Search PubMed⌕ Search

Biomedical subjects

M Kuwano

Publications and source records attributed to M Kuwano.

At least 235 records · Page 13Linked to original sources
PubMed1988

Novel feature of metabolism of low density lipoprotein receptor in a mouse macrophage-like cell line, J774.1.

Biosynthesis, processing, and degradation of low density lipoprotein (LDL) receptors were studied in a mouse macrophage-like cell line, J774.1, by immunoprecipitation and immunoblotting with an antibody directed against the COOH-terminal 14 amino acids of the LDL receptor. The molecular weight of the mature LDL receptor of J774.1 cells maintained in RPMI medium was 140,000 under nonreducing condition and 160,000 under reducing condition in sodium dodecyl sulfate-polyacrylamide gels. These sizes are 10,000-15,000 daltons larger than those of the receptor in other mouse fibroblastic cells or P388 leucocyte. However, when J774.1 cells were cultured in Dulbecco's modified Eagle's medium, the molecular weight of the mouse cell lines, 123,000 under nonreducing condition and 153,000 under reducing condition. The larger LDL receptor molecules produced by J774.1 cells cultured in RPMI were insensitive to the treatment with end-alpha-N-acetylgalactosaminidase (O-glycanase), suggesting that aberrant serine/threonine-linked (O-linked) glycosylation might account for the apparent large size. Pulse-chase experiments revealed that the rate of processing of the LDL receptor from precursor to mature form in J774.1 was similar to that in other mouse cell lines, but the rate of degradation was much faster: half-life of the LDL receptor of J774.1 was about 2 h. No significant difference in biological function or lifetime was observed between the normal and the larger LDL receptor. This novel character of molecular size and lifetime of the LDL receptor in J774.1 is discussed in relation to altered maturation and/or modification during receptor biosynthesis.

Animals↗
PubMed1988

Potentiation by a biscoclaurine alkaloid, cepharanthine, of the toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin in HeLa cells.

Cepharanthine, a biscoclaurine alkaloid, causes an 8-fold enhancement of the cytotoxic effect of a conjugate of epidermal growth factor (EGF) coupled with Pseudomonas exotoxin in HeLa cells. Cepharanthine also potentiates the effect of Pseudomonas exotoxin. Cepharanthine does not affect the binding and uptake of 125I-EGF by HeLa cells, but it delays the release of radioactivity associated with 125I-EGF into the medium. Analysis by colloidal silica gradients using cell homogenates suggests that 125I-EGF accumulates in the lysosomes of cells treated with cepharanthine and that [3H]cepharanthine accumulates in lysosomes. The pH in HeLa cell lysosomes is 5.2, and cepharanthine does not significantly increase the pH. Electron microscopy shows an increased number of electron-dense bodies and dilated Golgi apparatus after cepharanthine treatment. Cepharanthine appears to accumulate in lysosomes, and it may delay degradation of EGF-Pseudomonas exotoxin in the cells as does 125I-EGF.

ADP Ribose Transferases↗
PubMed1988

Estrogen inhibits the growth of MCF-7 cell variants resistant to transforming growth factor-beta.

Human breast cancer MCF-7 cells containing estrogen receptor are killed by transforming growth factor-beta (TGF-beta). We isolated variants of MCF-7 highly resistant to TGF-beta. Variants ES-1 and ES-4 were cloned, and the growth of ES-1 and ES-4 was found to be inhibited by estradiol, whereas estradiol stimulated the growth of the parental MCF-7 cells. ES-1 cells contained about 2-fold higher level of estradiol receptor than MCF-7 cells. Addition of estradiol to the culture medium for MCF-7 and the variant changed the expression of several secreted proteins. The repertoire of secreted proteins was markedly altered in the variant. Polypeptides of molecular weight 52,000 (52 K), 65 K and 160 K were increased about 10- to 50-fold in both estradiol-treated MCF-7 and ES-1 cells. Polypeptide of 130 K was decreased in estradiol-treated ES-1 cells while this polypeptide was increased about 4-fold in estradiol-treated MCF-7, as compared with untreated MCF-7. Polypeptide of 100 K was specifically secreted in ES-1 whether or not estradiol was present, but there appeared to be no significant amount of the 100 K protein in MCF-7. The estradiol-hypersensitive phenotype is discussed in relation to its aberrant expression of secreting proteins.

Breast Neoplasms↗
PubMed1988

Vincristine-resistant human cancer KB cell line and increased expression of multidrug-resistance gene.

A multidrug-resistant clone of human cancer KB cells was isolated by stepwise selection on exposure to increasing doses of vincristine. The final clone, VJ-300, obtained after ethylmethane sulfonate mutagenesis showed 400-fold higher resistance to vincristine than did KB cells. Cellular accumulation of vincristine in VJ-300 was decreased to less than one-tenth of that in KB. The cells were also cross-resistant to daunomycin, adriamycin, actinomycin D, colchicine and VP-16. During continuous culturing in the absence of any drug for several months, a different colchicine-resistant and multidrug-resistant clone, KB-C1, reverted almost completely to drug sensitivity, whereas drug resistance in VJ-300 was stably maintained. Amplification of the multidrug-resistance-1 (mdr-1) gene was more than 20-fold in KB-C1, but less than 2-fold in VJ-300. mdr-1 mRNA was, however, expressed in VJ-300 at a rate comparable to KB-C1. Acquisition of high multidrug resistance in VJ-300 might be correlated with both activated transcription of mdr-1 gene and amplification.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Transformation by viral and cellular oncogenes of a mouse BALB/3T3 cell mutant resistant to transformation by chemical carcinogens.

The mouse cell line MO-5 is resistant to transformation by various chemical carcinogens and also by UV irradiation (C. Yasutake, Y. Kuratomi, M. Ono, S. Masumi, and M. Kuwano, Cancer Res. 47:4894-4899, 1987). Northern (RNA) blot analysis showed active expression of ras and myc genes in MO-5 and BALB/3T3 cells. The effect of transfection of various oncogenes on transformation was compared in MO-5 cells and parental BALB/3T3 cells. Activated c-H-ras, c-N-ras, and v-mos gene induced transformation foci of MO-5 and BALB/3T3. Introduction of the polyomavirus middle T-antigen (mTag) or the Rous sarcoma virus-related oncogene v-src, however, efficiently transformed BALB/3T3 but not MO-5 cells. Expression and phosphorylation of mTag and the associated c-src proteins were observed in mTag-transfected clones of MO-5 as in BALB/3T3 and phosphorylation of the src protein was observed in v-src-transfected BALB/3T3 and MO-5 clones. Hybrids between mTag- or v-src-induced transformants of BALB/3T3 and untransformed MO-5 maintained the transformation phenotype, suggesting that no dominant suppressor of transformation exists in MO-5. A hybrid clone between BALB/3T3 and MO-5 induced efficient transformation foci after transfection with the mTag gene, suggesting that the deficient transformation phenotype of MO-5 was recessive. Instead, some other alteration of MO-5, plausibly membrane function, might lead to abortive transformation by chemical carcinogens and also by mTag and the v-src gene product.

Animals↗
PubMed1988

Most drugs that reverse multidrug resistance also inhibit photoaffinity labeling of P-glycoprotein by a vinblastine analog.

Multidrug-resistant human KB carcinoma cells express a 170,000-dalton membrane glycoprotein (P-glycoprotein) that can be photoaffinity labeled with the vinblastine analog N-(p-azido-[3-125I]salicyl]-N'-(beta-aminoethyl)vindesine. Several agents that suppress the multidrug-resistant phenotype, including N-solanesyl-N,N'-bis(3,4-dimethylbenzyl)ethylenediamine, cepharanthine, quinidine, and reserpine, were found to inhibit photolabeling of P-glycoprotein at doses comparable to those that reverse multidrug resistance. However, the phenothiazines chlorpromazine and trifluoperazine, which also effectively reverse multidrug resistance, were poor inhibitors of the photoaffinity labeling of P-glycoprotein. Chloroquine, propranolol, or atropine, which only partially reversed the drug resistance, also did not inhibit photolabeling. Naphthalene sulfonamide calmodulin inhibitors, W7 and W5, as well as many other drugs that did not circumvent multidrug resistance, did not inhibit photolabeling. These studies suggest that most, but not all, agents that phenotypically suppress multidrug resistance also inhibit drug binding to a site on P-glycoprotein with which a photoaffinity analog of vinblastine interacts.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1987

Altered interaction between Sendai virus and a Chinese hamster cell mutant with defective cholesterol synthesis.

An amphotericin B-resistant mutant (AMBr-1) isolated from the Chinese hamster V79 cell line is defective in a pathway for sterol synthesis and contains a much reduced free cholesterol level as compared with the parental V79. The character of the plasma membrane of AMBr-1 was compared with that of V79 by measuring the fusion with the envelope of the Sendai virus and also by measuring membrane fluidity: AMBr-1 was found to be more sensitive to Sendai virus-induced cytolysis than V79. Both assays for membrane-permeability change and electron spin resonance (ESR) study showed an enhanced response to the fusion between viral envelope and plasma membrane in AMBr-1 cells. Measurement of the fluorescence polarization for 1,6-diphenyl-1,3,5-hexatriene suggested that the membrane of AMBr-1 was more fluid than that of V79. This aberrant nature of the cell membrane of AMBr-1 might be caused by the altered membranous sterol content.

Animals↗
PubMed1987

Altered function and structure of low-density lipoprotein receptor in compactin (ML236B)-resistant mutants of Chinese hamster cells.

Mutants resistant to compactin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, have been previously isolated from the Chinese hamster V79 cell line. Two compactin-resistant mutants, MF-1 and MF-2, show altered responses to human low-density lipoprotein (LDL). Accumulation of fluorescent-labeled LDL was much reduced. Ligand blotting showed LDL receptor activity in MF-1 and MF-2 cells of about one half to one third that of V79. Internalization and degradation of LDL in MF-1 or MF-2 cells were about one tenth those in V79 cells, suggesting that the LDL binding as well as the LDL internalization of the compactin-resistant clones was altered. Down-regulation of LDL receptor activity as well as hydroxymethylglutaryl CoA reductase was observed in V79 cells treated with LDL, while there appeared to be much less down-regulation in MF-1 and MF-2 cells. Using anti-LDL receptor antibody, MF-1 and MF-2 cells were found to produce smaller-sized mature forms of LDL receptor: the molecular mass of the mutant LDL receptor was 3-5 kDa smaller than that of the parental LDL receptor. Altered O-linked oligosaccharides or amino acid sequence might account for the decreased molecular mass and aberrant properties of the LDL receptor in MF-1 and MF-2.

Animals↗
PubMed1987

Low binding capacity and altered O-linked glycosylation of low density lipoprotein receptor in a monensin-resistant mutant of Chinese hamster ovary cells.

We have studied function and structure of the low density lipoprotein (LDL) receptors in a monensin-resistant (Monr-31) mutant isolated from Chinese hamster ovary (CHO) cells. To assay the ability of the receptor to bind LDL, we employed three methods, 125I-LDL binding to the cells at 4 degrees C, 125I-LDL binding to the receptor-phospholipid complex (Schneider, W.J., Goldstein, J.L., and Brown, M.S. (1980) J. Biol. Chem. 255, 11442-11447), and ligand blotting (Daniel, T.O., Schneider, W.J., Goldstein, J.L., and Brown, M.S. (1983) J. Biol. Chem. 258, 4606-4611). The LDL receptor number was similar in both CHO and Monr-31, but the binding affinity was reduced in the mutant. The semi-quantitative immunoblotting assay with an antibody directed against the COOH-terminal 14 amino acids and the ligand-blotting assay with LDL also showed that the relative steady-state level of the receptor in Monr-31 was comparable to that in CHO, whereas the binding capacity of the receptor in Monr-31 was lower than that in CHO. The precursor and degradation forms of the LDL receptors produced in the mutant cells were similar in size to those in the parental cells, but the apparent molecular mass of the mature receptor protein in sodium dodecyl sulfate-polyacrylamide gels was reduced about 5000 daltons in the mutant. These results suggest a structural change at the NH2-terminal LDL binding domain. Tests of the effects of tunicamycin, endo-alpha-N-acetylgalactosaminidase (O-glycanase), and sialidase (neuraminidase) on the molecular size of the mature receptors indicated that the reduced size of the receptor in the mutant cells resulted from altered oligosaccharide chain(s) linked to serine/threonine residues in the binding domain. We compared the molecular sizes and binding activity of human LDL receptors in several clones derived from CHO and Monr-31 cells which were transfected with human LDL receptor cDNA. The human LDL receptors produced in the transfected clones of Monr-31 were also smaller in molecular size and lower in binding capacity than those produced in the transfected clones of CHO. These results suggest that both structural and functional alteration of the LDL receptor of Monr-31 is not caused by a mutation in the structural gene of the LDL receptor but by altered processing or maturation of the receptor. The correlation of the decrease in molecular size and reduced binding capacity of the LDL receptor is discussed.

Animals↗
PubMed1987

Effect of 5-azacytidine on malignant transformation of a mutant derived from the mouse BALB/c 3T3 cell line resistant to transformation by chemical carcinogens.

BALB/c 3T3 and its derivative MO-5, isolated as a monensin-resistant clone, showed a very low rate of spontaneous malignant transformation. Treatment of BALB/c 3T3 cells with benzo(alpha)pyrene, 4-nitroquinoline-N-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, or UV light irradiation significantly enhanced the rate of transformation, whereas the treatment of MO-5 cells with these carcinogens had only a slight if any effect. Exposure of MO-5 as well as BALB/c 3T3 to 5 or 10 microM 5-azacytidine for 3 to 7 days significantly increased the number of transformation foci. The Luria-Delbrück fluctuation test showed that spontaneous mutation frequency (mutants/cell/generation) was 1.2 X 10(-6) for BALB/c 3T3 and 7.1 X 10(-7) for MO-5, respectively, when appearance of cadmium-resistant clones was tested. N-Methyl-N'-nitro-N-nitrosoguanidine enhanced induced mutation frequency of ouabain-resistant and cadmium-resistant mutants of BALB/3T3 but it only slightly enhanced that of MO-5. Methylation status of DNA of MO-5 was compared with that of BALB/c 3T3 by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes HpaII and MspI. DNA of MO-5 was found to be more methylated than that of BALB/c 3T3 in the vicinity of c-myc as well as the metallothionein-I gene. Aberrant DNA methylation in MO-5 and the cellular sensitivity to transformation by chemical carcinogens or 5-azacytidine are discussed.

4-Nitroquinoline-1-oxide↗
PubMed1987

Effect of bisbenzylisoquinoline (biscoclaurine) alkaloids on multidrug resistance in KB human cancer cells.

Cepharanthine, a bisbenzylisoquinoline (biscoclaurine) alkaloid, completely overcomes resistance of a multidrug-resistant subline, ChR-24, derived from human KB carcinoma cells, to vincristine, actinomycin D, and daunomycin, and partially overcomes resistance to Adriamycin. Another biscoclaurine alkaloid, berbamine, partially overcomes resistance to these anticancer agents. Accumulation of [3H]daunomycin in ChR-24 cells is about 10% of that in both the parental KB and revertant cell line (Rev-2) which is derived from ChR-24. Cepharanthine prominently increases the accumulation of daunomycin in resistant ChR-24 cells, but not in parental KB and Rev-2 cells. Enhanced efflux of daunomycin from the resistant cells is completely inhibited by cepharanthine. Cellular uptake of [3H]daunomycin is not significantly affected in the resistant cells by cepharanthine. Accumulation of [3H]cepharanthine is observed at similar levels in both KB and ChR-24. Phosphatidylserine specifically inhibited the accumulation of [3H]cepharanthine in KB and ChR-24 cells when tested by adding various phospholipids such as phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin to culture medium. The enhanced accumulation of [3H]daunomycin in cepharanthine-treated ChR-24 cells is inhibited in the presence of 20 micrograms/ml phosphatidylserine. Cepharanthine may overcome multidrug resistance by binding to phosphatidylserine in the plasma membrane and perturbing membrane function.

Alkaloids↗
PubMed1987

Enhancement by a synthetic isoprenoid of the toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin.

A newly synthesized isoprenoid, N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine, has a verapamil-like structure but no calcium channel blocking activity. The isoprenoid enhanced the cytotoxic effect of a conjugate of epidermal growth factor coupled with Pseudomonas exotoxin in human KB cells. By using iodinated epidermal growth factor ([125I]EGF), the effect of the isoprenoid on intracellular transport of EGF was examined. The isoprenoid did not affect the binding and uptake of [125I]EGF by KB cells. The release of radioactivity associated with [125I]EGF into medium was slow in the presence of the isoprenoid. Density gradient fractionation studies using cell homogenates suggest that [125I]EGF accumulates in an undegraded form in lysosomes when cells are treated with the isoprenoid. The pH value in lysosomes of KB cells was 5.5, and SDB did not affect significantly the pH value at the concentrations used to potentiate the cytotoxicity of chimeric toxins. Electron microscopy showed an increased number of electron-dense bodies in KB cells grown for 24 hr with 17-51 micrograms/ml isoprenoid. The potentiating action of chimeric toxins by the isoprenoid is discussed in relation to the altered lysosomal function in treated cells.

ADP Ribose Transferases↗
PubMed1987

Chinese hamster ovary cell mutant with defective down-regulation of low density lipoprotein receptors.

A monensin-resistant clone (Monr-31) shows a related series of differences from its parental Chinese hamster ovary (CHO) cell line in the cellular response to several ligands. The uptake and metabolism of low density lipoprotein (LDL) in the mutant cells are defective. Accumulation of fluorescent-labeled LDL as well as internalization and degradation of 125I-LDL are greatly reduced in Monr-31 cells. The receptor number for LDL on the cell surface of Monr-31 is about one-third that for CHO cells, but affinity constants for both cell lines are similar. Electrophoretic analysis shows a slightly reduced molecular weight of LDL receptor in Monr-31 cells in comparison to that in CHO cells. The internalization index (internalization plus degradation per binding) of LDL of the mutant is about one-half that of CHO cells, suggesting a failure of internalization of LDL as well as LDL binding. Hybrids (hyb-1, -2, and -3) between CHO and Monr-31 cells show LDL binding and LDL internalization activities comparable to that of CHO cells, suggesting that the altered LDL response in Monr-31 cells is recessive. Addition of exogenous LDL to culture medium down-regulates the LDL receptor activity of CHO, hyb-2, and hyb-3 cells, whereas no such down-regulation is seen in Monr-31 cells. Probably as a result of the failure of down-regulation, the prominent inhibition of sterol synthesis from acetate and 3-hydroxy-3-methylglutaryl-coenzyme A reductase observed in CHO cells is scarcely detectable in Monr-31 cells. As a correlated result, sterol synthesis from acetate is 6-fold higher in the mutant. The failure of down-regulation of LDL receptors in Monr-31 cells is discussed in relation to the altered binding and internalization of LDL.

Animals↗
PubMed1987

Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor beta.

A mutant clone (MO-5) was originally isolated as a clone resistant to Na+/K+ ionophoric antibiotic monensin from mouse Balb/c3T3 cells. MO-5 was found to show low receptor-endocytosis activity for epidermal growth factor (EGF): binding activity for EGF in MO-5 was less than one tenth of that in Balb/c3T3. Anchorage-independent growth of MO-5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-beta efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing in soft agar of MO-5 or Balb/c3T3 was significantly enhanced by TGF-beta while EGF did not further enhance that of MO-5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF-beta showed low colony formation capacity in soft agar in the absence of TGF-beta. Colonies of MO-5 formed by TGF-beta in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-beta. Pretreatment of MO-5 with TGF-beta induced secretion of TGF-beta-like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF-beta-like activity. The loss of EGF-receptor activity in the stable expression and maintenance of the "transformed" phenotype in MO-5 is discussed.

Animals↗
PubMed1987

Potentiation of vincristine by vitamin A against drug-resistant mouse leukaemia cells.

Vitamin A has been shown to potentiate the cytotoxic action of anticancer agents like vincristine (VCR) against drug resistant mouse P388 leukaemia cells. In vitro tests showed enhancement by retinyl acetate of cytocidal activities of VCR against drug-sensitive leukaemia (P388/S) and VCR-resistant leukaemia (P388/VCR) cells in culture; retinyl acetate rather specifically potentiated VCR against cultured P388/VCR cells than P388/S cells. The cellular accumulation of radioactive VCR was significantly enhanced in cultured P388/VCR cells when retinyl acetate was present. The efflux of VCR from drug-resistant cells was blocked by retinyl acetate. The effect of the combination of vitamin A and VCR was also tested in vivo on the life-span of mice bearing P388/S or P388/VCR. Intraperitoneal administration of retinyl palmitate at 41.75 or 83.5 mg kg-1 was effective to potentiate the antileukaemic activity of VCR against P388/S bearing mice, and it also overcame vincristine-resistance in P388/VCR bearing mice.

Animals↗
PubMed1986

Cellular retinol-binding proteins in head and neck tumors and their adjacent tissues.

The levels of cellular retinol-binding protein (CRBP) were examined by Scatchard analysis in 17 surgically resected human head and neck tumors and their adjacent normal tissues (normal tissues corresponded to muscle tissue). The center area and the marginal area of tumors were tested for their levels of CRBP in 17 cases, and the adjacent tissue was tested in 13 of the 17 cases. Only a small difference (if any) in the dissociation constant (Kd values) was seen in some cases between tumor and adjacent tissue; in most cases the Kd values in both areas were similar, and these values were also similar between the central and marginal areas of tumors. The average level of CRBP in the marginal area of the tumors was higher than that in the center area of the tumors in all the 17 cases. Among the 13 tumor cases whose adjacent tissues were tested, 12 showed CRBP levels more than two times higher in the marginal area of tumors; whereas in only one case was the level in the marginal or the center area of tumor similar to that in the adjacent tissues.

Aged↗