Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A doxorubicin-resistant subline (5637/DR5.5) from human bladder cancer cells (5637) was induced by stepwise increase in the doxorubicin concentration. 5637/DR5.5 cells were cross-resistant to vinblastine and etoposide but not to mitomycin C and cisplatin. We analyzed the mdr1, MRP (multidrug resistance-associated protein), and DNA topoisomerase II gene expression using the reverse transcription polymerase chain reaction assay (RT-PCR) and investigated possible differences in the accumulation and efflux of radiolabeled daunorubicin. 5637/DR5.5 cells do not express the mdr1 gene, but the expression levels of MRP are markedly higher than in drug-sensitive 5637 cells. The intracellular accumulation of radiolabeled daunorubicin was markedly decreased in the 5637/DR5.5 cells in comparison with the parent cells. This reduced drug accumulation was associated with an enhanced drug efflux, but was reversed when cells were incubated with cyclosporin A. Cyclosporin A at the concentration of 5 microM caused 3.4-fold enhancement of daunorubicin-sensitivity in the 5637/DR5.5 cells. On the other hand, there was no difference in DNA-topoisomerase II activity between the parent and resistant cells. The resistance of the 5637/DR5.5 cells is therefore associated with an enhanced drug efflux mediated by the MRP gene overexpression, as distinct from P-glycoprotein, and is modulated by cyclosporin A.
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PMID:Multidrug resistance-associated protein-mediated multidrug resistance modulated by cyclosporin A in a human bladder cancer cell line. 749 17

We have analysed the contribution of several parameters, e.g. drug accumulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a large set of drug-resistant variants of the human non-small-cell lung cancer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine. Selection with either drug nearly always resulted in MDR clones. The resistance of these clones could be explained by reduced drug accumulation and was associated with a decrease rather than an increase in the low MDR1 mRNA level. To test whether a decrease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells. Although this led to a substantial reduction in MDR1 mRNA, it did not result in resistance. In all resistant clones we found an altered form of the multidrug resistance-associated protein (MRP), migrating slightly slower during SDS-polyacrylamide gel electrophoresis than MRP in parental cells. This altered MRP was also present in non-P-gp MDR somatic cell hybrids of the SW-1573 cells, demonstrating a clear linkage with the MDR phenotype. Treatment of crude cellular membrane fractions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translational modification. There was no detectable difference in sialic acid content. In most but not all doxorubicin-selected clones, this MDR phenotype was accompanied by a reduction in topo II alpha mRNA level. No reduction was found in the clones selected with vincristine. We conclude from these results that selection of the SW-1573 cell line for low levels of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein. This mechanism can be accompanied by other resistance mechanisms, such as reduced topo II alpha mRNA in case of doxorubicin selection.
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PMID:Altered MRP is associated with multidrug resistance and reduced drug accumulation in human SW-1573 cells. 764 Feb 9

The acquisition of the multidrug resistance phenotype in human tumours is associated with an overexpression of the 170 kDa P-glycoprotein encoded by the multidrug resistance 1 (MDR1) gene, and also with a 190 kDa membrane ATP-binding protein encoded by a multidrug resistance-associated protein (MRP) gene. Human bladder cancer is a highly malignant neoplasm which is refractory to anti-cancer chemotherapy. In order to understand the mechanism underlying multidrug resistance in bladder cancer, we established three doxorubicin-resistant cell lines, T24/ADM-1, T24/ADM-2 and KK47/ADM, and one vincristine-resistant cell line, T24/VCR, from human bladder cancer T24 and KK47 cells respectively. Both T24/ADM-1 and T24/ADM-2 cells which had elevated MRP mRNA levels showed both a cross-resistance to etoposide and a decreased intracellular accumulation of etoposide. T24/VCR cells which had elevated levels of MDR1 mRNA and P-glycoprotein but not of MRP mRNA, showed cross-resistance to doxorubicin. On the other hand, KK47/ADM cells, which had elevated levels of both MRP and MDR1 mRNA and a decreased level of topoisomerase II mRNA, were found to be cross-resistant to etoposide, vincristine and a camptothecin derivative, CPT-11. Our present study demonstrates a concomitant induction of increased levels of MRP mRNA, decreased levels of topoisomerase II mRNA and decreased drug accumulation during development of multidrug resistance in human bladder cancer cells. The enhanced expression of the MRP gene is herein discussed in a possible correlation with the decreased expression of the topoisomerase II gene.
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PMID:Expression of multidrug resistance-associated protein (MRP), MDR1 and DNA topoisomerase II in human multidrug-resistant bladder cancer cell lines. 773 14

A drug-resistant human small cell lung cancer cell line, H209/V6, selected in the presence of increasing concentrations of 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-demethylepipodophylloto xin (VP-16) from parental H209 cells, is 22-, 9-, and 4-fold resistant to VP-16, 4'-(9-acridinyl-amino)methanesulfon-m-anisidide, and doxorubicin, respectively, but not cross-resistant to 1,4-dihydroxy-5,8-bis((2-[(2-hydroxyethyl)amino] ethyl]-amino)-9,10-anthracenedione. These cells do not overexpress P-glycoprotein or the multidrug resistance-associated protein. Immunoblotting demonstrates that H209 cells contain the M(r) 170,000 isoform of topoisomerase II (topo II), while H209/V6 cells have a M(r) 160,000 enzyme but none of the M(r) 170,000 isoform. The cell lines have equal amounts of topo II beta. The H209/V6 cells have a 5-fold decrease in total immunoreactive topo II alpha. The catalytic and VP-16-induced DNA cleavage activities of the topo II present in 0.35 M NaCl nuclear extracts are decreased 2- to 3-fold in the drug-resistant cell line. This decrease in enzymatic activity is not consistent with either the 22-fold VP-16 resistance of the H209/V6 cell line or the approximately 5-fold decrease in immunoreactive topo II alpha in the cells. The M(r) 160,000 isoform from the H209/V6 cell line and the M(r) 170,000 enzyme from the parental cell line were purified so that the enzymatic activity of the 2 isoforms could be evaluated. The catalytic activities of the purified isoforms were found to be very similar. The drug-induced DNA cleavage activity of the M(r) 160,000 enzyme was reduced compared to the M(r) 170,000 enzyme. However, as with the nuclear extracts, the differences in enzymatic activity of the purified enzymes are considerably less than the level of drug resistance. Investigations of the subcellular localization of topo II by immunocytochemical techniques and cytoplasm/nuclear fractionation studies demonstrated that the M(r) 160,000 topo II alpha-related enzyme is primarily localized in the cytoplasm, while the M(r) 170,000 topo II alpha enzyme and topo II beta are located in the nucleus. These data imply that the deleted sequence in the M(r) 160,000 enzyme is not necessary for catalytic activity but is required to facilitate nuclear localization.
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PMID:Altered subcellular distribution of topoisomerase II alpha in a drug-resistant human small cell lung cancer cell line. 830 38

Previous structure-activity studies of the antitumor compound etoposide (VP-16) have suggested that replacement of the glycoside moiety could afford therapeutically active analogues with different biochemical determinants for cellular accumulation and drug resistance. In the present report, 10 analogues of VP-16 in which the glycosidyl moiety was replaced with alkyl or arylamino substituents exhibited 5-10-fold better binding affinity for topoisomerase II/DNA complex in human KB cells. A similar increase in the binding affinity was observed in an isolated-nuclei model. The analogues displayed greater or comparable potency to VP-16 in cell growth-inhibition studies and were less affected by cell membrane-associated drug resistance mechanisms, as exemplified by overexpressions of P-glycoprotein multidrug-resistance gene or multidrug resistance-associated protein. Interestingly, in animal studies, analogues least affected by the membrane transport-deficiency phenotypes exhibited low therapeutic index values, thus suggesting that highly efficient modulation of cellular membrane transport defects could perturb the selectivity of antitumor agents for cancer cells. This report also suggests a new method of quantifying drug-induced protein-linked DNA breaks by graphically determining the apparent dissociation-inhibition constant (Kdi) for the inhibitors.
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PMID:Activities of novel nonglycosidic epipodophyllotoxins in etoposide-sensitive and -resistant variants of human KB cells, P-388 cells, and in vivo multidrug-resistant murine leukemia cells. 860 2

The cytotoxic activity and cross-resistance pattern of the novel topoisomerase I inhibitor topotecan (Topo) were investigated in ten cell lines, representing different mechanisms of cytotoxic drug resistance, and in 218 fresh human tumour samples using the fluorometric microculture cytotoxicity assay (FMCA). Resistance to Topo in the cell lines was associated with expression of the multidrug resistance-associated protein (MRP), whereas the cell lines with P-glycoprotein (P-gp), topoisomerase II and glutathione-associated resistance did not show decreased sensitivity to the drug. Topo was more active in haematological than in solid tumour samples, but substantial activity was observed in carcinomas of the ovary and breast, sarcoma and childhood solid tumours. Cross-resistance to standard drugs representing different mechanisms of action was generally low in patient cells. The effect of Topo was better after longer exposure, but this time-dependent effect was largely abolished when adjustment for in vitro exposure was made. Topo showed activity both in proliferative and non-proliferative cell systems. The results indicate that Topo is insensitive to major mechanisms of resistance except for MRP. Proliferation does not seem to be necessary for the effect of Topo, and no superiority for protracted dosing schedules was observed. The results also suggest that, for example, leukaemias, lymphomas, sarcomas and childhood solid tumours may be suitable targets for future phase II trials.
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PMID:Cytotoxic activity of topotecan in human tumour cell lines and primary cultures of human tumour cells from patients. 923 21

Variants of the human ovarian carcinoma cell line, OAW42, exhibiting low-level intrinsic resistance (OAW42-SR) and drug-induced higher-level resistance (OAW42-A1 & OAW42-A), were studied along with a sensitive clonal population (OAW42-S) which was isolated from OAW42-SR. Expression of the MDR-associated protein P-170, the more recently discovered LRP (lung resistance-related protein) and MRP (multidrug resistance-associated protein), topoisomerase II alpha and beta, GST pi and the cytoskeletal proteins, cytokeratin 8 and vimentin, were studied (using immunocytochemistry and Western blotting techniques) in conjunction with drug (doxorubicin) accumulation and subcellular distribution. Expression of mRNA for P-170, MRP, topoisomerase 11 alpha and beta and GST pi was studied using RT-PCR (reverse transcriptase polymerase chain reaction). Results indicate differential co-expression of four MDR-associated parameters (P-170, MRP, LRP and reduced topoisomerase II alpha and beta) in the OAW42-SR and OAW42-A1 variants, whereas resistance in the OAW42-A variant appeared to be mainly P-170 mediated. Comparable amounts of MRP and greater amounts of LRP were detected in the OAW42-S cells compared to the OAW42-SR variant (which showed increased resistance compared to the OAW42-S cells), but all cell lines expressed similar low-level amounts of MRP mRNA (by RT-PCR). GST pi levels did not differ markedly between variants. Increased levels of the cytoskeletal proteins were observed with increasing levels of resistance. The relative resistance of the variants, OAW42-SR and OAW42-A1, compared with OAW42-S was seen to change during increased serial passaging of the cells. There was greater drug accumulation by the sensitive OAW42-S cell line compared with that of the resistant variants, particularly the most highly resistant OAW42-A cells. Both verapamil and cyclosporin A effectively restored the accumulation defects seen in the resistant variants, cyclosporin A being the more effective of the two. Sub-cellular location of drug was predominantly in the nucleus with maximum levels seen in the sensitive OAW42-S variant and minimum levels in the most resistant OAW42-A clone.
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PMID:Co-expression of MDR-associated markers, including P-170, MRP and LRP and cytoskeletal proteins, in three resistant variants of the human ovarian carcinoma cell line, OAW42. 927 50

A possible link between protein kinase C (PKC) and P-glycoprotein (P-gp)-mediated-multidrug resistance (MDR) was assumed from studies on MDR cell lines selected in vitro. The functional relevance of PKC for the MDR phenotype remains unclear, and the involvement of a particular PKC isozyme in clinically occurring drug resistance is not known. Recently, we have demonstrated significant correlations between the expression levels of the PKC eta isozyme and the MDR1 or MRP (multidrug resistance-associated protein) genes in blasts from patients with acute myelogenous leukaemia (AML) and in ascites cell aspirates from ovarian cancer patients. To extend these findings to further types of human tumours we analysed specimens from 64 patients with primary breast cancer for their individual expression levels of several MDR-associated genes (MDR1, MRP, LRP (lung cancer resistance-related protein), topoisomerase (Topo) II alpha/IIbeta, cyclin A and the PKC isozyme genes (alpha, beta1, beta2, eta, theta, and mu) by a cDNA-PCR approach. We found significantly enhanced mean values for MRP, LRP and PKC eta gene expression, but significantly decreased Topo II alpha and cyclin A gene expression levels in G2 tumours compared with G3. Remarkably, significant positive correlations between the MDR1, MRP or LRP gene expression levels and PKC eta were determined: MDR1/PKC eta (rs = +0.6451, P < 0.0001) n = 62; MRP/PKC eta (rs = +0.5454, P < 0.0001) n = 63; LRP/PKC eta (rs = +0.5436, P < 0.0001) n = 62; MRP/LRP (rs = +0.7703, P < 0.0001) and n = 62, MDR1/MRP (rs = +0.5042, P < 0.0001) n = 62. Our findings point to the occurrence of a multifactorial MDR in the clinics and to PKC eta as a possible key regulatory factor for up-regulation of a series of MDR-associated genes in different types of tumours.
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PMID:Multiple gene expression analysis reveals distinct differences between G2 and G3 stage breast cancers, and correlations of PKC eta with MDR1, MRP and LRP gene expression. 945 50

The efficacy of cancer treatment is limited by either intrinsic or acquired resistance to various chemotherapeutic agents. To evaluate the clinically important factors related to prognosis in primary breast cancer retrospectively, we investigated the expression of the following genes involving acquirement of drug resistance: multidrug resistance 1 (MDRl), multidrug resistance-associated protein (MRP), and topoisomerase (Topo) I, II alpha, and II beta. Using an RT-PCR method, we semiquantified the gene expression level in untreated stage II breast cancer tissue (n = 27) and noncancerous breast tissue (n = 10). Among the 27 cancer patients, who were all treated by adjuvant chemoendocrine therapy after surgery, 10 patients showed relapse within the following 10 years whereas 17 patients did not. The gene expression levels of MDRl, MRP, and Topo I, II alpha, and II beta were normalized to the level of the beta 2-microglobulin RT-PCR product. MRP mRNA expression was detected in 70% of the breast cancer tissues and its expression levels were significantly increased in the cancer group compared with the noncancerous breast tissues. Furthermore, the MRP level was much higher in the relapsed patient group. On the other hand, there were no significant differences in the MDRl mRNA levels between the noncancerous and cancer groups. Although Topo II alpha mRNA was not detected in noncancerous breast tissues, it was detected in 52% of the breast cancer tissues. In cancer patients, no significant difference in Topo II alpha mRNA levels was observed between the relapsed and nonrelapsed groups. These findings suggest that MRP might be used as one of the markers for poor prognosis in patients with breast cancer.
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PMID:Clinical significance of the increased multidrug resistance-associated protein (MRP) gene expression in patients with primary breast cancer. 966 17

Selection for in vitro drug resistance can result in a complex phenotype with more than one mechanism of resistance emerging concurrently or sequentially. We examined emerging mechanisms of drug resistance during selection with mitoxantrone in the human myeloma cell line 8226. A novel transport mechanism appeared early in the selection process that was associated with a 10-fold resistance to mitoxantrone in the 8226/MR4 cell line. The reduction in intracellular drug concentration was ATP-dependent and ouabain-insensitive. The 8226/MR4 cell line was 34-fold cross-resistant to the fluorescent aza-anthrapyrazole BBR 3390. The resistance to BBR 3390 coincided with a 50% reduction in intracellular drug concentration. Confocal microscopy using BBR 3390 revealed a 64% decrease in the nuclear:cytoplasmic ratio in the drug-resistant cell line. The reduction in intracellular drug concentration of both mitoxantrone and BBR 3390 was reversed by a novel chemosensitizing agent, fumitremorgin C. In contrast, fumitremorgin C had no effect on resistance to mitoxantrone or BBR 3390 in the P-glycoprotein-positive 8226/DOX6 cell line. Increasing the degree of resistance to mitoxantrone in the 8226 cell line from 10 to 37 times (8226/MR20) did not further reduce the intracellular drug concentration. However, the 8226/MR20 cell line exhibited 88 and 70% reductions in topoisomerase II beta and alpha expression, respectively, compared with the parental drug sensitive cell line. This decrease in topoisomerase expression and activity was not observed in the low-level drug-resistant, 8226/MR4 cell line. These data demonstrate that low-level mitoxantrone resistance is due to the presence of a novel, energy-dependent drug efflux pump similar to P-glycoprotein and multidrug resistance-associated protein. Reversal of resistance by blocking drug efflux with fumitremorgin C should allow for functional analysis of this novel transporter in cancer cell lines or clinical tumor samples. Increased resistance to mitoxantrone may result from reduced intracellular drug accumulation, altered nuclear/cytoplasmic drug distribution, and alterations in topoisomerase II activity.
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PMID:Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line. 1007 Sep 58


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