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T Tsuruo

Publications and source records attributed to T Tsuruo.

At least 415 records · Page 23Linked to original sources
PubMed1989

Effect of calcium influx blocker verapamil on adriamycin-induced cytotoxicity in leukemia cells in vitro.

The effect of verapamil (calcium influx blocker) on adriamycin-induced cytotoxicity against sensitive and resistant subline of K 562 acute myelogenous human leukemia cells has been evaluated. Verapamil by itself at a concentration of 0.5 microgram/ml did not affect the cell growth. It was found that the nontoxic concentration of verapamil moderately enhanced adriamycin cytotoxicity against K 562 cells, showing enhancement ratio of 1.3-1.4 according to the schedule used. A significant enhancement of adriamycin cytotoxicity (enhancement ratio of 5.8) was observed when verapamil and adriamycin were administered simultaneously against resistant subline of K 562/ADM cells. The results obtained give grounds to assume that verapamil could be used to overcome drug resistance in tumor cells.

Animals↗
PubMed1989

[Tumor metastasis and growth factors].

Massive investigations are being carried out on growth factors. Growth factors are an important determinant for the formation of successive tumor metastasis at target organs. We review the findings, including our data, of growth factors in relation to tumor metastasis.

Animals↗
PubMed1989

Expression of the mdr-1/P-170 gene in patients with acute lymphoblastic leukemia.

Increased expression of the multidrug resistance gene (mdr-1/P-170) and the dihydrofolate reductase (DHFR) gene have been implicated in the development of in vitro drug resistance. Overexpression, with or without gene amplification, is seen in the development of drug resistance in culture and it has been postulated that genetic modulation of mdr-1/P-170 and DHFR may also be involved in the development of clinical drug resistance. We screened lymphoblasts from 28 patients with acute lymphoblastic leukemia (ALL) for evidence of overexpression of mdr-1/P-170 using RNAse protection, RNA in situ hybridization and immunohistochemistry. Overexpression of mdr-1/P-170 without gene amplification was detected in samples from four patients (three after multiple relapses, one at presentation). Overexpression of mdr-1/P-170 was heterogeneous within the population of malignant lymphoblasts as demonstrated by RNA in situ hybridization, immunohistochemistry, and drug uptake using daunomycin autofluorescence analysis. There was no evidence of overexpression of DHFR in any of the eight patient samples tested by RNAse protection nor was there any evidence of gene amplification in 11 patient samples on Southern blot analysis. From these observations it appears that overexpression without gene amplification of mdr-1/P-170 may be one mechanism of clinical drug resistance in ALL.

Adolescent↗
PubMed1988

Mr 85,000 membrane protein specifically expressed in adriamycin-resistant human tumor cells.

For the characterization of membrane changes related to Adriamycin resistance in tumor cells, we have developed monoclonal antibodies against Adriamycin-resistant human myelogenous leukemia K562 (K562/ADM). In addition to the monoclonal antibodies which recognize P-glycoprotein, we have obtained two monoclonal antibodies (designated MRK4 and MRK20) which recognize an Mr 85,000 membrane protein. Using MRK20 as a probe, we have studied the expression of the Mr 85,000 protein in various human multidrug-resistant and -sensitive cell lines. The Mr 85,000 protein was overexpressed in K562/ADM and in a human ovarian cancer cell line resistant to Adriamycin, 2780AD. The protein, if any, was not detected in other drug-resistant human cell lines such as colchicine-resistant KB cells (KB-C4), vinblastine-resistant CEM cells (CEM/VLB100), and vincristine-resistant K562 cells (K562/VCR). We have isolated subclones of K562/ADM cells which express different amounts of the Mr 85,000 protein. The expression of the Mr 85,000 protein diminished when the cells were not kept in Adriamycin, and increased when the clones were kept in the presence of Adriamycin. In contrast, the expression of P-glycoprotein remained constant whether in the presence or absence of Adriamycin during these experiments. These findings suggest that the Mr 85,000 membrane protein is closely related to the resistant mechanism specific to Adriamycin resistance, which is different from that of the pleiotropic drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

MX2, a morpholino anthracycline, as a new antitumor agent against drug-sensitive and multidrug-resistant human and murine tumor cells.

MX2, a new morpholino anthracycline, showed similar or superior chemotherapeutic effects to Adriamycin (ADM) against several experimental murine tumors. i.v. administration of MX2 against L1210-bearing mice induced a prolongation of life-span by twice or more compared to ADM. MX2 was equally or slightly more effective against Lewis lung carcinoma and colon adenocarcinomas 26 and 38 than ADM when either drug was given i.v. The antitumor activity of MX2 against human tumor xenografts was similar to that of ADM, and the compound was effective against three out of four gastric adenocarcinomas, one out of two non-small-cell lung carcinomas, and two out of two mammary adenocarcinomas. In particular, this compound exhibited a marked effect against MX-1, a human mammary adenocarcinoma. MX2, in contrast to ADM, was effective against sublines of P388 leukemia resistant to ADM or aclacinomycin A in vivo as well as in vitro. A maximum percentage increase in life-span of about 90% was obtained in mice bearing these resistant tumors. MX2 is a unique anthracycline antibiotic effective on drug-sensitive as well as multidrug-resistant murine and human cells.

Animals↗
PubMed1988

Identification of a platelet-aggregating factor of murine colon adenocarcinoma 26: Mr 44,000 membrane protein as determined by monoclonal antibodies.

The interaction between platelets and tumor cells plays an important role in the hematogenous spread of certain malignant cancers. We found that metastatic clones of murine colon adenocarcinoma 26 induced platelet aggregation in a membrane protein-dependent manner. Two monoclonal antibodies (mAbs) of IgG2a class were generated against a highly metastatic colon 26 clone, NL-17. These two mAbs, designated 8F11 and 20A11, showed a two-fold higher level of NL-17 binding than a low metastatic clone, NL-14, which possesses low platelet-aggregating ability. Both mAbs retarded platelet aggregation induced by NL-17. Western blot analysis showed that both mAbs recognized the same Mr 44,000 membrane protein as antigen under reducing conditions. Trypsin treatment of NL-17 diminished the ability of the cells to induce platelet-aggregation and resulted in a decrease in the reactivity of the cells to 8F11. These results suggest that the Mr 44,000 membrane protein recognized by the two mAbs is a platelet-aggregating factor of colon 26 cells.

Adenocarcinoma↗
PubMed1988

Characterization of the ATPase activity of the Mr 170,000 to 180,000 membrane glycoprotein (P-glycoprotein) associated with multidrug resistance in K562/ADM cells.

The Mr 170,000 to 180,000 membrane glycoprotein associated with multidrug resistance (P-glycoprotein) is involved in drug transport mechanisms across the plasma membrane of multidrug-resistant cells. We have recently reported the purification of P-glycoprotein. The purified P-glycoprotein was found to have an ATPase activity, which might be coupled with the active efflux of anticancer drugs. In the present study, we have further studied the properties of the P-glycoprotein ATPase activity by an immobilized enzyme assay procedure using a P-glycoprotein-antibody-Protein A-Sepharose complex. GTP was also hydrolyzed by the P-glycoprotein, although less efficiently than ATP. The ATPase activity of P-glycoprotein had an optimal pH range around neutrality (pH 6.5-7.4). The detergent concentration of 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate used for protein solubilization was essential for enzyme recovery. Maximum activity was obtained when 0.1-0.2% 3-[(3-cholamidopropyl)dimethyl-ammonio]-propane sulfonate was used, while higher concentrations markedly inhibited the ATPase activity. The ATPase activity was dependent on Mg2+; maximum activity was obtained at 2-10 mM. Manganese and cobalt could substitute for magnesium as ionic cofactors. Divalent cations such as Ca2+, Zn2+, Ni2+, Cd2+, and Cu2+ inhibited the Mg2+-catalyzed ATP hydrolysis. N-Ethylmaleimide and vanadate inhibited the ATPase activity, while sodium azide or ouabain had no effect. Anticancer agents such as vincristine and Adriamycin did not affect the enzyme activity. In contrast, verapamil and trifluoperazine, agents which inhibit active drug efflux and restore drug sensitivity in resistant cells, caused an increase in the P-glycoprotein ATPase activity suggesting that P-glycoprotein might be the target molecule of these agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

ATP/Mg2+-dependent binding of vincristine to the plasma membrane of multidrug-resistant K562 cells.

To study the mechanism of active drug efflux in multidrug-resistant cells, the interaction between [3H] vincristine (VCR) and plasma membrane prepared from an adriamycin (ADM)-resistant variant (K562/ADM) of human myelogenous leukemia K562 cells was examined by filtration method. [3H]VCR bound to the plasma membrane prepared from K562/ADM cells, but not from parental K562 cells, depending on the concentrations of ATP and Mg2+. Adenosine 5'-O-(3-thio)triphosphate was not effective in the binding of [3H]VCR, indicating that ATP hydrolysis is required for this binding. Dissociation constant (Kd) of VCR binding was 0.24 +/- 0.04 microM in the presence of 3 mM ATP. In the absence of ATP, specific binding of VCR to K562/ADM membrane was also observed; however, the affinity (Kd = 9.7 +/- 3.1 microM) was 40 times lower than that observed in the presence of ATP. The high affinity VCR binding to K562/ADM membrane was dependent on temperature. The bound [3H]VCR molecules were rapidly released by unlabeled VCR added to the reaction mixture at 25 degrees C. The high affinity binding of [3H]VCR to K562/ADM membrane was inhibited by VCR, vinblastine, actinomycin D, and ADM, to which K562/ADM cells exhibit cross-resistance, whereas 5-fluorouracil and camptothecin, to which K562/ADM cells are equally sensitive as K562 cells, did not inhibit the [3H]VCR binding. Furthermore, verapamil and other agents, which are known to circumvent drug resistance by inhibiting the active efflux of antitumor agents from resistant cells, could also inhibit the high affinity [3H]VCR binding. These results indicate that ATP/Mg2+-dependent high affinity VCR binding to the membrane of resistant cells closely correlates with the active drug efflux of this resistant cell line.

Adenosine Triphosphate↗
PubMed1988

Apparent stronger expression in the human adrenal cortex than in the human adrenal medulla of Mr 170,000-180,000 P-glycoprotein.

A monoclonal antibody (MAb), MRK 16, specific to Adriamycin-resistant human myelogenous leukemia cell line K562, was used to examine whether the antigen molecules (P-glycoprotein) recognized by the MAb are present in the adrenals. The materials examined included 61 human adrenals and several cell lines. Immunohistochemical analysis revealed that almost all of the human adrenal specimens (59 out of 61) were stained positively with MAb MRK 16 and that the antigen was strongly expressed even in cases where anticancer agents had not been given. Immunoprecipitation showed that the Mr 170,000-180,000 glycoprotein was present in all of the adult adrenals but not in fetal and neonatal adrenals. Furthermore, fluorescence image analysis revealed that the P-glycoprotein was more strongly expressed in the cortex than in the medulla, showing a tendency to occur in cell clusters in the latter area. The cell lines derived from animal adrenals (SW-13, Y-1, and PC-12) showed no positive staining with MAb MRK 16. It is suggested that this glycoprotein may be related to maturation of the adrenal, in which it possibly plays a physiological role.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Increase of daunorubicin and vincristine accumulation in multidrug resistant human ovarian carcinoma cells by a monoclonal antibody reacting with P-glycoprotein.

An overexpression of plasma membrane 170-180 kDa P-glycoproteins is consistently found in multidrug-resistant (MDR) cell lines. In this study MRK-16, a monoclonal antibody (mAb) reacting with P-glycoprotein is used to study the putative functional role of this protein in vincristine (VCR) and daunorubicin (DNR) cellular accumulation in the MDR human ovarian carcinoma cell line 2780AD. We established that this cell line is highly cross-resistant to vincristine and daunomycin, related to a greatly reduced drug accumulation. Verapamil (Vp) (8 microM) caused a 3.6-fold increase in DNR as well as VCR accumulation. Exposition of 2780AD cells to MRK-16 led to an increase of 30% in cellular accumulation of VCR, both in normal growth medium as well as in medium without added glucose and with sodium azide, which largely depleted cellular ATP levels. No increase in DNR accumulation was found under these conditions. However, in the presence of 8 microM Vp, MRK-16 increased not only VCR but also DNR accumulation with about 30%. The relative increase of DNR accumulation was constant in a concentration range of 0.2-4 microM DNR. These data indicate that mAbs against P-glycoprotein might potentiate the action of calcium antagonists like Vp to increase cellular anthracycline accumulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Purification of the Mr 22,000 calcium-binding protein (sorcin) associated with multidrug resistance and its detection with monoclonal antibodies.

A low molecular weight cytoplasmic protein (Mr 19,000-22,000) has been reported to be overexpressed in some multidrug-resistant cells. We have found that a cytoplasmic protein with a molecular weight of 22,000 is highly expressed in the human myelogenous leukemia K562 cells resistant to Adriamycin (K562/ADM). The Mr 22,000 protein was shown to be one of the major calcium-binding proteins in the cytoplasmic extract from K562/ADM cells. The protein was purified to apparent homogeneity from K562/ADM cells using a four-step procedure including ammonium sulfate fractionation, anion-exchange chromatography, and gel filtration. 1.5 mg of the Mr 22,000 protein was purified from 3.0 x 10(9) of K562/ADM cells. The protein was acidic (pI 5.3) and exists as a homodimer (Mr 44,000) as revealed by gel filtration and sucrose density-gradient centrifugation. The purified protein appeared as a single band (Mr 22,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of reducing agents, suggesting that the homodimer was generated by noncovalent linkage. Monoclonal antibodies specific to the Mr 22,000 protein were raised by in vitro immunization with purified protein or by in vivo immunization with the crude membrane fraction of K562/ADM. These antibodies were used as probes for the detection of the protein. We have surveyed the expression of the Mr 22,000 protein in various multidrug-resistant and -sensitive cell lines, and found that the overexpression of the protein is not a sufficient nor a necessary condition for the acquisition of the multidrug-resistant phenotype.

Animals↗
PubMed1988

Tissue distribution of P-glycoprotein encoded by a multidrug-resistant gene as revealed by a monoclonal antibody, MRK 16.

A monoclonal antibody, MRK 16, specific to a human myelogenous leukemia cell line, K-562, and resistant to Adriamycin, was used to determine the localization of the antigen molecules (P-glycoprotein) recognized by the monoclonal antibody. P-glycoprotein was found to be expressed very strongly in the adrenal cortex and medulla of adults and strongly in the renal tubules of the kidney and the placenta. Interestingly, P-glycoprotein was not distributed in fetal and neonatal adrenals, and thus may be closely related to adrenal maturation. A high level of P-glycoprotein expression was also seen in one case each of untreated lung cancer (one of ten) and breast cancer (one of nine). Immunoelectron microscopically, the P-glycoprotein was distributed evenly on the membranes of K-562/ADM and 2780 cells. These results imply that the presence of the glycoprotein may be useful as a marker for in vitro studies of multidrug resistance in various malignancies and as an indicator of therapeutic efficacy of ex vivo eradication of multidrug-resistant cancer cells, although other mechanisms of drug resistance may exist, and there is a possibility that this MRK 16 monoclonal antibody may not recognize all P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance. 170- to 180-kilodalton membrane glycoprotein is an ATPase.

170-180-kDa membrane glycoprotein (P-glycoprotein) associated with multidrug resistance is involved in drug transport mechanisms across the plasma membrane of resistant cells. From sequence analysis of cDNAs of the P-glycoprotein gene, it is postulated that the active drug-efflux pump function may be attributable to the protein. However, purification of the P-glycoprotein while preserving its enzymatic activity has not been reported. In this study, we have purified the P-glycoprotein from the human myelogenous leukemia K562 cell line resistant to adriamycin (K562/ADM) by means of one-step immunoaffinity chromatography using a monoclonal antibody against P-glycoprotein. The procedure was simple and efficiently yielded an electrophoretically homogeneous P-glycoprotein sample. By solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified P-glycoprotein was found to have ATPase activity. This ATP hydrolysis may be coupled with the active efflux of anticancer drugs across the plasma membrane of multidrug-resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Comparison of glutathione S-transferase activity between drug-resistant and -sensitive human tumor cells: is glutathione S-transferase associated with multidrug resistance?

We have studied the levels of glutathione S-transferase in drug-resistant and -sensitive human tumor cell lines to examine a possible involvement of glutathione S-transferase (GST) in multidrug resistance mechanisms. No increase in the activity of glutathione S-transferase was detected in myelogenous leukemia K562 resistant to adriamycin (K562/ADM), ovarian carcinoma cell line A2780 resistant to adriamycin (2780AD), or acute lymphoblastic leukemia cell line CCRF-CEM resistant to vinblastine (CEM-VLB100), compared with the drug-sensitive parent tumor cells. The human breast cancer cell lines Hattori and MCF-7 had a 12- to 63-fold lower level of glutathione S-transferase activity than K562, A2780, CCRF-CEM, and their drug-resistant sublines. Induction of ADM resistance in Hattori did not increase the activity of glutathione S-transferase. However, induction of colchicine resistance in MCF-7 resulted in a 70-fold increase in the activity of glutathione S-transferase. A revertant of the colchicine-resistant MCF-7 contained a level of glutathione S-transferase activity similar to that of the resistant subline. The increase of glutathione S-transferase activity did not alter the sensitivity of the cell to cytotoxic drugs. The increased activity was due to the appearance of glutathione S-transferase pi, as shown by enzyme inhibition using anti-glutathione S-transferase pi antibody. Our findings indicate that increased cellular glutathione S-transferase activity is not associated with the development of multidrug resistance.

Antineoplastic Agents↗
PubMed1988

Antitumor effect of CPT-11, a new derivative of camptothecin, against pleiotropic drug-resistant tumors in vitro and in vivo.

CPT-11, a new derivative of camptothecin, was effective against tumor cells, especially vincristine (VCR)-and adriamycin (ADM)-resistant P388 leukemia, compared to either VCR or ADM. The drug showed superior chemotherapeutic effects over VCR and ADM in sensitive P388 leukemia-bearing mice, and was also effective in VCR- and ADM-resistant P388 leukemia-bearing mice. These latter survival advantages with CPT-11 were almost equal to those obtained by CPT-11 against sensitive P388 leukemia. CPT-11 was found to be effective against human tumor cells, especially various pleiotropically drug-resistant human tumor lines, compared to VCR and ADM. CPT-11 should be considered for further development as a new chemotherapeutic agent potentially effective against pleiotropically drug-resistant tumors.

Animals↗
PubMed1988

Inhibition of spontaneous and experimental metastasis by a new derivative of camptothecin, CPT-11, in mice.

The antimetastatic effect of a new water-soluble derivative of camptothecin, 7-ethyl-10-(4-(1-piperidino)-1-piperidino) carbonyloxy-camptothecin (CPT-11), were examined in several metastatic murine tumor systems. Intravenous (i.v.) injection of CPT-11 into BALB/c mice inhibited lung metastasis by i.v. inoculated, metastatic colonic adenocarcinoma 26 (C26) cells, C26NL-17, in BALB/c mice. This treatment was also effective in C57BL/6 mice against lung metastasis by i.v. inoculated B16-F10 and B16-BL6 cells, highly metastatic variants of the B16 melanoma. Furthermore, intraperitoneal (i.p.) injection of CPT-11 significantly inhibited the growth of C26NL-22 cells, a highly metastatic variant of C26, inoculated subcutaneously (s.c.) into the left front footpads of BALB/c mice. Also, i.p. or i.v. injection of CPT-11 effectively inhibited the growth of 3LL tumors inoculated s.c. into the hind footpads of C57BL/6 mice. Moreover, following s.c. inoculation of either C26NL-22 or 3LL cells, combined surgical excision of the primary tumor and either i.p. or i.v. CPT-11 injections given before or after surgery markedly inhibited the formation of pulmonary metastases. These results show that a new derivative of camptothecin, CPT-11, has a potent inhibitory effect against both spontaneous and experimental lung metastasis.

Animals↗
PubMed1988

Cellular and tissue distribution of MRK20 murine monoclonal antibody-defined 85-kDa protein in adriamycin-resistant cancer cell lines.

A murine monoclonal antibody (MAb) specific to adriamycin-resistant K-562 (K-562/ADM) cells, MRK20, was found to react strongly with an 85-kDa protein present in K-562/ADM and adriamycin-resistant ovarian cancer (2780AD) cells. This protein was present at only very low levels in parental cells (K-562 and A2780), methotrexate-resistant K-562 cells (K-562/MTX3, K-562/MTX4 and K-562/MTX5) and cisplatin-resistant ovarian cells (KFr). Immunoelectron microscopically, the protein was found to be located on the cell membrane of K-562/ADM and 2780AD cells. Furthermore, the presence of the protein in various cell lines, normal tissues and surgical materials from patients given no anti-cancer agents was examined by immunocytochemistry and flow cytometry. MRK20 reacted with granulocytes, monocytes and endothelial cells in various tissues, but did not react with tissue macrophages. This 85-kDa protein recognized by MRK20 seems to be the second multidrug-resistance gene-encoded product appearing in adriamycin-resistant cancer cells, following the characterization of 170-180-kDa glycoprotein, and may be important for elucidating the multidrug-resistance mechanism relevant to adrimycin and Vinca alkaloids.

ATP Binding Cassette Transporter, Subfamily B, Mem↗