Biomedical subjects

M Tatsuka

Publications and source records attributed to M Tatsuka.

34 records · Page 2Linked to original sources

PubMed1995

Requirement for tyrosine phosphorylation of Cdk4 in G1 arrest induced by ultraviolet irradiation.

Exposure to ultraviolet light arrests the function of mammalian fibroblasts in the G1 phase of the cell cycle, as well as the S and G2 phases. Although p21, an inhibitor of cyclin-dependent kinase (Cdk) that is induced by DNA damage may partly account for the arrest in G1 (ref. 1), the mechanism is little understood. Here we show that tyrosine phosphorylation of Cdk4 is required for this arrest. In rat fibroblast, Cdk4 is tyrosine-phosphorylated during G1 progression, and its dephosphorylation is required for S phase. When cells are ultraviolet-irradiated, their arrest in G1 is accompanied by an increase in phosphorylation level. Conversely, cells expressing unphosphorylatable Cdk4F17 fail to arrest in G1, and suffer significantly elevated chromosomal aberrations and cell death.

Animals↗

PubMed1995

Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations.

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.

3T3 Cells↗

PubMed1995

Structural organization of the mouse DNA repair gene, N-methylpurine-DNA glycosylase.

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removes N-alkylpurines and other purine lesions induced in DNA by simple alkylating carcinogens. A mouse MPG cDNA clone was isolated from a lambda recombinant phage library of BALB/c mouse lung cell and characterized. Using the mouse MPG cDNA as a probe, the complete mouse MPG gene was isolated in two overlapping lambda recombinant genomic clones. The 6-kb gene has four exons containing 1,002 bp of coding sequence. The transcription start site was identified in the genomic sequence by primer extension of MPG mRNA from a mouse lung fibroblast cell line. The location of this transcription start site was confirmed by in vitro transcription with the promoter-containing plasmid template. Promoter function of the sequence 5' upstream of the transcription initiation site was shown by transient expression of the firefly luciferase reporter gene under the control of this sequence in transfected human and mouse cells. The mouse MPG promoter contains no TATA box, but has a CAAT element and is G.C-rich with putative AP2 elements and SP1-complementary sequences.

3T3 Cells↗

PubMed1994

HPRT yeast artificial chromosome transfer into human cells by four methods and an involvement of homologous recombination.

The transfer of a yeast artificial chromosome (YAC) into mouse and human cell lines was effected by four methods, and the efficiency and integrity of the incorporated YAC DNA were compared. A 500 kb YAC containing the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene was transferred more efficiently by polyethylene glycol-mediated fusion than by lipofection, electrofusion, or electroporation. Southern blot analysis demonstrated that PEG fusion lines yielded fragments of the size of the original YAC clone, whereas lipofection and electroporation did not. Two of 53 fusion lines showed 6-thioguanine resistance and confirmatory disruption of the HPRT gene in the YAC DNA, suggesting that the YAC DNA was integrated by homologous recombination with the endogenous HPRT gene region.

Animals↗

PubMed1992

Elongation factor-1 alpha gene determines susceptibility to transformation.

Elongation factor-1 alpha (EF-1 alpha), an essential component of the eukaryotic translational apparatus, is a GTP-binding protein that catalyses the binding of aminoacyl-transfer RNAs to the ribosome. Expression of the EF-1 alpha gene decreases towards the end of the lifespans of mouse and human fibroblasts, but forced expression of EF-1 alpha prolongs the lifespan of Drosophila melanogaster. Eukaryotic initiation factor-4E, another component of the translational machinery, is mitogenic or oncogenic when constitutively expressed in some mammalian cells. Thus, components of the protein synthesis apparatus seem to be involved in the control of cell proliferation. Using expression cloning, we have isolated a complementary DNA clone from a BALB/c 3T3 mouse fibroblast variant, A31-I-13 (ref. 10), which specifies a factor determining the susceptibility of BALB/c3T3 to chemically and physically induced transformation. Here we report that the factor is EF-1 alpha and that its constitutive expression causes BALB/c 3T3 A31-I-1 (ref. 10), C3H10T1/2 (ref. 11) and Syrian hamster SHOK fibroblasts to become highly susceptible to transformation induced by 3-methylcholanthrene and ultraviolet light. EF-1 alpha messenger RNA is also constitutively expressed in a quiescent culture of the highly susceptible variant A31-I-13. We conclude that the removal of regulation of the expression of these components of the translational machinery may predispose cells to become more susceptible to malignant transformation.

3T3 Cells↗

PubMed1992

Expression of platelet-derived growth factor-independent phenotypes in BALB/c 3T3 cell variant with high susceptibility to chemically or physically induced neoplastic cell transformation: dissociation from activation of protein kinase C.

A31-I-13, a clonal cell variant of nontransformed BALB/c 3T3 that is highly susceptible to chemically or physically induced malignant cell transformation but is not sensitive to cell killing or susceptible to induced somatic cell mutation compared with another less transformation-susceptible A31-I-1 cell variant, was previously found to be constitutively competent [platelet-derived growth factor (PDGF)-independent] to synthesize DNA (M. Tatsuka et al., J. Cell. Physiol., 139: 18-23, 1989). The present study has demonstrated that density-arrested, quiescent A31-I-13 cells autonomously exhibit disruption of actin filamentous bundles and perturbations of dynamic morphology. PDGF induced these cytoskeletal modulations in quiescent A31-I-1 cells, which require PDGF for the induction of DNA synthesis. Furthermore, the cytoskeletal modulations of quiescent A31-I-13 cells were not accompanied by an increased production of plasminogen activators, activation of protein kinase C, or phosphorylation of a Triton X-100-soluble protein (molecular weight, 90,000) known as 80K, a major substrate for protein kinase C. However, these modulations were accompanied by the tyrosine phosphorylation of Triton X-100-insoluble (cytoskeletal) proteins with molecular weights of 24,000, 32,000-33,000, and 36,000. These Triton X-100-insoluble proteins, as well as the 80K protein, were phosphorylated by the exposure of quiescent A31-I-1 cells to PDGF. Thus the pathway for producing the transformation-susceptible phenotype in A31-I-13 appears to coincide with the PDGF signaling pathway but does not involve the protein kinase C pathway.

3T3 Cells↗

PubMed1989

High invasiveness associated with augmentation of motility in a fos-transferred highly metastatic rat 3Y1 cell line.

We previously reported that v-fos transfer to a src-transformed rat 3Y1 cell line enhanced lung metastasis. To clarify the mechanism of this enhancement, we compared various biological factors related to metastatic potential between a fos-transferred highly metastatic cell line (fos-SR-3Y1-202) and the control cell line transferred with genetic marker (pSV2-neo) plus pBR322, neo-SR-3Y1-200. Lung arrest, the effect of lung extract on cell growth, or sensitivity to natural killer cells could not explain the higher metastasis of fos-SR-3Y1-202, compared to findings with neo-SR-3Y1-200. The invasiveness, assessed by penetration through a Matrigel-coated filter was about 5 times higher in fos-SR-3Y1-202 than in neo-SR-3Y1-200; high invasiveness in vitro was also observed in a fos-transferred mixed-population cell line (fos-SR-3Y1-200) and fos-transferred highly metastatic clones. Histopathological evidence of an in vivo tumor also showed the high invasiveness of fos-SR-3Y1-202 cells. To elucidate the causes of the increased invasiveness of fos-SR-3Y1-202, attachment of the cells to Matrigel and its components, such as laminin and type IV collagen, type IV collagenase activity, and motility were examined. Attachment of the cells to the substrate coated on Petri dishes or the activity of type IV collagenase did not differ significantly. On the other hand, cell motility, determined by a new method to directly quantitate alteration of cell shape continuously, using video image analysis and computer techniques, was higher in fos-SR-3Y1-202 than in neo-SR-3Y1-200. Thus, the fos-transferred cell line, fos-SR-3Y1-202 has a high invasiveness, in association with augmentation of motility, hence the enhancement of metastatic potential.

Animals↗

PubMed1989

Specific inhibitors of tyrosine-specific protein kinases: properties of 4-hydroxycinnamamide derivatives in vitro.

Inhibition by seven synthetic 4-hydroxycinnamamide derivatives, ST 271, ST 280, ST 458, ST 494, ST 633, ST 638, and ST 642, of tyrosine-specific protein kinases (tyrosine kinase) of oncogene or proto-oncogene products (p130gag-v-fps, p70gag-actin-v-fgr, pp60v-src, pp60c-src) and epidermal growth factor (EGF) receptor kinase were investigated. ST 638 (alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide) strongly inhibited more of the tyrosine kinases than any of the other compounds. The susceptibilities of these tyrosine kinases to ST 638 increased in the following order: EGF receptor greater than p70gag-actin-v-fgr greater than pp60c-src greater than p130gag-v-fps, pp60v-src, with 50% inhibitory concentration values of 1.1, 4.2, 18, 70, and 87 microM, respectively. The phosphorylation of the tyrosine residues in particulate fractions from RR1022 cells expressing pp60v-src was inhibited by ST 638 in a dose-dependent way, while it had a negligible effect on the phosphorylations of threonine and serine residues. Kinetic analysis showed that ST 638 competitively inhibited the phosphorylation of an exogenous substrate by the EGF receptor kinase with a Ki of 2.1 microM. ST 638 noncompetitively inhibited autophosphorylation by EGF receptor kinase. These results indicate that ST 638 is a potent and specific inhibitor of tyrosine kinases in vitro, and that its inhibitory activity is caused by competing with the substrate protein for the tyrosine kinase binding site.

Adenosine Triphosphate↗

PubMed1989

Analysis of cell variants showing differential susceptibilities to radiation- or chemical-induced neoplastic transformation: differences in their responses to growth factors.

The induction of DNA synthesis in quiescent, density-arrested Balb/c 3T3 cells is known to be controlled by the sequential action of at least two functionally distinct sets of growth factors, so-called "competence factors" and "progression factors." Here we examined this induction pathway in Balb/c 3T3 A31-I variants, which showed differential susceptibilities to radiation- and chemical-induced neoplastic transformation despite their similar susceptibilities to radiation- or chemical-induced cell killing and mutagenesis. DNA synthesis was acquired only with the exposure to progression factors in a highly susceptible cell variant (A31-1-13) whereas both competence factors and progression factors were required for a less susceptible cell variant (A31-I-1). The competent state constitutively produced by an autologous mechanism in the highly transformation-susceptible A31-I-13 cells suggests the existence of an endogenous promoter that acts for the expression of the transformed phenotype in an autocrine fashion when the cells have been initiated by radiation or chemical carcinogens. The growth factor requirements acting as a determining factor for susceptibilities to transformation are discussed.

Animals↗

PubMed1989

A digital image processing system for quantitating dynamic morphology in cultured mammalian cells.

An automated, video-driven system has been developed which can quantitate dynamic cell morphology in cultured mammalian cells. This system is based upon the Personal Image Analysis System and is assisted by a video-enhanced contrast microscopy with a computer-aided digital image processing unit and a time-lapse video technique. Various parameters for cell motility including locomotion (vectorial translation) and accompanying shape changes can be simultaneously analyzed. Here, we describe this system and demonstrate its application in Balb/c 3T3 cell culture. This system represents a new tool for exploring subtleties of mammalian cell behavior.

Animals↗

PubMed1989

X-ray-induced G2 arrest in ataxia telangiectasia lymphoblastoid cells.

Sensitivity to X-ray-induced G2 arrest was compared between ataxia telangiectasia (AT) lymphoblastoid cells and normal human cells. Flow cytometrical analysis of cells following X-ray irradiation revealed that the fraction of cells with 4n DNA content was greater in AT cells than in normal cells as previously reported by other investigators. However, the other parameters for cell-cycle progression kinetics including mitotic indices, cumulative mitotic indices and cumulative labelled mitotic indices indicated that X-ray-induced G2 arrest as a function of dose in AT cells was indistinguishable from that in normal cells. Moreover, no significant difference in cell viability was noted between AT and normal cells until 48 h following X-irradiation up to 2.6 Gy, although X-irradiated AT cells, compared to normal cells, showed a significantly decreased survival in terms of cell multiplication in growth medium and colony formation in soft agar. These data collectively suggest that the greater accumulation of AT cells with 4n DNA content in flow cytometry cannot be attributed to more stringent irreversible blockage of cell-cycle progression at the G2 phase and eventual cell death there. The possible reasons for this greater accumulation are discussed.

Ataxia Telangiectasia↗

PubMed1989

Quantitative measurement of cell motility associated with transformed phenotype.

The motility of individual mammalian cells is crucial for many biological processes. This report describes a new technique to quantitate cell motility, momentary alterations of cell shape, based on trace images obtained by video-image analyses and computer techniques. By means of this system, quantitation of cell motility could be automatically done without human observation or subjective judgement. Quantitative data from transformed and nontransformed rodent fibroblasts revealed that the cell motility measured here was related to the expression of such transformed phenotypes as morphological changes and tumorigenicity.

Animals↗

PubMed1988

An improved method of electroporation for introducing biologically active foreign genes into cultured mammalian cells.

We have developed a modified, reproducible, and efficient method for introducing cloned genes into mammalian cells by using an electric field followed by treatment with sodium butyrate. Transfection frequencies with plasmid pSV2-neo, consisting of an antibiotic (G418) resistance gene and simian virus 40 (SV40) early promoter, by electroporation were higher than those by calcium phosphate DNA precipitation. Treatment with sodium butyrate following electroporation significantly increased the frequency of transfection in various types of cell lines and primary cultured cells including human skin fibroblasts. Treatment with sodium butyrate also increased the transient expression of the gene for chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol O3-acetyltransferase, CAT, EC 2.3.1.28) when the gene was introduced into BALB/c 3T3 cells by electroporation. Electroporation combined with sodium butyrate treatment is an improved method for stable and transient biochemical transformation of foreign genes in cultured mammalian cells.

Animals↗

PubMed1983

[An approach to prolongation of survival rate in tumor bearing mice using 5-fluorouracil in combination with various kinds of herb medicine].

The effect of 5-fluorouracil (5-FU) in combination with herb medicine on the prolongation of survival rate in tumor bearing mice was studied. 5-FU directly inhibited the growth of cancer cells in vitro. However, the herb medicines used in this experiment showed neither an inhibitory effect on the growth of cancer cells nor prevention of inhibitory effect of 5-FU on the growth of cancer cells. 5-FU in combination with various kinds of herb medicine prolonged the survival time of tumor bearing mice. 5-FU in combination with shosaiko-toh was most effective; its increase of life span (ILS) was by 56%. However, mitomycin C in combination with shosaiko-toh was not associated with the prolongation of the survival rate of tumor bearing mice. The long-term administration of 5-FU to rats resulted in decrease in body weight, food intake, and white blood cell count, and these symptoms were improved when 5-FU was used in combination with shosaiko-toh. However, the increase in A/G ratio due to a decrease in the serum globulin fraction and marked atrophy of the thymus were not improved even when shosaiko-toh was combined with 5-FU.

Animals↗

PubMedDate not supplied

A tyrosine-specific protein kinase inhibitor, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide, blocks the phosphorylation of tyrosine kinase substrate in intact cells.

Inhibition by alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638) of tyrosine-specific protein kinase was examined using epidermal growth factor (EGF)-treated A431 cells at the concentration of 25 to 100 microM. ST 638 had negligible effects on the growth and morphology of A431 cells and on EGF binding to its receptor, and subsequent down-regulation of the receptor. ST 638 specifically inhibited EGF-induced phosphorylation of tyrosine residues of whole cell proteins in a dose-dependent manner without affecting the phosphorylation of serine and threonine residues. ST 638 greatly inhibited the EGF-induced phosphorylation of lipocortin I at 25 microM, and yet had a negligible effect on the EGF-induced phosphorylation of EGF receptor. Neither the amount of [35S]methionine-labeled lipocortin I nor the serine/threonine phosphorylation level of fodrin beta-subunit was affected by the same concentration of ST 638. These results indicate that the phosphorylation of lipocortin I is not relevant to the transformation of A431 cells. In cell lines transformed by src or fgr oncogene encoding tyrosine kinase, ST 638 also inhibited phosphorylation of calpactin I (p36) without affecting that of the oncogene products. Two-dimensional polyacrylamide gel electrophoresis showed that ST 638 specifically inhibited the EGF-induced phosphorylation and dephosphorylation of cellular proteins in A431 cells.

Animals↗

PubMedDate not supplied

Functional suppression of integrin beta 4-mediated adhesion caused by in vivo sequential selection for cancer cell intravasation.

Intravasation is essential for hematogenous metastasis in cancer cells, but its cellular determinants have not been well elucidated because of a lack of suitable experimental cell systems. Int-3LL was originally developed by in vivo sequential selection for intravasation from Lewis lung carcinoma (3LL) cells. Here, we found that these variant cells showed a highly penetrating ability in vitro as well as an augmented intravasating potential in vivo. In three-dimensional collagen-gel, Int-3LL cells formed diffusive colonies with less plating efficiency than their parental cells. Despite these properties, Int-3LL cells showed an ability of invasive migration in vitro similar to parental cells. On the other hand, a reduced adhesiveness and less spreading on extracellular matrices were revealed in Int-3LL cells. Analyses using anti-integrin antibodies indicated that the dysadhesion phenotype in Int-3LL cells was associated with integrin beta 4 dysfunction, which is known to produce epithelial detachment. Also, the types and the levels of integrins were not indistinguishable between Int-3LL and parental 3LL cells. Thus, the impaired function of integrin beta 4-mediated adhesion is considered to be an important factor in intravasation during metastasis.

Animals↗