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Biomedical subjects

M Kuwano

Publications and source records attributed to M Kuwano.

At least 289 records · Page 16Linked to original sources
PubMed1983

Isolation of chloroquine-resistant Chinese hamster V79 cell variants that are also resistant to ammonium chloride.

Chloroquine-resistant (CQr) clones (CQ-21 and CQ-22) have been isolated from mutagenized hamster lung V79 cells by exposing the cells to a high dose of chloroquine. CQ-21 and CQ-22 showed about 3-fold higher resistance to chloroquine than the parental V79 cells, and they showed specific cross-resistance to another amine, NH4Cl, which is also concentrated in lysosomes. CQr clone showed no cross-resistance to other unrelated agents. Chloroquine-induced inhibition of [125I]ricin internalization was observed in both cell lines at neutral pH, but the inhibition of uptake was less in the variant. Also, the degradation of endogenous protein was slowed in the mutant; further, treatment of cells with 30 micrograms/ml of chloroquine inhibited the degradation of endogenous proteins in the parental V79, but not in CQ-22 cells. Similar levels of acid phosphatase, beta-glucuronidase and cathepsin D were observed in V79 and CQ-22 cells, but the level of cathepsin B was lower in the mutant. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes, in the mutant cells grown for 4 days with 5 micrograms/ml of chloroquine. Similar aberrant structures were observed in the parental V79 cells treated for only 3 h with 5 micrograms/ml of chloroquine.

Ammonium Chloride↗
PubMed1983

[Experimental and clinical studies on application of vitamin A to cancer treatment].

Retinol-type vitamin A enhances cytocidal effect of several anticancer agents or radiation in in vitro as well as in vivo. We describe therapeutic effect fo a combination therapy of vitamin A and other agents which has been applied to cancer patients since 1972. We also discuss a possible role of vitamin A in the efficient synergistic effect.

Antineoplastic Agents↗
PubMed1982

Chinese hamster cell mutant resistant to ML236B (Compactin) is defective in endocytosis of low-density lipo-protein.

A fungal metabolite, ML236B (Compactin), isolated from Penicillium citrinum, is a specific inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase (EC 1.1.1.34). Three ML236B-resistant (ML236Br) mutants, MF-1, MF-2, and MF-3, were isolated from V79 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The fluctuation test showed 2.2 X 10(-6) mutants per cell per generation of a spontaneous mutation frequency of ML236Br clones. These ML236Br clones showed a four- to fivefold-higher resistance to the drug than did their parental V79. Radioactive acetate, but not mevalonate, incorporation into the sterol fraction increased about 10-fold in ML236Br clones in comparison with that in V79. The cellular level of HMG-coenzyme A reductase in three ML236Br mutants was found to be a few-fold higher than that of V79 when cultured in the presence of lipoproteins. The 125I-labeled low-density lipoprotein-binding assay showed binding activity in three ML236Br clones comparable to that of the parental V79 cells. By contrast, an internalization assay of 125I-labeled low-density lipoprotein into the cells showed significantly reduced activity in three ML236Br clones in comparison with V79.

Acetates↗
PubMed1982

Chinese hamster cell variants resistant to the A chain of ricin carry altered ribosome function.

Ricin, a toxic lectin from Ricinus communis, is composed of two different polypeptide chains, A and B, and the ricin A chain (RA) blocks protein synthesis. We studied cell lines resistant to cytotoxic action of RA. One low-RA-resistant cell line, AR10, isolated from Chinese hamster ovary (CHO) cells, was resistant to a low dose of RA (1 microgram/ml) and showed a 10-fold-higher resistance to RA and ricin than that of CHO. We further mutagenized AR10 to isolate high-RA-resistant cell lines AR100-6, AR100-9, and AR100-13, which were resistant to higher doses of RA and ricin (100- to 1,000-fold) than CHO was. The binding of [125I]ricin to AR10, AR100-6, AR100-9, and AR100-13 cells was decreased to about 30% of that of CHO. The internalization of [125I]ricin in AR10 cells and in the high-RA-resistant clones was the same. Polyuridylate-dependent polyphenylalanine synthesis, using S-30 extracts from either AR100-9 or AR100-13, was about 100-fold more resistant to the inhibitory action of RA than when CHO, AR10, and AR100-6 cells extracts were used. The protein synthesis with ribosomes (80S) from AR100-9 or AR100-13 was 10- to 100-fold more resistant to RA than it was with parental ribosomes when combined with the S-100 fraction of CHO cells. The polyphenylalanine synthesis assay using the ribosomes constituted from the 60S subunit of AR100-9 and the 40S subunit of CHO indicated that the resistant phenotype of AR100-9 cells is due to an alteration of the 60S ribosomal subunit.

Animals↗
PubMed1982

Potentiation of antifungal effect of amphotericin B by squalene, an intermediate for sterol biosynthesis.

The antifungal effect of a polyene antibiotic amphotericin B (AMB) was almost completely abrogated by exogenous addition of 0.1 microgram/ml ergosterol in the medium. The cytocidal effect of AMB on Saccharomyces cerevisiae was synergistically enhanced when cultured for more than 4 hours with squalene, an obligatory intermediate molecule for sterol formation. However, we could not find significant increase in cellular level of ergosterol content in the yeast cells fed with squalene.

Amphotericin B↗
PubMed1982

Differential effect of imidazole antibiotics on untransformed and virus-transformed rat cell lines.

The imidazole antimycotics clotrimazole and miconazole were tested on untransformed rat cell line 3Y1-B clone 1-6 (3Y1) and six transformed cell lines, which were independently isolated from 3Y1 or 3Y1-B clone 1 after infection with adenovirus 12 (AD-12) or with SV40, to determine their sensitivities to these drugs. The relative plating efficiency of three cell lines (T3, W4, and W5) transformed with AD-12 was reduced to 10(-1( of the initial value by clotrimazole (2 to 4 microgram/ml), whereas that of the parental cell line 3Y1 was reduced to 10(-1) of the initial value by clotrimazole (20 to 25 microgram/ml). By contrast, the differential effect of miconazole on 3Y1 and AD-12-transformed cell lines was found to be a factor of 2. The sensitivity of the SV40-transformed 3Y1 and the AD-12-transformed cell lines. The cellular sensitivity of untransformed 3Y1 cells to clotrimazole was significantly enhanced when exposed to various doses of the unsaturated fatty acid, linoleic acid. The untransformed and transformed cell lines showed sensitivities similar to the cytocidal activity of sterol-binding antimycotics, amphotericin B and filipin.

Amphotericin B↗
PubMed1981

The synthesis of ribosomal 5 S RNA in cultured hamster cells during the inhibition of protein synthesis.

Ribosomal 5 S RNA synthesis in Chinese hamster V79 cells treated with an inhibitory antibiotic of protein synthesis, cycloheximide, was quantitated by hybridization of RNA preparations with plasmid ColE1-pSC101 DNA carrying Xenopus 5 S DNA. In V79 cells, cycloheximide produced a notable decrease in the production of the higher molecular weight ribosomal RNA (rRNA) fraction, whereas the accumulation of both 5 S RNA and polyadenylate-containing messenger RNA was much less affected. When the cellular protein synthesis was inhibited by over 85% of the control, accumulation of 5 S RNA and of rRNA was respectively 40-50% and about 10% of the control. The size distribution analysis of RNA species revealed that 5 S RNAs obtained from V79 cells after treatment with 0.1 microgram/ml or 2.0 microgram/ml cycloheximide were indistinguishable from control. Inhibition of precursor uptake into 5 S RNA by cycloheximide was apparently phase-specific during the cell cycle and was 44-67% and 78-85% of the control for the S and the M phase, respectively. The responses of RNA polymerase III activities to cycloheximide in isolated nuclei from each phase of the cell cycle correlated very well with those of precursor uptake into 5 S RNA observed in vivo, while total solubilized RNA polymerase activities showed no inhibition by the drug at any phase of the cell cycle. Cytosine arabinoside, a specific inhibitor of DNA synthesis, did not cause any decrease in the cellular level of 5 S RNA.

Animals↗
PubMed1981

Inhibition of DNA synthesis and cell cycle in Chinese hamster cells by sterol-binding polyene amphotericin B.

Amphotericin B, a sterol-binding polyene antibiotic, was found to inhibit DNA synthesis more than protein or RNA synthesis of asynchronous cultures of Chinese hamster V79 cells. DNA synthesis in the asynchronous V79 cells was inhibited to 40--60% of the control activity in the presence of 50 micrograms/ml amphotericin B. However, addition of 50 micrograms/ml of polyene immediately after to onset of DNA synthesis (early S phase) caused a drastic reduction of DNA synthesis (below 10--20% of the control in synchronized V79 cells, whereas the inhibition was much lessened when the polyene was added 1 h later (middle S phase). In contrast, there was no inhibition of DNA synthesis by amphotericin B in an amphotericin-B-resistant (AMBR) clone that was derived from V79. Flow microfluorometry analysis confirmed that a large number of asynchronous V79 cells were arrested in the G1 phase of the cell cycle when treated with lower dose of amphotericin B. A higher dose of the polyene antibiotic also accumulated cells at the G2 (or at both S and G2) phase as well as the G1 phase. Morphological studies by scanning electron microscope showed an increased number of V79 cells with decreased microvilli in V79 cells treated with amphotericin B.

Amphotericin B↗
PubMed1981

Amphotericin B resistance is recessive in Chinese hamster hybrid cells.

Previous studies from our laboratory have shown that Chinese hamster V79 cells mutated to high level resistance to amphotericin B have a lower cellular level of cholesterol, the target molecule for the polyene antibiotic. Two amphotericin B-resistant (AMBR) mutants were each hybridized to their parental amphotericin B-sensitive (AMBS) V79 cells. All the hybrids derived from AMBR/AMBS fusions were as sensitive to polyene antibiotics (amphotericin B, filipin, and pimaricin) as AMBS cells or AMBS/AMBS hybrids. The AMBR/AMBS hybrids were found to contain cholesterol per phospholipids that is comparable to those in AMBS or AMBS/AMBS. The analysis of hybrids formed between mutant and wild-type cells thus indicated that resistance to amphotericin B is a recessive marker, and that the cellular level of cholesterol is compensated in the AMBS/AMBR hybrids. Hybrids of AMBR and AMBR cells were all resistant, so that the three AMBR mutants all fell into a single complementation group.

Amphotericin B↗
PubMed1981

Isolation and preliminary characterization of bleomycin-resistant mutants from Chinese hamster ovary cells.

Stable mutants resistant to an anticancer antibiotic, bleomycin-A2, were selected in Chinese hamster ovary (CHO) cell either spontaneously or after ethylmethane sulfonate mutagenesis. Fluctuation analysis showed that bleomycin resistance occurs in CHO at a rate of 6.50--6.58 x 10(-7) mutations per cell per generation. Bleomycin-A2-resistant cell lines exhibited increased resistance to bleomycin analogs--bleomycin-A5, -B2, -B4, and pepleomycin. Colchicine, mitomycin C, and ultraviolet light irradiation inhibited colony formation equally in CHO cells and in bleomycin-resistant mutants. Cell-cell hybridization tests showed that bleomycin-resistance behaves as a dominant trait. Bleomycin-inactivating activity in the mutant cell extracts was three to fourfold higher than that in extracts of the parental CHO cell.

Animals↗
PubMed1981

Isolation and characterization of Chinese hamster ovary cell mutants with altered sensitivity to high doses of tunicamycin.

A mutant, CTM422, resistant to low dose of tunicamycin (TM) was isolated from Chinese hamster ovary (CHO) cells, and it showed 7- to 10-fold higher resistance to TM than CHO. We further mutagenized CTM422, to isolate TM-high-resistant mutants which were resistant to about 100-fold higher dose of TM than CHO. The TM-high-resistant mutants (N101 and N102) acquired about 4-fold higher cross-resistance to 2-deoxy-d-glucose than CHO or CTM422, while both CHO and CTM422 showed similar sensitivity to 2-deoxy-d-glucose. TM-low-resistance appeared to be codominant, and TM-high-resistance was partially codominant against TM sensitivity, respectively. The transfer activity of N-acetylglucosamine from UDP-N-acetylglucosamine into the lipid fraction with CHO and CTM422 cell extracts was inhibited by TM to a similar extent, while the extract of N102 cells showed about 10-fold higher resistance to TM than CHO or CTM422.

Animals↗
PubMed1981

Enhancement of the antitumor effect of 6-mercaptopurine by vitamin A.

The inhibition of the growth of HeLa cells in culture by 6-mercaptopurine was greatly enhanced in combination with retinol, retinal or retinol acetate, but not in combination with retinoic acid or retinol palmitate. The antitumor effect of retinol palmitate combined with 6-mercaptopurine was examined in BDF1 mice with L1210 leukemia. The blood level of retinol, a strong potentiator of 6-mercaptopurine among vitamin A compounds, was increased in BDF1 mice after retinol palmitate injection. Retinol palmitate was found to enhance the antitumor effect of 6-mercaptopurine against murine L1210 leukemia to a significant extent (69% increase in life span).

Animals↗
PubMed1980

Antitumor effect of a combination of 6-methylthioinosine and amphotericin B on mouse leukemia L1210.

Antileukemic activity of 5 kinds of sulfur-containing purine ribonucleosides were examined in the presence and absence of amphotericin B against L1210 in mice. Among these compounds, 6-methylthioninosine was potentiated by amphotericin B. 6-Methylthioinosine in combination with amphotericin B produced a 75% increase in the lifespans, which was greater than the increase in lifespans by 6-methylthioinosine (38%) or amphotericin B alone (2%). Antitumor effects of other sulfur-containing ribonucleosides, such as 6-thiocyanatoguanine, 6-thiocyanatopurine, 6-thiocyanatoinosine, and 6-methylthiopurine, were not augmented by amphotericin B.

Adjuvants, Immunologic↗
PubMed1980

Classification of polyene antibiotics according to their synergistic effect in combination with bleomycin A2 or fusidic acid.

Five polyene antibiotics were compared for their effects on colony formation of either Chinese hamster V79 or Saccharomyces cerevisiae cells. A 10 to 40 times higher concentration of amphotericin B (heptaene) or nystatin (degenerated heptaene) was necessary to inhibit colony formation of hamster cells than that needed to inhibit colony formation of yeast cells. In contrast, colony formation of both hamster and yeast cells was inhibited to the same extent by similar concentrations of filipin (pentaene), pentamycin (pentaene), or pimaricin (tetraene). The five polyene antibiotics were also compared for their effects on colony formation of either V79 or S. cerevisiae cells when combined with a nonpolyene antibiotic, fusidic acid or bleomycin A2. Amphotericin B or nystatin could augment the cytocidal effect of fusidic acid but not that of bleomycin A2, whereas pentamycin or pimaricin could augment the cytocidal effect of both fusidic acid and bleomycin A2 against hamster and yeast cells. Filipin was found to enhance the action of fusidic acid and bleomycin upon growth of mammalian cells, whereas the pentaene polyene significantly potentiated the action of fusidic acid, but not that of bleomycin A2, against S. cerevisiae. It was therefore suggested that these polyene antibiotics be classified into two groups: group 1 (pimaricin, pentamycin, and filipin) and group 2 (amphotericin B and nystatin).

Amphotericin B↗