.

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Influence of p53 mutations on prognosis of patients with glioblastoma

Shiraishi Shoji, Tada Kenji, Nakamura Hideo, Makino Keishi, Kochi Masato, Saya Hideyuki, Kuratsu Jun Ichi, Ushio Yukitaka

Cancer 95 ( 2 ) 249 - 257 2002.07

ISSN 0008-543X

BACKGROUND. The influence of p53 mutations on the biology of astrocytic tumors is controversial. p53 is thought to be inactivated in the early stage of gliomagenesis; however, what role its inactivation plays in the malignancy of gliomas remains unknown. To understand the significance of p53 inactivation, the authors identified the locus of p53 gene mutation in glioma samples at different stages of progression and studied the correlation between the mutation and clinical behavior. METHODS. Samples from newly diagnosed gliomas, including pure and mixed astrocytomas, were analyzed for p53 mutations using a yeast functional assay. To determine the locus of the gene mutations, DNA sequencing was performed. RESULTS. The incidence of p53 mutations was higher in anaplastic astrocytomas (AA, 48%) than glioblastomas (GBM, 31%). There was no significant difference in the average ages of GBM patients with and without p53 mutations (54.9 years ± 2.3 and 53.2 years ± 4.6, respectively). In GBM patients, the mutation did not affect progression free survival or overall survival. Astrocytomas and GBM differed in the distribution of p53 mutation loci. CONCLUSIONS. The p53 gene mutation does not markedly affect the survival of GBM patients. The difference in the location of p53 mutations between AA and GBM suggests that in gliomas, the p53 mutation may contribute not only to tumorigenesis (as an early event) but also to progression to malignancy (as a late event).

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Cooperation of HECT-domain ubiquitin ligase hHYD and DNA topoisomerase II-binding protein for DNA damage response

Honda Yoshiomi, Tojo Masahide, Matsuzaki Kazuhito, Anan Tadashi, Matsumoto Mitsuhiro, Ando Masayuki, Saya Hideyuki, Nakao Mitsuyoshi

Journal of Biological Chemistry 277 ( 5 ) 3599 - 3605 2002.02

ISSN 0021-9258

Ubiquitin ligases define the substrate specificity of protein ubiquitination and subsequent proteosomal degradation. The catalytic sequence was first characterized in the C terminus of E6-associated protein (E6AP) and referred to as the HECT (homologous to E6AP C terminus) domain. The human homologue of the regulator of cell proliferation hyperplastic discs in Drosophila, designated hHYD, is a HECT-domain ubiquitin ligase. Here we show that hHYD provides a ubiquitin system for a cellular response to DNA damage. A yeast two-hybrid screen showed that DNA topoisomerase IIβ-binding protein 1 (TopBP1) interacted with hHYD. Endogenous hHYD bound the BRCA1 C-terminus domains of TopBP1 that are highlighted in DNA damage checkpoint proteins and cell cycle regulators. Using an in vitro reconstitution, specific E2 (ubiquitin-conjugating) enzymes (human UbcH4, UbcH5B, and UbcH5C) transferred ubiquitin molecules to hHYD, leading to the ubiquitination of TopBP1. TopBP1 was usually ubiquitinated and degraded by the proteosome, whereas X-irradiation diminished the ubiquitination of TopBP1 probably via the phosphorylation, resulting in the stable colocalization of up-regulated TopBP1 with γ-H2AX nuclear foci in DNA breaks. These results demonstrated that hHYD coordinated TopBP1 in the DNA damage response.

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Analysis of homozygous deletion of the p16 gene and correlation with survival in patients with glioblastoma multiforme

Kamiryo Takanori, Tada Kenji, Shiraishi Shoji, Shinojima Naoki, Nakamura Hideo, Kochi Masato, Kuratsu Jun Ichi, Saya Hideyuki, Ushio Yukitaka

Journal of Neurosurgery 96 ( 5 ) 815 - 822 2002

ISSN 0022-3085

Object. One of the most frequent genetic abnormalities found in patients with glioblastoma multiforme (GBM) is homozygous deletion of the p16 tumor suppressor gene. The authors investigated whether this deletion is associated with prognosis in patients with GBM. Methods. In 46 adult patients with supratentorial GBM, homozygous deletion of the p16 gene in tumor DNA was examined using the multiplex polymerase chain reaction assay. The deletion was confirmed in 14 (30.4%) of 46 patients, eight (30.8%) of 26 men and six (30.0%) of 20 women. Cox proportional hazard regression analysis, adjusted for age at surgery, the Karnofsky Performance Scale score, extent of resection, and the MIB-1 labeling index, revealed that homozygous deletion of the p16 gene was significantly associated with overall survival and progression-free survival in men, but not in women. Conclusions. The results of this study suggest that p16 homozygous deletion is a significant unfavorable prognostic factor in male patients with GBM.

Molecular mechanism for development of malignant tumors

Saya Hideyuki

Nishinihon Journal of Urology 64 ( 3 ) 127 - 131 2002

ISSN 0029-0726

Maintenace of genomic integrity following DNA damage depends upon the coordinated action of DNA repair and checkpoint systems. Checkpoints control a signaling system that changes the activity of cyclin-dependent kinases (cdks), resulting in delayed cell cycle progression. Arrest in G1 is thought to prevent aberrant replication of damaged DNA, while arrest in G2 allows cells to avoid segregation of defective chromosomes. The failure of the checkpoint system leads to genomic instability, which causes multiple DNA sequence alterations and chromosomal aberrations, resulting in a disposition to develop cancer. On the other hand, it has been elucidated that checkpoint abnormality is the major reason why cancer cells are sensitive to genotoxic agents, such as anti-cancer drugs and X-radiation. In normal cells, DNA damage activates checkpoints, resulting in cell cycle arrest. However, cancer cells which have checkpoint abnormalities cannot be arrested and the abrogation of the checkpoint system induces cell death via an unknown mechanism called "biochemical crush". Tumor cells which have more malignant phenotypes are considered to have defects in cell death signals which are induced by biochemical crush, and such cells are able to survive even with severe DNA damage. Accumulation of basic knowledge regarding checkpoint regulation and the cell death system is imortant for developing new therapeutic approaches for dealing with malignant tumors.

Hyperploidy induced by drugs that inhibit formation of microtubule promotes chromosome instability

Nitta Masayuki, Tsuiki Hiromasa, Arima Yoshimi, Harada Kei, Nishizaki Takahumi, Sasaki Kousuke, Mimori Tatsuyuki, Ushio Yukitaka, Saya Hideyuki

Genes to Cells 7 ( 2 ) 151 - 162 2002

ISSN 1356-9597

Background: Antimicrotubule drugs (AMDs), such as taxol and vincristine, are the most important addition to the chemotherapeutic armamentarium against human cancers. It has been shown that prolonged AMD treatment induces hyperploidy in G1-checkpoint-defective cancer cells and that these hyperploid cells subsequently undergo apoptosis. However, a fraction of these hyperploid cells are able to survive the prolonged mitotic stress and resume cell-cycle progression. Results: We established hyperploid clones that escaped from cell death after AMD treatment from two glioma cell lines, U251MG and U87MG. Subtractive comparative genomic hybridization (CGH) analysis revealed that clones derived from U87MG mainly had chromosome number changes, but that those from U251MG showed both numerical and structural chromosomal changes. Furthermore, numerous aberrations identified in U251MG clones were remarkably chromosome-specific, which may have been due to clonal selection for cells that have an advantage in growth and/or survival. All clones derived from both cell lines had abnormalities in chromosome segregation, and karyotypes of clones were more heterogeneous than those of parental cells, suggesting that cells having a higher chromosome number are subject to asymmetric chromosome segregation, resulting in a heterogeneous karyotype. All clones derived from U87MG and U251MG increased both centric and acentromeric micronuclei, suggesting the presence of chromosome structural abnormality. Conclusions: AMD treatment induces hyperploid formation and chromosome instability in checkpoint-deficient cancer cells.

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Usefulness of sampling with cotton swab for PCR-diagnosis of cutaneous leishmaniasis in the New World

Mimori T., Matsumoto T., Calvopiña M. H., Gomez E. A., Saya H., Katakura K., Nonaka S., Shamsuzzaman S. M., Hashiguchi Y.

Acta Tropica 81 ( 3 ) 197 - 202 2002

ISSN 0001-706X

In this study, we tested the polymerase chain reaction (PCR)-method to diagnose cutaneous leishmaniasis (CL) by taking exudate materials from lesions with cotton swabs, using our previously tested (PCR) panel comprised of Leishmania (Viannia) panamensis, L. (V.) braziliensis, L. (V.) guyanensis, L. (Leishmania) mexicana and L. (L.) amazonensis. The objectives of the present study were to improve the sampling method convenient for the patients and to test the usefulness of samples taken with cotton swabs. Sixteen patients were clinically diagnosed to have CL including one case of diffuse cutaneous leishmaniasis (DCL) in Ecuador and the causative Leishmania parasites were identified by PCR. All the 12 samples from CL patients of La Mana, positive for Leishmania DNA, were identified as L. (V.) panamensis, while two from CL of Huigra and one from DCL of San Ignacio were L. (L.) mexicana. In the field condition, taking biopsy material is not only painful but sometimes causes iatrogenic bacterial infections. Considering the sensitivity of the test, and convenient sampling procedure, it may be suggested that collection of exudates using cotton swabs may be a better alternative to biopsy sample for PCR-diagnosis of CL.

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Proteolytic cleavage of the CD44 adhesion molecule in multiple human tumors

Okamoto Isamu, Tsuiki Hiromasa, Kenyon Lawrence C., Godwin Andrew K., Emlet David R., Holgado-Madruga Marina, Lanham Irene S., Joynes Christopher J., Vo Kim T., Guha Abhijit, Matsumoto Mitsuhiro, Ushio Yukitaka, Saya Hideyuki, Wong Albert J.

American Journal of Pathology 160 ( 2 ) 441 - 447 2002

ISSN 0002-9440

Cell surface adhesion molecules are crucial for the development and/or pathogenesis of various diseases including cancer. CD44 has received much interest as a major adhesion molecule that is involved in tumor progression. We have previously demonstrated that the ectodomain of CD44 undergoes proteolytic cleavage by membrane-associated metalloproteases in various tumor cell lines. The remaining membrane-bound CD44 cleavage product can be detected using antibodies against the cytoplasmic domain of CD44 (anti-CD44cyto antibody). However, the cleavage of CD44 in primary human tumors has not been investigated. Using Western blots with anti-CD44cyto antibody to assay human tumor tissues, we show that the CD44 cleavage product can be detected in 58% (42 of 72) of gliomas but not in normal brain. Enhanced CD44 cleavage was also found in 67% (28 of 42) of breast carcinomas, 45% (5 of 11) of non-small cell lung carcinomas, 90% (9 of 10) of colon carcinomas, and 25% (3 of 12) of ovarian carcinomas. Tumors expressing a CD44 splice variant showed a significantly higher incidence of enhanced CD44 cleavage. The wide prevalence of CD44 cleavage suggests that it plays an important role in the pathogenesis of human tumors.

Study of autoantibodies against advanced glycation endproducts of the Maillard reaction

Araki Norie, Shibayama Rie, Ejima Yumiko, Nagai Ryoji, Araki Tomohiro, Saya Hideyuki, Horiuchi Seikoh

International Congress Series 1223 ( C ) 49 - 58 2001.12

ISSN 0531-5131

Long-term incubation of proteins with reducing sugar proceeds to advanced glycation end products (AGEs). We previously demonstrated the presence of AGE structures in human and animal tissues, and suggested the implication of AGEs in aging and diabetic complications. In this study, we tested whether AGEs present in vivo, such as N^ε-(carboxymethyl) lysine (CML) adduct, could serve as immunogens for generating autoantibodies. Plasma samples from STZ-induced diabetic rats reacted positively with AGE-proteins as well as CML-BSA, suggesting the presence of autoantibodies against AGE-structures, particularly CML in diabetic rats; the activity of the autoantibody increased with the duration of diabetic states, reflecting the accumulation of AGE proteins in these diabetic rats. An autoantibody fraction purified from the plasma of diabetic patients showed a positive reaction to AGE-proteins as well as to CML proteins, and also to human lens proteins which are known to undergo CML modification in vivo. Patients with renal failure caused by diabetes or nondiabetic pathologies had a higher autoantibody activity against AGE structures than that in normal subjects or diabetic patients without renal failure. These results suggest that AGEs accumulated in vivo serve as immunological epitopes for developing autoantibodies against AGEs, particularly against a CML structure, which might be used as an indicator of diabetic nephropathy or chronic renal failure.

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Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway

Okamoto Isamu, Kawano Yoshiaki, Murakami Daizo, Sasayama Takashi, Araki Norie, Miki Toru, Wong Albert J., Saya Hideyuki

Journal of Cell Biology 155 ( 5 ) 755 - 762 2001.11

ISSN 0021-9525

CD44 is a widely distributed cell surface adhesion molecule and is implicated in diverse biological processes. However, the nature of intracellular signaling triggered by CD44 remains to be elucidated. Here, we show that CD44 undergoes sequential proteolytic cleavage in the ectodomain and intracellular domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. Consequently, CD44ICD acts as a signal transduction molecule, where it translocates to the nucleus and activates transcription mediated through the 12-O-tetradecanoylphorbol 13-acetate-responsive element, which is found in numerous genes involved in diverse cellular processes. Expression of an uncleavable CD44 mutant as well as metalloprotease inhibitor treatment blocks CD44-mediated transcriptional activation. In search of the underlying mechanism, we have found that CD44ICD potentiates transactivation mediated by the transcriptional coactivator CBP/p300. Furthermore, we show that cells expressing CD44ICD produce high levels of CD44 messenger RNA, suggesting that the CD44 gene is one of the potential targets for transcriptional activation by CD44ICD. These observations establish a novel CD44 signaling pathway and shed new light on the functional link between proteolytic processing of an adhesion molecule at the cell surface and transcriptional activation in the nucleus.

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Activation of Cdh1-dependent APC is required for G₁ cell cycle arrest and DNA damage-induced G₂ checkpoint in vertebrate cells

Sudo Tamotsu, Ota Yosuke, Kotani Shuji, Nakao Mitsuyoshi, Takami Yasunari, Takeda Shunichi, Saya Hideyuki

EMBO Journal 20 ( 22 ) 6499 - 6508 2001.11

ISSN 0261-4189

Anaphase-promoting complex (APC) is activated by two regulatory proteins, Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (Cdh1-APC) activity targets mitotic cyclins from the end of mitosis to the G₁ phase. To investigate the function of Cdh1 in vertebrate cells, we generated clones of chicken DT40 cells disrupted in their Cdh1 loci. Cdh1 was dispensable for viability and cell cycle progression. However, similarly to yeast and Drosophila, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G₁, resulting in abrogation of G₁, arrest caused by treatment with rapamycin, an inducer of p27^Kipl. Furthermore, we found that Cdh1^-1- cells fail to maintain DNA damage-induced G₂ arrest and that Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G₁ arrest and DNA damage-induced G₂ arrest.

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Calpain-dependent proteolysis of merlin occurs by oxidative stress in meningiomas

Kaneko Takuro, Yamashima Tetsumori, Tohma Yasuo, Nomura Motohiro, Imajoh-Ohmi Shinobu, Saido Takaomi C., Nakao Mitsuyoshi, Saya Hideyuki, Yamamoto Hiroshi, Yamashita Junkoh

Cancer 92 ( 10 ) 2662 - 2672 2001.11

ISSN 0008-543X

BACKGROUND. The purpose of this study is to indicate that oxidative stress may contribute to occurrence of meningiomas. Recently, it was reported that aside from the neurofibromatosis type 2 (NF2) gene mutations, the calpain-dependent proteolysis of the NF2 gene product, merlin might be closely related to the development of certain NF2-related tumors. Although meningiomas are well known to occur more frequently in aged persons, it still remains unknown why calpain activation occurs predominantly in them. Because the production of free radicals with aging might be one of the causes of calpain activation especially in leptomeningeal cells being devoid of blood supply, the authors examined the relations between μ-calpain activation and merlin proteolysis induced by the oxidative stress. METHODS. The authors examined 12 patient-derived sporadic meningiomas and their primary cultured cells. Malignant glioma cell line (U-251MG), which had no relation to NF2, was used as a control. They were exposed to hydrogen peroxide (H₂O₂) for 1 hour. After oxidative stress, they were examined by Western blot and immunofluorescence microscopic analyses. RESULTS. Despite the consistent expressions of activated μ-calpain in 11 of 12 meningioma tissues, this calpain activation completely disappeared after culture; instead the full-length merlin appeared again in 8 of 11 cases. The treatment of cultured cells with hydrogen peroxide induced both μ-calpain-dependent cleavage of merlin and reduction of an intrinsic calpain inhibitor calpastatin. Such proteolysis was significantly blocked by a specific calpain inhibitor, Z-LLal. The full-length merlin was immunocytochemically colocalized with activated μ-calpain at the plasma membrane, and, after μ-calpain activation, the fragment of merlin translocated to the perinuclear cytoplasm or into the nucleus. CONCLUSIONS. These findings suggest that oxidative stress-induced activation of μ-calpain causes proteolysis of merlin conceivably to impair cell adhesion and/or contact inhibition of meningioma cells.

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Restoration of endogenous wild-type p53 activity in a glioblastoma cell line with intrinsic temperature-sensitive p53 induces growth arrest but not apoptosis

Ikeda Jun, Tada Mitsuhiro, Ishii Nobuaki, Saya Hideyuki, Tsuchiya Kazuhiko, Okaichi Kumio, Mishima Kazuhiko, Sawamura Yutaka, Fulci Giulia, Liu Ta Jen, Van Meir Erwin G.

International Journal of Cancer 94 ( 1 ) 35 - 43 2001.10

ISSN 0020-7136

p53 protein is a transcription factor involved in multiple tumor-suppressor activities including cell cycle control and apoptosis. TP53 gene is frequently mutated in glioblastoma, suggesting the importance of inactivation of this gene product in gliomagenesis. Restoration of p53 function in glioblastoma cell lines deficient for p53 has shown that p53 induces growth arrest or apoptosis depending on the cell line and vector used to transduce wild-type TP53 alleles. Considering that astrocytes grow and express p53, it is not clear whether these results reflect physiologic responses or the result of p53 overexpression in combination with cellular responses to viral vector infection. Here, we reassessed this issue using a glioblastoma cell line (LN382) that expresses an endogenous temperature-sensitive mutant p53. This cell line expresses TP53 alleles (100% as determined by a p53 transcriptional assay in yeast) mutated at codon 197 GTG (Val) > CTG (Leu). We found that the p53 protein in these cells acted as an inactive mutant at 37°C and as a functional wild-type p53 below 34°C as demonstrated by several lines of evidence, including (i) restoration of transactivating ability in yeast, (ii) induction of p53-modulated genes such as CDKN1^p21 and transforming growth factor-α, (iii) disappearance of accumulated p53 protein in the nucleus and (iv) decrease in steady state p53 protein levels. This temperature switch allowed p53 levels, which were close to physiological levels to dramatically reduce LN382 cell proliferation by inducing a G₁/S cell cycle block, but not to induce apoptosis. The lack of apoptosis was considered to be a result of the low level p53 expression, because increasing wild-type p53 levels by adenoviral-mediated gene transfer caused apoptosis in these cells. The LN382 cell line will be extremely useful for investigations into the roles of p53 in cellular responses to a variety of stimuli or damages.

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Phosphorylation of neurofibromin by cAMP-dependent protein kinase is regulated via a cellular association of N^G,N^G-dimethylarginine dimethylaminohydrolase

Tokuo Hiroshi, Yunoue Shunji, Feng Liping, Kimoto Masumi, Tsuji Hideaki, Ono Tomomichi, Saya Hideyuki, Araki Norie

FEBS Letters 494 ( 1-2 ) 48 - 53 2001.04

ISSN 0014-5793

The neurofibromatosis type 1 (NF1) tumor suppressor (neurofibromin) is thought to play crucial roles in cellular Ras- and cAMP-dependent kinase (PKA)-associated signals. In this study, we identified a cellular neurofibromin-associating protein, N^G,N^G-dimethylarginine dimethylaminohydrolase (DDAH) that is known as a cellular NO/NOS regulator. The interaction of DDAH was mainly directed to the C-terminal domain (CTD) and to the cysteine/serine-rich domain (CSRD) of neurofibromin, coinciding with the regions containing specific PKA phosphorylation sites. DDAH increased PKA phosphorylation of native neurofibromin in a dose-dependent manner, especially affecting the phosphorylation of CSRD. These findings suggest that the PKA accessibility of neurofibromin was regulated via DDAH interaction, and this regulation may modulate the cellular function of neurofibromin that is implicated in NF1-related pathogenesis.

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A novel tetracycline-dependent transactivator with E2F4 transcriptional activation domain.

Akagi K., Kanai M., Saya H., Kozu T., Berns A.

Nucleic Acids Research 29 ( 4 ) 2001.02

ISSN 1362-4962

A tetracycline-controlled gene expression system provides a powerful tool to dissect the functions of gene products. However, it often appears difficult to establish cell lines or transgenic animals stably expressing tetracycline-dependent transactivators, possibly as a result of toxicity of the transactivator domains used. In order to overcome this problem, we developed a novel tetracycline-dependent transactivator that works efficiently in mammalian cells. This transactivator is a fusion of the tet reverse repressor mutant and the transcriptional activating domain of human E2F4, which is ubiquitously expressed in vivo. We demonstrate here that this tetracycline-regulated gene expression system provides a two log transcriptional activation in mammalian cells as assessed by northern blot and luciferase analyses. Combining this system with green fluorescent protein reporter systems or microarray gene expression profiling will facilitate the study of gene function.

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