Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 300-fold adriamycin resistant variant (DLKP-A) of the human lung squamous cell carcinoma line DLKP was established by stepwise selection in increasing concentrations of adriamycin. Different levels of cross-resistance were observed towards VP-16, VM-26, colchicine, vincristine and, somewhat unexpectedly, cis-platin. Resistance was stable for at least 3 months in culture in the absence of drug. P-glycoprotein overexpression was detected by immunofluorescence and Western Blotting, and a direct causal role for P-glycoprotein overexpression in the resistant phenotype was established by transfection with an mdr1 specific antisense oligonucleotide. A modified cryopreservation procedure was necessary for the resistant variant line. The resistant population displays clonal heterogeneity with respect to resistance level. A higher frequency of double minute chromosomes was observed in DLKP-A when compared with the parental cell line.
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PMID:Multiple drug-resistance in variant of a human non-small cell lung carcinoma cell line, DLKP-A. 136 13

The classical multidrug resistance (MDR) phenotype is characterized by cross-resistance between a number of chemically unrelated drugs due to an increased efflux across the plasma membrane via a P-glycoprotein-mediated mechanism. The epipodophyllotoxin derivatives etoposide (VP-16) and teniposide (VM-26) are usually included among the drugs recognized by this MDR phenotype, and the MDR EHR2/DNR cell line is greater than 50-fold cross-resistant to VP-16. The steady-state accumulation of VP-16 in EHR2/DNR cells is only half that of wild-type EHR2 cells, and deprivation of energy by sodium azide surprisingly increased accumulation to a similar extent in both sublines. Efflux was rapid (halflife of 32-35 s) and similar in both sublines, while initial influx was markedly lower in the resistant cells. The temperature coefficients over 10 degrees C for VP-16 in- and efflux indicated passive transport in both sublines. In agreement with this finding, up to 10-fold molar excess (50 microM) VM-26 had no effect on VP-16 accumulation in MDR cells. VP-16 at a 100-fold molar excess inhibited azidopine photoaffinity labeling of P-glycoprotein by only 30% and vincristine binding to plasma membrane vesicles from EHR/DNR cells by 45%. However, VP-16 itself did not differentially bind to plasma membrane vesicles from EHR2 and EHR2/DNR cells. Finally, neither VP-16 accumulation nor cytotoxicity in EHR2/DNR cells were increased to the same degree as for daunorubicin and vincristine by verapamil, and the modulation was similar in wild-type and resistant cells. Thus, although VP-16 may be a substrate for P-glycoprotein, its other transport characteristics such as rapid diffusion and sensitivity to membrane perturbation in wild-type cells lessen any effect of P-glycoprotein-mediated efflux, resulting in a lack of differential modulation by verapamil. These results may be considered when planning clinical trials involving MDR modulators and epipodophyllotoxin derivatives.
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PMID:Relationship of VP-16 to the classical multidrug resistance phenotype. 158 2

We have previously shown that the multidrug-resistant EHR2/DNR+ cells, which overexpress P-glycoprotein, accumulate only about 20-30% of daunorubicin at steady state compared to the sensitive cells. These cells have been thought to be a "pure" P-glycoprotein cell line. We now report that the EHR2/DNR+ cells exhibit decreased DNA topoisomerase II catalytic activity. We also found that the amount of immunoreactive DNA topoisomerase II from these cells is about one-third that seen in the drug-sensitive cell line. In agreement with the decreased activity and amount of topoisomerase II, the number of DNA-protein complexes stabilized by teniposide (VM-26) was reduced by about 50% in nuclear extracts from EHR2/DNR+ cells. Furthermore, using an intact cell assay for DNA protein complexes, we found that the VM-26-stimulated complexes formed in the drug-resistant cells never reached the level seen in the drug-sensitive cells. Verapamil and Cremophor EL block P-glycoprotein-mediated efflux of "natural product" drugs and increase their accumulation in resistant cells. Coincubation of the EHR2/DNR+ cells with VM-26 and either of these modulators increased the number of complexes formed in the resistant cells. However, neither modulator increased the number of topoisomerase II-DNA complexes in the drug-resistant cells to the level seen in the EHR2 cells. We conclude that the resistance of EHR2/DNR+ cells is due in part to reduced amounts of DNA topoisomerase II. Furthermore, we note that a single cell line can express features of both P-glycoprotein-associated multidrug resistance and altered topoisomerase II-associated multidrug resistance.
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PMID:Decreased DNA topoisomerase II in daunorubicin-resistant Ehrlich ascites tumor cells. 167 12

Stable acquired resistance to etoposide (VP-16) or teniposide (VM-26) in HCT116 human colon carcinoma cells and A549 human lung adenocarcinoma cells, was previously obtained by weekly 1-h exposures to either drug (B. H. Long, Natl. Cancer Inst. Monogr., 4: 123-127, 1987). The purpose of this study was to identify possible mechanisms of resistance present in these cells by using human mdr1 and topoisomerase II DNA probes, antibodies to these gene products, and P4 phage unknotting assay for topoisomerase II activities. HCT116(VP)35 cells were 9-, 7-, and 6-fold resistant to VP-16, VM-26, and Adriamycin, respectively, and showed no cross-resistance to colchicine and actinomycin D. These cells had no differences in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels but displayed decreased levels of topoisomerase II mRNA and enzyme activity without any alteration of drug sensitivity displayed by the enzyme. HCT116(VM)34 cells were 5-, 7-, and 21-fold resistant to VP-16, VM-26, and Adriamycin; were cross-resistant to colchicine (7-fold) and actinomycin D (18-fold); and possessed a 9-fold increase in mdr1 mRNA and increased P-glycoprotein without evidence of mdr1 gene amplification. No alterations in topoisomerase II gene or mRNA levels, enzyme activity, or drug sensitivity were observed. A549(VP)28 and A549(VM)28 cells were 8-fold resistant to VP-16 and VM-26 and 3-fold resistant to Adriamycin. Both lines were not cross-resistant to colchicine or actinomycin D but were hypersensitive to cis-platinum. No alterations in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels, but lower topoisomerase II mRNA levels and decreased enzyme activities, were observed. Of the four acquired resistant cell lines, resistance is likely related to elevated mdr1 expression in one line and to decreased topoisomerase II expression in the other three lines.
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PMID:Mechanisms of resistance to etoposide and teniposide in acquired resistant human colon and lung carcinoma cell lines. 171 44

We established an etoposide (VP-16)-resistant human small-cell lung cancer cell line (H69/VP) by stepwise exposure to VP-16. The resistance of H69/VP to VP-16 was 9.4-fold that of the parent cell line (H69/P). H69/VP showed cross-resistance to Adriamycin (ADM), (4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinopiperidino) carbonyloxy]-1H-pyrano[3',4':6,7]indolizino [1,2-b]quinoline-3,14(4H,12H)-dionehydrochloride trihydrate (CPT-11), teniposide (VM-26), vindesine (VDS) and vincristine (VCR). The amount of DNA topoisomerase II (topo.II) was nearly the same in H69/P and H69/VP cells. The catalytic activity of topo.II in H69/VP cells was lower than that in the H69/P line. Accumulation of [3H]-VP-16 in H69/VP was 6.1-7.5 times lower than that in H69/P. According to Northern blot analysis, the mdr-1 mRNA level in H69/VP was markedly higher than that in H69/P. These findings suggest that H69/VP has a typical multidrug resistance (MDR) phenotype and that alteration of the drug accumulation mediated by P-glycoprotein may play an important role in resistance to VP-16. Reduced topo.II activity may also be associated with VP-16 resistance.
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PMID:Characterization of an etoposide-resistant human small-cell lung cancer cell line. 197 50

Our human T-cell leukemia line, CEM/VM-1, selected for resistance to VM-26 (teniposide), is cross-resistant to several drugs that interact with topoisomerase II, including VP-16 (etoposide), 4'-(9-acridinylamino)methanesulphon-m-anisidide, daunorubicin, and mitoxantrone. However, in contrast to cell lines exhibiting multidrug resistance (MDR) associated with overexpression of P-glycoprotein, this line is not cross-resistant to the Vinca alkaloids, is not impaired in drug accumulation, and does not overexpress the mdrl gene (Cancer Res., 47: 1297, 5455, 1987). More recently we found that nuclear extracts of these cells exhibit decreased topoisomerase II catalytic and cleavage activity, compared to the drug-sensitive line (Biochemistry, 1988). These results suggest that an alteration in topoisomerase II or a modulator of this enzyme may be responsible for this altered topoisomerase II-form of multidrug resistance (at-MDR). In the present work, we studied the somatic cell genetics of at-MDR. We produced hybrid cell lines by polyethylene glycol-mediated fusion of the CEM/VM-1 line with a hypoxanthine-guanine phosphoribosyl transferase-deficient, ouabain-resistant CEM line (CEM.AG1.OU1.5) that exhibits VM-26 sensitivity. Ten of the hybrid lines that grew in selective medium were randomly chosen for expansion and four were analyzed for both DNA content by flow cytometry and VM-26 sensitivity in a 72-h growth inhibition assay. The hybrid lines all contained approximately 2x DNA compared to unfused controls, indicating that the fusions were successful. The IC50 for VM-26 in 3 of the 4 lines was the same as that of the sensitive controls, ranging from 4.7 to 7.4 x 10(-8) M, and another was 76 x 10(-8) M. These data indicate that drug sensitivity was reconstituted by the hybridization procedure. By comparison, the VM-26 IC50 values in the CEM/VM-1 cells and CEM/VM-1 x CEM/VM-1 control "fusions" were 360 and 750 x 10(-8) M, respectively. To determine whether a topoisomerase II-mediated function was reconstituted in the hybrids, we measured drug-stimulated DNA cleavage ("cleavable complex formation"). Using 32P-labeled pBR322 DNA as substrate with nuclear extracts from drug sensitive cells, 100 microM VM-26 maximally stimulated DNA cleavage by approximately 11-fold compared to no-drug controls.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Genetic characterization of the multidrug-resistant phenotype of VM-26-resistant human leukemic cells. 253 2

The simultaneous development of resistance to the cytotoxic effects of several classes of natural product anticancer drugs, after exposure to only one of these agents, is referred to as multiple drug resistance (MDR). At least two distinct mechanisms for MDR have been postulated: that associated with P-glycoprotein and that thought to be due to an alteration in DNA topoisomerase II activity (at-MDR). We describe studies with two sublines of human leukemic CCRF-CEM cells approximately 50-fold resistant (CEM/VM-1) and approximately 140-fold resistant (CEM/VM-1-5) to VM-26, a drug known to interfere with DNA topoisomerase II activity. Each of these lines is cross-resistant to other drugs known to affect topoisomerase II but not cross-resistant to vinblastine, an inhibitor of mitotic spindle formation. We found little difference in the amount of immunoreactive DNA topoisomerase II in 1.0 M NaCl nuclear extracts of the two resistant and parental cell lines. However, topoisomerase II in nuclear extracts of the resistant sublines is altered in both catalytic activity (unknotting) of and DNA cleavage by this enzyme. Also, the rate at which catenation occurs is 20-30-fold slower with the CEM/VM-1-5 preparations. The effect of VM-26 on both strand passing and DNA cleavage is inversely related to the degree of primary resistance of each cell line. Our data support the hypothesis that at-MDR is due to an alteration in topoisomerase II or in a factor modulating its activity.
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PMID:Altered catalytic activity of and DNA cleavage by DNA topoisomerase II from human leukemic cells selected for resistance to VM-26. 285 72

To study the mechanisms of the acute induction of drug resistance in cancer cells, we have established a model system in which adriamycin (ADM) induces immediate drug resistance. In this system, human colon carcinoma HT-29 cells were pretreated for 1 h with a subtoxic dose of ADM (0.3 micrograms/ml) and incubated for 24 h in drug-free medium. Then the cells were treated for 1 h with ADM, and the cell survival was determined in terms of colony-forming ability. The survival of the pretreated cells was increased up to 100-fold, as compared with that of untreated cells. Such increased survival, however, was observed only after high doses of ADM (2 to 8 micrograms/ml); more than 99% of the cells were killed. These results indicate that only a small fraction of ADM-pretreated cells acquire the ADM-resistant phenotype. Similar induced resistance was observed in five of seven subclones isolated from HT-29 cells by limiting dilution, suggesting that the majority of cells in the parental HT-29 population could acquire the ADM-resistant phenotype. In the subclone HT-29T9, the ADM pretreatment induced concomitant resistance to daunomycin, VP-16, and VM-26 but not to agents other than topoisomerase II inhibitors. The ADM-induced drug resistance did not accompany MDR1 gene expression and could not be overcome by verapamil, a P-glycoprotein inhibitor. The present system could be useful to study the acute induction mechanism(s) of ADM-resistance, which could be relevant to clinical resistance in patients.
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PMID:Acute induction of adriamycin-resistance in human colon carcinoma HT-29 cells exposed to a sublethal dose of adriamycin. 773 Jan 48

Over the past decade, DNA topoisomerase I and II appeared to be the targets of some antitumor agents: CPT-11 and Topotecan derived from Camptothecin which interact with topoisomerase I; Actinomycin D, Adriamycin and Daunorubicin, Elliptinium Acetate, Mitoxantrone, Etoposide and Teniposide, Amsacrine which interact with topoisomerase II. The multiple functions of these enzymes are important as they play a role during replication, transcription, recombination, repair and chromatine organisation. Particularly, they relax torsional constraints which appear when intertwined DNA strands are separated while replication fork or RNA polymerases are moving. To some extent, topoisomerase I and II are structurally and functionally different. Moreover, topoisomerase I is not indispensable for a living cell whereas topoisomerase II is. Drug-topoisomerase interaction which probably leads to antitumoral effect of the compounds studied in this review is not a trivial inhibition of the enzyme but rather a poisoning due to stabilization of cleavable complexes between the enzyme and DNA. These stabilized complexes are likely to induce apoptosis-like programmed cell death, which is characterised by DNA fragmentation. However, it appears that it is the collision of the replication fork with the drug-stabilized cleavable complex that is responsible for the cytotoxicity of the drug: poisoning of topoisomerases by antitumor agents leads to a new concept of "dynamic toxicity". Although they interact with a common target, topoisomerase II poisons have differential effects on macromolecules syntheses, cell cycle and chromosome fragmentation; a few compounds may produce free radicals. Because of these differential effects in addition to quantitative and qualitative variations of stabilized cleavable complexes, in particular DNA sequences on which topoisomerase II is stabilized, these antitumor agents do not resemble each other. Cellular resistance to topoisomerases poisons results of two principal types of alteration: target and/or drug transport modification. Decreased ability to form the cleavable complex in resistant cells may be the consequence of both decreased amount of topoisomerase or altered enzyme. On the other hand, overexpression of membrane P-glycoprotein, which pumps drugs out of the cell by an energy dependent process provokes a decreased accumulation of these drugs. Cross resistances to other drugs are mainly under control of these two different mechanisms of resistance. A complete knowledge of their individual effects and mechanisms of resistance would allow a better clinical use of topoisomerases poisons, especially when administered in combination chemotherapy.
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PMID:[Poisons of DNA topoisomerases I and II]. 808 Oct 34

Drug resistance to inhibitors of DNA topoisomerase II can result from qualitative or quantitative alterations in the target enzyme, topoisomerase II, or from perturbations in drug transport that may or may not involve P-glycoprotein. In the present study, a drug-resistant Chinese hamster ovary cell line, SMR16, was selected in the presence of an epipodophyllotoxin (VP-16) and was found to be cross-resistant to all classes of topoisomerase II inhibitors (3-35-fold). The 3-fold level of resistance of these cells to vincristine is likely due to diminished uptake of this drug, and this is not mediated by overexpression of P-glycoprotein. No alteration in transport of VP-16 was observed. Immunoblotting with several polyclonal anti-topoisomerase II antibodies demonstrated that the resistant cells contain approximately two-thirds of the parental enzyme amount. The topoisomerase II catalytic activity present in 0.35 M NaCl nuclear extracts paralleled this decrease. VP-16- and 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced DNA damage, mediated by topoisomerase II, was found to be decreased 10-12-fold in both intact SMR16 cells and nuclei isolated from these cells, when measured by alkaline filter elution. However, the VP-16-induced DNA cleavage activity present in 0.35 M NaCl nuclear extracts of the resistant cells was attenuated only 2-fold, relative to wild-type cells. Homogeneous preparations of the enzyme obtained from resistant cells demonstrated the same cleavage and catalytic activity as purified wild-type topoisomerase II. Analysis by pulse-field gel electrophoresis of the DNA isolated from VM-26- and 4'-(9-acridinylamino)methanesulfon-m-anisidide-treated sensitive and resistant cells demonstrated significantly less conversion of SMR16 chromosomal DNA into 50-150-kilobase DNA fragments. Chinese hamster ovary SMR16 cells are apparently resistant to topoisomerase II poisons because the topoisomerase II that defines the DNA topological domains is either decreased in amount or insensitive to drug action.
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PMID:Topoisomerase II activity involved in cleaving DNA into topological domains is altered in a multiple drug-resistant Chinese hamster ovary cell line. 809 26


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