[A case of ectopic gastric mucosa in the cervical esophagus].
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Choristoma↗
Biomedical subjects
H Hamada
Publications and source records attributed to H Hamada.
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↗
Multivariate analysis for clinical prognostic factors in 163 patients with soft tissue sarcoma.
Clinical and histologic findings in 163 patients with localized soft tissue sarcoma (STS) in the extremities and trunk were reviewed. There were 91 male patients and 72 female patients ranging in age from 2 to 84 years (median, 46 years). The histologic status of the tumors was as follows: low grade, 29 cases; intermediate grade, 52 cases; and high grade, 82 cases. The primary tumors were treated by intralesional excision (two cases), marginal (88), wide local (52), or amputation (21). Subsequent adjuvant therapy was given to 61 patients; 17 had radiotherapy (RT), 27 had chemotherapy (CH), and 17 had combined RT and CH. The overall survival (P less than 0.1) and disease-free survival (P less than 0.001) were better in the group that received multimodal treatment (radical surgery and adjuvant chemotherapy with or without radiotherapy) than in the group treated only by surgery. This suggests the favorable role of adjuvant therapy. The univariate and the Cox multivariate analysis for prognosis revealed that sex, tumor-related symptoms, tumor size, tumor depth, and histologic grade were the significant factors. Among the treatment schemes, adjuvant chemotherapy was the only one that affected survival, especially for the intermediate-grade tumors. Initial surgical treatment (marginal versus wide local excision) significantly contributed to the local control of the primary tumors.
Actuarial Analysis↗
Strong activity of acid-stable trypsin inhibitor in bovine thrombin for clinical use.
Strong activity of acid-stable trypsin inhibitor (ASTI) was confirmed in some clinical thrombin preparations. Thrombin preparations of human plasma origin had no detectable ASTI activity, whereas some preparations of bovine plasma origin revealed more than 5,000 U/vial (5,000 thrombin units), indicating a higher content of ASTI than of thrombin in terms of protein concentration. Contamination by other biologically active substances was also suggested by variations in amidolytic activity with several synthetic substrates (S-2238, S-2251, S-2444, S-2266 and Bz-L-Arg-pNA). On isoelectric focussing, the ASTI activities migrated in acidic positions with pI values of 3.9, 4.5, 5.0, 5.9 and 6.5, respectively. They were almost parallel to the thrombin Bz-L-Arg-pNA hydrolytic activity, and differed from that of the purified thrombin preparation (pI = 7.0). By gel filtration on Sephadex G-100, the molecular weights of the inhibitors as calculated using standard proteins were 140,000 (main), 70,000 and less than 10,000 (minor), respectively. An immunological difference between the main inhibitor (pI = 3.9, mol wt 140,000) and previously reported plasma ASTI was also confirmed with goat anti-UTI serum by the double immunodiffusion and ELISA methods. The inhibitor exerted a strong inhibitory effect not only on trypsin and chymotrypsin, but also on non-plasmic fibrinolysis with human leukocyte elastase, and to a lesser extent on the blood coagulation system (lengthening of APTT and PT). Clearly, when using thrombin preparations and analyzing the data obtained after their administration, the effects of this and other contaminant biologically active substances must be taken into account.
Animals↗
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↗
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↗
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↗
Acid-stable trypsin-plasmin inhibitors formed enzymatically from plasma precursor protein.
Enzymatic formation of acid-stable trypsin-plasmin inhibitors (ASTPIs) in human plasma with several proteinases, particularly SH-proteinases, was demonstrated. The maximal activity obtained with bromelain was 40 U/ml plasma, which corresponded to about a 10-fold increase as compared to the untreated control plasma (4.2 U/ml). Gel filtration revealed at least two ASTPI activity peaks of molecular weight 16,000 (main peak) and 8000 (minor peak). The main ASTPI was further purified by trypsin-Sepharose affinity chromatography, isoelectric focusing and gel filtration on Sephadex G-75 superfine. The purified inhibitor was found to be identical to the active fragment of plasma ASTPI or urinary trypsin inhibitor (UTI) formed by bromelain treatment. It had an isoelectric point (pI) of 3.7, a molecular weight of 16,000 by SDS-polyacrylamide gel electrophoresis and was a glycine- and glutamic acid-rich protein lacking histidine. The NH2-terminal amino acid sequence was H2N-(Lys)-Glu-Asp-Ser-X-Gln-Leu-Gly-Tyr-Ser-Ala-Gly-Pro-X-Met-Gly-Met-Th r-X-Arg - Tyr-Phe-Tyr-... COOH, which was homologous to the Lys22-Met36 part (or Glu23-Met36 part; 30% of the total) of the plasma ASTPI or UTI molecule (molecular weight 70,000-80,000 by gel filtration). The purified ASTPI displayed the same antigenicity as UTI and exerted strong inhibitory effects on trypsin, chymotrypsin and plasmin amidolysis, but had a much lesser effect on plasmin fibrinolysis. It also strongly inhibited non-plasmic fibrinolysis with human leukocyte proteinase and earthworm proteinase.
Amino Acid Sequence↗
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↗
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↗
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↗
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↗
Disease and pregnancy in adolescent girls.
The frequency of visits to the outpatient clinic, chief complaints, and pregnancies of adolescent girls were analyzed. The age distribution of adolescent girls who visited the clinic showed that the higher the age, the higher the frequency of menstrual cycle abnormalities. Recently, increased sexual activity has been seen in teenagers and has caused problems, especially the increase in teenage pregnancies, contributing to increases in induced abortion. To avoid unwanted pregnancy of unmarried teenagers, intensive sex education is essential, as well as provision of contraceptives.
Adolescent↗
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↗
Functional cloning of mouse chromosomal loci specifically active in embryonal carcinoma stem cells.
Chromosomal loci that are specifically active in embryonal carcinoma stem cells were cloned from the mouse genome by functional selection. P19 cells, a pluripotent embryonal carcinoma cell line, were transfected with an enhancer trap (a plasmid containing an enhancerless inactive neo gene), and NEO+ transformants were isolated. All of the NEO+ cell lines retained pluripotency and expressed the neo gene. When the cells were induced to differentiate, most of the cell lines continued to express the neo gene, while the neo gene in some cell lines became repressed. From the latter group of cell lines, we have cloned the integrated neo gene plus the flanking cellular DNA sequences. Three of the six cloned DNAs possessed a high NEO+-transforming activity in undifferentiated P19 cells. Among these three, two (015 and 052) were inactive in differentiated P19 cells and NIH 3T3 cells, while the remaining one was active in these differentiated cells. Deletion analysis suggested that both 015 and 052 contain two regulatory elements (promoter and enhancer) of cellular DNA origin. The putative enhancer and promoter are separated by at least 6 kilobases in 015 and 1 kilobase in 052. Therefore, 015 and 052 cloned fragments contain regulatory DNA elements that are specifically active in the embryonal carcinoma stem cells.
Animals↗
Effect of methanol on the frequency of respiration-deficient mutation induced by ethidium bromide in yeast under growing and non-growing conditions.
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DNA-Directed DNA Polymerase↗
[Detection of multidrug resistant phenotype in leukemia and lymphoma by monoclonal antibodies].
Two monoclonal antibodies, MRK16 and MRK20 that recognize P-glycoprotein and P-85 kd protein on the surface of adriamycin (ADM) resistant cells, respectively, were tested for the reactivity with 40 cultured leukemia/lymphoma cell lines. F(ab')2 form is essential to avoid false reaction through Fc gamma-R. Drug sensitivity of 19 representative cell lines were also examined in vitro. From this study, it was found that these cell lines were classified into 4 groups. Group 1 (4 cell lines) was insensitive to ADM, mitoxantron (MXT), etoposide (VP-16) and vincristine (VCR), and reactive to MRK16 and MRL20. Group II (1 cell line) was insensitive to the 4 drugs, but not reactive to both antibodies. Group III (3 cell lines) was insensitive to ADM, MXT and VP-16, but sensitive to VCR, and reactive to MRK20, but not to MRK16. Group IV (all other cell lines) was sensitive to these drugs, and not reactive to both antibodies. From these results, MRK16 detects P-glycoprotein-associated multidrug resistance (MDR), while MRK20 does P 85-kd-associated another type MDR (cross resistance to ADM, MXT and VP-16, but not to VCR). MRK20 reacted with monocytes, but MRK16 did not with any WBC type. One hundred and ninety eight clinical samples obtained from blood cancer were tested for the reactivity with MRK16. MRK16 did not react with any of 98 samples obtained before treatment, but did with 9 of 100 obtained at relapse or refractory stage after chemotherapy. The results indicate that MRK16 is useful to detect drug resistance phenotype of leukemia and lymphoma.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Detection of drug resistance in human hematologic malignancy by monoclonal antibodies.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗