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Biomedical subjects

M Kuwano

Publications and source records attributed to M Kuwano.

At least 199 records · Page 11Linked to original sources
PubMed1991

Counteraction of estradiol-induced activation of tissue-type plasminogen activator in a human breast cancer cell line by an anti-estrogen, LY117018.

LY117018 is a non-steroid anti-estrogen which exhibits about 100 times higher affinity for estrogen receptor than tamoxifen, another anti-estrogen. The cell line ES-1, which was isolated from human breast cancer MCF-7 cells, was highly sensitive to the cytocidal action of estradiol. Growth of ES-1 cells was inhibited by 10(-8)M 17 beta-estradiol, a concentration that stimulated the growth of parental MCF-7 cells. The estradiol-induced growth inhibition of ES-1 cells was almost completely reversed by treatment with LY117018, but not by treatment with tamoxifen. The relative binding affinity of LY117018 for estradiol receptor was equal to that of estradiol in both MCF-7 and ES-1 cells. Treatment of ES-1 cells with estradiol specifically induced tissue-type plasminogen activator (t-PA), whereas such estradiol-induced activation was not observed in parental MCF-7 cells. Quantitative immunoreactive assays and Northern blot analysis showed that estradiol-induced expression of t-PA was blocked by LY117018 in ES-1 cells. The inhibitory effect of tamoxifen was about 100 times lower than that of LY117018. The inhibition of t-PA gene expression by LY117018 might be due to competitive inhibition with estradiol in estradiol receptor binding.

Breast Neoplasms↗
PubMed1991

Tumor necrosis factor and epidermal growth factor modulate migration of human microvascular endothelial cells and production of tissue-type plasminogen activator and its inhibitor.

Epidermal growth factor (EGF) induces tubular formation of cultured human microvascular endothelial (HME) cells in the gel matrix containing collagen, and tumor necrosis factor (TNF) disrupts the tubular formation (Mawatari et al. (1989) J. Immunol. 143, 1619-1627). Here we studied the effects of EGF and TNF on endothelial cell migration and on the production of proteases. Confluent HME cells, when wounded with a razor blade, moved into the denuded space. This migration was stimulated by EGF and inhibited by TNF in this assay and in the Boyden chamber assay. Antibody against tissue-type plasminogen activator (t-PA) inhibited the EGF-stimulated cell migration in both assays by approximately 70%, but antibody against urokinase-type plasminogen activator (u-PA) could not inhibit its migration. Quantitative immunoreactive assays showed an approximately three- to fourfold increase of t-PA at 6 to 12 h after EGF addition, and TNF inhibited the production of t-PA by 50%. Northern blot analysis showed increased expression of t-PA mRNA by EGF alone in a time- and dose-dependent manner, whereas TNF alone inhibited its expression in a time- and dose-dependent manner. Northern blot analysis showed a significant increase of plasminogen activator inhibitor-1 (PAI-1) mRNA when EGF or TNF was present. Stimulation by EGF of cell migration of HME cells and its inhibition by TNF appear to be closely correlated with the cellular modulation of t-PA and PAI-1 activities.

Cell Movement↗
PubMed1991

Hyperthermic sensitivity and cholesterol levels of mammalian cell lines in culture.

In this study, we asked whether cholesterol contents were specifically correlated with cellular sensitivity to hyperthermia. To examine this possibility, we employed two isogenic mammalian cell lines, Chinese hamster V79 cell line and its amphotericin B-resistant (AMBr) cell line. AMBr cells had a decreased content of membrane sterols in comparison with V79 cells. Clonogenic assays showed that AMBr cells were more sensitive to hyperthermic treatment at 45 degrees C than V79 cells. Cholesterol contents were increased in AMBr cells by exposure to liposomes containing 1:1 lecithin-cholesterol, and the sterol level was comparable to that of V79 cells. In comparison with untreated AMBr cells, AMBr cells were more resistant to hyperthermia at 45 degrees C when incubated with liposomes containing cholesterol. Treatment of V79 cells with oxygenated sterol, a potent inhibitor of endogeneous sterol synthesis, sensitized the hyperthermic cytotoxicity. Our present data present the hypothesis that cellular cholesterol contents are closely correlated with cellular heat toxicity.

Animals↗
PubMed1991

Enhancement of antitumour activity of etoposide by dihydropyridines on drug-sensitive and drug-resistant leukaemia in mice.

We recently reported that six 1,4-dihydropyridine derivatives out of 57 screened effectively over-came vincristine (VCR)-resistance in VCR-resistant (P388/VCR) leukaemia-bearing mice when the dihydropyridines and VCR were administered intraperitoneally (i.p.). Furthermore, among the six dihydropyridine derivatives, two compounds, NK-250 and NK-252, most effectively overcame VCR-resistance while exhibiting relatively low calcium antagonistic activity and toxicity. In this study, we examined whether NK-250 and NK-252 could potentiate the antitumour activities of etoposide in mice with drug-sensitive (P388/S) or VCR-resistant (P388/VCR) leukaemia cells when the anticancer agents and tumour cells were administered by various routes. In both groups of mice inoculated i.p. with P388/S- and P388/VCR-leukaemia cells, the oral (p.o.) administration of NK-250 combined with i.p. or intravenously (i.v.) administration of etoposide (ip-po-ip trials and ip-po-iv trials) dramatically potentiated the antitumour activity of etoposide. Although etoposide alone was less effective in treating mice inoculated i.v. with P388/S- and P388/VCR-leukaemia cells, p.o. administration of NK-250 combined with i.p. or i.v. administration of etoposide (iv-po-ip trials and iv-po-iv trials) potentiated the antitumour activity of etoposide to similar levels as in treating mice inoculated i.p. with leukaemia cells. These 1,4-dihydropyridines were therefore highly effective in potentiating anticancer drugs against both drug-sensitive and drug-resistant tumours.

Administration, Oral↗
PubMed1991

[Mechanisms of resistance to DNA topoisomerase II inhibitors].

Cytotoxicity of anticancer drugs are depended on cellular differences of drug transport processes, drug metabolism or proliferation process including DNA synthesis. Thus, resistance to anticancer drugs is likely due to many cellular changes. Mechanisms of resistance to anticancer drugs have been studied with cells selected for their ability to grow in the presence of drugs. In this paper, the mechanisms of cellular resistance to DNA topoisomerase II inhibitors was described.

Animals↗
PubMed1991

Malignant transformation of mouse BALB/3T3 cells by polyoma middle T antigen requires epidermal growth factor receptor expression.

The mouse cell line MO-5, which is defective in receptor-binding activity of epidermal growth factor (EGF), is very poorly transformed by polyoma middle T antigen or v-src gene, but activated c-H-ras and v-mos gene can induce the transformation (M. Ono, M. Yakushinji, K. Segawa, and M. Kuwano, Mol. Cell. Biol., 8: 4190-4196, 1988). We established clones of MO-5 expressing a functional EGF receptor (EGF-R) after introduction of the human EGF-R complementary DNA into MO-5 (MNER23 and MNER31), and we also established a clone (BNER4) expressing human EGF-R from the parental cell line, BALB/3T3. MNER23, MNER31, and BNER4 expressed EGF-R activity at about 2- to 6-fold higher levels than did control BALB/3T3 cells. A marked increase in DNA synthesis in response to EGF was observed in these BNER4, MNER23, and MNER31 cell lines compared to BALB/3T3 cells; however, there was little if any increase in DNA synthesis of MO-5 in the presence of EGF. Introduction of the polyoma middle T antigen gene into BALB/3T3, BNER4, MNER23, and MNER31 resulted in the appearance of transformation foci, but MO-5 again showed little response. We purified clones B4-mT-2, M23-mT-1, M23-mT-2, M23-mT-3, and M31-mT-13 from transformation foci of BNER4, MNER23, and MNER31 cells, which were respectively transfected with the middle T antigen. All of the middle T antigen-positive transfectants demonstrated abilities to form both colonies in soft agar and tumors in nude mice. The presence of EGF-R appears to be indispensable for malignant transformation by polyoma middle T antigen.

Animals↗
PubMed1991

Potentiation of etoposide and vincristine by two synthetic 1,4-dihydropyridine derivatives in multidrug-resistant and atypical multidrug-resistant human cancer cells.

Newly synthesized 1,4-dihydropyridine derivatives had been screened to determine whether they could overcome vincristine (VCR)-resistance in VCR-resistant (P388/VCR) leukemia-bearing mice, and six compounds had strong reversing ability among the screened compounds. We further determined whether NK-250 and NK-252 among the six compounds could potentiate cytocidal activities of etoposide (VP16) as well as VCR against both multidrug-resistant (MDR) cell line (VJ-300) and atypical MDR cell line (KB/VM-4). Both VJ-300 and KB/VM-4 were derived from the same parental human cancer KB cell line: VJ-300 cells showed enhanced expression of a MDR-specific glycoprotein of molecular weight of 170,000 Da (gp170) while KB/VM-4 cells were selected as teniposide (VM26)-resistant cell line with no expression of gp170. NK-250 and NK-252 potentiated the cytotoxic action of VCR about 2- to 10-fold against KB and KB/VM-4 cells, and they almost completely reversed VCR-resistance in VJ-300 cells. By contrast, NK-250 and NK-252 potentiated the cytotoxic action of VP16 about 2-fold against KB cells while they reversed 5- to 10-fold VP16-resistance in both VJ-300 and KB/VM-4 cells. The reversal effect by NK-250 and NK-252 of VCR-resistance in VJ-300 cells appeared to be due to enhanced cellular accumulation of radioactive VCR through interaction to 170-kDa P-glycoprotein. The potentiation effects by these dihydropyridines of VCR and VP16 on KB or KB/VM-4 cells also appeared to be due to enhanced accumulation of radioactive VP16 or VCR, but the effects might be mediated through other mechanisms, plausibly enhanced cellular uptake of the drugs.

Antineoplastic Agents↗
PubMed1990

Tissue-specific enhancer of the human multidrug-resistance (MDR1) gene.

Identification of cis-regulatory sequences is a first step in analyzing the regulation of the human multidrug-resistant 1 (MDR1) gene which encodes the 170-kilodalton membrane P-glycoprotein in normal tissues and tumor cells. We have studied several overlapping genomic clones containing the 5'-flanking region of the gene. These clones span about 30 kilobases (kb) of contiguous DNA containing 10 kb of the gene and 20 kb of the 5'-flanking sequence. The nucleotide sequence of the first exon and the 2 kb preceding the exon were determined. DNA sequences containing the 5'-flanking regions were linked to the chloramphenicol acetyltransferase (CAT) gene. For transient CAT assay, we have employed six cell lines, including human cancer KB, vincristine-resistant VJ-300 derived from KB, mouse adrenal tumor Y-1, African green monkey kidney CV-1, mouse fibroblast NIH3T3, and human adrenal carcinoma SW-13 cells. Promoter activity was very weak regardless of the length of the promoter region in mouse adrenal tumor Y-1 and monkey kidney CV-1 cells, in which endogenous P-glycoprotein was expressed. Introduction of a 700-base genomic DNA fragment from a site located at 10 kb far upstream of the initiation site increased the transcription of the CAT gene in Y-1, CV-1, and SW-13 cells. However, no significant increase in the CAT activity could be observed in NIH3T3, KB, and VJ-300 cells. This fragment markedly augmented the expression of the CAT gene regardless of orientation or position, and it acted in a cell type-specific manner even with heterogenous promoters. Our present study suggests that the 700-base pair fragment may carry a tissue-specific transcriptional enhancer that is active in at least some adrenal and kidney-derived cell lines.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Differential effects of brefeldin A on sialylation of N- and O-linked oligosaccharides in low density lipoprotein receptor and epidermal growth factor receptor.

Low density lipoprotein receptor (LDL-R) is a membrane glycoprotein carrying both N- and O-linked oligosaccharides, processing of which is reflected in conversion from a precursor to mature form during its synthesis and intracellular transport. Treatment with brefeldin A (BFA) of mouse macrophage-like J774 cells, Chinese hamster ovary cells, and two human cancer cell lines (A431 and IMC-2) resulted in production of LDL-R with a molecular size 5-10 kDa smaller than that of the mature form in the control cells. Treatment with sialidase caused apparent reduction in the molecular size of LDL-R synthesized in all BFA-treated J774, Chinese hamster ovary, A431, and IMC-2 cell lines as observed for the mature form of the control cells. Thus, O-linked sugar chains of LDL-R were apparently sialylated in the BFA-treated cells. We also examined the effect of BFA on the processing of another membranous glycoprotein, epidermal growth factor receptor (EGF-R) carrying only N-linked oligosaccharides. EGF-R synthesized in the presence of BFA was found to have no response to sialidase treatment, suggesting that the drug blocks the sialylation of EGF-R. The results indicate that BFA causes different effects on the sialylation of LDL-R and EGF-R depending upon linkage types of their oligosaccharides.

Animals↗
PubMed1990

Reduction of drug accumulation and DNA topoisomerase II activity in acquired teniposide-resistant human cancer KB cell lines.

We have isolated stable teniposide (VM26)-resistant cell lines from human cancer KB cells by stepwise exposure to increasing doses of the drug. At each step, we have purified VM26-resistant cell lines. KB/VM-a, KB/VM-b, KB/VM-1, KB/VM-2, KB/VM-3, and KB/VM-4 showed 3-, 6-, 12-, 16-, 74-, and 95-fold higher resistance to VM26 than did KB. We have further characterized KB/VM-2 and KB/VM-4 which showed about 15- and 100-fold higher resistance to VM26 or etoposide (VP16) than did KB. Both VM26-resistant cell lines showed 4- to 11-fold higher relative resistance to daunomycin and Adriamycin than did KB. Steady-state levels of the cellular accumulation of radioactive VP16 in KB/VM-2 and KB/VM-4 cells were about 40% of that of KB cells, whereas similar levels of radioactive daunomycin accumulation were observed in KB/VM-2 and KB/VM-4 cells as KB cells. Topoisomerase II activity of nuclear extracts of both KB/VM-2 and KB/VM-4 assayed by decatenation of kinetoplast DNA was consistently two-thirds or less the activity of KB cells. A similar reduction was seen in both immunoblot assays with specific anti-topoisomerase II antibody and Northern blot analysis with specific human DNA topoisomerase II complementary DNA. DNA topoisomerase I activity, however, was similar between the mutants and their parent. Furthermore, cell growth of KB/VM-2 and KB/VM-4 was more thermolabile than that of KB, while KB/VM-b already showed temperature-sensitive growth. KB/VM-1 did show reduced accumulation of VP16 as in KB/VM-2 or KB/VM-4, but it had a normal level of topoisomerase II content as in KB cells. These data suggest that the reduced expression of DNA topoisomerase II, possibly combined with decreased permeability to the drugs, can account for the acquired VM26 resistance of KB/VM-2 and KB/VM-4 cells and also that the temperature-sensitive phenotype might not be obligatorily coupled with the reduced expression of topoisomerase II or the decreased permeability.

DNA Topoisomerases, Type I↗
PubMed1990

Rapid turnover of low-density lipoprotein receptor by a non-lysosomal pathway in mouse macrophage J774 cells and inhibitory effect of brefeldin A.

The low-density lipoprotein (LDL) receptor of molecular mass 155 kDa was expressed on the cell surface of cultured mouse macrophage J774 cells. The conversion rate of precursor to mature form of LDL receptor in J774 cells was comparable to that in mouse fibroblast L cells. The half-life of the LDL receptor of J774 cells was about 2 h, that of L cells was about 11 h. The rapid degradation of LDL receptor was not significantly inhibited by the lysosomotropic agents, chloroquine and NH4Cl, nor by the thiol-protease inhibitors leupeptin and E-64. By contrast, incubation at 18 degrees C retarded the degradation of LDL receptor. Treatment of J774 cells with brefeldin A, an inhibitor of membrane transport between the endoplasmic reticulum and the Golgi apparatus, inhibited the rapid turnover of the LDL receptor. Even after a 9-h chase in the presence of brefeldin A, LDL receptor 5-10 kDa smaller than the normal mature form was found to be stable. Rapid turnover of the LDL receptor in the macrophages appeared to occur after exit from the Golgi apparatus, possibly during transport of the LDL receptor to the plasma membrane.

Animals↗
PubMed1990

Enhanced production of tissue-type plasminogen activator by estradiol in a novel type variant of human breast cancer MCF-7 cell line.

ES-1 cells, which showed a higher sensitivity to the cytocidal action of estradiol were isolated from a human breast cancer MCF-7 cell line. Growth of ES-1 cells was inhibited by a dose of 17-beta estradiol that stimulated the growth of the parental MCF-7 cells. Proteins secreted from MCF-7 and ES-1 cells when cultured with 17-beta estradiol were compared by sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis (SDS-PAGE). Addition of estradiol to culture medium enhanced secretion of a protein of molecular mass of 52 kDa in media for both MCF-7 and ES-1 cell lines, but the secretion of a second 67 kDa protein was enhanced about 10-fold only in ES-1 cells. The analysis by SDS-PAGE of culture medium immunoprecipitated with anti-tissue-type plasminogen activator (t-PA) antibody demonstrated that the band of 67 kDa protein specifically secreted from estradiol-treated ES-1 cells contained t-PA. Zymography assays, quantitative immunoreactive assays, and Northern analysis showed about 5-fold specific increase by estradiol of t-PA with molecular mass of 65-70 kDa in ES-1 but not in its parental MCF-7 cells. Cellular level of the plasminogen activity was also specifically enhanced in ES-1 cells by estradiol, but only a slightly in MCF-7 cells. By contrast, another urokinase-type PA (u-PA) with molecular weight of 55 kDa showed very low level activity in both MCF-7 and ES-1 cell lines in the presence of estradiol. Formation of t-PA mRNA was specifically enhanced in ES-1 cells when ES-1 cells were treated for more than 12 h with 10(-8) M 17-beta estradiol. Estradiol did not elongate the lifetime of t-PA mRNA in ES-1 cells. A unique phenotype of ES-1 cells in response to estradiol is discussed in relation to activating expression of the t-PA gene.

Breast Neoplasms↗
PubMed1990

Inverse correlation between expression of multidrug resistance gene and N-myc oncogene in human neuroblastomas.

Genomic amplification of N-myc is an important prognostic indicator in neuroblastoma. The tumors with amplified N-myc are initially sensitive to chemotherapy but often acquire resistance to therapy, recur, and ultimately kill the patients. We measured amplification and expression of N-myc and expression of mdr-1 in 35 surgically resected neuroblastomas, before acquisition of drug resistance and in 4 recurrent tumors resistant to chemotherapy. The mdr-1 mRNA expression was found to be inversely correlated with the N-myc expression. The mdr-1 gene expression was at a low level in advanced stage and histologically undifferentiated neuroblastomas, the same group of tumors in which N-myc expression is elevated. A significantly better prognosis was noted in those patients whose tumors had a high level of mdr-1 expression and a low level of N-myc expression. The role, if any, of increased expression of mdr-1 in the acquisition of multidrug resistance in neuroblastoma remains unclear. However, the aggressive clinical behavior associated with N-myc amplification and/or expression appears to be linked to down-regulation of mdr-1 expression.

Cell Differentiation↗
PubMed1990

Drug resistance to cis-diamminedichloroplatinum (II) in Chinese hamster ovary cell lines transfected with glutathione S-transferase pi gene.

Establishment of Chinese hamster ovary (CHO) cell lines expressing human glutathione S-transferase-pi (GST-pi) was performed after cotransfection of pSV2-neo and human GST-pi cDNA-carrying plasmid p beta actGPi-2. About 30 G418-resistant clones were tested for their expression of GST-pi by Northern blot analysis. Two clones, beta 2-3 and beta 2-5, expressed a significant amount of GST-pi mRNA; and one clone, beta 1-1, that did not was also used for further study. Western blot analysis with anti-GST-pi antibody showed significant increases of GST-pi in beta 2-3 and beta 2-5, but not in beta 1-1. Northern blot analysis with the human GST-pi cDNA probe showed that the increase in the expression of GST-pi-mRNA in beta 2-3 and beta 2-5 was respectively 2- and 4-fold higher than that in beta 1-1. Southern blotting analysis showed that beta 1-1, beta 2-3 and beta 2-5 contained about one copy of the human GST-pi cDNA sequence. beta 2-3 and beta 2-5 were resistant to 1.4- and 3.0-fold higher doses of CDDP than CHO, respectively, but beta 1-1 was not. Increased expression of GST-pi might be associated with CDDP-resistance in CHO cells.

Animals↗