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Biomedical subjects

M Ohtsu

Publications and source records attributed to M Ohtsu.

At least 37 records · Page 2Linked to original sources
PubMed1999

Extensive degradation of mutant-type D123 protein is responsible for temperature-sensitive proliferation inhibition in 3Y1tsD123 cells.

A temperature-sensitive mutant of 3Y1, 3Y1tsD123, reversibly arrested in G1 phase of cell cycle at the restrictive temperature of 39.8 degrees C, shows a single amino acid exchange in the D123 protein. In this study, we found that the D123 protein level in 3Y1tsD123, which was 1/8 of that in 3Y1 compared at the permissive temperature of 33.9 degrees C, lowered to 1/4 after a shift to the restrictive temperature. During inhibition of protein synthesis with cycloheximide, the D123 protein level in 3Y1tsD123 decreased markedly depending on the incubation temperature, compared with that in 3Y1, indicating that the lowered levels of D123 protein in 3Y1tsD123 are due to its degradation. Unexpectedly, 2 stably temperature-resistant clones were isolated after transfection of SV-3Y1tsD123 (SV40-transformed 3Y1tsD123, which shows cell death instead of G1 arrest at the restrictive temperature) with the cDNA of the mutant-type (3Y1tsD123-derived) D123 protein. The D123 protein in both clones degraded extensively at both temperatures, suggesting that the overexpression of the mutant-type D123 protein exceeds its degradation. Both temperature-resistant clones contained higher levels of D123 protein at the restrictive temperature than did SV-3Y1tsD123 at the permissive temperature. We concluded that the lowered D123 protein level at the restrictive temperature induces the temperature-sensitive characteristics of 3Y1tsD123 and SV-3Y1tsD123.

Animals↗
PubMed1998

A cloning method for caspase substrates that uses the yeast two-hybrid system: cloning of the antiapoptotic gene gelsolin.

Caspase-mediated proteolysis is a critical and central element of the apoptotic process; therefore, it is important to identify the downstream molecular targets of caspases. We established a method for cloning the genes of caspase substrates by two major modifications of the yeast two-hybrid system: (i) both large and small subunits of active caspases were expressed in yeast under ADH1 promoters and the small subunit was fused to the LexA DNA-binding domain; and (ii) a point mutation was introduced that substituted serine for the active site cysteine and thereby prevented proteolytic cleavage of the substrates, possibly stabilizing the enzyme-substrate complexes in yeast. After screening a mouse embryo cDNA expression library by using the bait plasmid for caspase-3, we obtained 13 clones that encoded proteins binding to caspase-3, and showed that 10 clones including gelsolin, an actin-regulatory protein implicated in apoptosis, were cleaved by recombinant caspase-3 in vitro. Using the same bait, we also isolated human gelsolin cDNA from a human thymus cDNA expression library. We showed that human gelsolin was cleaved during Fas-mediated apoptosis in vivo and that the caspase-3 cleavage site of human gelsolin was at D352 of DQTD352G, findings consistent with previous observations on murine gelsolin. In addition, we ascribed the antiapoptotic activity of gelsolin (which we previously reported) to prevention of a step leading to cytochrome c release from the mitochondria into the cytosol. Our results indicate that this cloning method is useful for identification of the substrates of caspases and possibly also of other enzymes.

Apoptosis↗
PubMed1998

Successful peripheral T-lymphocyte-directed gene transfer for a patient with severe combined immune deficiency caused by adenosine deaminase deficiency.

Ten patients with adenosine deaminase deficiency (ADA-) have been enrolled in gene therapy clinical trials since the first patient was treated in September 1990. We describe a Japanese ADA- severe combined immune deficiency (SCID) patient who has received periodic infusions of genetically modified autologous T lymphocytes transduced with the human ADA cDNA containing retroviral vector LASN. The percentage of peripheral blood lymphocytes carrying the transduced ADA gene has remained stable at 10% to 20% during the 12 months since the fourth infusion. ADA enzyme activity in the patient's circulating T cells, which was only marginally detected before gene transfer, increased to levels comparable to those of a heterozygous carrier individual and was associated with increased T-lymphocyte counts and improvement of the patient's immune function. The results obtained in this trial are in agreement with previously published observations and support the usefulness of T lymphocyte-directed gene transfer in the treatment of ADA-SCID.

Adenosine Deaminase↗
PubMed1998

Elevation of serum IgE level and peripheral eosinophil count during T lymphocyte-directed gene therapy for ADA deficiency: implication of Tc2-like cells after gene transduction procedure.

We have successfully carried out T-cell-directed gene therapy for a boy with severe combined immunodeficiency due to adenosine deaminase deficiency (ADA SCID) and unexpectedly found an elevation of serum IgE level and peripheral eosinophil count during the course. More than 90% of transduced cells cultured for 7-11 days before infusion into the patient were positive for CD8 and expressed Th2-type cytokine genes such as IL-4, IL-5 and IL-13. Furthermore, CD4(+) T-depleted PBMC (peripheral blood mononuclear cells) from the patient synthesized IgE in vitro by stimulation with IL-4. Collectively, these results suggested that Tc2-like cells in the transduced cells have distinct immunological functions to help IgE synthesis and activate eosinophils.

Adenosine Deaminase↗
PubMed1997

Inhibition of apoptosis by the actin-regulatory protein gelsolin.

Gelsolin is an actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin-regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti-Fas antibody, C2-ceramide or dexamethasone, without changing the F-actin morphology or the levels of Fas or Bcl-2 family proteins. Upon the induction of apoptosis, an increase in CPP32(-like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro-CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti-Fas treatment in the gelsolin transfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(-like) proteases strongly inhibited Fas-mediated apoptosis, but only partially suppressed both C2-ceramide- and dexamethasone-induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(-like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.

Actins↗
PubMed1993

Induction of interleukin 2-responsiveness in thymocytes of the transgenic mice carrying lck-transgene.

The role of lck gene in T cell proliferation and differentiation was investigated with transgenic mice carrying human lck cDNA whose expression was regulated by the promoter of mouse H-2Kb and the enhancer element of mouse IgH. RNase protection assay revealed that the lck transgene was expressed in the thymus and spleen, whereas immunoblot analysis demonstrated that amounts of p56lck in freshly isolated lymphoid organs were almost equal between transgenic mice and negative littermates. Cell-surface marker analyses of the thymocytes and peripheral lymphocytes revealed no remarkable difference between both groups. Notable finding is that the thymocytes from transgenic mice showed a significant proliferative response to the stimulation with IL-2, but not the thymocytes from negative littermates. Further analysis revealed that CD4+8- single positive thymocytes proliferated in response to IL-2. While surface expression levels of IL-2R alpha and IL-2R beta of these CD4+8- thymocytes from transgenic and control mice were almost equal before stimulation with IL-2, the expression of IL-2R beta was induced only in transgenic thymocytes after stimulation with IL-2. Immunoblot analysis demonstrated that the expression of p56lck of transgenic thymocytes was not down-regulated at 4 hr after stimulation with IL-2, whereas p56lck of control ones were not detectable any more at 4 hr after stimulation with IL-2. Moreover, in vitro kinase assay substantiated such unchanged expression of p56lck in the thymocytes from transgenic mice: the kinase activities of p56lck did not decrease in thymocytes from transgenic mice after stimulation with IL-2, while kinase activities of control ones were significantly down-regulated by stimulation of IL-2. These results suggested that a significant proliferative response found in the thymocytes from lck-transgenic mice after the stimulation with IL-2 was caused by a constitutive expression of p56lck in these thymocytes even after the stimulation. Our findings, therefore, support a possibility that p56lck may play a role in the IL-2R-mediated signaling system in CD4+8- thymocytes.

Animals↗
PubMed1992

Effects of butyrate on cell cycle progression and polyploidization of various types of mammalian cells.

We studied the effect of butyrate on cell cycle progression and polyploidization in three fibroblast (rat 3Y1, human IMR-90, and human embryo lung HEL) and two epithelial (human embryo kidney HEK and monkey kidney BSC-1) cells. In these cells, except for 3Y1, G1 arrest with butyrate was incomplete, and the production of tetraploid cells was detectable in the presence of butyrate. G2 arrest with butyrate was also incomplete in HEL and BSC-1 cells, and the number of HEL cells increased in the presence of butyrate. On the contrary, most BSC-1 cells that divided in the presence of butyrate were unstable and the number of attached cells decreased. These results indicate that the effect of butyrate on cell cycle progression varies with the cell type and that polyploidization can be induced by a single treatment with butyrate.

Animals↗
PubMed1991

Accumulation of p60lck in HTLV-I-transformed T cell lines detected by an anti-lck monoclonal antibody, MOL 171.

The lck gene encodes a protein tyrosine kinase of nonreceptor type, p56lck, whose expression occurs almost exclusively in T lymphocytes. MOL 171, an anti-p56lck monoclonal antibody, was produced by using a 25-amino-acid synthetic polypeptide as the antigen, its structure corresponding to the N-terminal region deduced from the lck cDNA sequence. Immunoblot analysis with MOL 171 showed the accumulation of 60 kD form of Lck protein, p60lck, and the decrease of p56lck in human T cell leukemia virus type I (HTLV-I)-transformed T cell lines. Another anti-Lck monoclonal antibody, MOL 294, raised by using a synthetic peptide corresponding to the C-terminal region deduced from the lck cDNA sequence, also detected the accumulation of p60lck in those HTLV-I-transformed T cell lines. The appearance of p60lck with the decrease of p56lck in normal T lymphocytes after stimulation suggested the origin of p60lck in HTLV-I-transformed T cells.

Antibodies, Monoclonal↗
PubMed1989

Mechanism of selective killing by dilinoleoylglycerol of cells transformed by the E1A gene of adenovirus type 12.

Rat 3Y1 fibroblasts transformed by the E1A gene of adenovirus type 12 (E1A-3Y1 cells) are highly sensitive to the cell-killing effect of 1,3-dilinoleoylglycerol (DLG) administered in a culture medium, whereas the parental 3Y1 cells are less sensitive (H. Shimura et al., Cancer Res., 48: 578-583, 1988). The selective cytotoxicity of DLG to E1A-3Y1 cells was markedly reduced by the simultaneous administration of nonspecific antioxidants such as vitamin E, butylated hydroxytoluene, and ascorbic acid. Specific scavengers for oxygen radicals had no effect. Lipoxygenase inhibitors (nordihydroguaiaretic acid, esculetin, and baicalein) reduced the DLG-mediated selective cytotoxicity, whereas cyclooxygenase inhibitors (acetylsalicylic acid and indomethacin) showed no effect. The intracellular and extracellular contents of the products from lipid peroxidation as measured by the thiobarbituric acid test were significantly greater in E1A-3Y1 cells than in the parental 3Y1 cells. In comparison with DLG, linoleic acid and monolinoleoylglycerol were equally toxic to E1A-3Y1 and parental 3Y1, and trilinoleoylglycerol was weakly toxic to both types of cells. Scanning electron microscopy revealed that numerous holes about 0.2 micron in diameter were scattered all over the surface of the E1A-3Y1 cells after treating the cultures with DLG. These results suggest that; (a) the DLG-mediated cytotoxicity to the E1A-transformed cells is attributable to lipid peroxidation; (b) the structural property of DLG is essential to the E1A specificity of cytotoxicity; and finally (c) the destruction of the cell membrane is the basis of cytotoxicity of DLG.

Adenovirus Early Proteins↗
PubMed1989

The high sensitivity of cells transformed by E1A gene of adenovirus type 12 to diacylglycerol-mediated cell killing.

Viability of rat 3Y1 fibroblasts transformed by adenovirus type 12 (Ad12) was markedly impaired by the administration of dilinoleoylglycerol (DLG) to the culture medium. To identify the gene(s) of Ad12 responsible for the high sensitivity to DLG, we established several transformed sublines of 3Y1 induced by the viral E1A gene or by the mutants of Ad12 which have mutations in the E1A region. All of the transformed sublines of 3Y1 expressing either the 12S or the 13S message from the E1A region were highly sensitive to the cytotoxicity of DLG. We propose that the high sensitivity of Ad12-transformed cells to the DLG mediated cytotoxicity is attributable to the common function of E1A-12S and E1A-13S mRNA products.

Adenoviridae↗
PubMed1989

Frequency of cell transformation by the small DNA tumor viruses: infection of proliferating cells and quiescent cells.

Small DNA-containing tumor viruses (simian virus 40, mouse polyoma-virus, and adenoviruses) malignantly transform fibroblasts of the susceptible rodents. Fibroblasts can exist, in vitro and in vivo, in either of the two states: the proliferating state or the quiescent state. In the present study, we examined whether the state of fibroblasts at the time of exposure to these DNA viruses affects the frequency of transformation. Dense-focus formation in monolayer culture of rat 3Y1 fibroblasts was used to quantitate transformation. Results show that the frequency of transformation by simian virus 40 and mouse polyomavirus was reduced when cells were in the proliferating state at the time of virus inoculation as compared to cells in the quiescent state, whereas that by adenovirus type 12 was similar in the two cellular states. The reduction of the frequency of transformation in proliferating cells infected with simian virus 40 was also observed in BALB/c 3T3 mouse cells. Mechanisms underlying the difference between the two cellular states and the difference between the papovavirus and adenovirus in this aspect of transformation remain to be investigated.

Adenoviridae↗
PubMed1989

Induction of DNA synthesis by cholera toxin in the temperature-sensitive cell-cycle mutants of rat 3Y1 fibroblasts at a restrictive temperature.

Four temperature-sensitive mutants of rat 3Y1 fibroblasts representing separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125 and 3Y1tsH203) are arrested at a restrictive temperature of 39.8 degrees C mainly with a G1-phase DNA content (temperature arrest). Cholera toxin (CT) (3 micrograms ml-1) induced DNA synthesis at 39.8 degrees C in the temperature-arrested cultures of two mutants (3Y1tsD123 and 3Y1tsG125). This effect of CT was not mimicked by other agents known to elevate the cellular level of cyclic AMP, such as dibutyryl-cyclic AMP, prostaglandin E1 and forskolin, suggesting that the elevation of cellular cyclic AMP level per se is not responsible for the induction of DNA synthesis by CT. Addition of the B subunit of CT to the temperature-arrested cultures of 3Y1tsD123 and 3Y1tsG125 did not induce DNA synthesis at 39.8 degrees C, indicating that the binding of CT to the cell surface alone is insufficient for the induction. The CT-treated cell membrane fraction prepared from temperature-arrested 3Y1tsG125 cells had similar activity for [32P]ADP-ribosylation of the 45 X 10(3) Mr protein to that prepared from cells proliferating at a permissive temperature of 33.8 degrees C. All these results suggest that 3Y1tsG125 cells utilize a CT-responsive signal transduction pathway, different from adenylate cyclase cascade, for preparation for entry into S phase in the temperature-arrested 3Y1tsG125.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Functional rescue of temperature-sensitive defects in the cell-cycle mutants of rat 3Y1 fibroblasts by the small t-antigen deletion mutant of simian virus 40.

We examined the effects of large T antigen of simian virus 40 (SV40) on the proliferation phenotypes of temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, which cease proliferating in the G1 phase of the cell cycle at a restrictive temperature (39.8 degrees C). Four ts mutants, each representing independent complementation groups, were transformed with the dl-884 mutant of SV40 which lacks the unique coding region for small t antigen. In the case of two ts mutants, their transformed derivatives did not cease proliferation at 39.8 degrees C. In the other two mutants, the transformed cells continued to enter the S phase but the cells became detached from the dishes thereafter, at 39.8 degrees C. The proliferation phenotypes of the dl-884-transformed cells at 39.8 degrees C were quite similar with those of the same mutants transformed with the wild-type SV40. These results indicate that large T antigen alone is sufficient to overcome the inhibition of cellular entry into S phase caused by four different ts defects and determines the proliferation phenotypes of the cells after entering the S phase at a restrictive temperature, and that small t antigen does not alter the cellular phenotypes determined by large T antigen.

Animals↗
PubMed1988

Selective cytotoxicity of phospholipids and diacylglycerols to rat 3Y1 fibroblasts transformed by adenovirus type 12 or its E1A gene.

The colony-forming ability of rat 3Y1 fibroblasts transformed by adenovirus type 12 (Ad12) was drastically reduced when the cells were cultivated for 18 h in medium augmented with 300 micrograms/ml of liposomes composed of either phosphatidylcholine (PC) or phosphatidylinositol. In contrast, those of untransformed 3Y1 cells and simian virus 40-transformed and polyomavirus-transformed 3Y1 cells were not. The cytotoxicity of PC liposomes was also observed in 3Y1 cells transformed by plasmid DNA containing Ad12-E1A gene but not in those transformed by adenovirus type 2, Rous avian sarcoma virus, or plasmid DNA carrying v-Ha-ras oncogene. The extensive killing of Ad12-transformed and E1A-transformed 3Y1 cells occurred in liposomes of dioleoyl-PC and of dilinoleoyl PC but not those of dipalmitoyl PC, distearoyl-PC, or diarachidonyl PC, suggesting that the acyl groups of phospholipids play an important role in cytotoxicity. Dilinoleoylglycerol, 60 micrograms/ml, was also cytotoxic selectively to Ad12-transformed and E1A-transformed 3Y1 cells, although the toxicity of lysophosphatidylcholine or linoleic acid was not specific to these transformants. These results suggest that cell transformation by Ad12 is characterized by a high sensitivity to exogenously administered phospholipids and diacylglycerol that contain oleoyl or linoleoyl acyl groups and that the sensitivity is attributable to the expression of E1A gene of Ad12.

Adenovirus Early Proteins↗
PubMed1985

Isolation of tetraploid clones with high efficiency from diploid 3Y1 rat fibroblasts treated with sodium butyrate.

Sodium butyrate causes proliferation arrest with a G2 (4C) DNA content and induces formation of tetraploid cells upon removal of the inhibitor, in rat 3Y1 diploid fibroblasts. We isolated tetraploid clones from the butyrate-treated 3Y1 cells with high efficiency; among 21 clones randomly isolated, 5 were pure diploid, 7 were mainly tetraploid with a small contaminating diploid population, and 7 were pure tetraploid. Among the pure tetraploid clones, two showed doubled chromosome numbers with slightly broader distributions than that seen in parental 3Y1 cells. Butyrate further induced polyploid formation in the tetraploid cells thus produced, but octaploid cells that resulted could not be maintained for prolonged cultivation. We found no difference between the tetraploid and the (parental and parallel isolated) diploid clones in terms of colony-forming ability, proliferation rate, and sensitivity to density-dependent inhibition of proliferation. These results suggest that doubling of chromosome number by itself does not cause a change in proliferation property. The tetraploid clones had lower average saturation densities possibly due to enlargement of cell size represented by higher cellular protein content.

Animals↗