Details of a Researcher - Saya, Hideyuki

Papers - Saya, Hideyuki

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Identification of a human homolog of the Drosophila neuralized gene within the 10q25.1 malignant astrocytoma deletion region

Nakamura Hideo, Yoshida Mitsuhiro, Tsuiki Hiromasa, Ito Kiyoharu, Ueno Mikako, Nakao Mitsuyoshi, Oka Koji, Tada Mitsuhiro, Kochi Masato, Kuratsu Jun Ichi, Ushio Yukitaka, Saya Hideyuki

Oncogene 16 ( 8 ) 1009 - 1019 1998.02

ISSN 0950-9232

The loss of chromosome 10 is the most frequent genetic alteration found in malignant astrocytomas. In particular, the long arm of chromosome 10 was previously reported to have two or more common deletion regions where tumor suppressor genes may be located. In this study, we performed deletion mapping of 44 malignant astrocytomas using 12 microsatellite markers on chromosome 10q and demonstrated that the minimal common region of loss of heterozygosity (LOH) was present between D10S192 and D10S566 localized at 10q25.1. Subsequently, we have identified a novel gene, termed h-neu, within the region frequently deleted and found that h-neu encodes a protein with strong homology to the Drosophila neuralized (D-neu) protein. Northern blot and RT-PCR analyses revealed that h-neu mRNA was expressed at very low levels in human malignant astrocytoma tissues and the majority of glioma cell lines examined, while normal brains expressed h-neu transcript. Furthermore, DNA sequencing analysis of the h-neu transcript revealed one of the glioma cell lines, U251MG, had a single nucleotide substitution which resulted in an amino acid change from glycine (GGC) to serine (AGC) at codon 253. The D-neu gene is known to serve a critical function in neurogenesis in Drosophila, and loss-of-function mutations produce hyperplasia of primitive neuronal cells. These observations led us to hypothesize that h-neu gene plays a role in determination of cell fate in the human central nervous system and may act as a tumor suppressor whose inactivation could be associated with malignant progression of astrocytic tumors.

Overexpression of Bcl-2 in bladder cancer cells inhibits apoptosis induced by cisplatin and adenoviral-mediated p53 gene transfer

Miyake Hideaki, Hanada Norihisa, Nakamura Hideo, Kagawa Shunsuke, Fujiwara Toshiyoshi, Hara Isao, Eto Hiroshi, Gohji Kazuo, Arakawa Soichi, Kamidono Sadao, Saya Hideyuki

Oncogene 16 ( 7 ) 933 - 943 1998.02

ISSN 0950-9232

To investigate the effects of the expression of Bcl-2 protein in bladder cancer on the apoptosis induced by cisplatin or adenoviral-mediated p53 gene (Ad5CMV-p53) transfer, we transfected the bcl-2 gene into KoTCC-1, a human bladder cancer cell line that does not express the Bcl-2 protein. The Bcl-2-transfected KoTCC-1 (KoTCC-1/B) exhibited significantly higher resistance to both cisplatin and Ad5CMV-p53 transfer than did either the parental KoTCC-1 (KoTCC-1/P) or the vector-only transfected cell line (KoTCC-1/C). The flow cytometric analysis of the propidium iodide-stained nuclei and DNA fragmentation analysis after cisplatin or Ad5CMV-p53 treatment revealed DNA degradation in both KoTCC-1/P and KoTCC-1/C, whereas KoTCC1/B showed a marked inhibition of DNA degradation. Following the treatment with cisplatin or Ad5CMV-p53, the accumulation of p53 protein was highly detectable for a long period in KoTCC-1/B compared to that in KoTTC-1/P and KoTCC-1/C. Furthermore, the cisplatin and Ad5CMV-p53 treatments each reduced the volume of the subcutaneous tumors established in nude mice formed by KoTCC-1/P or KoTCC-1/C; in contrast, their reductive effects on the tumors formed by KoTCC-1/B were significantly suppressed. The intraperitoneal tumor cell implantation model revealed that the prognoses of mice injected with KoTCC-1/B were significantly inferior to those of the mice injected with either KoTCC-1/P or KoTCC-1/C after treatment with cisplatin or Ad5CMV-p53. These findings suggest that the expression of Bcl-2 in bladder cancer cells interferes with the therapeutic effects of cisplatin and Ad5CMV-p53 through the inhibition of the apoptotic pathway.

Molecular detection of cancer cells by competitive reverse transcription-polymerase chain reaction analysis of specific CD44 variant RNAs

Okamoto Isamu, Morisaki Tetsuro, Sasaki Ji Ichiro, Miyake Hideaki, Matsumoto Mitsuhiro, Suga Moritaka, Ando Masayuki, Saya Hideyuki

Journal of the National Cancer Institute 90 ( 4 ) 307 - 315 1998.02

ISSN 0027-8874

Background: CD44 is a cell surface glycoprotein implicated in such diverse biologic processes as lymphocyte activation and homing, extracellular matrix adhesion, and cellular migration. Primary transcripts of the CD44 gene can be alternatively spliced to produce a variety of messenger RNA (mRNA) species. The standard form of CD44 mRNA contains sequences from at least 20 genomic exons; variant mRNAs contain sequences from one or more additional exons (v1-10). Predominant expression of a specific CD44 variant, i.e., CD44v8-10, in several human carcinomas has been described previously. In this study, we developed a novel molecular approach for detecting cancer cells that overexpress CD44v8-10 mRNA. Methods: After finding that CD44v8-10 was predominantly expressed in non-small-cell lung and bladder carcinomas and that CD44v10 was predominantly expressed in leukocytes, we developed a competitive reverse transcription-polymerase chain reaction assay (CC-RT- PCR) that allows quantification of the relative expression of these two mRNA species in clinical specimens (i.e., determination of a v8-10/v10 ratio). CC- RT-PCR analysis was applied to pleural effusion specimens from patients with benign or malignant lung diseases as well as to spontaneously voided urine samples from patients with benign or malignant urologic diseases. Results: Fifty two of 54 samples from patients with benign diseases expressed CD44v10 predominantly (v8-10/v10 ratio ≤0.65), whereas 46 of 61 samples from patients with malignant diseases expressed CD44v8-10 predominantly (v8- 10/v10 ratio >1.00) (two-sided P<.001). CC-RT-PCR detected predominant expression of CD44v8-10 in cytologically negative samples from 11 patients who were later diagnosed with malignant disease. Conclusions: CC-RT-PCR analysis of CD44v8-10 expression could be an important adjunct to cytologic examination in cancer diagnosis, especially in detecting exfoliated cancer cells in pleural effusions and urine.

Evidence that HLA class II-restricted human CD4⁺ T cells specific to p53 self peptides respond to p53 proteins of both wild and mutant forms

Fujita Hiroshi, Senju Satoru, Yokomizo Hiroshi, Saya Hideyuki, Ogawa Michio, Matsushita Sho, Nishimura Yasuharu

European Journal of Immunology 28 ( 1 ) 305 - 316 1998.01

ISSN 0014-2980

By stimulating peripheral blood mononuclear cells of four healthy donors with a mixture of overlapping peptides representing the core domain of p53, we established two CD4⁺ αβ T cell clones and four lines that recognized wild-type and mutant p53 proteins as well as p53 self peptides in an HLA class II-restricted fashion. Two T cell lines established from two unrelated donors reacted to the p53 peptide (p)153-166 and p108-122, respectively, in the context of DP5 molecules. Two T cell clones established from two other unrelated donors were specific for p193-204 in the context of DRB1*1401 and for p153-165 in the context of DP5, respectively. These two T cell clones responded almost equally to both wild-type and four mutant recombinant p53 proteins. The proliferative responses of these T cell clones to p53 recombinant proteins were augmented by heat denaturing, thereby suggesting that altered conformation of the protein facilitates proteolytic processing to produce antigenic peptides. The DRB1*1401-restricted T cell clone specific for p193-204 killed a B lymphoblastoid cell line homozygous for HLA-DRB1*1401 when the cell line was pre-pulsed with p53 protein as well as peptide. These results indicate that CD4⁺ T cells reactive to p53 do exist in healthy individuals and the epitopes are probably ignored by the immune system under physiological conditions. It is suggested that such epitopes stimulate T cells to induce anti-p53 antibody production in cancer patients as previously reported by others. The possible involvement of p53-reactive T cells in anti-tumor immunity is discussed.

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Monitoring adenoviral p53 transduction efficiency by yeast functional assay

Tada M., Sakuma S., Iggo R. D., Saya H., Sawamura Y., Fujiwara T., Roth J. A.

Gene Therapy 5 ( 3 ) 339 - 344 1998

ISSN 0969-7128

Monitoring the transduction efficiency is of paramount importance in gene therapy. To monitor adenovirus-mediated wild-type p53 gene transfer, we have used a quantitative assay which tests the ability of human p53 to activate transcription in yeast. Selective amplification of cellular and viral p53 transcripts followed by quantitative assessment of mutant p53 content with the assay permits measurement of the wild-type p53 transduction efficiency into SF-188, U251MG and HUG31 glioblastoma cells. One reverse transcription primer tracks the wild-type/mutant ratio of endogenous p53 mRNA (P2), and the other the wild-type/mutant ratio of both endogenous and exogenous p53 mRNA (P1). Following infection of cell lines homozygous for mutant p53, the apparent transduction efficiency calculated (τ(o) = [P1 - P2]/[1 + P2]) correlated with the level of p21 expression. Transduction efficiency in heterozygous wild-type/mutant HUG31 cells increased linearly with multiplicity of infection (MOI) for τ(o) value, in keeping with theoretical predictions. These results suggest that the yeast p53 functional assay may be a useful tool for monitoring p53 gene therapy.

Highly specific and sensitive detection of malignancy in urine samples from patients with urothelial cancer by CD44v8-10/CD44v10 competitive RT-PCR

Miyake Hideaki, Okamoto Isamu, Hiara Isao, Gohji Kazuo, Yamanaka Kazuki, Arakawa Soichi, Kamidono Sadao, Saya Hideyuki

International Journal of Cancer 79 ( 6 ) 560 - 564 1998

ISSN 0020-7136

CD44 is a widely expressed cell surface adhesion molecule in which various isoforms arise from alternative RNA splicing mechanism. Overexpression of specific CD44 splice variant, i.e., CD44v8-10, has been found in several human malignancies and is considered to be associated with tumor progression and metastasis. We have demonstrated a novel molecular approach, CD44v8-10/CD44v10 competitive reverse transcription-polymerase chain reaction (CC-RT-PCR) for the detection of cancer cells overexpressing CD44v8-10 by the measurement of the transcriptional level of CD44v8-10 relative to that of CD44v10 (v8-10/v10 ratio). In this study, we initially examined the expression of CD44 splice variants in human urothelial cancers and their adjacent normal urinary tissues by RT-PCR. Any CD44 variant isoforms were barely detectable in normal urinary tissues, whereas CD44v8-10 was predominantly expressed in 23 of the 30 (77%) urothelial cancer specimens. We then applied CC-RT-PCR to spontaneously voided urine samples from patients with 80 urothelial cancer and 50 various benign urologic diseases. The CC-RTPCR analysis revealed that all of the samples associated with benign diseases presented a predominant expression of the CD44v10 transcript (the v8-10/v10 ratios = 0.00-0.87), whereas 62 of the 80 samples associated with urothelial cancers mostly expressed the CD44v8-10 transcript (the v8-10/v10 ratios > 1.00). In addition, the positivity rate obtained by the CC-RT-PCR analysis was high regardless of the pathological grade of the urothelial cancers, although the sensitivity of the cytological examination declined with decreasing tumor grade. Our findings suggest strongly that CC- RT-PCR is a non-invasive useful tool for the diagnosis of urine samples from patients with urothetial cancer.

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Interaction between CD44 and hyaluronic acid regulates human prostate cancer development

Miyake Hideaki, Hara Isao, Okamoto Isamu, Gohji Kazuo, Yamanaka Kazuki, Arakawa Soichi, Saya Hideyuki, Kamidono Sadao

Journal of Urology 160 ( 4 ) 1562 - 1566 1998

ISSN 0022-5347

Purpose: This study was designed to investigate the significance of the interaction between CD44 and hyaluronic acid in the development of human prostate cancer. Materials and Methods: We transfected the standard CD44 isoform (CD44s) cDNA into PC3, a human prostate cancer cell line that barely expresses CD44s protein. The effects of the reintroduction of CD44s into PC3 cells on the ability to bind hyaluronic acid (HA) were analyzed by the cell adhesion assay and by the cell migration assay. The in vitro growth rate of CD44s transfected PC3 was measured by using the MTT assay. We then evaluated the in vivo tumor development of CD44s transfected PC3 cells by subcutaneous, intravenous, and intraperitoneal injection models in athymic nude mice. Results: The introduction of CD44s in PC3 cells markedly enhanced the binding and migration of these cells to HA, but not to other extracellular matrix molecules. In vitro growth of CD44s-transfected PC3 was found to be significantly decreased. In addition, the CD44s-transfected PC3 cells also demonstrated reduced tumorigenicity and metastatic potential in in vivo experimental models. Conclusions: These findings suggest that the CD44s downregulation plays an important role in the development of human prostate cancer, in part through reduction of the ability to bind HA.

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Growth inhibition of experimental glioma by human interferon-β superinduced by cationic liposomes entrapping polyinosilic

Kuratsu J. I., Saya H., Tabuchi K.

Neurologia Medico-Chirurgica 38 ( 8 ) 473 - 474 1998

ISSN 0387-2572

Loss of merlin-p85 protein complex in NF2-related tumors

Takeshima Hideo, Nishi Toru, Yamamoto Keizo, Kino Takeshi, Nakamura Hideo, Saya Hideyuki, Kochi Masato, Kuratsu Jun Ichi, Ushio Yukitaka

International Journal of Oncology 12 ( 5 ) 1073 - 1078 1998

ISSN 1019-6439

The NF2 tumor suppressor gene product, designated merlin, belongs to the family of molecules that links membranous protein with the cytoskeleton. We have previously shown that merlin was co-immunoprecipitated with a cellular protein, p85, in cultured cell. To analyze the alteration of merlin and associated proteins in surgical specimens, we developed a new method for biotin-labeling of whole cellular proteins. Screening of tumor tissues using our method showed that none of malignant gliomas and half of the NF2-related tumors had altered p85 and merlin. Our detection method seems useful for the screening of merlin alterations in NF2-related tumors.

Molecular biology of tumor suppressor genes related to cell adhesion signalings

Saya H., Makino K., Kuwahara H., Masuko N., Nishiyama Y., Morisaki T., Sasaki J., Nakao M., Ushio Y.

International Journal of Pediatric Hematology/Oncology 5 ( 1 ) 44 - 45 1998

ISSN 1070-2903

Human ubiquitin-protein ligase Nedd4

Anan Tadashi, Nagata Yoichi, Koga Hisashi, Honda Yoshiomi, Yabuki Nami, Miyamoto Chikara, Kuwano Akira, Matsuda Ichiro, Endo Fumio, Saya Hideyuki, Nakao Mitsuyoshi

Genes to Cells 3 ( 11 ) 751 - 763 1998

ISSN 1356-9597

Background: Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport. Results: Three mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, ≃120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed that both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme. Conclusion: Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.

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Identification of a novel, tissue-specific calpain htra-3; a human homologue of the Caenorhabditis elegans sex determination gene

Mugita Norifumi, Kimura Yoriyoshi, Ogawa Michio, Saya Hideyuki, Nakao Mitsuyoshi

Biochemical and Biophysical Research Communications 239 ( 3 ) 845 - 850 1997.10

ISSN 0006-291X

We cloned a novel human gene encoding a tissue-specific calpain, termed htra-3, which is highly homologous to the tra-3 sex determination gene of Caenorhabditis elegans. The predicted htra-3 polypeptide had similarity to the calpain large subunits in domain organization throughout domains I to III, but the sequences of domain IV lacked calcium-binding motifs. Northern blot analysis revealed high expression in the colon, small intestine and testis. Radiation hybrid mapping localized the htra-3 gene to chromosome 11q14 (2.53cR apart from WI-3895). Western blot analysis demonstrated that the approximately 73-kDa htra-3 protein was transiently expressed in COS-7 cells. These observations, together with the genetic information in C. elegans, suggest a unique function for htra-3 protein.

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Alternative splicing of the erythropoietin receptor gene correlates with erythroid differentiation in rat hematopoietic and leukemic cells

Fujita Masayuki, Takahashi Rei, Kitada Kazuhiro, Watanabe Rei, Kitazawa Sohei, Ashoori Faramarz, Liang Ping, Saya Hideyuki, Serikawa Tadao, Maeda Sakan

Cancer Letters 112 ( 1 ) 47 - 55 1997.01

ISSN 0304-3835

An alternative splicing of the rat erythropoietin receptor (EpoR) gene; was identified in normal and erythroleukemia cells. A 105 bp insert was found at a region corresponding to the extracellular domain of EpoR. The alternative transcript was translated to a soluble EpoR (EpoR-S) expressed in spleen, bone marrow, and cultured erythroleukemia cells in addition to the full-length EpoR (EpoR-F). One of the rat erythroleukemia sublines, K4DT, which partially lost erythroid phenotypes and manifested monocyte/macrophage characteristics also lacked EpoR-S expression. Thus, expression of EpoR-S may play an important role in differentiation of rat erythroid cells.

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Role of alternative splicing of the rat erythropoietin receptor gene in normal and erythroleukemia cells

Fujita M., Takahashi R., Liang P., Saya H., Ashoori F., Tachi M., Kitazawa S., Maeda S.

Leukemia 11 ( SUPPL. 3 ) 444 - 445 1997

ISSN 0887-6924

An alternatively splicing of the rat erythropoietin receptor (EpoR) gene was identified in normal and erythroleukemia cells by the reverse transcription PCR method. Insertion of a 105 bp fragment at the region corresponding to the extracellular domain of rat EpoR was found. The insert sequence, which encodes additional 21 amino acids, is similar to that previously found in the mouse EpoR gene, however, has an additional 27 bp direct repeat. Due to the presence of a stop codon in the insert, the alternative transcript is translated to a truncated and soluble form of EpoR which is preferentially expressed in liver, spleen, kidney, heart, and bone marrow cells as well as cultured erythroleukemia cells. These findings suggest that alternative splicing of the EpoR gene may play an important role in proliferation and differentiation of rat erythroid cells.

Cloning and characterization of NE-dlg

Makino Keishi, Kuwahara Hiroaki, Masuko Norio, Nishiyama Yasuyuki, Morisaki Tetsurou, Sasaki Ji Ichiro, Nakao Mitsuyoshi, Kuwano Akira, Nakata Motomi, Ushio Yukitaka, Saya Hideyuki

Oncogene 14 ( 20 ) 2425 - 2433 1997

ISSN 0950-9232

We have cloned a cDNA for a novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein, termed NE-dlg (neuronal and endocrine dlg). Northern blot analysis revealed that the gene is highly expressed in neuronal and endocrine tissues. Fluorescence in situ hybridization (FISH) and radiation hybrid mapping studies localized the NE-dlg gene to chromosome Xq13. We also found that the NE-dlg gene encoded a 100 kDa protein. Immunolocalization studies using an NE-dlg antibody showed that the protein tended to be expressed in non-proliferating cells, such as neurons, cells in Langerhans islets of the pancreas, myocytes of the heart muscles, and the prickle and functional layer cells of the esophageal epithelium. Proliferative cells, including various cultured cancer cell lines and basal cells in the esophageal epithelium, showed little expression of the NE-dlg protein. In addition, yeast two-hybrid screening and in vitro binding assays revealed that the NE-dlg interacted with the carboxyl-terminal region of the APC tumor suppressor protein. These data suggest that NE-dlg negatively regulates cell proliferation through its interaction with the APC protein.

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Human nedd-4 as a family member of ubiquitin ligases

Anan Tadashi, Nagata Yoichi, Honda Yoshiomi, Kimura Yoriyoshi, Kuwano Akira, Nomura Nobuo, Matsuda Ichiro, Saya Hideyuki, Nakao Mitsuyoshi

Japanese Journal of Human Genetics 42 ( 1 ) 38 1997

ISSN 0916-8478

Ubiquitin-proteasome proteolytic pathway is involved in a number of cellular physiology. In this system, a ubiquitin ligase, or E3 enzyme, is a major component which recognizes a specific substrate protein for targeted degradation. E6-associated protein (E6-AP) has been recently found to be a member of the E3 enzyme family that is characterized by a conserved carboxy-terminal sequence, termed Aectf-domain. Using human EST data bases, we identified five uncharacterized genes of this family. By FISH and radiation hybrid panel these genes were mapped to subchromosomal locations as follows; E6-AP (15ql2-13), NEDD-4 (15q22; D15S209), D28476 (2q35-36; D2S396), D13635 (7q36; FB16B4),and D25215 (4q21-22; WI-6336). NEDD-4 gene was mapped close to the translocation breakpoint of acute promyelocytic leukemia. Interestingly, NEDD-4 was highly conserved among species from yeast to human, suggesting its significant function on basic biomodulation. Northern blot analysis showed that NEDD-4 mRNA was much higher expressed in lung carcinoma A549 and chronic myelogenous leukemia K562 than in other cancer cell lines tested. The rest of the members, except D25215, were ubiquitously expressed in all cells. In immunoblot analysis, human NEDD-4 protein was down-regulated during neural differentiation in BDNFtreated neuroblastoma cells. Collectively, our data suggest that NEDD-4 protein may function as a ubiquitin ligase closely related to development, differentiation and tumorigenesis.

Neurofibromatosis type 2 (NF2)

Araki N., Takeshima H., Saya H.

Japanese Journal of Cancer and Chemotherapy 24 ( 11 ) 1427 - 1431 1997

ISSN 0385-0684

Neurofibromatosis type 2 (NF2) is an autosomal dominantly inherited disorder, strongly associated with development of benign intracranial tumors, including bilateral vestibular schwannomas and meningiomas. The NF2 gene located on the human chromosome 22q12 was recently cloned, and the protein it encodes termed merlin/schwannomin was found to be strikingly similar to the moesin-ezrin-radixin (MER) family of cytoskeleton-associated proteins. Mutations of NF2 gene have been found not only in the NF2 patient but also in NF2-related sporadic tumors such as acoustic schwannomas and meningiomas, suggesting that the NF2 gene functionally acts as a tumor suppressor gene. To elucidate the biological function of merlin, we have detected five merlin binding cellular proteins. The N-terminal region of merlin, the entire merlin-ezrin-radixin-moesin (MERM) homology domain, was found to be essential for to all five binding proteins. Since most reported NF2 mutations in the region were determined to be necessary for binding, the mutations probably impair binding. The majority of NF2 mutations are nonsense mutations or frameshifts that resulted in premature termination of merlin. The lack of these interactions caused by such mutations of NF2 gene may affect the cellular signals, resulting in benign intracranial tumors in NF2 patients.

Functional analysis of tumor suppressor p53 protein regulated by ubiquitin-proteasome system

Nagata Yoichi, Anan Tadashi, Honda Yoshiomi, Kimura Reiji, Saya Hidevuki, Nakao Mitsuvoshi, Nakata Motomi, Shinbara Naoki, Tanaka Keiii, Omura Satoshi

Japanese Journal of Human Genetics 42 ( 1 ) 38 1997

ISSN 0916-8478

The tumor suppressor p53 protein is most important for cell regulation such as cell cycle and apoptosis. In HPV-infected cells including HeLa cells, a viral oncpprptein E6 and a cellular E6-associated protein (E6AP) function together as a ubiquitin ligase targetting for p53. The complex plays a major role in the tumorigenesis by rapid turnover of p53. However, the mechanism of p53 degradation in normal cells remains unclear. 1) Using proteasome inhibitors, p53 protein was significantly accumulated in primary nbroblasts as well as HeLa cells in vivo. 2) Recombinant p53 proteins destined for wild- or mutant(273His)-type were analysed in an appropriate buffer and rabbit reticulpcyte lysates in vitro. Much slower degradation of mutant-p53 was found compared with the case of wild-p53. Interestingly, bpth-types of p53 were multiubiquitinated in a similar manner. 3) In fibroblasts infected with p53-expressing adenovirus, wild-p53 protein was co-immunoprecipitated with anti-E6AP antibody. In summary, E6AP may also work in the p53 ubiquitination in normal cells adding to E6-dependent case. Uoiguitinated mutant-p53 becomes resistant to degradation, suggesting that mutant-p53 is hardly recognized and/or degraded by proteasome due to unknown mechanism.

Neurofibromatosis 2 gene has novel alternative splicings which controls intracellular protein binding

Nishi Toru, Takeshima Hideo, Hamada Kazuya, Yoshizato Kimio, Koga Hisashi, Sato Kyoichi, Yamamoto Keizo, Kitamura Isao, Kochi Masato, Kuratsu Jun Ichi, Saya Hideyuki, Ushio Yukitaka

International Journal of Oncology 10 ( 5 ) 1025 - 1029 1997

ISSN 1019-6439

Three novel isoforms of the neurofibromatosis 2 (NF2) gene transcripts generated from alternative splicing were identified from normal human brain, schwannoma and glioma tissues. The 3 novel transcripts lack exon 2, exons 2 and 3, exons 2-4, respectively. Recombinant isoform proteins encoded by those new transcripts have lost the previously reported ability to bind ³⁵S-methionine labeled cellular proteins. Two of seven glioblastoma tissues expressed significantly high levels of the shorter transcripts whereas low grade astrocytomas expressed levels similar to those found in normal brain, suggesting that genomic mutation or aberrant alternative splicing of the NF2 gene may contribute to the progression of malignant gliomas.

Molecular biology of malignant brain tumors

Saya H.

Japanese Journal of Neurosurgery 6 ( 5 ) 306 - 310 1997

ISSN 0917-950X

Molecular genetic analyses have revealed that the genesis and progression of tumors are the result of accumulated changes in positive and negative growth regulatory genes. Several sequential genetic alterations are considered to be required to direct cells toward the malignant phenotype. Recent studies have demonstrated that glial oncogenesis is also involved in multiple, but relatively consistent genetic alterations. The most frequent chromosomal alterations observed in astrocytic tumors include amplification of chromosome 7 and losses on chromosomes 9p, 10, and 17p. The p53 gene and the CDKN2/p16 gene have been indicated as target genes for losses of chromosome 17p and 9p, respectively. The biochemical characters of these tumor suppressor proteins are discussed in this review.

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