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D Ayusawa

Publications and source records attributed to D Ayusawa.

At least 19 recordsLinked to original sources
PubMed1999

The introduction of dominant-negative p53 mutants suppresses temperature shift-induced senescence in immortal human fibroblasts expressing a thermolabile SV40 large T antigen.

Immortal human fibroblasts, SVts8 cells, which express a heat-labile SV40 large T antigen, induces a senescence-like phenomenon in response to upward shift in temperature. Cells with arrested division show strong induction of senescence-associated beta-galactosidase. We examined how p53 and pRB are involved in this phenomenon since they are major targets of the T antigen. Transfection of cells with plasmids encoding the wild-type T antigen or human papilloma virus type 16 E6/E7 proteins completely abolished the arrest in cell division, a plasmid encoding the E6 protein suppressed it markedly, while a plasmid encoding E7 had no effect. Plasmids encoding dominant-negative p53 mutants also suppressed the arrest in cell division to various degrees. Upon temperature shift, p21 mRNA was upregulated 10-fold in SVts8 cells, but only slightly in clones expressing the wild-type T antigen or dominant-negative p53 mutants. These data demonstrate that p53 plays a major role in this senescence-like phenomenon.

Antigens, Polyomavirus Transforming↗
PubMed1998

DNA topoisomerase inhibitors induce reversible senescence in normal human fibroblasts.

Inhibitors of DNA topoisomerases I and II induced arrest in cell division in normal human fibroblasts depending on cell divisions. Arrested cells showed morphology similar to those of normally senesced cells and strongly induced senescence-associated beta-galactosidase. In these cells, p16ink4a was upregulated, whereas p21waf1 or p53 was not altered. Upon removal of the inhibitors, the cells resumed growth but their cumulative population doublings were reduced dose dependently. Accelerated telomere shortening was not observed in the arrested cells. These results suggest that DNA topoisomerase inhibitors are efficient and reversible inducers of premature senescence in normal human cells.

Cellular Senescence↗
PubMed1998

Clustering of human chromosome fragments on the mouse genome by chromosome-mediated gene transfer.

Pieces of metaphase chromosomes prepared from mouse cells containing neo-tagged human chromosome 7 were transferred to mouse cells with calcium phosphate to isolate G418-resistant clones. FISH analysis revealed that the majority of them contained human DNA at a single site on their genome. These transformants contained STS markers mapped to various regions of chromosome 7. It is thus suggested that pieces of human chromosomes tend to assemble and integrate on the mouse genome.

Animals↗
PubMed1998

A mutation in succinate dehydrogenase cytochrome b causes oxidative stress and ageing in nematodes.

Much attention has focused on the aetiology of oxidative damage in cellular and organismal ageing. Especially toxic are the reactive oxygen byproducts of respiration and other biological processes. A mev-1(kn1) mutant of Caenorhabditis elegans has been found to be hypersensitive to raised oxygen concentrations. Unlike the wild type, its lifespan decreases dramatically as oxygen concentrations are increased from 1 to 60%. Strains bearing this mutation accumulate markers of ageing (such as fluorescent materials and protein carbonyls) faster than the wild type. We show here that mev-1 encodes a subunit of the enzyme succinate dehydrogenase cytochrome b, which is a component of complex II of the mitochondrial electron transport chain. We found that the ability of complex II to catalyse electron transport from succinate to ubiquinone is compromised in mev-1 animals. This may cause an indirect increase in superoxide levels, which in turn leads to oxygen hypersensitivity and premature ageing. Our results indicate that mev-1 governs the rate of ageing by modulating the cellular response to oxidative stress.

Aging↗
PubMed1998

Release of telomeric DNA from chromosomes in immortal human cells lacking telomerase activity.

Some immortal human cell lines lack telomerase activity. These cell lines were found to contain small dispersed DNA hybridizing to TTAGGG repeats. Such DNA was located in their cytoplasm and nuclei. Normal human fibroblasts or telomerase-positive cell lines did not contain such DNA. Upon cloning and sequencing, it was shown to consist of TTAGGG repeats. When electrophoresed on neutral and alkaline agarose gels, it behaved as double-stranded and linear DNA. These results suggest that telomeric DNA is released from chromosomes in association with maintenance of telomeres in telomerase-negative cell lines.

Cell Line↗
PubMed1998

A novel superoxide dismutase gene encoding membrane-bound and extracellular isoforms by alternative splicing in Caenorhabditis elegans.

We have identified a novel Cu/Zn superoxide dismutase gene (termed SOD-4) in Caenorhabditis elegans. Characterization of its complementary DNA revealed that the gene encodes two isoforms by alternative splicing, SOD4-1 and SOD4-2 which differ in their C-terminal exons. Their predicted amino acid sequences include a consensus signal peptide at their N-termini and are homologous to the extracellular-types of Cu/Zn superoxide dismutase in mammals. In addition, SOD4-2 possesses a putative transmembrane domain at the C-terminal region. When transiently expressed in Chinese hamster ovary cells, both types were found in the membranes and SOD4-1 also in the culture fluid. It is, therefore, indicated that SOD4-1 is an extracellular form and SOD4-2 a membrane-bound form, the latter representing a novel type of SOD. In C. elegans, SOD4-2 mRNA was found to be preferentially expressed in eggs.

Alternative Splicing↗
PubMed1998

Suppression of senescence in normal human fibroblasts by introduction of dominant-negative p53 mutants or human papilloma virus type 16 E6 protein.

Transfection of nearly senesced human fibroblasts with plasmids encoding HPV16 E6 protein or dominant-negative p53 mutants greatly increased their colony-forming ability. Isolated colonies with these plasmids showed extension of lifespan compared to those with a control plasmid. These data demonstrate that p53 plays a major role in senescence in normal human fibroblasts.

Cellular Senescence↗
PubMed1997

Decrease in amplified telomeric sequences and induction of senescence markers by introduction of human chromosome 7 or its segments in SUSM-1.

Introduction of human chromosome 7 by microcell-mediated chromosome transfer suppresses indefinite division of SUSM-1, an in vitro established human fibroblast line. This cell line has unusually long telomeric sequences although it lacks detectable telomerase activity. Thus, we examined whether such telomeric sequences change upon introduction of chromosome 7 or its segments. In the microcell hybrids that stopped dividing by introduction of chromosome 7, the telomeric sequences were found to be lost or markedly diminished. Introduction of various fragments (2-40 Mb) of chromosome 7 contained in radiation hybrids gave similar results. On the other hand, the telomeric sequences were not altered significantly in the unsuppressed hybrids, a revertant of one suppressed clone, or subclones of SUSM-1 used as controls. In the suppressed microcell hybrids, the distribution of a mortality marker, mortalin, was changed to the cytosolic type of mortal cells from the immortal type of perinuclear fibres. Also, senescence-associated beta-galactosidase was induced to a level similar to that of normally senesced diploid fibroblasts. These results suggest that human chromosome 7 induces senescence in SUSM-1 by suppressing its telomere maintenance mechanism, which does not depend on telomerase.

Animals↗
PubMed1996

N-Shc: a neural-specific adapter molecule that mediates signaling from neurotrophin/Trk to Ras/MAPK pathway.

Shc has been implicated in a variety of growth factor- and cytokine receptor-signaling through its specific binding to phosphotyrosine residues of the activated receptors. In neuronal cells, such as PC12, Shc has been shown to be involved in Ras-dependent MAP kinase activation following Trk receptor stimulation with NGF. While the ubiquitous role of Shc as an adaptor molecule in signal transduction is increasing in both neuronal and non-neuronal cells and tissues, the expression level of Shc is surprisingly low in the brain. We demonstrated here the isolation of a neural-specific member of the Shc family. This novel protein, named N-Shc (neuronal Shc), contains two potential phosphotyrosine-binding domains, PTB and SH2, and is expressed exclusively in the brain; whereas Shc is present in all other non-neuronal tissues. As in Shc, N-Shc can bind activated EGF receptor, become tyrosine phosphorylated, and form a complex with Grb2 adapter protein following EGF stimulation. Furthermore, N-Shc can bind activated TrkB receptor following the stimulation with brain-derived neurotrophic factor (BDNF), which is the most abundant neurotrophin in the brain. These data suggest that N-Shc, rather than Shc, mediates neurotrophin and other neuronal signalings in the central nervous system.

3T3 Cells↗
PubMed1996

A panel of radiation hybrids defining the 7q31-q32 region of human chromosome 7.

Mouse A9 cells containing human chromosome 7 tagged with pSV2neo were irradiated with X-rays and fused to A9 cells to isolate G418-resistant clones. From these clones, we selected radiation hybrids that contained 10-40 Mb of human DNA apparently at a single site of their genome by FISH analysis using human repetitive sequences as a probe. Then we made a panel of hybrids that contained various fragments of the 7q31-q32 region and cover its entire region altogether by PCR with STS markers of human chromosome 7. This panel is useful in chromosome transfer experiments since the dominant selective marker neo gene is attached to human DNA.

Animals↗
PubMed1996

Human UDP-galactose translocator: molecular cloning of a complementary DNA that complements the genetic defect of a mutant cell line deficient in UDP-galactose translocator.

We have cloned a cDNA that codes for a putative human UDP-galactose translocator (UGT) protein. The cDNA contained an open reading frame of 1,179 base pairs encoding a novel protein of 393 amino acids. Introduction of the open reading frame sequence into a UGT-deficient mouse cell line, Had-1, complemented the genetic defect of the mutant, namely the inability to transport UDP-galactose from the cytosol to the Golgi lumen, as judged from the lectin-sensitivity spectrum of the transformant. To our knowledge, this is the first mammalian nucleotide-sugar translocator whose cDNA sequence has been described.

Amino Acid Sequence↗
PubMed1995

Isolation of human purH gene expressed in the rodent transformant cells by subtractive enrichment of 3'-untranslated region of human transcript.

A subtraction procedure was developed for identification and isolation of a human gene transcribed in mouse transformant cells. The procedure was based on subtractive enrichment of the products that were amplified by the combination of reverse transcription and polymerase chain reaction from the 3'-untranslated region (3'-UTR) of human poly(A)+ RNA expressed in the mouse transformant cells. To assess the ability and usefulness of the procedure, we attempted to recover the human purH gene from a mouse transformant cell line, which was originally established by functional complementation using the human metaphase chromosome-mediated gene transfer technique from a mouse purH-negative mutant cell line. Using our procedure, a part of the human transcript in the transformant cells was successfully identified and isolated. The full-length cDNA was isolated using the 3'-UTR clone as a probe, and its biological activity was confirmed by introducing it into the mouse purH-negative mutant cells.

Acyltransferases↗
PubMed1995

Expression of the human cGMP-dependent protein kinase II gene is lost upon introduction of SV40 T antigen or immortalization in human cells.

We have cloned a human cGMP-dependent protein kinase type II cDNA to examine its gene expression in terms of cellular senescence and/or immortalization. The genetic locus was mapped to band 4q21 by FISH. Northern blot analysis revealed that expression of the type II gene was markedly decreased or lost in mortal or immortal human fibroblasts producing SV40 T antigen. Also in various immortalized cell lines tested, the gene was not expressed. In normal diploid fibroblasts, the gene was constitutively expressed during cell-cycle and population doubling levels (PDLs).

Amino Acid Sequence↗
PubMed1995

Inhibitors of cGMP-dependent protein kinase block senescence induced by inactivation of T antigen in SV40-transformed immortal human fibroblasts.

Immortal human fibroblasts isolated following transfection with thermolabile simian virus 40 T antigen lost division potential upon shift up in temperature due to heat inactivation of the antigen. Such cells showed a concomitant change in the distribution of a mortality marker, mortalin, from a juxtanuclear cap like distribution of immortal cells to a uniform cytosolic distribution of mortal cells. We made an attempt to modulate the above inducible system of cellular senescence using various protein kinase inhibitors. Among the indolocarbazole type inhibitors tested, only KT5823, defined as a specific inhibitor of cGMP-dependent protein kinase, blocked the loss of division potential as determined by cell growth and colony forming ability. This inhibitor also prevented the above change in mortalin distribution due to temperature shift. In addition, the isoquinoline sulfonamide derivatives H8, H9, H88 and H89, all shown to inhibit cGMP-dependent protein kinase, suppressed the senescence. Inhibitors specific to other types of protein kinases, protein phosphatases or tyrosine kinases tested had no effect. Since there was no difference between the effective and non-effective inhibitors in their effects on cell cycle progression, cell cycle arrest by itself cannot account for the above phenomenon. These results suggest that a signaling pathway possibly mediated by cGMP-dependent protein kinase is involved in the induction of cellular senescence.

Alkaloids↗
PubMed1995

Cyclin I: a new cyclin encoded by a gene isolated from human brain.

A new member of the cyclin family has been isolated from an equalized cDNA library derived from human forebrain cortex. This putative cyclin, designated cyclin I, contains a typical cyclin box near the N-terminus and a PEST sequence near the C-terminus. Cyclin I shows the highest sequence similarity in the cyclin box to cyclins G and E, while the similarity between cyclins I and G also extends toward the C-terminus from the cyclin box. Cyclin I mRNA was expressed at high levels in postmitotic tissues, including skeletal muscle, heart, and brain, and was expressed constantly during cell cycle progression. The expression of cyclin I mRNA does not correlate directly to the cell cycle, and therefore cyclin I may be a novel cyclin member that functions independently of the cell cycle control.

Adult↗
PubMed1995

Genetic complementation of the immortal phenotype in group D cell lines by introduction of chromosome 7.

Human immortal cell lines have been classified into at least four (A-D) genetic complementation groups by cell-cell hybrid analysis, i.e., a hybrid derived from different groups becomes mortal. Recently we have demonstrated that introduction of human chromosome 7 suppresses indefinite division potential in the non-tumorigenic human immortalized fibroblast lines KMST-6 and SUSM-1, both assigned to complementation group D. By extending our microcell-mediated chromosome transfer, we found that chromosome 7 also suppresses division potential in the human hepatoma line HepG2 (again, assigned to group D). Chromosome 7 was thus shown to suppress indefinite growth in the above group D cell lines irrespective of their cell types, or whether they are tumorigenic or not. Since chromosome 7 had no such effect on representative cell lines derived from complementation group A, B or C, these results indicate that the senescence gene(s) commonly mutated in the group D cell lines is located on chromosome 7.

Base Sequence↗
PubMed1994

A novel regulatory sequence affecting the constitutive expression of tissue plasminogen activator (tPA) gene in human melanoma (Bowes) cells.

In order to identify a possible enhancer-like regulatory sequence for the human tissue plasminogen activator (tPA) gene, various DNA segments in the 5'-flanking region were ligated to the chloramphenicol acetyltransferase (CAT) reporter gene driven by the tPA gene promoter, and their CAT enhancing activities were measured following transfection to human melanoma-derived Bowes cells that highly express the gene. Major and minor activities were detected in two adjacent upstream sequences, 160 base pairs (bp) (-2288 to -2129) and 102 bp (-2390 to -2289), respectively, and the former was subjected to further analysis. The CAT enhancing activity of the 160-bp sequence was greatly affected by its position and orientation in the constructs and the sequence also functioned weakly with the SV40 promoter. Deletion of any small portion from the sequence abolished the CAT enhancing activity, suggesting that the entire sequence is required for the activity. This sequence did not show a further CAT enhancing activity in Bowes cells treated with the inducers phorbol 12 myristate 13-acetate and dexamethasone and did not function in HeLa or HT1080 cells under any conditions. Taken together, the 160-bp sequence is likely to be responsible for the constitutive and/or cell type-specific expression of the tPA gene in human cells.

Base Sequence↗