116 results on '"Yoshiyuki Kuchino"'
Search Results
2. Achyranthoside H methyl ester, a novel oleanolic acid saponin derivative from Achyranthes fauriei roots, induces apoptosis in human breast cancer MCF-7 and MDA-MB-453 cells via a caspase activation pathway
- Author
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Yoshiyuki Kuchino, Yoshiteru Ida, Yasuaki Hirai, Motonori Fukumura, Kazuo Toriizuka, and Hidehiro Ando
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Time Factors ,Population ,Apoptosis ,Breast Neoplasms ,Biology ,Plant Roots ,Inhibitory Concentration 50 ,chemistry.chemical_compound ,Glucuronides ,Cell Line, Tumor ,Humans ,Cytotoxic T cell ,MTT assay ,Cytotoxicity ,education ,Oleanolic acid ,Achyranthes ,Cell Proliferation ,education.field_of_study ,Dose-Response Relationship, Drug ,Hydrolysis ,Saponins ,Enzyme Activation ,chemistry ,MCF-7 ,Biochemistry ,Caspases ,Cancer cell ,Molecular Medicine ,Female ,Drug Screening Assays, Antitumor ,Poly(ADP-ribose) Polymerases - Abstract
Achyranthoside H methyl ester (AH-Me) is an oleanolic acid saponin derivative isolated from the roots of Achyranthes fauriei through diazomethane treatment. AH-Me exhibited significant cytotoxicity against human breast cancer MCF-7 and MDA-MB-453 cells, with respective ID(50) values of 4.0 and 6.5 muM: in the MTT assay. AH-Me is a unique saponin containing three methoxycarbonyl groups in the sugar moiety linked to the C-3 position of oleanolic acid. The demethylation of these methoxycarbonyl groups by alkaline hydrolysis caused a marked reduction of the cytotoxicity of AH-Me, suggesting that the methoxycarbonyl groups of AH-Me are key groups for the acquisition of cytotoxicity against human cancer cells. The staining of cancer cells with 4',6'-diamidino-2-phenylindole (DAPI) showed that the population of cells with altered nuclear morphology, for example chromatin condensation and fragmentation, increased markedly after AH-Me treatment. Exposure of MCF-7 and MDA-MB-453 cells to AH-Me resulted in a dose-dependent and time-dependent increase in the sub-G1 population, and in the cleavage of poly-ADP-ribose polymerase (PARP) followed by the formation of an 89 kD peptide. Pretreatment of the cells with the pan-caspase inhibitor z-VAD-fmk abolished the cleavage of PARP by AH-Me treatment and suppressed the antiproliferative effect of AH-Me on tumor cell growth. These results together led to the suggestion that AH-Me induces apoptosis via the caspase activation pathway in human breast cancer cells, and apoptosis is the major mode of the cytotoxic effect triggered by AH-Me.
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- 2009
3. Two new glucuronide saponins, Achyranthosides G and H, from Achyranthes fauriei root
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Yumiko Hori, Yasuaki Hirai, Yoshiteru Ida, Yoshiyuki Kuchino, Motonori Fukumura, Kazuo Toriizuka, and Hidehiro Ando
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Magnetic Resonance Spectroscopy ,Molecular Structure ,biology ,Stereochemistry ,Saponins ,biology.organism_classification ,Plant Roots ,chemistry.chemical_compound ,Glucuronides ,chemistry ,Molecular Medicine ,Achyranthes ,Oleanolic Acid ,Glucuronide ,Oleanolic acid - Abstract
Two oleanolic acid saponins named achyranthosides G (1) and H (2) were newly isolated from Achyranthes fauriei root as methyl esters in addition to methyl esters of achyranthosides A - F and five oleanolic acid glucuronides (chikusetsusaponins IVa, V, 28-deglucosyl chikusetsusaponin V, pseudoginsenoside RT(1), and oleanolic acid 3-O-beta-D-glucuronopyranoside) as well as oleanolic acid 28-O-beta-D-glucopyranoside, beta-ecdysterone, and polypodine B. Their structures were characterized as follows on the basis of the chemical and spectroscopic evidences.
- Published
- 2007
4. Guanine Nucleotide Exchange Factor, Tiam1, Directly Binds to c-Myc and Interferes with c-Myc-mediated Apoptosis in Rat-1 Fibroblasts
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Masamitsu Tanaka, Yoshiro Otsuki, Haruhiko Sugimura, Chifumi Kitanaka, Takaharu Kamo, and Yoshiyuki Kuchino
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Cell ,Regulator ,Apoptosis ,RAC1 ,Biology ,Biochemistry ,Cell Line ,Proto-Oncogene Proteins c-myc ,medicine ,Animals ,Guanine Nucleotide Exchange Factors ,Humans ,T-Lymphoma Invasion and Metastasis-inducing Protein 1 ,Luciferase ,Molecular Biology ,Transcription factor ,Cell Nucleus ,Cell growth ,Proteins ,Cell Biology ,Fibroblasts ,Molecular biology ,Neoplasm Proteins ,Rats ,Cell biology ,medicine.anatomical_structure ,Guanine nucleotide exchange factor - Abstract
The transcription factor c-Myc is important for the control of cell growth, cell cycle progression, neoplasia, and apoptotic cell death. Recently, c-Myc-binding proteins, which bind either to the N-terminal domain or the C-terminal domain of c-Myc, have been proposed as the key molecules to realize the mechanisms of these multiple c-Myc functions. We report in the present study on another protein, Tiam1, which is a specific guanine nucleotide exchange factor of Rac1 and which binds to c-Myc and modulates several of its biological functions. We were able to detect the direct binding and in vivo association between c-Myc and Tiam1. The necessary role in this interaction of the Myc box II of c-Myc was revealed in the cell extracts. The additional discovery of the intranuclear localization of Tiam1 in Rat1 cells and in neuronal cells of the mouse brain suggests this interaction may occur in the nucleus. Overexpression of Tiam1 repressed the luciferase activity of c-Myc and also inhibited the c-Myc apoptotic activity through this protein-protein interaction. Taken together, we concluded that Tiam1 is another c-Myc regulator, working in the nuclei to control c-Myc-related apoptosis.
- Published
- 2003
5. A Cellular Mechanism That Reversibly Inactivates Pancaspase Inhibitor zAsp-CH2-DCB: A Potential Pitfall Causing Discrepancy between in Vitro and in Vivo Caspase Assays
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Akiko Kokubu, Takamasa Kayama, Jun Sunayama, Yoshiyuki Kuchino, Chifumi Kitanaka, Arata Tomiyama, and Kaori Sakurada
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Cytoplasm ,Biophysics ,Apoptosis ,Biochemistry ,Amino Acid Chloromethyl Ketones ,HeLa ,Neuroblastoma ,Tumor Cells, Cultured ,medicine ,Humans ,Staurosporine ,Protease Inhibitors ,Molecular Biology ,Caspase ,Aspartic Acid ,Enzyme Precursors ,Dose-Response Relationship, Drug ,biology ,Cell Biology ,biology.organism_classification ,Caspase Inhibitors ,Molecular biology ,In vitro ,Cell biology ,Kinetics ,Research Design ,Cell culture ,Caspases ,biology.protein ,Caspase 10 ,lipids (amino acids, peptides, and proteins) ,Poly(ADP-ribose) Polymerases ,Intracellular ,HeLa Cells ,medicine.drug - Abstract
Cell-permeable pancaspase inhibitors such as zAsp-CH2-DCB and zVAD-fmk are widely used to examine the involvement of caspases in cell death models. While examining the caspase-dependence of staurosporine (STS)-induced neuroblastoma cell death, we found that zVAD-fmk but not zAsp-CH2-DCB inhibits apoptosis. Time course analysis revealed that, in contrast to zVAD-fmk which constantly inhibited the processing of endogenous caspase substrates, zAsp-CH2-DCB inhibited substrate processing only for the first few hours after its addition to the culture medium. However, when the caspase activity in lysates prepared from cells treated with STS and zAsp-CH2-DCB was measured in vitro, quite unexpectedly, it was found that zAsp-CH2-DCB completely inhibits the STS-mediated activation of caspases throughout the observation period even when it apparently failed to inhibit the processing of caspase substrates within intact cells. These findings together suggest that there exists a cellular mechanism that inactivates zAsp-CH2-DCB in a reversible manner. This reversible inactivation was an active, intracellular process requiring de novo protein synthesis and was observed in another cell line HeLa and with different apoptotic stimuli such as ultraviolet irradiation. Our results have important implications that require consideration when designing experiments involving the use of caspase inhibitors as well as interpreting their results.
- Published
- 2002
6. Akt Protein Kinase Inhibits Non-apoptotic Programmed Cell Death Induced by Ceramide
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Toshihiro Mochizuki, Makoto Nakane, Nobuhito Saito, Akio Asai, Hideki Katagiri, Akira Tamura, Takaaki Kirino, Tomoichiro Asano, Chifumi Kitanaka, Sakae Tanaka, and Yoshiyuki Kuchino
- Subjects
Programmed cell death ,Ceramide ,Cell Survival ,Immunoblotting ,bcl-X Protein ,Apoptosis ,Cytochrome c Group ,Cysteine Proteinase Inhibitors ,Protein Serine-Threonine Kinases ,Mitochondrion ,Ceramides ,Biochemistry ,Adenoviridae ,Amino Acid Chloromethyl Ketones ,Membrane Potentials ,chemistry.chemical_compound ,Cytosol ,Proto-Oncogene Proteins ,Tumor Cells, Cultured ,Humans ,Enzyme Inhibitors ,Molecular Biology ,Protein kinase B ,Caspase ,Cell Death ,biology ,Kinase ,Cell Membrane ,Glioma ,Cell Biology ,Mitochondria ,Cell biology ,Microscopy, Electron ,Proto-Oncogene Proteins c-bcl-2 ,chemistry ,Caspases ,biology.protein ,Signal transduction ,Proto-Oncogene Proteins c-akt ,Protein Binding ,Signal Transduction - Abstract
A growing body of evidence now suggests that programmed cell death (PCD) occurs via non-apoptotic mechanisms as well as by apoptosis. In contrast to apoptosis, however, the molecular mechanisms involved in the regulation of non-apoptotic PCD remain only poorly understood. Here we show that ceramide induces a non-apoptotic PCD with a necrotic-like morphology in human glioma cells. Characteristically, the cell death was not accompanied by loss of the mitochondrial transmembrane potential, cytosolic release of cytochrome c from mitochondria, or the activation of the caspase cascade. Consistent with these characteristics, this ceramide-induced cell death was inhibited neither by the overexpression of Bcl-xL nor by the pan-caspase inhibitor zVAD-fmk. However, strikingly, the ceramide-induced non-apoptotic cell death was inhibited by the activation of the Akt/protein kinase B pathway through the expression of a constitutively active version of Akt. The results for the first time indicate that the Akt kinase, known to play an essential role in survival factor-mediated inhibition of apoptotic cell death, is also involved in the regulation of non-apoptotic PCD.
- Published
- 2002
7. The ciliate Euplotes octocarinatus expresses two polypeptide release factors of the type eRF1
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Tomonari Muramatsu, Aihua Liang, Claudia Brünen-Nieweler, Hildburg Beier, Klaus Heckmann, and Yoshiyuki Kuchino
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Genetics ,Sequence Homology, Amino Acid ,biology ,Molecular Sequence Data ,Gene Dosage ,Protein primary structure ,Tetrahymena ,Euplotes ,Nuclear Proteins ,General Medicine ,biology.organism_classification ,Stop codon ,Open reading frame ,Codon usage bias ,Complementary DNA ,Codon, Terminator ,Animals ,Eukaryotic release factors ,Amino Acid Sequence ,Cloning, Molecular ,Gene ,Peptide Termination Factors - Abstract
Amplification of macronuclear DNA of the ciliate Euplotes octocarinatus revealed the presence of two genes encoding putative polypeptide release factors (RFs) of the codon specific class-I type. They are named eRF1a and eRF1b, respectively. cDNA amplification revealed that both eRF1 genes are expressed. Determination of their copy numbers showed that they are similarly amplified to a level of about 27,000. The deduced protein sequences of the two genes are 57 and 58% identical with human eRF1 and 79% identical to each other. The gene encoding eRF1b possesses three in-frame UGA codons. This codon is known to encode cysteine in Euplotes; only UAA and UAG are used as stop codons in this organism. The primary structure of the two release factors is analyzed and compared with the primary structure of other eukaryotic release factors including the one of Tetrahymena thermophila which uses only UGA as a stop codon. eRF1a and eRF1b of Euplotes as well as eRF1 of Tetrahymena differ from human eRF1 and other class-I release factors of eukaryotes in a domain recently proposed to be responsible for codon recognition. Based on the changes which we observe in this region and the differential use of the stop codons in these two ciliates we predict the amino acids participating in stop codon recognition in eRF1 release factors.
- Published
- 2001
8. Actinomycin D and staurosporine, potent apoptosis inducers in vitro, are potentially effective chemotherapeutic agents against glioblastoma multiforme
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Yoshitaka Narita, Yoshiyuki Kuchino, Takaaki Kirino, and Akio Asai
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Cancer Research ,Programmed cell death ,Antineoplastic Agents ,Apoptosis ,Biology ,Toxicology ,Therapeutic index ,Tumor Cells, Cultured ,medicine ,Humans ,Staurosporine ,Pharmacology (medical) ,Viability assay ,Enzyme Inhibitors ,Pharmacology ,Cisplatin ,Antibiotics, Antineoplastic ,Dactinomycin ,Brain Neoplasms ,In vitro ,Nimustine ,Oncology ,Biochemistry ,Cancer research ,Drug Screening Assays, Antitumor ,Glioblastoma ,medicine.drug - Abstract
Purpose: Although chemotherapeutic protocols that include chloroethylnitrosoureas (CENUs), such as 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), have been a mainstay of treatment for glioblastomas, the clinical outcomes have been unsatisfactory. More effective chemotherapeutic protocols for these tumors will require clear delineation of more cytocidal and cytostatic chemotherapeutic drugs. Methods and Results: In this study, we measured the cytocidal effects of ACNU, cisplatin, actinomycin D, and staurosporine, administered within their therapeutic dose ranges, in the treatment of glioblastoma cells. As assessed by WST-1 colorimetric assay, the number of viable cells decreased markedly in T98G cultures treated with actinomycin D or staurosporine, to less than 20% of the level in control cultures at 72 h, but did not decrease or even increased after 6 days of treatment with ACNU. After treatment with cisplatin for 5 days, cell viability decreased to 30% of control. As assessed by fluorescence microscopic examination of nuclear staining by Hoechst 33258 and by electron microscopy, the majority of dead cells treated with actinomycin D, staurosporine, or cisplatin had morphologic features of apoptosis. Caspase-3 activity increased more than 20-fold in cells treated with actinomycin D, staurosporine, or cisplatin but increased less than fivefold in ACNU-treated cells. In addition to caspase-3 activation, western blot analysis demonstrated cleavage of caspase-2 during the apoptotic process. These findings indicate that actinomycin D and staurosporine potently induce apoptosis, whereas ACNU exerts mainly a cytostatic rather than a cytocidal effect. Conclusion: Actinomycin D and staurosporine and their derivatives are potentially effective chemotherapeutic agents against glioblastoma cells at least in vitro.
- Published
- 2000
9. Molecular cloning of the Lon protease gene fromThermus thermophilusHB8 and characterization of its gene product
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Yoshiyuki Kuchino, Hiroko Ao, Masaru Tanokura, Kenji Takahashi, Satoru Watanabe, Tomonari Muramatsu, and Yoshie Hirayama
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Proteases ,Protease ,Sequence analysis ,medicine.medical_treatment ,Nucleic acid sequence ,Biology ,Thermus thermophilus ,Molecular cloning ,biology.organism_classification ,Biochemistry ,Molecular biology ,Gene product ,medicine ,bacteria ,Peptide sequence - Abstract
The gene encoding Lon protease was isolated from an extreme thermophile, Thermus thermophilus HB8. Sequence analysis demonstrated that the T. thermophilus Lon protease gene (TT-lon) contains a protein-coding sequence consisting of 2385 bp which is ≈ 56% homologous to the Escherichia coli counterpart. As expected, the G/C content of TT-lon was 68%, which is significantly higher than that of the E. coli lon gene (52% G/C). The amino acid sequence of T. thermophilus Lon protease (TT-Lon) predicted from the nucleotide sequence contained several unique sequences conserved in other Lon proteases: (a) a cysteine residue at the position just before the putative ATP-binding domain; (b) motif A and B sequences required for composition of the ATP-binding domain; and (c) a serine residue at the proteolytic active site. Expression of TT-lon under the control of the T7 promoter in E. coli produced an 89-kDa protein with a yield of ≈ 5 mg·L−1. Recombinant TT-Lon (rTT-Lon) was purified to homogeneity by sequential column chromatography. The peptidase activity of rTT-Lon was activated by ATP and α-casein. rTT-Lon cleaved succinyl-phenylalanyl-leucyl-phenylalanyl-methoxynaphthylamide much more efficiently than succinyl-alanyl-alanyl-phenylalanyl-methoxynaphthylamide, whereas both peptides were cleaved with comparable efficiencies by E. coli Lon. These results suggest that there is a difference between TT-Lon and E. coli Lon in substrate specificity. rTT-Lon most effectively cleaved substrate peptides at 70 °C, which was significantly higher than the optimal temperature (37 °C) for E. coli Lon. Together, these results indicate that the TT-lon gene isolated from T. thermophilus HB8 actually encodes an ATP-dependent thermostable protease Lon.
- Published
- 1999
10. Regulation of c-Myc through Phosphorylation at Ser-62 and Ser-71 by c-Jun N-Terminal Kinase
- Author
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Hironobu Yamana, Kohji Noguchi, Chifumi Kitanaka, Toshihiro Mochizuki, Yoshiyuki Kuchino, and Akiko Kokubu
- Subjects
Paclitaxel ,Ultraviolet Rays ,Recombinant Fusion Proteins ,Apoptosis ,Biochemistry ,3T3 cells ,Proto-Oncogene Proteins c-myc ,Mice ,medicine ,Animals ,Humans ,Phosphorylation ,Molecular Biology ,Kinase ,Cell growth ,Chemistry ,c-jun ,JNK Mitogen-Activated Protein Kinases ,Wild type ,3T3 Cells ,Cell Biology ,Molecular biology ,Cell biology ,medicine.anatomical_structure ,Gene Expression Regulation ,Mutagenesis ,Mitogen-Activated Protein Kinases ,Signal transduction ,HeLa Cells ,Protein Binding ,Signal Transduction - Abstract
The expression of c-myc promotes cell proliferation and also sensitizes cells to various extracellular apoptotic stimuli. However, signal pathways regulating the function of Myc proteins during apoptosis are unknown. c-Jun N-terminal kinase (JNK) is activated by various apoptotic stimuli, but neither the target molecule(s) or the action of JNK has been identified in Myc-mediated apoptosis. Here, we found that JNK selectively interacted with, and phosphorylated, c-Myc at Ser-62 and Ser-71 as confirmed with phospho-c-Myc-specific antibodies. Interestingly, dominant negative mutant JNK(APF) impaired the c-Myc-dependent apoptosis, but not mutated c-Myc (S62A/S71A)-dependent apoptosis triggered by UV irradiation. Furthermore, c-Myc (S62A/S71A)-expressing NIH3T3 cells were not sensitized like wild type c-Myc-expressing NIH3T3 cells to JNK-activating apoptotic stimuli, such as UV and Taxol. These results indicate that the JNK pathway is selectively involved in the c-Myc-mediated apoptosis and that the apoptotic function of c-Myc is directly regulated by JNK pathway through phosphorylation at Ser-62 and Ser-71.
- Published
- 1999
11. Caspase-independent programmed cell death with necrotic morphology
- Author
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Yoshiyuki Kuchino and Chifumi Kitanaka
- Subjects
Intracellular Fluid ,Programmed cell death ,Necrosis ,Cell Death ,biology ,Necroptosis ,Intrinsic apoptosis ,Autophagy ,Apoptosis ,Cell Biology ,Cell biology ,Paraptosis ,Caspases ,ras Proteins ,medicine ,biology.protein ,Animals ,Humans ,medicine.symptom ,Molecular Biology ,Caspase ,Signal Transduction - Abstract
Cell death is generally classified into two large categories: apoptosis represents active, programmed cell death, while necrosis represents passive cell death without underlying regulatory mechanisms. Recent progress revealed that caspases, a family of cysteine proteases, play a central role in the regulation of apoptosis. Unexpectedly, however, caspase inhibition occasionally turns the morphology of programmed cell death from apoptotic into necrotic without inhibiting death itself. In this article, we review different models of caspase-independent programmed cell death showing necrotic-like morphology, including our Ras-mediated caspase-independent cell death. Based on these findings, we suggest the existence of a necrotic-like cell death regulated by cellular intrinsic death programs distinct from that of apoptosis. Even though type 2 physiological cell death, or autophagic degeneration, has been recognized as a necrotic-like programmed cell death for a long time, the underlying molecular mechanisms have not been identified despite its physiological significance. This has been in part due to the previous absence of adequate caspase-independent cellular models to study, recent efforts may now help to elucidate these mechanisms.
- Published
- 1999
12. Physical and Functional Interactions between Pim-1 Kinase and Cdc25A Phosphatase
- Author
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Akio Asai, Chifumi Kitanaka, Yoshiyuki Kuchino, Kohji Noguchi, Tomonari Muramatsu, and Toshihiro Mochizuki
- Subjects
Serine/threonine-specific protein kinase ,Cyclin-dependent kinase 1 ,MAP kinase kinase kinase ,Cell Biology ,Mitogen-activated protein kinase kinase ,Biology ,Biochemistry ,MAP2K7 ,Cell biology ,hemic and lymphatic diseases ,ASK1 ,Cyclin-dependent kinase 9 ,c-Raf ,Molecular Biology - Abstract
The pim-1 oncogene encodes a serine/threonine kinase (Pim-1) involved in the transduction of cytokine-triggered mitogenic signals. Pim-1 is unique in that it closely cooperates with c-Myc not only in oncogenesis, but also in apoptosis induction. However, the molecular basis of Pim-1 function remains poorly understood, largely because the downstream effector molecule(s) for Pim-1 kinase has not been identified. Here we provide several lines of evidence that Cdc25A cell cycle phosphatase, a direct transcriptional target for c-Myc, is a substrate for Pim-1 kinase and functions as an effector for Pim-1. We found that Pim-1 physically interacts with Cdc25A both in vitro and in vivoand phosphorylates Cdc25A. We also observed that Pim-1-mediated phosphorylation of Cdc25A increases its phosphatase activity. In addition, wild-type Pim-1, but not kinase-inactive Pim-1, enhanced Cdc25A-mediated cellular transformation and apoptosis. Our results indicate that Cdc25A might be a key molecule that links Pim-1 and c-Myc and that also ties Pim-1-mediated mitogenic signals to cell cycle machinery.
- Published
- 1999
13. Oncogenic Ras triggers cell suicide through the activation of a caspase-independent cell death program in human cancer cells
- Author
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Shunji Chi, Kohji Noguchi, Wenbin Chen, Hideaki Fujita, Shigeyuki Yokoyama, Masaru Himeno, Toshihiro Mochizuki, Yoshiyuki Kuchino, Yohji Nagashima, Chifumi Kitanaka, Midori Yoshida, Akio Asai, and Mikako Shirouzu
- Subjects
Cancer Research ,Programmed cell death ,Recombinant Fusion Proteins ,DNA Fragmentation ,Cysteine Proteinase Inhibitors ,Transfection ,medicine.disease_cause ,Proto-Oncogene Proteins p21(ras) ,Phagocytosis ,Stomach Neoplasms ,Genetics ,medicine ,Humans ,Molecular Biology ,Caspase ,Cell Nucleus ,Cell Death ,biology ,Oncogene ,Brain Neoplasms ,Autophagy ,Glioma ,Neoplasm Proteins ,Gene Expression Regulation, Neoplastic ,Cell Transformation, Neoplastic ,Genes, ras ,Proto-Oncogene Proteins c-bcl-2 ,Urinary Bladder Neoplasms ,Cell culture ,Apoptosis ,Caspases ,Vacuoles ,Neoplastic Stem Cells ,biology.protein ,Cancer research ,Signal transduction ,Glioblastoma ,Lysosomes ,Carcinogenesis ,Signal Transduction - Abstract
To prevent neoplasia, cells of multicellular organisms activate cellular disposal programs such as apoptosis in response to deregulated oncogene expression, making the suppression of such programs an essential step for potentially neoplastic cells to become established as clinically relevant tumors. Since the mutation of ras proto-oncogenes, the most frequently mutated proto-oncogenes in human tumors, is very rare in some tumor types such as glioblastomas and gastric cancers, we hypothesized that mutated ras genes might activate a cell death program that cannot be overcome by these tumor types. Here we show that the expression of oncogenically mutated ras gene induces cellular degeneration accompanied by cytoplasmic vacuoles in human glioma and gastric cancer cell lines. Cells dying as a result of oncogenic Ras expression had relatively well-preserved nuclei that were negative for TUNEL staining. An immunocytochemical analysis demonstrated that the cytoplasmic vacuoles are derived mainly from lysosomes. This oncogenic Ras-induced cell death occurred in the absence of caspase activation, and was not inhibited by the overexpression of anti-apoptotic Bcl-2 protein. These observations suggested that oncogenic Ras-induced cell death is most consistent with a type of programmed cell death designated 'type 2 physiological cell death' or 'autophagic degeneration', and that this cell death is regulated by a molecular mechanism distinct from that of apoptosis. Our findings suggest a possible role for this non-apoptotic cell death in the prevention of neoplasia, and the activation of the non-apoptotic cell death program may become a potential cancer therapy complementing apoptosis-based therapies. In addition, the approach used in this study may be a valuable way to find genetically-regulated cell suicide programs that cannot be overcome by particular tumor types.
- Published
- 1999
14. Molecular cloning and chromosomal mapping of mouse intronless myc gene acting as a potent apoptosis inducer
- Author
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Akinori Sugiyama, Michihiro C. Yoshida, Naoko Katou, Yoshiyuki Kuchino, Fumio Tashiro, Chifumi Kitanaka, Takeo Ono, and Kohji Noguchi
- Subjects
Transcriptional Activation ,Programmed cell death ,Molecular Sequence Data ,Genes, myc ,Apoptosis ,In situ hybridization ,Molecular cloning ,Biology ,Homology (biology) ,Cell Line ,Mice ,Open Reading Frames ,Transactivation ,Genetics ,Animals ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Gene ,In Situ Hybridization, Fluorescence ,MYC Family Gene ,Base Sequence ,Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ,Chromosome Mapping ,DNA ,General Medicine ,Transfection ,Molecular biology ,Introns ,Rats ,DNA-Binding Proteins ,Basic-Leucine Zipper Transcription Factors ,Dimerization ,HeLa Cells ,Transcription Factors - Abstract
Our previous findings suggest that the activation of the rat intronless myc gene provides a selective advantage in tumor suppression through apoptosis induction. In the present study, to examine whether intronless myc gene acting as an apoptosis inducer is evolutionarily conserved in mammalian cells, we isolated the mouse intronless myc gene and characterized it. A sequence analysis demonstrated that mouse intronless myc gene, ms- myc , has a linearly opened translatable frame consisting of 1293 bp with 90% homology with that of rat s- myc . The chromosomal locus of ms- myc was identified on chromosome 19B by a fluorescent in situ hybridization (FISH) analysis. Gene transfection experiments showed that the transient overexpression of ms-Myc with transactivation activity effectively induces cell death in a wild-type p53-independent manner. In addition, cells stably expressing transfected ms- myc became more susceptible to apoptosis induced by genotoxic stress such as UV-irradiation and hydrogen peroxide compared with untransfected control cells. These observations suggest that the rodents commonly contain an s- myc -type of intronless myc gene with apoptosis-inducing activity.
- Published
- 1999
15. Modified Nucleosides in the First Positions of the Anticodons of tRNA and tRNA from Escherichia coli
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Emanuel Goldman, Tatsuo Miyazawa, Susumu Nishimura, Shigeyuki Yokoyama, Kazuyuki Takai, Ziro Yamaizumi, Yoshiyuki Kuchino, and Nobuyuki Horie
- Subjects
chemistry.chemical_classification ,RNA ,Nuclear magnetic resonance spectroscopy ,Biology ,medicine.disease_cause ,Biochemistry ,chemistry ,Transfer RNA ,medicine ,Nucleotide ,Leucine ,Nucleoside ,Escherichia coli ,Gene - Abstract
Minor leucine tRNA species, tRNA and tRNA , from Escherichia coli B have been reported to recognize leucine codons UUA and UUG [Goldman, E., Holmes, W. M., and Hatfield, G. W. (1979) J. Mol. Biol. 129, 567−585]. In the present study, these two tRNALeu species were purified from E. coli A19, and the nucleotide sequences were determined by a post-labeling method. tRNA was found to correspond to the tRNA gene reported as su°6 tRNA [Yoshimura, M., Inokuchi, H., and Ozeki, H. (1984) J. Mol. Biol. 177, 627−644]. The first letter of the anticodon was identified to be 2‘-O-methylcytidine (Cm). tRNA was identified as the minor leucine tRNA that has been sequenced previously (tRNA ) [Yamaizumi, Z., Kuchino, Y., Harada, F., Nishimura, S., and McCloskey, J. A. (1980) J. Biol. Chem. 255, 2220−2225]. There was an unidentified modified nucleoside (N*) in the first position of the anticodon of tRNA . Nucleoside N* was isolated to homogeneity (1 A260 unit). By 1H NMR spectroscopy, nucleoside N* was found to be a 2‘-O-meth...
- Published
- 1998
16. A Functional Role for Death Proteases in s-Myc- and c-Myc-Mediated Apoptosis
- Author
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Yoshihide Tsujimoto, Yoshiyuki Kuchino, Chifumi Kitanaka, Yutaka Eguchi, Akinori Sugiyama, Akio Asai, Noriko Yasuhara, Shigehide Kagaya, Kohji Noguchi, and Toshihiro Mochizuki
- Subjects
Proteases ,Serine Proteinase Inhibitors ,medicine.medical_treatment ,Genes, myc ,Apoptosis ,Caspase 8 ,Culture Media, Serum-Free ,Proto-Oncogene Proteins c-myc ,chemistry.chemical_compound ,AEBSF ,medicine ,Animals ,Sulfones ,Molecular Biology ,Cells, Cultured ,Caspase ,Serine protease ,Protease ,biology ,Caspase 3 ,Intrinsic apoptosis ,Cell Biology ,Fibroblasts ,Molecular biology ,Recombinant Proteins ,Rats ,Cell biology ,Enzyme Activation ,Cysteine Endopeptidases ,chemistry ,Caspases ,Enzyme Induction ,biology.protein ,Signal Transduction ,Research Article - Abstract
Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 beta-converting enzyme (ICE)/CED-3 family protease (caspase) cascade. However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli such as Myc and p53. In this study, we provide lines of evidence that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc were induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate, Ac-DEVD-MCA, appeared in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death was effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Furthermore, exposing the cells to a serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), also significantly inhibited s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, AEBSF did not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the AEBSF-sensitive step in the signaling pathway of Myc-mediated apoptosis.
- Published
- 1997
17. Pim-1 kinase stimulates c-Myc-mediated death signaling upstream of caspase-3 (CPP32)-like protease activation
- Author
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Akinori Sugiyama, Shigehide Kagaya, Akio Asai, Shunji Chi, Chifumi Kitanaka, Yoshiyuki Kuchino, Kohji Noguchi, and Toshihiro Mochizuki
- Subjects
Cancer Research ,Recombinant Fusion Proteins ,Apoptosis ,Protein Serine-Threonine Kinases ,Biology ,Mitogen-activated protein kinase kinase ,Transfection ,Polymerase Chain Reaction ,Cell Line ,Proto-Oncogene Proteins c-myc ,Proto-Oncogene Proteins c-pim-1 ,Proto-Oncogene Proteins ,hemic and lymphatic diseases ,Endopeptidases ,Genetics ,Animals ,Kinase activity ,Protein kinase A ,Molecular Biology ,Serine/threonine-specific protein kinase ,Enzyme Precursors ,MAP kinase kinase kinase ,Caspase 3 ,Kinase ,Oncogenes ,Rats ,Cysteine Endopeptidases ,Cell Transformation, Neoplastic ,Caspases ,Cancer research ,Cyclin-dependent kinase 9 ,Signal transduction ,Signal Transduction - Abstract
Pim-1 oncoprotein is a serine/threonine kinase that can closely cooperate with c-Myc in lymphomagenesis, as does Bcl-2. Although the molecular mechanism of this cooperative transformation remains unknown, it is speculated that, similar to Bcl-2, Pim-1 contributes to transformation by inhibiting apoptosis. In this study, therefore, we examined the effect of Pim-1 expression on c-Myc-mediated apoptosis of Rat-1 fibroblasts triggered by serum deprivation. Our results showed that, rather than inhibiting apoptosis, Pim-1 expression stimulated c-Myc-mediated apoptosis in Rat-1 fibroblasts. Pim-1 stimulated c-Myc-mediated apoptosis through an enhancement of the c-Myc-mediated activation of caspase-3 (CPP32)-like proteases, since the suppression of this activity by a specific caspase inhibitor abolished the apoptosis stimulation by Pim-1. A kinase-defective Pim-1 mutant failed to stimulate c-Myc-mediated apoptosis, and Pim-1 expression alone in the absence of c-Myc overexpression did not induce apoptosis of serum-deprived Rat-1 cells, indicating that the kinase activity of Pim-1 and the activated c-Myc signaling pathway were required for apoptosis stimulation by Pim-1. Together, these results suggest that Pim-1 oncoprotein stimulates as a serine/threonine kinase the death signaling elicited by c-Myc at a step upstream of caspase-3-like protease activation in Rat-1 fibroblasts. Our results also suggest that Pim-1 kinase might function cooperatively with c-Myc through the phosphorylation of a factor(s) which regulates the common signaling pathway involved in c-Myc-mediated apoptosis and transformation.
- Published
- 1997
18. Primer/Template-Independent Synthesis of Poly d(A-T) by Taq Polymerase
- Author
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Takashi Odawara, Tomonari Muramatsu, Yoshiyuki Kuchino, Kenji Yamamoto, Chikateru Nozaki, Kenichi Hanaki, Kyosuke Mizuno, Hiroshi Yoshikura, and Michiaki Masuda
- Subjects
DNA polymerase ,DNA polymerase II ,Biophysics ,DNA-Directed DNA Polymerase ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Deoxyadenine Nucleotides ,Poly dA-dT ,DNA Nucleotidylexotransferase ,Thymine Nucleotides ,Taq Polymerase ,Molecular Biology ,Polymerase ,DNA Primers ,DNA clamp ,biology ,fungi ,Templates, Genetic ,Cell Biology ,Molecular biology ,Kinetics ,chemistry ,biology.protein ,Primase ,DNA polymerase mu ,Hot start PCR ,Taq polymerase - Abstract
Taq DNA polymerase polymerized dATP and dTTP to poly d(A-T) without requiring added primer/template in the temperature range of 60-70 degrees C. Tth DNA polymerase also catalyzed the reaction, while delta Tth, Vent, Vent(exo-), Pfu, Ultma, BcaBEST, and KOD DNA polymerases did not. The reaction was distinct from the template-nonrequiring terminal deoxynucleotidyl transferase reaction which absolutely required primers.
- Published
- 1997
19. Possible role of splice acceptor site in expression of unspliced gag-containing message of Moloney murine leukemia virus
- Author
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Aikichi Iwamoto, Yoshiyuki Kuchino, Masamichi Oshima, H Sakahira, Takashi Odawara, Hiroshi Yoshikura, and Tetsuro Matano
- Subjects
Gene Expression Regulation, Viral ,RNA Splicing ,viruses ,Molecular Sequence Data ,Immunology ,Mutant ,Gene Products, gag ,Regulatory Sequences, Nucleic Acid ,Microbiology ,Frameshift mutation ,Mice ,Virology ,Murine leukemia virus ,Animals ,Coding region ,splice ,Nucleotide ,Sequence Deletion ,chemistry.chemical_classification ,Messenger RNA ,Base Sequence ,biology ,Gene Products, env ,3T3 Cells ,biology.organism_classification ,Molecular biology ,Mutagenesis, Insertional ,chemistry ,Insect Science ,RNA splicing ,RNA, Viral ,Moloney murine leukemia virus ,Research Article - Abstract
Moloney murine leukemia virus (MLV) having the gag coding region alone, G3.6, produced a low level of mRNA (1/10 of the wild-type level). Ligation of 441 nucleotides (nt) containing a splice acceptor (SA) site to the downstream portion of the remaining gag region restored the level of the unspliced message, simultaneously activating a cryptic splice donor (SD) site in the middle of the p30 coding region (between nt 1596 and 1597). Ligation of the 441 nt in the same site in the inverted orientation also increased the level of the unspliced message, activating the same SD site (between nt 1596 and 1597) and a new SA site just in front of the inserted 441 nt (between nt 4770 and 4771). Deletion or inversion of the 441-nt SA sequence from the wild-type MLV or from int in-frame deletion or int frameshift mutant MLVs of nearly full size resulted in the loss of spliced mRNA and concomitantly in a severe reduction of the unspliced mRNA, particularly at 37 degrees C. Deletion of the 5' SD site did not result in the reduction of the unspliced-mRNA level. When the gag region in G3.6 was replaced with a Neo(r) coding region, the level of expression was high. The data taken together suggest that the presence of an SA signal is necessary for high-level expression of unspliced mRNA encoding Gag or Gag-Pol.
- Published
- 1996
20. Investigation of chemoresistance-related genes mRNA expression for selecting anticancer agents in successful adjuvant chemotherapy for a case of recurrent glioblastoma
- Author
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Motoo Nagane, Kazuhiro Nomura, Soichiro Shibui, Akio Asai, Yoshiyuki Kuchino, and Hiroshi Oyama
- Subjects
Pathology ,medicine.medical_specialty ,Methyltransferase ,medicine.medical_treatment ,Cellular detoxification ,Antineoplastic Agents ,Drug resistance ,O(6)-Methylguanine-DNA Methyltransferase ,Maintenance therapy ,medicine ,Humans ,Neoplasm Invasiveness ,RNA, Messenger ,RNA, Neoplasm ,Northern blot ,Cerebellar Neoplasms ,Etoposide ,Glutathione Transferase ,Chemotherapy ,Brain Neoplasms ,business.industry ,Methyltransferases ,Middle Aged ,Blotting, Northern ,Drug Resistance, Multiple ,Multiple drug resistance ,Chemotherapy, Adjuvant ,Drug Resistance, Neoplasm ,Cancer research ,Female ,Metallothionein ,Surgery ,Neurology (clinical) ,Neoplasm Recurrence, Local ,Glioblastoma ,business ,Brain Stem ,medicine.drug - Abstract
BACKGROUND Glioblastoma multiforme represents one of the most malignant forms of primary intracranial tumors, often intractable to multimodality of treatment including chemotherapy. The unsatisfactory results of chemotherapy are chiefly attributed to chemoresistance. Since various molecules that could confer drug resistance have been elucidated, screening of the amount of such molecules in the tumor cells could provide possibilities for predicting their chemoresistance beforehand and help select more effective drugs. METHODS We present a 45-year-old woman with recurrent glioblastoma multiforme in the cerebellum and invading the brain stem, treated successfully by postoperative chemotherapy. In this patient, anticancer drugs were determined by measurements of mRNA expression of chemoresistance-related genes, such as O6-methylguanine-DNA methyltransferase (MGMT), mdr1, glutathione S-transferase (GST)-pi, and metallothionein (MT) in the resected tumor. RESULTS Northern blot analysis demonstrated the moderate mRNA level of MGMT, a major molecule causing ACNU (1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride) resistance. On the other hand, expression levels of mdr1 which codes the P-glycoprotein responsible for multidrug resistance, and GST-pi, a detoxification enzyme, were low. Transcript of MT, another thiol containing molecule for cellular detoxification possibly associated with cisdiamminedichloroplatinum(II) (CDDP) resistance, was only faintly detectable. Postoperatively, the patient was treated initially with intravenous administration of ACNU and etoposide (VP16), resulting in a minor response of tumor regression. For maintenance therapy, we changed ACNU to CDDP according to the findings of the Northern blot analysis. Consequently, the residual tumor showed a marked response and almost disappeared after two courses of systemic chemotherapy with CDDP and VP16. CONCLUSIONS The successful tumor regression in this case suggests that Northern blot analysis on expression of these chemoresistance-related genes in tumor tissues could provide beneficial information for determination of optimal anticancer agents to improve the efficacy of chemotherapy.
- Published
- 1995
21. Establishment of Two Rat Osteosarcoma Cell Lines (YROS-1 and YROS-2) Induced by Radioactive Phosphorus
- Author
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Nobuyuki Yoshida, Ichiro Aoki, Y. Nagashima, Jiro Machida, Toru Hiruma, Kazuaki Misugi, Tomihisa Koshino, Yoshiyuki Kuchino, Y. Miyagi, and Shin-ichiro Watanabe
- Subjects
Pathology ,medicine.medical_specialty ,Lung Neoplasms ,Mice, Nude ,Matrix (biology) ,Pathology and Forensic Medicine ,Flow cytometry ,Mice ,Polysome ,Tumor Cells, Cultured ,medicine ,Animals ,Rats, Wistar ,Osteosarcoma ,medicine.diagnostic_test ,Chemistry ,Cell Biology ,Molecular biology ,In vitro ,Rats ,Cell culture ,Female ,Rat Osteosarcoma ,Phosphorus Radioisotopes ,Reticulum ,Cell Division ,Injections, Intraperitoneal ,Neoplasm Transplantation ,Radioactive phosphorus - Abstract
Summary Two rat osteosarcoma cell lines, YROS-1 and YROS-2, were established from two experimental osteosarcomas and induced by internal irradiation with radioactive phosphorus. Both cell lines formed a monolayer cell sheet in vitro with focal piling. The YROS-1 cells were re fractile and spindle or polygonal in shape, whereas the YROS-2 cells were flat, spread and polygonal in shape. Ultrastructurally, the YROS-1 cells had well-developed rough-surfaced endo plasmic reticulum with focal pericellular deposition of calcified matrix, whereas YROS-2 had abundant polysomes and intracytoplasmic filaments. Both cell lines grew stably with population doubling times o f 23 and 39 h, respectively. Flow cytometry revealed that YROS1 was rich in proliferating cells compared to YROS-2, with a higher colony-forming efficiency. YROS-1 showed high alkaline phosphatase activity, while YROS-2 possessed low activity. When subcutaneously transplanted into lumbodorsal area o f athymic nude mice, only YROS-1 formed tumors with frequent lung metastasis.
- Published
- 1995
22. Elevated levels of eukaryotic translation initiation factor eIF-4E, mRNA in a broad spectrum of transformed cell lines
- Author
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Yoshiyuki Kuchino, Sylvia J. Kerr, Akio Asai, Tomoko Okazaki, Yohei Miyagi, and Akinori Sugiyama
- Subjects
Cancer Research ,Molecular Sequence Data ,macromolecular substances ,In Vitro Techniques ,Biology ,environment and public health ,Cell Line ,Eukaryotic translation ,Peptide Initiation Factors ,Protein biosynthesis ,Animals ,Initiation factor ,heterocyclic compounds ,RNA, Messenger ,RNA, Neoplasm ,DNA Primers ,Base Sequence ,EIF4E ,Translation (biology) ,EIF4A1 ,Molecular biology ,Eukaryotic translation initiation factor 4 gamma ,Rats ,Gene Expression Regulation, Neoplastic ,EIF4EBP1 ,Cell Transformation, Neoplastic ,Eukaryotic Initiation Factor-4E ,Oncology - Abstract
Translation initiation factor eIF-4E, which binds to the 5′ cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Over-expression of eIF-4E in fibroblasts results in their malignant transformation. However, no information on eIF-4E expression in established transformed cell lines has been available. We report here that a variety of tumor cell lines, chemically, virally and oncogenically transformed, exhibit elevated levels of eIF-4E mRNA expression as compared to their normal counterparts. Overexpression of eIF-4E, which is normally rate-limiting in protein synthesis, may stimulate the translation of regulatory and oncogenic proteins involved in transformation.
- Published
- 1995
23. Establishment and Characterization of a Malignant Melanoma Cell Line (YP-MEL) Derived from a Patient with Neurocutaneous Melanosis
- Author
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Kazuaki Misugi, Makoto Umeda, Ichiro Aoki, Yohei Miyagi, Koichiro Ikuta, Yoshiyuki Kuchino, Y. Nagashima, and Tetsunori Funabiki
- Subjects
Male ,Pathology ,medicine.medical_specialty ,Skin Neoplasms ,Biopsy ,Nervous System Neoplasms ,Biology ,Pathology and Forensic Medicine ,Melanin ,Tumor Cells, Cultured ,medicine ,Humans ,Doubling time ,Melanoma ,neoplasms ,Melanins ,Staining and Labeling ,medicine.diagnostic_test ,Histocytochemistry ,Brain ,Chromosome Mapping ,Cell Biology ,medicine.disease ,Stimulation, Chemical ,Microscopy, Electron ,Neurocutaneous melanosis ,Giant cell ,Cell culture ,Child, Preschool ,Immunohistochemistry - Abstract
A cell line, YP-MEL, was established from an intracranial malignant melanoma occurring in a neurocutaneous melanosis (NCMsis) patient. The established cell line was successfully cultured in serum-free medium with a doubling time of 41 h. The cells were refractile and small in size, with occasional pigmented giant cells. Histochemical and immunohistochemical features were compatible with common malignant melanoma and its cell line. Chromosome analysis revealed many supernumerary chromosomes and marker chromosomes including double minutes (DMs). When transplanted into nude mice, YP-MEL formed tumors histologically consistent with the original tumor. Addition of sera to the medium caused cellular spreading and elongation of cytoplasmic processes with an increase of melanin contents and tyrosinase activity. Because there was no melanoma cell line derived from a NCMsis patient, YP-MEL might be a beneficial tool for study on NCMsis.
- Published
- 1994
24. Elevation of nerve growth factor synthesis by constitutive expression of v-src oncogene in cultured rat fibroblasts
- Author
-
Yoshio Ueno, Yoshiyuki Kuchino, Fumio Tashiro, Shoei Furukawa, Akiko Kitamura, Kazuko Shiroki, Kenji Ishii, Tatsuya Hagiwara, Wataru Habano, Naomitsu Hirano, Kiyomitsu Nemoto, and Fumio Omae
- Subjects
Transcription, Genetic ,Biology ,Gene expression ,Animals ,Nerve Growth Factors ,Northern blot ,Cells, Cultured ,Repetitive Sequences, Nucleic Acid ,Rous sarcoma virus ,Oncogene ,General Neuroscience ,Oncogenes ,Transfection ,Fibroblasts ,Protein-Tyrosine Kinases ,Blotting, Northern ,biology.organism_classification ,Molecular biology ,Rats ,Gene Expression Regulation, Neoplastic ,Cell Transformation, Neoplastic ,Nerve growth factor ,Avian Sarcoma Viruses ,nervous system ,Cell culture ,v-Src - Abstract
Rat fibroblast 3Y1 cells transformed by Rous sarcoma virus (RSV) or transfected with the v-src gene showed a highly constitutive v-src gene expression. Simultaneously, marked increases in the cellular level of nerve growth factor (NGF) mRNA and NGF content in the culture medium were observed. The levels of NGF mRNA and NGF secreted into the medium were correlated with the expression level of v-src mRNA gene in both transformants and control 3Y1 cells. These results suggest that v-src gene expression is relevant to regulation of NGF synthesis in rat 3Y1 fibroblasts.
- Published
- 1991
25. Point Mutation of theneuGene in Rat Neural Tumor RT4-AC Cells: Suppression of Tumorigenicity by s-Myc
- Author
-
Yoshiyuki Kuchino, Noboru Sueoka, Akinori Sugiyama, and Soo-Yong Lee
- Subjects
Cancer Research ,neu gene ,Guinea Pigs ,Molecular Sequence Data ,Cell ,Genes, myc ,Biology ,medicine.disease_cause ,Polymerase Chain Reaction ,s‐myc gene ,Peripheral Nervous System Neoplasms ,Sequence Homology, Nucleic Acid ,Gene expression ,medicine ,Animals ,Gene ,Tumor suppression ,Regulation of gene expression ,Mutation ,Point mutation ,DNA ,Blotting, Northern ,Molecular biology ,Neoplasm Proteins ,Rats ,Gene Expression Regulation, Neoplastic ,Blot ,Transmembrane domain ,Cell Transformation, Neoplastic ,medicine.anatomical_structure ,Oncology ,RNA ,Electrophoresis, Polyacrylamide Gel ,RT4‐AC cells ,Rapid Communication - Abstract
Forced expression of the s-myc gene suppressed the tumorigenicity of rat RT4-AC tumor cells in nude mice, as reported previously. Polymerase chain reaction (PCR) analysis indicated that RT4-AC cells established from a tumor of the peripheral nervous system contain an activated neu gene with a T----A transversion in the transmembrane domain. Synthesis of a protein of 60 kd kd in RT4-AC cells was specifically inhibited by expression of the s-myc gene. These results strongly suggest that s-Myc suppresses the transforming activity of rat neural cells transformed by expression of the activated neu gene, and plays an important role in regulating expression of a cellular gene contributing to cell transformation, such as the gene that encodes the p60 protein.
- Published
- 1990
26. Evidence for involvement of a nuclear envelope-associated RNA helicase activity in nucleocytoplasmic RNA transport
- Author
-
Durdica Ugarkovic, Werner E.G. Müller, Yoshiyuki Kuchino, Peter Langen, Michael Bachmann, Heinz C. Schröder, and August Dorn
- Subjects
Physiology ,Clinical Biochemistry ,RNA ,RNA-dependent RNA polymerase ,RNA transport ,Cell Biology ,Biology ,Non-coding RNA ,RNA Helicase A ,Biochemistry ,RNA polymerase I ,Biophysics ,Degradosome ,Small nuclear RNA - Abstract
It seems well established that translocation of at least some mRNAs through the nuclear pore is (1) an energy-dependent process, and (2) dependent on the presence of the poly(A) segment attached to most mRNA species. We describe that RNA helicase (RNA duplex unwindase) activity is present in a nuclear envelope (NE) preparation, which also appears to be involved in nucleocytoplasmic RNA transport. This activity unwinds RNA: RNA hybrids. The helicase has a pH optimum of 7.5 and a temperature optimum of 30 degrees C. Applying the sealed NE vesicle system, it was shown that duplex RNA species are readily released from the vesicles in an unidirectional manner, in contrast to single-stranded RNA, which is much slower transported into the extravesicular space. Attachment of a poly(A) segment to the RNA duplex additionally increases the efflux rate of this RNA. Efflux of duplex RNA but not efflux of single-stranded RNA was strongly inhibited by formycin B 5'-triphosphate. Our results suggest that, besides poly(A), duplex structures, if present in a given RNA, modulate and control the export of RNA.
- Published
- 1990
27. Protection of HeLa‐T4 + cells against human immunodeficiency virus (HIV) infection after stable transfection with HIV LTR‐2‘,5‘‐oligoadenylate synthetase hybrid gene 1
- Author
-
Yoshiyuki Kuchino, Durdica Ugarkovic, Werner E.G. Müller, Heinz C. Schröder, Takashi Okamoto, and Helmut Merz
- Subjects
Expression vector ,2'-5'-Oligoadenylate ,viruses ,Transfection ,Biology ,Biochemistry ,Virology ,Molecular biology ,Virus ,Long terminal repeat ,Gene product ,chemistry.chemical_compound ,chemistry ,Genetics ,Molecular Biology ,Hygromycin B ,Selectable marker ,Biotechnology - Abstract
An expression vector (pU3R-III/2-5AS) of human 2',5'-oligoadenylate (2-5A) synthetase was constructed in which a cDNA encoding an active form of the enzyme was located 3' to a 3'-long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). The LTR-directed expression of this hybrid DNA could be activated in trans by the HIV tat gene product. This vector was used for transfection of HeLa-T4+ cells, which are permissive to HIV infection, as well as of normal HeLa cells. HIV replication after infection of the CD4-receptor-bearing HeLa-T4+ cells with HIV-1 was found to be strongly reduced when drug-selected cells cotransfected with pU3R-III/2-5AS and a hygromycin B resistance gene containing plasmid were used. In nontransfected cultures or after transfection with the selectable marker plasmid only, about 60% p17- and p24-positive cells were found 5 days after infection. However, after stable transfection with pU3R-III/2-5AS the number of positive cells was decreased to about 2%. The reverse tran...
- Published
- 1990
28. 8-Hydroxydeoxyguanosine in dna Inhibits Restriction Endonuclease Digestion
- Author
-
Eiko Ohtsuka, Susumu Nishimura, Yoshiyuki Kuchino, Fumiko Nemoto, and David S. Jones
- Subjects
biology ,Chemistry ,Phagemid ,Biochemistry ,Molecular biology ,Restriction fragment ,Restriction enzyme ,chemistry.chemical_compound ,Recognition sequence ,Genetics ,biology.protein ,Replisome ,Primer (molecular biology) ,In vitro recombination ,DNA - Abstract
A DNA fragment containing 8-hydroxydeoxyguanosine was used to prime second strand synthesis of a recombinant pTZI9 phagemid single stranded DNA. The resultant double stranded closed circular product and also the annealed product of the single stranded DNA and the fragment were found to be resistant to cleavage by the restriction enzyme ApaLI in whose recognition sequence the 8-hydroxydeoxyguanosine had been inserted.
- Published
- 1990
29. Critical role for mitochondrial oxidative phosphorylation in the activation of tumor suppressors Bax and Bak
- Author
-
Ken Tachibana, Chifumi Kitanaka, Shinobu Serizawa, Arata Tomiyama, Yoshiyuki Kuchino, Hirotsugu Samejima, and Kaori Sakurada
- Subjects
Cancer Research ,Programmed cell death ,Oligomycin ,Cell Survival ,Protein Conformation ,Blotting, Western ,Respiratory chain ,Apoptosis ,Oxidative phosphorylation ,Antimycin A ,Biology ,Mitochondrion ,Cell Fractionation ,Endoplasmic Reticulum ,Oxidative Phosphorylation ,Mitochondrial Proteins ,chemistry.chemical_compound ,Confidence Intervals ,Tumor Cells, Cultured ,Animals ,Humans ,Immunoprecipitation ,bcl-2-Associated X Protein ,Cell growth ,Tunicamycin ,Fibroblasts ,Molecular biology ,Mitochondria ,Rats ,Cross-Linking Reagents ,bcl-2 Homologous Antagonist-Killer Protein ,Oncology ,chemistry ,Ultraviolet Therapy ,Reactive Oxygen Species ,Dimerization ,Glycolysis ,DNA Damage - Abstract
Background: Activation of Bax and Bak, which act to permeabilize the mitochondrial membrane, is an essential step in the cell death response and therefore in the suppression of tumorigenesis. However, the mechanisms that regulate activation are poorly understood. Methods: Bax and Bak activation (conformational change and dimerization) was monitored in Rat-1 fi broblasts and human cancer cells subjected to endoplas mic reticulum (ER) stress, DNA damage, or tumor necrosis factor- α (TNF- α ) treatment. Pharmacologic inhibitors of reactive oxygen species production, electron transport in the respiratory chain, oxidative phosphorylation, and appropriate controls were used to identify potential modes by which Bax and Bak activation and the cell death response are controlled. The oligomerization state of Bax and Bak was determined by cross-linking and sub sequent immunoblot analysis; Bax conformational change was analyzed by immunoprecipitation and immunoblotting with an antibody specifi c for the active conformation. Cell death was evaluated by dye exclusion. Results: In both fi broblasts and human cancer cells subjected to cell death stimuli, inhibition of oxidative phosphorylation by use of antimycin A or oligomycin prevented ER stress – , DNA damage – , and TNF- α – induced Bax and Bak activation and cell death (UV-induced Rat-1 cell death at 15 hours: control, mean = 33.6%, 95% confi dence interval [CI] = 18.8% to 48.4%; antimycin A, mean = 10.0%, 95% CI = 0% to 21.7%; oligomycin, mean = 13.1%, 95% CI = 5.7% to 20.5%; tunicamycin-induced MCF-7 cell death at 9 hours: control, mean = 29.2%, 95% CI = 21.6% to 36.8%; antimycin A, mean = 15.3%, 95% CI = 0.8% to 29.8%; oligomycin, mean = 11.5%, 95% CI = 3.9% to 19.1%; TNF- α – induced MCF-7 cell death at 6 hours: control, mean = 24.0%, 95% CI = 12.6% to 35.4%; antimycin A, mean = 8.9%, 95% CI = 3.9% to 13.9%; oligomycin, mean = 13.3%, 95% CI = 10.4% to 16.2%). Increasing and decreasing glycolytic adenosine triphosphate production, by adding glucose and 2-deoxy- D glucose to the cell growth medium, respectively, neither reversed nor recapitulated, respectively, the effect of compromised oxidative phosphorylation on Bax and Bak activation. Conclusion: Oxidative phosphorylation is required for the activation of Bax and Bak and cell death triggered by disparate death stimuli. The reliance of tumor cells on glycolysis in preference to oxidative phosphorylation even under normoxic conditions (Warburg effect) may therefore be a potential means by which these cells evade programmed cell death. [J Natl Cancer Inst 2006;98: 1462 – 73 ]
- Published
- 2006
30. Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32
- Author
-
Kaori Sakurada, Fumio Tashiro, Dongchon Kang, Yukiko Gotoh, Yoshiyuki Kuchino, Jun Sunayama, Chifumi Kitanaka, Arata Tomiyama, N Itoh, Akinori Sugiyama, and Y Ando
- Subjects
Signal peptide ,Recombinant Fusion Proteins ,Mutant ,Apoptosis ,Mitochondrion ,Biology ,Astrocytoma ,Pore forming protein ,Mitochondrial Proteins ,Transduction (genetics) ,Cell Line, Tumor ,Proto-Oncogene Proteins ,Two-Hybrid System Techniques ,Chlorocebus aethiops ,Animals ,Humans ,Amino Acid Sequence ,RNA, Small Interfering ,Molecular Biology ,Conserved Sequence ,Fluorescent Dyes ,Glutathione Transferase ,Sequence Deletion ,Inhibitor of apoptosis domain ,Binding Sites ,Rhodamines ,Neuropeptides ,Colocalization ,Nuclear Proteins ,Cell Biology ,Immunohistochemistry ,Precipitin Tests ,Peptide Fragments ,Cell biology ,body regions ,Microscopy, Fluorescence ,COS Cells ,Apoptosis Regulatory Proteins ,Carrier Proteins ,Fluorescein-5-isothiocyanate ,Protein Binding - Abstract
Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74–282)) and the conserved C-terminal region (p32 (1–221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.
- Published
- 2004
31. Forced expression of antisense 14-3-3beta RNA suppresses tumor cell growth in vitro and in vivo
- Author
-
Akinori Sugiyama, Chifumi Kitanaka, Nobuya Kurabe, Fumio Tashiro, Yohei Miyagi, Yoji Nagashima, Naoko Kato, Yoshiyuki Kuchino, and Yuko Komiya
- Subjects
Cancer Research ,Telomerase ,Tyrosine 3-Monooxygenase ,Mice, Nude ,Biology ,medicine.disease_cause ,Transfection ,Mice ,Proto-Oncogene Proteins c-myb ,Liver Neoplasms, Experimental ,Gene expression ,medicine ,Tumor Cells, Cultured ,Animals ,RNA, Antisense ,Oncogene ,Cell growth ,General Medicine ,Molecular biology ,Rats ,Proto-Oncogene Proteins c-raf ,14-3-3 Proteins ,Liver ,Cell culture ,Signal transduction ,Carcinogenesis ,Cell Division ,Neoplasm Transplantation - Abstract
The 14-3-3 family proteins are key regulators of various signal transduction pathways including malignant trans- formation. Previously, we found that the expression of the 14-3-3b gene is deregulated as well as c-myc gene in afla- toxin B1 (AFB1)-induced rat hepatoma K1 and K2 cells. To elucidate the implication of 14-3-3b in tumor cell growth, in this paper we analyzed the effect of forced expression of antisense 14-3-3b RNA on the growth and tumorigenicity of K2 cells. K2 cells transfected with antisense 14-3-3b cDNA expression vector diminished their growth ability in monolayer culture and in semi-solid medium. Expres- sion level of vascular endothelial growth factor mRNA was also reduced in these transfectants. Tumors that formed by the transfectants in nude mice were much smaller and histologically more benign tumors, because of their decreased level of mitosis compared with those of the par- ental cells. Frequency of apoptosis detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was increased in the transfectant-derived tumors accompanying the inhibition of angiogenesis. In addition, over-expression of 14-3-3b mRNA was observed in various murine tumor cell lines. These results suggest that 14-3-3b gene plays a pivotal role in abnormal growth of tumor cells in vitro and in vivo. (3-6), Bcr/Bcr-Abl (7), polyoma middle T antigen (8), phos- phatidylinositol 3-kinase (9), protein kinase C (10), ASK-1 (11), BAD (12), Cdc25 (13,14), FKHRL1 transcription factor (15), keratin cytoskelton (16) and TERT telomerase subunit (17). However, the functional analysis of the 14-3-3 proteins in oncogenic transformation in vitro and in vivo is very limited. Recently Takihara et al. (18) reported that the enforced expression of 14-3-3b RNA in NIH3T3 cells confers on them a tumorigenicity in nude mice through the stimulation of mitogen-activated protein kinase (MAPK) cascade. Nonethe- less, there are conflicting reports that 14-3-3s related proteins are strongly down-regulated in SV40-transformed keratino- cytes, and in SV40- and v-Ha-ras-transformed epithelial cells (19,20). Moreover, the enforced expression of 14-3-3z in combination with ras or raf-1 cannot transform normal mouse fibroblasts (21). Thus, the real function of 14-3-3 pro- teins in oncogenic transformation is still vague. In the course of the study of aflatoxin B1 (AFB1) hepatocar- cinogenesis, we found that the expression of the 14-3-3b gene was deregulated together with the c-myc gene, which is known to cooperate with Raf-1 kinase for a cellular transformation, in AFB1-induced rat hepatocellular carcinoma K1 and K2 cells (22,23). Mutation of 14-3-3b gene locus was also detected in these cells (24). On the other hand, the mutation and/or deregu- lated expression of ras family oncogenes and suppressive oncogene such as p53 and Rb were not detected in K1 and K2 cells (24,25). Therefore, it is highly possible that 14-3-3b gene cooperates with c-myc gene in cellular transformation. In this paper, to investigate whether the over-expression of the 14-3-3b gene implicates in neoplastic phenotype of K2 cells, we established K2 cells expressing reduced level of 14-3-3b mRNA by the stable transfection with antisense 14-3-3b cDNA expression vector and analyzed their growth ability in vitro and in vivo. As we would expect, the antisense transfectants diminished their growth ability in the soft agar medium as well as in the monolayer culture. Tumors that formed by these transfectants in nude mice were much smaller and histologically more benign tumors. In these tumors, high frequency of apoptosis and inhibition of angiogenesis were also observed. Moreover, by the simultaneous addition of antisense 14-3-3b and c-myc oligodeoxynucleotides (ODNs) the K2 cell growth was diminished in a synergistic manner. We also found that the 14-3-3b gene was over-expressed in various tumor cell lines. Thus, it seems likely that the 14-3-3b gene plays an important role in tumor cell growth perhaps through the cooperation with the c-myc gene.
- Published
- 2003
32. Increased Ras expression and caspase-independent neuroblastoma cell death: possible mechanism of spontaneous neuroblastoma regression
- Author
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Hisato Kigasawa, Yukichi Tanaka, Takashi Momoi, Toshiji Nishi, Shigeyuki Yokoyama, Akira Nakagawara, Keisuke Kato, Chifumi Kitanaka, Yoshiyuki Kuchino, Arata Tomiyama, Yasunori Toyoda, Mikako Shirouzu, Yohji Nagashima, Kohji Noguchi, Rieko Ijiri, and Kaori Sakurada
- Subjects
Cancer Research ,Programmed cell death ,Pathology ,medicine.medical_specialty ,Apoptosis ,In Vitro Techniques ,Transfection ,Immunoenzyme Techniques ,Neuroblastoma ,Predictive Value of Tests ,medicine ,Biomarkers, Tumor ,Tumor Cells, Cultured ,Humans ,Receptor, trkA ,Child ,Caspase ,biology ,Apoptotic DNA fragmentation ,Infant ,Membrane Proteins ,DNA, Neoplasm ,medicine.disease ,Prognosis ,Up-Regulation ,Tissue Degeneration ,Gene Expression Regulation, Neoplastic ,Microscopy, Electron ,Genes, ras ,Oncology ,Neoplasm Regression, Spontaneous ,Caspases ,Child, Preschool ,biology.protein ,Cancer research ,ras Proteins ,Immunohistochemistry ,Carrier Proteins ,Signal Transduction - Abstract
Background: Neuroblastoma undergoes spontaneous regression frequently during its natural course. Although programmed cell death (PCD) has been implicated in this process, accumulating evidence suggests that apoptosis, a form of PCD that is regulated by caspases, may not play a major role. We examined the mechanism(s) of spontaneous regression of neuroblastoma, focusing on the role of Ras, a favorable prognostic marker of neuroblastoma. Methods: Tumor tissues were analyzed by light microscopy, electron microscopy, and immunohistochemistry to examine cell degeneration and expression of Ras and several indicators of PCD. Cell degeneration was also studied in vitro in neuroblastoma cells transfected with the H-ras gene. All statistical tests were two-sided. Results: Immunohistochemical analyses revealed that Ras expression was increased in areas of cellular degeneration lacking apoptotic characteristics. The degenerating cells were fragmented without nuclear condensation and, essentially, lacked caspase-3 activation and apoptotic DNA fragmentation. These cells had ultrastructural features of autophagic degeneration, another form of PCD that is distinct from apoptosis. Focal areas of degeneration associated with Ras expression were seen more frequently in tumors from patients detected in a mass-screening program (53 [60.9%] of 87) than in tumors from clinically detected, advanced-stage patients over 1 year of age (7 [29.2%] of 24) (P = .006; chi-square test), suggesting a positive relationship between Ras-associated degeneration and probability of spontaneous regression/favorable prognosis. The characteristic features of Ras-associated nonapoptotic degeneration observed in tumor samples were recapitulated in vitro by transfection-mediated Ras expression, and Ras-mediated degeneration was augmented by TrkA, another favorable prognostic marker. Conclusions: High-level expression of H-Ras in neuroblastoma cells is associated with caspase cascade-independent, nonapoptotic PCD. This Ras-mediated nonapoptotic tumor cell death may play a key role in spontaneous regression of neuroblastoma.
- Published
- 2002
33. ASK1-signaling promotes c-Myc protein stability during apoptosis
- Author
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Akiko Kokubu, Yoshiyuki Kuchino, Chifumi Kitanaka, Kohji Noguchi, and Hidenori Ichijo
- Subjects
Biophysics ,Regulator ,Apoptosis ,Transfection ,Biochemistry ,3T3 cells ,Proto-Oncogene Proteins c-myc ,Mice ,Phosphoserine ,Ubiquitin ,medicine ,Tumor Cells, Cultured ,Animals ,Humans ,ASK1 ,Mitogen-Activated Protein Kinase 8 ,Phosphorylation ,Molecular Biology ,Ubiquitins ,Plant Proteins ,biology ,Arabidopsis Proteins ,Cell Biology ,3T3 Cells ,Cell biology ,Kinetics ,medicine.anatomical_structure ,biology.protein ,Signal transduction ,Mitogen-Activated Protein Kinases ,Signal Transduction - Abstract
We previously reported that JNK is involved in the regulation of c-Myc-mediated apoptosis triggered by UV irradiation and anticancer drug treatment. Here we show that ASK1 is an upstream regulator for c-Myc-mediated apoptosis triggered by UV, and we found a direct role for Ser-62 and Ser-71 in the regulation of protein stability and function of c-Myc. The ASK1-JNK pathway enhanced the protein stability of c-Myc through phosphorylation at Ser-62 and Ser-71, which was required for c-Myc-dependent apoptosis by ASK1-signaling. Interestingly, ASK1-signaling attenuated the degradation of ubiquitinated c-Myc without affecting the ubiquitination process. Together, these findings indicate that the ASK1-JNK pathway promotes the proapoptotic activity of c-Myc by modulating c-Myc protein stability through phosphorylation at Ser-62 and Ser-71.
- Published
- 2001
34. Molecular mechanism of stop codon recognition by eRF1: a wobble hypothesis for peptide anticodons
- Author
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Tomonari Muramatsu, Klaus Heckmann, Chifumi Kitanaka, and Yoshiyuki Kuchino
- Subjects
Silent mutation ,Models, Molecular ,Eukaryotic release factor 1 ,Conventional ,Molecular Sequence Data ,Biophysics ,Reading frame ,Biology ,Biochemistry ,Substrate Specificity ,Tetrahymena thermophila ,Sense Codon ,Evolution, Molecular ,Start codon ,Structural Biology ,Genetics ,Anticodon ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Codon recognition mechanism ,Euplotes octocarinatus ,Models, Genetic ,Cell Biology ,Genetic code ,Stop codon ,Protein Structure, Tertiary ,Open reading frame ,Eukaryotic Cells ,Genetic Code ,Codon usage bias ,Protein Biosynthesis ,Codon, Terminator ,Nucleic Acid Conformation ,Unconventional eukaryotic class I release factor ,Sequence Alignment ,Peptide Termination Factors - Abstract
We propose that the amino acid residues 57/58 and 60/61 of eukaryotic release factors (eRF1s) (counted from the N-terminal Met of human eRF1) are responsible for stop codon recognition in protein synthesis. The proposal is based on amino acid exchanges in these positions in the eRF1s of two ciliates that reassigned one or two stop codons to sense codons in evolution and on the crystal structure of human eRF1. The proposed mechanism of stop codon recognition assumes that the amino acid residues 57/58 interact with the second and the residues 60/61 with the third position of a stop codon. The fact that conventional eRF1s recognize all three stop codons but not the codon for tryptophan is attributed to the flexibility of the helix containing these residues. We suggest that the helix is able to assume a partly relaxed or tight conformation depending on the stop codon recognized. The restricted codon recognition observed in organisms with unconventional eRF1s is attributed mainly to the loss of flexibility of the helix due to exchanged amino acids.
- Published
- 2001
35. Expression pattern of chemoresistance-related genes in human malignant brain tumors: a working knowledge for proper selection of anticancer drugs
- Author
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Hiroshi Oyama, Motoo Nagane, Yoshiyuki Kuchino, Akio Asai, Kazuhiro Nomura, and Soichiro Shibui
- Subjects
Cancer Research ,Brain tumor ,Antineoplastic Agents ,Pharmacology ,Tetraspanin 29 ,Antigens, CD ,Glioma ,Tumor Cells, Cultured ,Medicine ,Humans ,Radiology, Nuclear Medicine and imaging ,Northern blot ,RNA, Messenger ,Etoposide ,Cisplatin ,Membrane Glycoproteins ,business.industry ,Brain Neoplasms ,O-6-methylguanine-DNA methyltransferase ,General Medicine ,medicine.disease ,Chemotherapy regimen ,Nimustine ,Oncology ,Doxorubicin ,Cancer cell ,Cancer research ,Genes, MDR ,business ,medicine.drug - Abstract
Background: In addition to traditional modalities such as surgical intervention and radiother apy, chemotherapy is a common therapeutic method for human malignant brain tumors. How ever, the effectiveness of chemotherapy is frequently hampered by cancer cell chemoresistance, resulting in an unsatisfactory outcome. To overcome this disadvantage, the proper selection of efficacious anticancer agents is required. Methods: The expression levels of chemoresistance-related genes, MGMT, mdr1, MRP, MTIIA and GST-n, in 28 surgical specimens of human brain tumors and in 10 human glioma cell lines were examined by Northern blot analysis. In addition, the SD1Q values of human gli oma cell lines against ACNU, COOP, ADM and VP16 were estimated by a cell survival assay. Results: The expression levels of each of the chemoresistance-related genes, except MRP, were generally higher in brain tumors than those in non-neoplastic brain tissues. MGMT expression correlated exclusively with ACNU resistance in all glioma cell lines examined (p = 0.0002). The transcriptional level of mdr1 in the tumor cells correlated with the 8010 values of VCR (p = 0.04) and ADM (p = 0.034). In contrast, the expression levels of MTIIA and GST-n did not correlate with resistance to any of the drugs tested. A correlation of MRP mRNA expres sion with multidrug resistance was not apparent in the 10 cell lines tested. Conclusions: The data indicate that knowledge of the expression levels of MGMTand mdr1 may be particularly useful for a more rational selection of drugs which are not influenced by these resistance genes and which have improved efficacy against human brain tumors.
- Published
- 2000
36. Differential role of the JNK and p38 MAPK pathway in c-Myc- and s-Myc-mediated apoptosis
- Author
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Yoshiyuki Kuchino, Chifumi Kitanaka, Toshihiro Mochizuki, Akiko Kokubu, Hironobu Yamana, and Kohji Noguchi
- Subjects
Chloramphenicol O-Acetyltransferase ,Ultraviolet Rays ,p38 mitogen-activated protein kinases ,Mutant ,Biophysics ,Apoptosis ,MAP Kinase Kinase 6 ,Transfection ,Biochemistry ,p38 Mitogen-Activated Protein Kinases ,Cell Line ,Proto-Oncogene Proteins c-myc ,Transactivation ,Genes, Reporter ,Animals ,Humans ,Molecular Biology ,Caspase ,Mitogen-Activated Protein Kinase Kinases ,COS cells ,biology ,Chemistry ,JNK Mitogen-Activated Protein Kinases ,Cell Biology ,Recombinant Proteins ,Cell biology ,Rats ,Kinetics ,Cell culture ,Caspases ,COS Cells ,Calcium-Calmodulin-Dependent Protein Kinases ,biology.protein ,Mitogen-Activated Protein Kinases ,HeLa Cells - Abstract
The s-Myc is similar to c-Myc in its ability to induce apoptosis requiring caspase activation. However, s-Myc is distinct from c-Myc in that it has activity to suppress tumor growth and does not require wild-type p53 to induce apoptosis. These facts suggest differential regulation between s-Myc and c-Myc. Here we showed that s-Myc-mediated apoptosis triggered by UV was not inhibited by the inactive form mutant JNK (APF), though c-Myc-mediated apoptosis was. Moreover, we found that JNK did not affect the transactivation activity of s-Myc, but stimulated that of c-Myc. In contrast, both Myc-mediated apoptosis and caspase-3-like protease activation were suppressed by kinase-negative MKK6 and an inactive form mutant p38(AGF). Our results indicate that s-Myc does not require the JNK signaling unlike c-Myc during UV-triggered apoptosis, but the MKK6/p38MAPK pathway might regulate common apoptotic machinery for both s-Myc and c-Myc upstream of caspase.
- Published
- 2000
37. High level calcineurin activity predisposes neuronal cells to apoptosis
- Author
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Jian-hua Qiu, Nobuhito Saito, Yoshiyuki Kuchino, Akio Asai, Nobusada Shinoura, Shunji Chi, Yoshitaka Narita, Takaaki Kirino, and Hirofumi Hamada
- Subjects
Recombinant Fusion Proteins ,Phosphatase ,Gene Expression ,Caspase 3 ,Apoptosis ,Cysteine Proteinase Inhibitors ,Transfection ,Biochemistry ,PC12 Cells ,Tacrolimus ,Cell Line ,Viral Proteins ,Cyclosporin a ,Nerve Growth Factor ,Animals ,Humans ,Genetic Predisposition to Disease ,Rats, Wistar ,Molecular Biology ,Calcimycin ,Serpins ,Cerebral Cortex ,Neurons ,biology ,Ionophores ,L-Lactate Dehydrogenase ,Cytochrome c ,Adenoviruses, Human ,Calcineurin ,Cell Biology ,Cell biology ,Rats ,Enzyme Activation ,Proto-Oncogene Proteins c-bcl-2 ,Cell culture ,Caspases ,biology.protein ,Cyclosporine ,Calcium - Abstract
Calcineurin is a Ca(2+)/calmodulin-dependent protein phosphatase that is abundantly expressed in several specific areas of the brain, which are exceptionally vulnerable to stroke, epilepsy, and neurodegenerative diseases. In this study, we assessed the effects of high level activity of calcineurin on neuronal cells. Virus-mediated high level constitutive activity of calcineurin rendered neuronal cells susceptible to apoptosis induced by serum reduction or by a brief exposure to calcium ionophore. Adenovirus-mediated, high level forced activity of calcineurin induced cytochrome c/caspase-3-dependent apoptosis in neurons. Preincubation with the calcineurin inhibitors cyclosporin A and FK506 reduced susceptibility to apoptosis. High level constitutive expression of Bcl-2 or CrmA or incubation with a specific caspase-3 inhibitor inhibited the calcineurin-induced apoptosis. These data indicate that high level constitutive activity of calcineurin predisposes neuronal cells to cytochrome c/caspase-3 dependent apoptosis even under sublethal conditions.
- Published
- 1999
38. Heparin-binding epidermal growth factor-like growth factor stimulates mitogenic signaling and is highly expressed in human malignant gliomas
- Author
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Yoshiyuki Kuchino, Y. Nagashima, Naoyuki Taniguchi, Chifumi Kitanaka, Takaaki Kirino, Kazuhiko Mishima, Akio Asai, Shigeki Higashiyama, and Kazuko Yamaoka
- Subjects
medicine.medical_specialty ,EGF Family of Proteins ,Heparin-binding EGF-like growth factor ,medicine.medical_treatment ,Basic fibroblast growth factor ,Mitosis ,Biology ,Amphiregulin ,Pathology and Forensic Medicine ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Epidermal growth factor ,Transforming Growth Factor beta ,Internal medicine ,Glioma ,medicine ,Tumor Cells, Cultured ,Humans ,Growth factor receptor inhibitor ,RNA, Messenger ,Autocrine signalling ,Growth Substances ,Glycoproteins ,Epidermal Growth Factor ,Growth factor ,Transforming Growth Factor alpha ,medicine.disease ,Recombinant Proteins ,ErbB Receptors ,Endocrinology ,chemistry ,Cancer research ,Intercellular Signaling Peptides and Proteins ,Neurology (clinical) ,Glioblastoma ,hormones, hormone substitutes, and hormone antagonists ,Cell Division ,Heparin-binding EGF-like Growth Factor ,Signal Transduction - Abstract
We previously reported that schwannoma-derived growth factor (SDGF), a member of heparin-binding epidermal growth factor (EGF) family, participates in autocrine pathways and promotes rat glioma cell growth. To investigate the potential role of similar molecules in human gliomas, we examined 7 human glioma cell lines and 11 glioblastoma specimens for expression of the human homologue of SDGF, amphiregulin (AR), as well as heparin-binding EGF-like growth factor (HB-EGF). Northern blot analysis revealed that only one cell line and no tumor specimens expressed AR mRNA. In contrast, HB-EGF mRNA was expressed in all human glioma cell lines and its level of expression was two- to five-fold higher than that of control brain tissues in 8 of 11 glioblastoma cases. Immunohistochemistry demonstrated that membrane-anchored HB-EGF (proHB-EGF) and EGFR were co-expressed in 44% of 34 human malignant gliomas. Introduction of exogenous HB-EGF (10 ng/ml) increased human glioma cell proliferation, and anti-HB-EGF blocking antibodies reduced the growth of glioma cells by 30-40%, confirming the presence of an autocrine loop. When added to the medium, transforming growth factor-alpha, basic fibroblast growth factor, or HB-EGF rapidly induced HB-EGF mRNA expression. These results indicate that HB-EGF and proHB-EGF contribute to the growth of human malignant glioma cells, most likely through autocrine and juxtacrine mechanisms.
- Published
- 1998
39. Two different reactions involved in the primer/template-independent polymerization of dATP and dTTP by Taq DNA polymerase
- Author
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N. Nakajima, Chikateru Nozaki, Kyosuke Mizuno, Kenichi Hanaki, Tomonari Muramatsu, Hiroshi Yoshikura, Takashi Odawara, Yoshiyuki Kuchino, and Yohko K. Shimizu
- Subjects
Exonuclease ,DNA polymerase ,Polymers ,Biophysics ,Oligonucleotides ,DNA-Directed DNA Polymerase ,Biochemistry ,Deoxyadenine Nucleotides ,Thymine Nucleotides ,Taq Polymerase ,Molecular Biology ,Polymerase ,DNA Primers ,chemistry.chemical_classification ,biology ,Oligonucleotide ,Temperature ,Nucleic Acid Precursors ,Cell Biology ,DNA ,Templates, Genetic ,Amino acid ,Molecular Weight ,chemistry ,Polymerization ,Ethylmaleimide ,biology.protein ,Primer (molecular biology) ,Elongation - Abstract
Taq and Tth DNA polymerases catalyzed polymerization of dATP and dTTP into poly d(A-T) without requiring added primer/template (Hanaki et al., Biochem. Biophys. Res. Commun. 238, 113-118), while the Stoffel fragment of Taq DNA polymerase and delta Tth DNA polymerase with respective deletions of ca. 290 and 250 N-terminal amino acids did not. The primer/template-independent polymerization appeared to proceed via two reactions, the slow process of formation of 16-19 nt long oligo d(A-T) without primer/template and the rapid process of elongation of the oligo d(A-T) by self-priming. As the former step was more sensitive to N-ethylmaleimide than the elongation reaction, probably the formation of the oligonucleotide preceded the elongation. But when the substrates were depleted, Taq DNA polymerase degraded the high molecular weigh d(A-T) polymer to the oligomers which were resistant to the further digestion by the 5'--3' exonuclease activity of Taq DNA polymerase. Probably, the elongation and the degradation reactions proceeded simultaneously, the former process being faster than the latter in the presence of enough dATP and dTTP.
- Published
- 1998
40. Caspase-dependent apoptosis of COS-7 cells induced by Bax overexpression: differential effects of Bcl-2 and Bcl-xL on Bax-induced caspase activation and apoptosis
- Author
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Kohji Noguchi, Yoshihide Tsujimoto, Yoshiyuki Kuchino, Akemi Hayashi, Shigehide Kagaya, Toshihiro Mochizuki, Takahiro Namiki, Chifumi Kitanaka, Shunji Chi, and Akio Asai
- Subjects
Cancer Research ,Proteases ,Programmed cell death ,biology ,Gene Expression ,Bcl-xL ,Apoptosis ,Molecular biology ,Caspase-Dependent Apoptosis ,Cell biology ,Enzyme Activation ,Cysteine Endopeptidases ,Cell culture ,Proto-Oncogene Proteins ,Gene expression ,COS Cells ,Genetics ,biology.protein ,Animals ,Protease Inhibitors ,Molecular Biology ,Caspase - Abstract
Bcl-2 family proteins and ICE/CED-3 family proteases (caspases) are regarded as the basic regulators of apoptotic cell death. They are evolutionarily conserved and implicated in a variety of apoptosis. However, the precise mechanism by which these two families interact to regulate cell death is not yet known. In this study, we found that the overexpression of the Bcl-2 family member Bax induced apoptotic cell death in COS-7 cells through the activation of CPP32 (caspase-3)-like proteases that cleaved the DEVD tetrapeptide. This apoptotic cell death was suppressed by the viral proteins CrmA and p35, as well as by the chemically synthesized caspase inhibitors Z-Asp-CH2-DCB and zVAD-fmk. We also found that the Bax-induced apoptosis of COS-7 cells was suppressed by Bcl-xL and Bcl-2, though both Bcl-xL and Bcl-2 similarly prevented etoposide-induced apoptosis in COS-7 cells. In addition, Bcl-xL inhibited the activation of caspase-3-like proteases accompanying Bax-induced COS-7 cell death but Bcl-2 did not. These results indicate that the caspase activation is essential for Bax-induced apoptosis, and that the ability of Bcl-2 and Bcl-xL to prevent the Bax-induced caspase activation and apoptosis in COS-7 cells could be differentially regulated. Our results also suggest that Bcl-2 family proteins function upstream of caspase activation and control apoptosis through the regulation of caspase activity.
- Published
- 1997
41. Partial purification and characterization of a CAAX-motif-specific protease from bovine brain using a novel fluorometric assay
- Author
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Tomonari Muramatsu, Wataru Nishii, Yoshiyuki Kuchino, Shigeyuki Yokoyama, and Kenji Takahashi
- Subjects
Proteolysis ,medicine.medical_treatment ,Size-exclusion chromatography ,Detergents ,Cysteine Proteinase Inhibitors ,Biochemistry ,Sensitivity and Specificity ,Sepharose ,Gel permeation chromatography ,Microsomes ,medicine ,Animals ,Molecular Biology ,Chelating Agents ,chemistry.chemical_classification ,Dansyl Compounds ,Chromatography ,Protease ,medicine.diagnostic_test ,Molecular mass ,Proteolytic enzymes ,Brain ,General Medicine ,Hydrogen-Ion Concentration ,Chromatography, Ion Exchange ,Peptide Fragments ,Cysteine Endopeptidases ,Enzyme ,Spectrometry, Fluorescence ,chemistry ,Solubility ,Chromatography, Gel ,Cattle ,Oligopeptides ,Phenanthrolines - Abstract
Proteolytic trimming of isoprenylated proteins, including Ras, at the C-terminal CAAX motifs is a key event in their activation. However, the protease responsible for the proteolysis has not been well characterized yet. In this study, we established a novel assay method for the enzyme using a fluorescent substrate, dansyl(Dns)-KSKTKC(S-farnesyl)VIM, with which we can assess the proteolytic activity with high sensitivity and more easily than by the former assay methods using radio-labeled substrates. Using this assay method, we purified the protease 104-fold from bovine brain microsomal membranes by Sepharose CL-6B gel filtration and DE-52 chromatography. The partially purified enzyme was shown to be an endoprotease specific to the farnesylated peptide and to have a K(m) value of 1.0 microM for Dns-KSKTKC(S-farnesyl)VIM. o-Phenanthroline and zinc chloride strongly inhibited the activity. Interestingly, however, m- and p-phenanthrolines were as effective as o-phenanthroline, indicating that the inhibition by o-phenanthroline is not simply due to its chelating action. The molecular mass of the protease was deduced to be 480 kDa by gel filtration. The enzymatic activity was lost during further attempts at chromatographic purification, but was partially recovered by mixing the chromatographic fractions which had apparently lost the activity. These results suggest that this protease consists of multiple subunits.
- Published
- 1997
42. Application of antisense ribonucleic acid complementary to O6-methylguanine-deoxyribonucleic acid methyltransferase messenger ribonucleic acid for therapy of malignant gliomas
- Author
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Motoo Nagane, Yoshiyuki Kuchino, Soichiro Shibui, Kazuhiro Nomura, and Akio Asai
- Subjects
Genetic enhancement ,Antineoplastic Agents ,Biology ,Transfection ,RNA, Complementary ,O(6)-Methylguanine-DNA Methyltransferase ,Transcription (biology) ,Glioma ,medicine ,Tumor Cells, Cultured ,Neoplasm ,Animals ,RNA, Messenger ,neoplasms ,Messenger RNA ,O-6-methylguanine-DNA methyltransferase ,RNA ,Methyltransferases ,medicine.disease ,Molecular biology ,digestive system diseases ,Antisense RNA ,Rats ,Antisense Elements (Genetics) ,Nimustine ,Drug Resistance, Neoplasm ,Protein Biosynthesis ,Surgery ,Neurology (clinical) - Abstract
Objective A derivative of chloroethylnitrosoureas, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), is a drug of choice for the chemotherapy of human malignant brain tumors. However, the cytocidal effect of ACNU is effectively repressed through repair of ACNU-mediated deoxyribonucleic acid lesions by O6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT). Because a variety of human tumors, including brain tumors, contain high levels of MGMT activity, we investigated the effect of antisense ribonucleic acid (RNA) complementary to MGMT messenger RNA on ACNU resistance in tumor cells. Methods We established a stable ACNU-resistant clone, C6AR, from the rat glioma cell line C6 exposed to a stepwise increasing concentration of ACNU. We transfected a plasmid deoxyribonucleic acid-encoding antisense MGMT RNA under the control of the human metallothionein promoter into C6AR cells and determined the effect of the antisense RNA on ACNU resistance of tumor cells by a colony-forming efficiency assay. Results C6AR cells expressed abundant MGMT messenger RNA, although the transcription level of the MGMT gene in parental C6 cells was below the lower limits of detection under the same assay conditions. ACNU resistance of C6AR cells was significantly repressed by transfected gene-dependent antisense MGMT RNA expression that resulted in decreased survival of the tumor cells. Conclusion ACNU resistance resulting from the expression of MGMT in rat glioma cells is significantly overcome by the expression of antisense MGMT RNA. This result suggests that the antisense MGMT RNA system might be a useful strategy for overcoming ACNU resistance in the treatment of intractable malignant gliomas.
- Published
- 1997
43. Up-regulation of c-myc gene expression following focal ischemia in the rat brain
- Author
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Tadayoshi Nakagomi, Hideaki Kanemitsu, Akio Asai, Akira Tamura, Yoshiyuki Kuchino, Kouji Narita, and Takaaki Kirino
- Subjects
Male ,Cerebellum ,Thalamus ,Ischemia ,Hippocampus ,Apoptosis ,Arterial Occlusive Diseases ,Biology ,Brain Ischemia ,Proto-Oncogene Proteins c-myc ,Rats, Sprague-Dawley ,Downregulation and upregulation ,Gene expression ,medicine ,Animals ,Northern blot ,RNA, Messenger ,Brain ,General Medicine ,medicine.disease ,Blotting, Northern ,Molecular biology ,Rats ,Up-Regulation ,medicine.anatomical_structure ,nervous system ,Neurology ,Gene Expression Regulation ,Proto-Oncogene Proteins c-bcl-2 ,Neurology (clinical) ,Tumor Suppressor Protein p53 ,Proto-Oncogene Proteins c-fos - Abstract
Changes in gene expression including that of c-fos occur following cerebral ischemia. Proto-oncogenes c-myc and s-myc and oncosuppressor gene p53 are known to induce apoptosis in some types of cells, whereas proto-oncogene bcl-2 inhibits apoptosis. Possible induction of mRNAs for c-myc, N-myc, s-myc, c-fos, p53 and bcl-2 was examined following focal ischemia in the rat anterior cortex, hippocampus, thalamus and cerebellum by Northern blot analysis. Animals were decapitated 1, 2, 6, 12, and 24 hours following the left middle cerebral artery (MCA) occlusion. In sham-operated control rats, the mRNAs for c-myc, N-myc, c-fos and p53 were present in the anterior cortex, hippocampus, thalamus on both sides, and in the cerebellum, whereas those for s-myc and bcl-2 were not. The c-myc gene expression was rapidly and markedly induced by the MCA occlusion in the ipsilateral anterior cortex, hippocampus and thalamus in a time-dependent manner. In these regions, the c-fos gene expression was also induced as early as 1 hour after the MCA occlusion. The p-53 mRNA was induced in the ipsilateral hippocampus at 24 hours after MCA occlusion. In contrast, mRNAs for N-myc, s-myc and bcl-2 were not induced following MCA occlusion. These results indicate a possibility that high-level expression of the c-myc gene may be involved in the ischemic cellular events including apoptosis.
- Published
- 1996
44. Regional distribution of heparin-binding epidermal growth factor-like growth factor mRNA and protein in adult rat forebrain
- Author
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Y. Nagashima, Akira Tamura, Nobutaka Kawahara, Akio Asai, Shigeki Higashiyama, Naoyuki Taniguchi, Yohei Miyagi, Takaaki Kirino, Yoshiyuki Kuchino, and Kazuhiko Mishima
- Subjects
Male ,medicine.medical_specialty ,DNA, Complementary ,Heparin-binding EGF-like growth factor ,medicine.medical_treatment ,Gene Expression ,In situ hybridization ,Biology ,Polymerase Chain Reaction ,Rats, Sprague-Dawley ,Prosencephalon ,Epidermal growth factor ,Internal medicine ,medicine ,Animals ,ERBB3 ,RNA, Messenger ,Protein Precursors ,Receptor ,Cellular localization ,In Situ Hybridization ,Epidermal Growth Factor ,Heparin ,General Neuroscience ,Growth factor ,Membrane Proteins ,Immunohistochemistry ,Cell biology ,Rats ,Endocrinology ,Antisense Elements (Genetics) ,nervous system ,Forebrain ,Intercellular Signaling Peptides and Proteins ,hormones, hormone substitutes, and hormone antagonists ,Heparin-binding EGF-like Growth Factor - Abstract
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a recently described member of the EGF family that binds to and stimulates phosphorylation of the EGF receptor (EGFR). In this study, we examined the cellular localization of HB-EGF gene transcripts and protein in adult rat forebrain. In situ hybridization studies showed that neurons in various regions, including cortex, hippocampus, and deep structures, express HB-EGF mRNA. Positively labeled cells were also present in white matter, which suggests that both neurons and glia express HB-EGF mRNA. Immunohistochemical studies with an antibody specific to proHB-EGF, a transmembrane form of HB-EGF, demonstrated ubiquitous immunoreactivity in neurons and glial cells in white matter. In view of the wide expression of its cognitive receptor, EGFR, in central nervous system neurons, our results suggest that HB-EGF is an endogenous ligand for EGFR in the central nervous system and may play an important role in physiological conditions.
- Published
- 1996
45. Increased expression of schwannoma-derived growth factor (SDGF) mRNA in rat tumor cells: involvement of SDGF in the growth promotion of rat gliomas
- Author
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Takaaki Kirino, Shigehide Kagaya, Akinori Sugiyama, Kazuhiko Mishima, Akio Asai, Chifumi Kitanaka, Yoshiyuki Kuchino, and Yohei Miyagi
- Subjects
Cancer Research ,medicine.medical_specialty ,EGF Family of Proteins ,medicine.medical_treatment ,Molecular Sequence Data ,Gene Expression ,Biology ,Amphiregulin ,Polymerase Chain Reaction ,Liver Neoplasms, Experimental ,Epidermal growth factor ,Internal medicine ,Glioma ,Gene expression ,medicine ,Animals ,RNA, Antisense ,Northern blot ,RNA, Messenger ,Growth Substances ,Lung ,DNA Primers ,Glycoproteins ,Base Sequence ,Cell growth ,Growth factor ,medicine.disease ,Sciatic Nerve ,Rats ,ErbB Receptors ,Kinetics ,Endocrinology ,Oncology ,Cell culture ,Protein Biosynthesis ,Cancer research ,Intercellular Signaling Peptides and Proteins ,DNA Probes ,Cell Division ,Neurilemmoma - Abstract
Schwannoma-derived growth factor (SDGF) is a member of the epidermal growth factor (EGF) family, having mitogenic activity on rat astrocytes, fibroblasts and Schwann cells. The SDGF gene is significantly expressed in the newborn rat lung and in the adult rat sciatic nerve. However, except for one rat schwannoma cell line, from which SDGF and its cDNA were isolated, nothing is known about SDGF expression in established tumor cell lines. We examined the expression level of the SDGF gene in a variety of rat tumor cell lines by Northern blotting and found that it was increased in 11 of 25 established lines. The most abundant SDGF mRNA, which was about 50-fold higher than in the newborn rat lung, was expressed in rat liver adenoma dRLa74 cells. In rat glioma cell lines, such as C6, 9L and T9, and in the rat hepatoma dRLh84 and H411E cells, the SDGF expression level was about 10-fold higher than in the newborn rat lung. In 8 of 13 cell lines expressing SDGF mRNA, the EGF receptor (EGFR) gene, the product of which is regarded as a functional receptor of SDGF, was co-expressed. In addition, transfected gene-dependent anti-sense SDGF RNA expression under the control of the human metallothionein promoter significantly suppressed the in vitro growth as well as in vivo tumorigenicity of 9L glioma cells. Our results suggest that SDGF acts as an autocrine growth factor in the development and growth of rat tumors such as gliomas.
- Published
- 1996
46. Structural feature of the initiator tRNA gene from Pyrodictium occultum and the thermal stability of its gene product, tRNA(imet)
- Author
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James A. McCloskey, Karl O. Stetter, Yoshiyuki Kuchino, T. Muramatsu, Takuya Ueda, Kimitsuna Watanabe, C. Ushida, Pamela F. Crain, and H. Mizushima
- Subjects
DNA, Bacterial ,RNA, Transfer, Met ,Base pair ,Inverted repeat ,Molecular Sequence Data ,Nucleic Acid Denaturation ,Biochemistry ,Heating ,Coding region ,Pyrodictium ,Peptide Chain Initiation, Translational ,Promoter Regions, Genetic ,Gene ,biology ,Base Sequence ,Intron ,Nucleic acid sequence ,General Medicine ,biology.organism_classification ,Archaea ,Introns ,RNA, Bacterial ,Genes, Bacterial ,Transfer RNA ,Nucleic Acid Conformation - Abstract
Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105 degrees C. To study how tRNA molecules in P occulrum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3'CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal rRNA genes, 450 bp upstream of the 5' end of the tRNA coding region. In the region adjacent to the 3' end of the tRNA coding region, there was a sig G-C base pair inverted repeat followed by a C-rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (Tm) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 degrees C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the Tm of the natural initiator tRNA isolated from P occultum was over 100 degrees C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [32P] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(Met)i is due to posttranscriptional modification.
- Published
- 1996
47. Myc-Mediated Apoptosis
- Author
-
Yoshiyuki Kuchino, Akio Asai, and Chifumi Kitanaka
- Subjects
biology ,UVB-induced apoptosis ,Apoptosis ,Glioma ,Intrinsic apoptosis ,biology.protein ,medicine ,Embryo ,medicine.disease ,Gene ,Peptide sequence ,Caspase ,Cell biology - Abstract
Mammalian cells contain an intron-less myc gene, such as the rat s-myc gene and human myc L2 gene, which are expressed in rat embryo chondrocytes and human testis, respectively. Our recent findings demonstrated that s-Myc expression suppresses the growth activity and tumorigenicity of glioma cells, indicating that s-Myc acts as a negative regulator in tumor growth. In addition, we found that s-Myc overexpression can effectively induce apoptotic cell death in human and rat glioma cells without serum deprivation, which is distinct from c-Myc-mediated apoptosis.
- Published
- 1996
48. Application of the Apoptotic Gene to Gene Therapy of Malignant Gliomas
- Author
-
Kazuhiko Mishima, Yoshiyuki Kuchino, Akinori Sugiyama, Chifumi Kitanaka, and Akio Asai
- Subjects
Exon ,Programmed cell death ,Glioma ,Genetic enhancement ,Gene expression ,medicine ,Cancer research ,Transfection ,Biology ,medicine.disease ,Gene ,MYC Family Gene - Abstract
The s-myc gene is a unique myc family gene without introns that was isolated from a rat genomic library using the –-myc exon 3 region as a probe. Significant expression of the s-myc gene was detected in rat embryo chondrocytes committed to programmed cell death. Gene transfection experiments showed that s-myc expression in rat and human glioma cells killed the cells by apoptosis induction. These obser–a-tions, indicating that s-Myc can act as an apoptosis inducer in –i–o and in –itro, suggest that the s-myc gene might be useful for gene therapy of gliomas. To test this possibility, we introduced the s-myc gene (pCEP4smyc), linked to the cytomegalo–i-rus promoter, into glioma-bearing rats. Surprisingly, injection of pCEP4smyc plasmid DNA together with rat 9L or C6 glioma cells completely pre–ented 9L- or C6-deri–ed tumor formation in the brain or the hind leg of rats. A similar dramatic antitumor effect induced by s-myc gene expression was obser–ed at a site distant from the original site at which the s-myc gene and glioma cells were coinjected. These results indicated that s-myc expression has the potential to elicit a host antitiumor immune response.
- Published
- 1996
49. Nonsense suppression in mammalian cells
- Author
-
Yoshiyuki Kuchino and Tomonari Muramatsu
- Subjects
Transcription, Genetic ,media_common.quotation_subject ,Nonsense mutation ,Nonsense ,Molecular Sequence Data ,Biology ,Biochemistry ,Suppression, Genetic ,RNA, Transfer ,RNA, Transfer, Gln ,Translational regulation ,Transcriptional regulation ,Protein biosynthesis ,Animals ,RNA, Messenger ,Codon ,Gene ,media_common ,Genetics ,Mammals ,Base Sequence ,Translation (biology) ,General Medicine ,Rats ,Protein Biosynthesis ,Transfer RNA ,Nucleic Acid Conformation - Abstract
Mammalian cells contain suppressor tRNAs that can translate nonsense codons such as UAG and UGA localized at a specific site of natural mRNAs. For translation of these nonsense codons, a specific secondary or tertiary structure of mRNAs located in the region surrounding the translatable nonsense codon is required. In mammalian cells, transcriptional expression of the tRNA gene encoding UAG suppressor glutamine tRNA is repressed by the binding of a nuclear protein to a specific site in the 5'-flanking region of the gene. Based on these findings, we discuss the translational regulation of nonsense codons in mammalian mRNAs.
- Published
- 1996
50. Molecular cloning and functional expression of a cDNA for rat phospholipid hydroperoxide glutathione peroxidase: 3'-untranslated region of the gene is necessary for functional expression
- Author
-
Nobuyoshi Chiba, Akiharu Hanamoto, Yasuhito Nakagawa, Akinori Sugiyama, Masayoshi Arai, Hirotaka Imai, Yoshiyuki Kuchino, and Daigo Sumi
- Subjects
Untranslated region ,Male ,DNA, Complementary ,GPX3 ,Molecular Sequence Data ,Restriction Mapping ,Biology ,Biochemistry ,GPX5 ,GPX6 ,Gene Expression Regulation, Enzymologic ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Complementary DNA ,Animals ,Northern blot ,Amino Acid Sequence ,Phospholipid-hydroperoxide glutathione peroxidase ,Cloning, Molecular ,Molecular Biology ,Cells, Cultured ,chemistry.chemical_classification ,Glutathione Peroxidase ,Base Sequence ,Glutathione peroxidase ,General Medicine ,Phospholipid Hydroperoxide Glutathione Peroxidase ,Molecular biology ,Rats ,chemistry ,Protein Biosynthesis - Abstract
A full-length cDNA clone for phospholipid hydroperoxide glutathione peroxidase (PHGPx) was isolated from a rat brain. The cDNA was 0.761 kb in length and encoded 170 amino acids, which included a TGA-encoded selenocysteine at residue 46. The protein has a calculated molecular mass of 19,473 Da. We succeeded in the transient functional expression of a full-length cDNA for PHGPx, which includes the 3'-UTR, in COS-7 cells at the first attempt. Deletion of the 3'-UTR prevented the expression of the PHGPx activity and the incorporation of [75Se]selenious acid into the monomeric 19.7 kDa PHGPx protein. Thus, the 3'-UTR of the cDNA for PHGPx was required for the functional expression of PHGPx. Northern blot analysis demonstrated that the mRNA for PHGPx was widely expressed in normal rat tissues, especially in the testis. The mRNA levels of PHGPx in the cultured cells such as hepatomas, neuronal cells, nephroblastoma, and mammary myo-epithelial cells were higher than those of the tissues. The ratio of PHGPx to cytosolic glutathione peroxidase (cGPx) was significantly high in the testis and relatively high in the cultured cells. The mRNA levels of PHGPx in tissues were lower than those of cGPx.
- Published
- 1995
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