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Biomedical subjects

Y Ikawa

Publications and source records attributed to Y Ikawa.

At least 37 records · Page 2Linked to original sources
PubMed2000

Fas drives cell cycle progression in glioma cells via extracellular signal-regulated kinase activation.

Recent studies have revealed that a variety of malignant tumors express Fas and/or its ligand FasL. However, tumor cells expressing Fas are not always susceptible to Fas-mediated cell death, and the biological significance of simultaneous expression of Fas and FasL in the same tumor is not known. In the present study, we addressed this question in three glioma cells lines, A-172, T98G, and YKG-1, which express both Fas and FasL endogenously and their Fas transfectants. We report here that: (a) in gliomas, [3H]TdR incorporation was enhanced by anti-Fas IgM monoclonal antibody CH-11 and conversely inhibited by anti-FasL monoclonal antibody NOK-2; (b) cross-linking of Fas with CH-11 drove both cell cycle progression and apoptosis as demonstrated by the induction of the S-G2 phase of DNA and RNA and fragmented nuclei; (c) phosphorylation of extracellular signal-regulated kinase (ERK), but not of c-Jun NH2-terminal kinase or p38, was induced by cross-linking of Fas; (d) a mitogen-activated protein kinase/ERK kinase 1 (MEK1) inhibitor PD98059 completely blocked CH-11-induced ERK phosphorylation as well as cell cycle progression without affecting induction of apoptosis; and (e) a broad-spectrum caspase inhibitor Z-Asp-CH2-DCB inhibited CH-11-induced ERK phosphorylation, cell cycle progression, and apoptosis. These results indicate that Fas-mediated caspase activation elicits two independent cellular responses; one is to induce apoptosis and another is to promote cell cycle progression; the latter is closely linked to the MEK-ERK pathway. Together, our data strongly suggest that FasL may play a role as an autocrine growth factor in gliomas.

Animals↗
PubMed2000

Ultraviolet-irradiated apoptotic lymphocytes produce interleukin-10 by themselves.

Since inflammatory responses are rarely associated with apoptotic cell death, it is plausible that cells undergoing apoptosis may signal the immune system to suppress inflammatory responses. By employing intracytoplasmic cytokine staining in conjunction with annexin V-binding, we examined the representative pro-inflammatory cytokine tumor necrosis factor-alpha (TNFalpha) and anti-inflammatory cytokine interleukin-10 (IL-10) expression in ultraviolet (UV)-irradiated lymphocytes and analyzed them with apoptosis induction at a single cell level. We show here that lymphocytes exposed to UV resulted in IL-10 expression with marginal TNFalpha expression, and these IL-10-expressing cells underwent apoptosis. Addition of inhibitors for caspases blocked UV-induced apoptosis but not IL-10. These results indicate that UV elicited at least two types of signals: one which was caspase dependent, leading to apoptosis; and another which was caspase independent, leading to IL-10 production. Lymphocyte apoptosis was thus found to link anti-inflammatory cytokine secretion, and thereby may contribute to preventing unwanted immune responses.

Apoptosis↗
PubMed2000

Characterization of P8 and J8/7 elements in the conserved core of the tetrahymena group I intron ribozyme.

The universally conserved core region in the group I intron ribozymes is responsible for its catalytic activity. The structural elements in this region have been known to organize the active site of this class of ribozymes. However, it has been unclear whether all elements are requisite or some elements are dispensable for conducting the catalysis. To investigate the necessity of these elements in the catalysis, we prepared and examined a series of mutants having a nick or deletion in these elements. In this report, we show that two elements, P8 and 5' portion of J8/7, are nonessential for activity.

Animals↗
PubMed2000

Minimal catalytic domain of a group I self-splicing intron RNA.

The self-splicing intron ribozymes have been regarded as primitive forms of the splicing machinery for eukaryotic pre-mRNAs. The splicing activity of group I self-splicing introns is dependent on an absolutely conserved and exceptionally densely packed core region composed of two helical domains, P3-P7 and P4-P6, that are connected rigidly via base triples. Here we show that a mutant group I intron ribozyme lacking both the P4-P6 domain and the base triples can perform the phosphoester transfer reactions required for splicing at both the 5' and 3' splice sites, demonstrating that the elements required for splicing are concentrated in the stacked helical P3-P7 domain. This finding establishes that the conserved core of the intron consists of two physically and functionally separable components, and we present a model showing the architecture of a prototype of this class of intron and the course of its molecular evolution.

Base Sequence↗
PubMed2000

Analysis of the P7 region within the catalytic core of the Tetrahymena ribozyme by employing in vitro selection.

The highly conserved P7 region is generally believed to act as a major portion of the catalytic site in the Group I intron ribozyme. However, its functions have not been elucidated except for the fact that it specifically binds a cofactor guanosine required for self-splicing reaction. We attempted an in vitro selection experiment to determine the sequence requirements of this region in the mechanism of catalysis by using the Tetrahymena ribozyme. We found that the selected active clones have the secondary structure similar to that of the wild type with few exceptions. However, their primary sequences were not conserved except G264 and C311 that are the major elements of the binding site for the guanosine. Our results suggest that the unique secondary structure of the P7 region is a primary requisite for the catalytic function of this class of ribozymes.

Animals↗
PubMed2000

Synthesis and tumor-promoting activities of 12-Epi-phorbol-12,13-dibutyrate.

12-Epi-phorbol-12,13-dibutyrate (1), the C12-epimer of the most frequently used phorbol ester probe, phorbol-12,13-dibutyrate (PDBu), has been synthesized from phorbol in 9 steps in order to investigate the structural requirements for tumor-promoting activity. Compound 1 showed about 100-fold weaker in vitro biological activities related to in vivo tumor promotion, Epstein-Barr virus early antigen (EBV-EA)-inducing ability, superoxide (O2-) generation-inducing ability, and binding to the protein kinase C (PKC) regulatory domain surrogate peptides. The results indicated that the beta-stereochemistry at position 12 of the phorbol skeleton is important for optimal activity. Binding selectivity to each PKC C1 domain of 1 was almost equal to that of PDBu.

Antigens, Viral↗
PubMed1999

Induction of manganese-superoxide dismutase in MRC-5 cells persistently infected with an alphavirus, sindbis.

Sindbis virus (SV), a single-stranded positive-sense RNA virus, multiplies in a variety of cells and causes various outcomes of infection. As we described acute infection of SV induces stress response of small heat shock protein HSP27 and activation of mitogen-activated protein kinase (MAP) signaling pathway (Nakatsue, T., et al., Biochem. Biophys. Res. Commun. 253, 59-64, 1998). In contrast to lytic infection in Vero cells, MRC-5 cells, a human fetus lung cell line, resulted in persistent infection by SV. Here we investigated a cellular factor involved in persistent infection of MRC-5 cells infected with SV. Partial sequence analysis of a 25 kilodalton (kDa) protein, accumulated in large amounts in the cells, showed that manganese-superoxide dismutase (Mn-SOD) was induced during the infections. When Mn-SOD was overexpressed in Vero cells, 20% of the cells survived more than one month, in contrast with the death of 99% of the vehicle-transfected Vero cells at 48 h after infection with SV. These data strongly suggest that a cellular factor which regulates the oxidative pathway modulates the outcome of SV infection.

Amino Acid Sequence↗
PubMed1999

Mutational analysis of p51A/TAp63gamma, a p53 homolog, in non-small cell lung cancer and breast cancer.

p51, a novel family member of human p53, is a recently identified candidate tumor suppressor gene mapped at chromosome 3q28. Like p53, p51 was found to activate p21Waf1/Cip1 and to induce apoptosis. Since the DNA loss at 3q is reported in several cancers including non-small cell lung cancer (NSCLC), we screened for mutations in p51A (TAp63gamma), an isoform of p51 with short C-terminal region, in 80 NSCLCs as well as 85 breast cancers by RT-PCR single strand conformation polymorphism (SSCP) analysis and DNA sequencing. In NSCLCs, p51 was expressed in most tumors at variable levels and we found three missense and one silent mutations: Gln31His (transactivation domain) in two tumors, Ala148Pro (DNA-binding domain) and Leu248Leu (DNA-binding domain). In the tumor with Ala148Pro or the silent mutation, only the mutant gene appeared to be expressed. The modified FASAY method to test the ability of yeast expressing p51A cDNA to grow in medium lacking histidine has revealed that Ala148Pro results in a loss of function, while Gln31His does not. In contrast to NSCLC, no mutation was observed in all 85 breast cancers by the similar method. Our results suggest that, because of infrequent mutation, p51 may not be a Knudson type tumor suppressor in most NSCLCs and breast cancers. Nevertheless, in at least a part of NSCLC, p51 may play a certain role in carcinogenesis in a tissue-specific manner.

Adult↗
PubMed1999

The transcriptional activities of p53 and its homologue p51/p63: similarities and differences.

p51/p63 is a novel p53 homologue that has been shown to act as a transcriptional activator through the p53-binding sequence of the p21/WAF1 promoter and to induce apoptosis when it is expressed transiently in a human tumor cell line. We developed transcription assay systems for these two related genes in both Saccharomyces cerevisiae and mammalian cells and used them to investigate the functional similarities and differences of these genes. We found that p51/p63 trans-activated the previously identified p53 target genes, but the degree of the transactivation by p51/p63 differed from that by p53. These results suggest that the cellular signal on p51/p63 cross-talks partially but not completely with that of the p53 pathway.

Cyclin-Dependent Kinase Inhibitor p21↗
PubMed1999

A conserved motif in group IC3 introns is a new class of GNRA receptor.

Terminal tetraloops consisting of GNRA sequences are often found in biologically active large RNAs. The loops appear to contribute towards the organization of higher order RNA structures by forming specific tertiary interactions with their receptors. Group IC3 introns which possess a GAAA loop in the L2 region often have a phylogenetically conserved motif in their P8 domains. In this report, we show that this conserved motif stands as a new class of receptor that distinguishes the sequences of GNRA loops less stringently than previously known receptors. The motif can functionally substitute an 11 nt motif receptor in the Tetrahymena ribozyme. Its structural and functional similarity to one class of synthetic receptors obtained from in vitro selection is observed.

Animals↗
PubMed1999

Trans-activation of the Tetrahymena group I intron ribozyme via a non-native RNA-RNA interaction.

The peripheral P2.1 domain of the Tetrahymena group I intron ribozyme has been shown to be non-essential for splicing. We found, however, that separately prepared P2.1 RNA efficiently accelerates the 3' splice-site-specific hydrolysis reaction of a mutant ribozyme lacking both P2.1 and its upstream region in trans. We report here the unusual properties of this trans-activation. Compensatory mutational analysis revealed that non-native long-range base-pairings between the loop region of P2.1 RNA and L5c region of the mutant ribozyme are needed for the activation in spite of the fact that P2.1 forms base-pairings with P9.1 in the Tetrahymena ribozyme. The trans -activation depends on the non-native RNA-RNA interaction together with the higher order structure of P2.1 RNA. This activation is unique among the known trans-activations that utilize native tertiary interactions or RNA chaperons.

Animals↗
PubMed1999

p53 family genes: structural comparison, expression and mutation.

The p53-related genes, p51/p63 and p73, have been isolated respectively from cDNA libraries of skeletal muscle and the brain, and their structural features and biological functions have been compared. High expression of p51A (TAp63gamma) in the skeletal muscle tissue drove us to investigate a differentiation-inducible myoblastic cell line which showed increased p51A expression after differentiation induction. Tissue-specific expression was further confirmed by reverse transcriptase-polymerase chain reaction (RT - PCR) using primers specific for DeltaN (TA-domain lacking p51), p51A, and p51B expression. p51A alone induced erythrodifferentiation when expressed in the erythroleukemia line (Tg-gp55-1-2-3) expressing a temperature-sensitive mutant of p53, and induced remarkable apoptosis when wild-type p53 expression was induced by the temperature shift to 32 degrees C. Human p51A and p53 were introduced exogenously into the above erythroleukemia cells, and although their expression was rather low, both p51A and p53 proteins were induced by DNA-damaging treatment with UV and ActinomycinD. However, the protein-protein interactions analyzed by a yeast two-hybrid assay between p51 and p53, between p51 and p73, and between p51 and oncoproteins showed that p51 is functionally rather distant from p53. Extensive mutation analysis of p51/p63 in human tumors revealed only four mutations in 80 non-small cell lung carcinomas; two adenocarcinoma cases possessing Glu31His mutations in the transactivation domain (TA) domain, suggesting that p51/p63 is not a Knudson type tumor suppressor gene. Mutation and loss of heterozygosity (LOH) of p73, deregulated expression of p73 and loss of imprinting of p73 are also discussed.

Amino Acid Sequence↗
PubMed1999

Self-renewal and differentiation of a basic fibroblast growth factor-dependent multipotent hematopoietic cell line derived from embryonic stem cells.

Despite the accumulation of informat on on the origin of hematopoietic stem cells, it is still unclear how these cells are generated in ontogeny. Isolation of cell lines equivalent to early embryonic hematopoietic progenitor cells can be helpful. A multipotent hematopoietic progenitor cell line, A-6, was isolated from H-1 embryonic stem (ES) cells. The self-renewal of A-6 cells was supported by basic-fibroblast growth factor (b-FGF) and their differentiation into definitive erythroid cells, granulocytes and macrophages was induced after co-culture with ST-2 stromal cells. A-6 cells were positive for the surface markers of hematopoietic stem cell, c-kit, CD31, CD34, Flt3/Flk2, PgP-1, and HSA, but were negative for that of the differentiated cells. Reverse transcription-polymerase chain reaction analysis showed that A-6 cells produced mRNA from SCL/tal-1 and GATA-2 genes. Among various cytokines examined, on y stem cell factor (SCF) and Flt3/Flk2 ligand (FL) supported the proliferation of A-6 cells instead of b-FGF. The FL, as well as b-FGF, supported the self-renewal of A-6 cells, whereas SCF induced differentiation into myeloid cells. A-6 cells will be useful for the characterization of hematopoietic progenitor cells derived from ES cells and provide a model system to realize the control mechanisms between self-renewal and different ation of hematopoietic stem cells.

Animals↗
PubMed1999

Apoptotic changes precede mitochondrial dysfunction in red cell-type pyruvate kinase mutant mouse erythroleukemia cell lines.

Two erythroleukemia cell lines have been established from the splenic lesions of red blood cell-type pyruvate kinase (R-PK) activity-deficient mice of CBA/N origin infected with a polycythemic strain of Friend leukemia virus complex (FVp). Ten to 30% of the cells of these cell lines undergo apoptotic changes in routine passage, as shown by nuclear fragmentation, DNA laddering, DNA content (propidium iodide (PI) staining), and annexin V binding assay. In these cells, however, although adenosine 5'-triphosphate (ATP) levels were lower than in the control cells, the mitochondrial inner transmembrane potential (delta psi m), detected by rhodamine 123 (R123) and diSC3(5) staining, remained unchanged until the final stage of apoptosis. No evidence was obtained to relate this finding to R-PK mutation due to difficulty in cloning stable, conditionally inducible R-PK gene transfectants. However, low delta psi m in the apoptotic cell population of the control T3-K-1 (K-1) and T3-CI-2-0 (2-0) Friend erythroleukemia cells supports a possible relationship, as do results obtained in two Friend erythroleukemia cells recently isolated from normal CBA/N mice. These cell lines are expected to be useful for clarifying both the primary apoptotic changes independent of mitochondrial dysfunction and the PK-isozyme changes during erythrodifferentiation, for example, the decreased muscle type 2 (M2) PK level. Modification of growth signals in these cell lines may modulate differentiation and/or apoptosis and allow further elucidation of the signaling networks.

Adenosine Triphosphate↗
PubMed1999

Human p53-p51 (p53-related) fusion protein: a potent BAX transactivator.

We recently discovered human p51, a new gene structurally and functionally related to human p53. This gene encodes two major splicing variants, p51A and p51B, which differ in their carboxyl-terminal structure. However, p51A shows strong transactivation potential, while p51B has only weak potential. To clarify the reason for this difference, we made chimeric gene constructs expressing fusion proteins of p53-p51A and p53-p51B, having an N-terminus of p53 and a C-terminus of p51A or p51B, respectively. In a BAX promoter-luciferase assay using p53-deficient SAOS-2 cells, they exhibited up to 30-fold stronger transactivation potential than p53 and p51A themselves, suggesting that the C-terminus of p51B does not simply serve as a repressor. We obtained similar results with p21WAF1 promoter-reporter plasmids. These chimeras will be valuable tools for gene therapy.

Cyclin-Dependent Kinase Inhibitor p21↗
PubMed1998

Acute infection of Sindbis virus induces phosphorylation and intracellular translocation of small heat shock protein HSP27 and activation of p38 MAP kinase signaling pathway.

In general, viral infection is supposed to induce stress responses in the host cell. However, very few detailed observations about virus-induced stress responses have been reported. Here we investigated specific stress responses in Vero cells infected with Sindbis virus (SV), a single-stranded RNA virus, acute infection with which is known to cause apoptotic cell death in the host cells. Prior to the onset of apoptosis, p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinases (JNKs) were activated. Subsequently, a 27-kDa heat shock protein (HSP27) became phosphorylated, and intracellular distribution of HSP27 was changed from the cytoplasm to the perinuclear region. These results indicate that the cellular signaling cascades activated by pro-inflammatory cytokines and environmental stresses are also activated as a result of lytic infection with SV. These responses may contribute to the delayed onset of apoptosis in the host cells and the facilitation of viral replication.

Animals↗
PubMed1998

Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK.

A human fibroblast cDNA expression library was screened for cDNA clones giving rise to flat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.

3T3 Cells↗