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:3.6.3.44 (P-glycoprotein)
13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2',2'-Difluorodeoxycytidine (gemcitabine; dFdC) is a nucleoside analog with promising antitumor activity. To be active it must be phosphorylated by deoxycytidine kinase (dCK). We induced resistance to gemcitabine in the human ovarian carcinoma cell line A2780 by exposure to increasing concentrations of gemcitabine. At 72 hours' exposure the IC50, defined as the concentration of gemcitabine causing 50% growth inhibition, increased from 0.6 nmol/L gemcitabine in A2780 to 92 mumol/L in the resistant variant, AG6000. AG6000 is cross-resistant to other drugs that require activation by dCK, such as I-beta-D-arabinofuranosylcytosine, 5-aza-2'-deoxycytidine, and 2-chlorodeoxyadenosine. AG6000 was also cross-resistant to 2',2'-difluorodeoxyuridine (dFdU), the deamination product of gemcitabine. In addition, cross-resistance to the multidrug-resistance drugs doxorubicin and vincristine was observed. This was not associated with induction of P-glycoprotein. No accumulation of gemcitabine triphosphate could be detected in AG6000 cells, in contrast to the parental A2780 cells. There was no specific dCK activity in extracts from AG6000 cells. Western blot analysis using a polyclonal anti-dCK antibody did not reveal any dCK protein in AG6000 cell extracts. Reverse transcribed and polymerase chain reaction amplified mRNA, using specific dCK primers, demonstrated that AG6000 expressed a normal length amplicon of 701 base pairs, besides an aberrant amplicon of 500 base pairs. Although the resistant cell line is routinely cultured in 6 mumol/L gemcitabine, the resistant phenotype can be maintained for at least 10 passages without gemcitabine. These results indicate that the gemcitabine resistance phenotype is stable and mainly due to dCK deficiency.
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PMID:Induction of resistance to 2',2'-difluorodeoxycytidine in the human ovarian cancer cell line A2780. 748 43

2',2'-Difluorodeoxycytidine (gemcitabine, dFdCyd) is a deoxycytidine analogue with promising antitumor activity. In order to be active it must be phosphorylated by deoxycytidine kinase (dCK). We induced resistance to dFCyd in the human ovarian carcinoma cell line A2780 by exposure to increasing concentrations of dFdCyd. The IC50, defined as the concentration of dFdCyd causing 50% growth inhibition, at 72 h exposure increased from 0.6 nM dFdCyd in A2780 to 92 microM in the resistant variant, named AG6000. Although the resistant cell line is routinely cultured in 6 microM dFdCyd, the resistant phenotype can be maintained for at least 10 passages without dFdCyd. AG6000 is cross-resistant to other drug which require activation by dCK, such as 1-beta-D-arabinofuranosylcytosine, 5-aza-2'-deoxycytidine, and 2-chlorodeoxyadenosine. There was no specific dCK activity in extracts from AG600 cells. Western blot analysis using a polyclonal anti-dCK antibody did not reveal any dCK protein in AG6000 cell extracts. Reverse-transcribed and PCR-amplified mRNA, using specific dCK primers, demonstrated that AG6000 expressed a normal length amplicon of 701 base pairs, besides an aberrant amplicon of 500 base pairs. Chromosome spreads from the cell lines showed no major differences between A2780 and AG6000. The latter cell line was also cross-resistant to 2',2'-difluorodeoxyurdine, the deamination product of dFdCyd. Additionally, cross-resistance to the multidrug resistant drugs doxorubicin and vincristine was observed. This was not associated with the induction of P-glycoprotein, as determined by the RNase protection assay. Injection of AG6000 cells s.c. into nude mice demonstrated that the cell line had retained its tumorigenicity; AG6000 xenografts were not sensitive to dFdCyd treatment, in contrast to the parental A2780 tumors. No dFdCyd triphosphate accumulation was found in the resistant tumors, in contrast to the parental A2780 tumors. These results indicate that the dFdCyd resistance phenotype is stable, and mainly due to dCK deficiency.
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PMID:Development and molecular characterization of a 2',2'-difluorodeoxycytidine-resistant variant of the human ovarian carcinoma cell line A2780. 803 47

A new human myeloid leukemia cell line, designated KF-19, and its drug resistant sublines have been established. The KF-19 cell line was established from the pericardial effusion of a patient with acute myeloid leukemia clinically resistant to chemotherapy and KF-19 cells were characterized by expression of myeloid markers and differentiation into neutrophil- and macrophage-like cells upon optimal stimulations. KF-19AraC, KF-19ADR and KF-19VCR were established as sublines resistant to cytosine arabinoside (AraC), adriamycin (ADR) and vincristine (VCR), respectively. Efflux of the corresponding drugs was documented in each cell line. Expression of the MDR1 gene and the P-glycoprotein was found only in KF-19ADR, which showed a cross resistance to anthracyclines and vinca alkaloids; this resistance was reversed by verapamil or cyclosporin A. KF-19VCR lacking MDR1 gene and P-glycoprotein expression showed only resistance to vinca alkaloids, which was partially reversed by verapamil and cyclosporin A. Unexpectedly, KF-19ADR and KF-19VCR displayed cross resistance to AraC, despite lack of alterations of deoxycytidine kinase (dCK) and deaminase (dA) activities. KF-19AraC showed an efflux of AraC as well as a decreased level of dCK, but not of dA. In addition, KF-19AraC showed cross resistance to VCR in the efflux assay. The cell lines reported herein will provide new aspects on the mechanisms of drug resistance in leukemic cells.
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PMID:Characterization of newly established human myeloid leukemia cell line (KF-19) and its drug resistant sublines. 900 51

Gemcitabine (2'-2'-difluorodeoxycytidine; dFdC) is a deoxycytidine analogue which is effective against solid tumours, including lung cancer and ovarian cancer. dFdC requires phosphorylation by deoxycytidine kinase (dCK) for activation. In the human ovarian cancer cell line A2780 and its 30,000-fold dFdC-resistant variant AG6000 (P<0.001), we investigated the cross-resistance profile to several drugs. AG6000, which has a complete dCK deficiency, was approximately 1000-10,000-fold resistant to other deoxynucleoside analogues such as 1-beta-D-arabinofuranosyl cytosine, 2-chloro-deoxyadenosine, aza-deoxycytidine and 2', 2'-difluorodeoxyguanosine (dFdG) (P<0.001). dFdG can be activated by dCK and deoxyguanosine kinase (dGK), but the latter enzyme was not altered in AG6000 cells. Thus dFdG resistance was only due to dCK deficiency. AG6000 was 1.6- and 46.7-fold resistant to 5-fluorouracil (5-FU) and ZD1694, respectively (the latter was significant; P<0.01), which may be due to the 1.7-fold higher thymidylate synthase (TS) activity, but AG6000 cells were also 2. 7-fold resistant to the lipophilic TS inhibitor AG337 (P<0.05). Remarkably, AG6000 cells were 2.5-fold more sensitive to methotrexate (MTX) (P<0.01) than A2780 cells, but 1.6-fold more resistant to trimetrexate (TMQ) (P<0.10). However, no differences in reduced folate carrier activity, folylpolyglutamate synthetase (FPGS) activity and polyglutamation of MTX were found between the cell lines. AG6000 cells were approximately 2 to 7.5-fold more resistant to doxorubicin (DOX), daunorubicin (DAU), epirubicin and vincristine (VCR) (the latter was significant; P<0.02) and approximately 4-fold more resistant to the microtubule inhibitors paclitaxel and docetaxel (P<0.001). Fluorescent activated cell sorter (FACS) analysis revealed no P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP) expression, but less fluorescence of intercalated DAU in AG6000 cells. An approximately 2-fold resistance to the topoisomerase I and II inhibitors etoposide, CPT-11 and SN38 was found in AG6000 cells. Topoisomerase I and IIalpha RNA expression was decreased in AG6000 cells. AG6000 was 2.4, 2.4, 2.3 and 3.7-fold more resistant to EO9 (P<0.02), mitomycin-C (MMC) (P<0.05), cisplatin (CDDP) (P<0.10) and maphosphamide (MAPH), respectively. DT-diaphorase (DTD), which activates EO9, was 2.2-fold lower in AG6000 cells. CDDP resistance might be related to a reduced retention of DNA adducts in AG6000. However, glutathione levels were equal in A2780 and AG6000 cells. A 24 h exposure to DOX, VCR and paclitaxel at equimolar and equitoxic concentrations, resulted in more double-strand breaks (1.5- to 2-fold) in A2780 than in AG6000 cells. MAPH at 1120 nM and 17 nM of EO9 did not cause DNA damage in either cell line. In conclusion, AG6000 is a cell line highly cross-resistant to a wide variety of drugs. This cross-resistance might be related to altered enzyme activities and/or increased DNA repair.
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PMID:Cross-resistance in the 2',2'-difluorodeoxycytidine (gemcitabine)-resistant human ovarian cancer cell line AG6000 to standard and investigational drugs. 1100 May 80

We have established a human myelogenous leukemia cell line (HL60/AD) that is 10-fold cross-resistant to both 1-beta-D-arabinofuranosylcytosine (ara-C) and daunorubicin; the cell line was isolated from HL60 by simultaneous treatment with these two agents at low drug concentrations attainable in clinical trials. HL60/AD was found to have multiple resistance mechanisms. With regard to ara-C, HL60/AD cells showed decreased deoxycytidine kinase activity but did not show elevation of cytidine deaminase activity or a decrease in ara-C influx. With regard to daunorubicin, a decrease in topoisomerase II activity was found. A decrease in intracellular accumulation of daunorubicin was also found. P-glycoprotein was not detected, but the multidrug resistance-associated protein was expressed. Furthermore, an increase of total cellular glutathione (GSH) content was found. Interestingly, the resistance of HL60/AD cells not only to daunorubicin but also to ara-C was markedly reversed by treatment with L-buthionine-(S,R)-sulfoximine (BSO), a potent inhibitor of GSH synthesis. After exposure of HL60/AD to ara-C, mitochondrial membrane potential and reactive oxygen intermediates showed no significant change, but a considerable loss of mitochondrial membrane potential and an increase in reactive oxygen intermediate generation were caused by pre-incubation with BSO. Neither elevation of GSH nor reversal of resistance by BSO was found in ara-C-resistant HL60 cells that were selected only with ara-C. These findings suggest that in addition to the summation of the mechanisms of resistance to each agent reported previously, an increased level of GSH plays an important role in the cross-resistance induced in HL60/AD cells by simultaneous exposure to both drugs.
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PMID:Simultaneous treatment with 1-beta-D-arabinofuranosylcytosine and daunorubicin induces cross-resistance to both drugs due to a combination-specific mechanism in HL60 cells. 1119 56

Gemcitabine is phosphorylated by deoxycytidine kinase and thymidine kinase 2 and during S-phase incorporated into DNA. The steroids cortisol and dexamethasone, which regulate cell proliferation and gene expression, are pumped out of the cell by the membrane efflux pumps P-glycoprotein and multidrug resistance-associated protein (MRP), which are blocked by verapamil. In parental non-small cell lung cancer (NSCLC) cells (SW1573), 5 microM cortisol and 100 nM dexamethasone decreased sensitivity to gemcitabine. However, both cortisol and dexamethasone only decreased sensitivity with verapamil in MRP (2R120) and P-glycoprotein (2R160) overexpressing variants. Cortisol decreased deoxycytidine kinase activity in SW1573 cells and cortisol with verapamil in 2R120 and 2R160 cells. Dexamethasone with verapamil decreased deoxycytidine kinase activity in 2R160. Cortisol decreased thymidine kinase 2 activity in 2R120 and 2R160 cells. Dexamethasone decreased thymidine kinase 2 activity in SW1573, 2R120 and 2R160 cells. In conclusion, since dexamethasone is frequently used to treat side effects of oncolytic therapy, a decrease of sensitivity to gemcitabine by steroids might be clinically relevant.
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PMID:Steroids affect collateral sensitivity to gemcitabine of multidrug-resistant human lung cancer cells. 1128 8

Multidrug resistance (MDR), characterized by a cross-resistance to many natural toxin-related compounds, may be caused either by overexpression of a drug efflux pump such as P-glycoprotein, (P-gP), multidrug resistance proteins MRP1-3, or BCRP/MXR or, in the case of DNA topoisomerase II active drugs, by a decrease in the enzymatic activity of the target molecule termed altered topoisomerase MDR (at-MDR). However, human small cell lung carcinoma (SCLC) cell lines showed a collateral sensitivity to 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) and 1-beta-D-arabinofuranosylcytosine (ara-C). H69/DAU, a daunorubicin (DAU)-resistant variant of H69 with a P-gP overexpression, and NYH/VM, a VM-26 (teniposide)-resistant variant of NYH with an at-MDR, were both 2-fold more sensitive to gemcitabine and 7- and 2-fold more sensitive to ara-C, respectively. MDR variants had a 4.3- and 2.0-fold increased activity of deoxycytidine kinase (dCK), respectively. dCK catalyzes the first rate-limiting activation step of both gemcitabine and ara-C. In addition, deoxycytidine deaminase, responsible for inactivation of dFdC and ara-C, was 9.0-fold lower in H69/DAU cells. The level of thymidine kinase 2, a mitochondrial enzyme that can also phosphorylate deoxycytidine and gemcitabine, was not significantly different between the variants. These differences most likely caused an increased accumulation of the active metabolites (dFdCTP, 2.1- and 1.6-fold in NYH/VM and H69/DAU cells, respectively) and of ara-CTP (1.3-fold in NYH/VM cells). Ara-CTP accumulation was not detectable in either H69 variant. The pools of all ribonucleoside and deoxyribonucleoside triphosphates were at least 3- to 4-fold higher in the NYH variants compared to the H69 variants; for dCTP and dGTP this difference was even larger. The higher ribonucleotide pools might explain the >10-fold higher accumulation of dFdCTP in NYH compared to H69 variants. Since dCTP is low, H69 cells might not need a high ara-CTP accumulation to inhibit DNA polymerase. This might be related to the lack of ara-CTP in H69 variants. In addition, the increased CTP, ATP, and UTP pools in the MDR variants might explain the increased ara-CTP and dFdCTP accumulation. In conclusion, the MDR variants of the human SCLC cell lines were collaterally sensitive due to an increased dCK activity, and consequently an increased ara-CTP and dFdCTP accumulation.
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PMID:Collateral sensitivity to gemcitabine (2',2'-difluorodeoxycytidine) and cytosine arabinoside of daunorubicin- and VM-26-resistant variants of human small cell lung cancer cell lines. 1133 Oct 76

Cross-resistance between different classes of anti-neoplastic agents can jeopardize successful combination cancer chemotherapy. In this study, we observed an unexpected cross-resistance between the podophyllotoxine derivative etoposide (VP) and the nucleoside analogue cladribine (CdA) in CCRF-CEM cells developed for resistance to VP. The resistant cells also displayed 14- and twofold resistance to cytarabine (ara-C) and gemcitabine respectively. Closer analysis of these cells showed that they contained lower amounts of topoisomerase (topo) IIalpha (P < 0.001) and beta protein (P < 0.026), formed substantially lower amounts of the topo II-DNA complex, and had a markedly decreased level of Fas (CD95/APO-1)-ligand mRNA expression. Interestingly, Fas expression in the resistant cells did not differ from that in the parental cell line. No differences were observed in the accumulation/efflux of daunorubicin or in the gene expressions of P-glycoprotein, multidrug resistance-associated protein and the lung resistance-related protein. The activity of deoxycytidine kinase (dCK), responsible for activation of CdA and ara-C, was the same for resistant and wild-type cells. However, there was an increase in the activity of the cytosolic 5'-nucleotidases (5'-NT), responsible for deactivation of nucleotides, amounting to 206% (P < 0.001) for the high Km and 134% (P < 0.331) for the low Km 5'-NT in resistant cells. The high Km 5'-NT is probably responsible for the decreased amount of the active metabolite CdA 5'-triphosphate [40% decreased (P < 0.045)], as well as for other purine ribonucleosides and deoxyribonucleosides triphosphates in the resistant cells. In contrast, a significantly higher deoxycytidine triphosphate (dCTP) level (167%, P < 0.001) was observed in the resistant cells. Thus, this study suggests that the major cause of resistance to the nucleoside analogues CdA and ara-C in cells selected for resistance to VP is a result of metabolic alterations producing increased activity of 5'-NT and higher dCTP levels. Furthermore, these results indicate that there is a common factor in the regulation of nucleotide-degrading enzymes and DNA topoisomerases, which may be altered in cross-resistant cells.
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PMID:Pharmacological basis for cladribine resistance in a human acute T lymphoblastic leukaemia cell line selected for resistance to etoposide. 1138 Mar 97

The pyrimidine analogue cytosine arabinoside (AraC) is one of the most effective drugs used in the treatment of acute leukaemia. Overexpression of the multidrug resistance (MDR-1) gene and its product, P-glycoprotein (P-gp), is associated with cellular resistance to drugs, such as anthracyclines and vinca alkaloids. This resistance can be reversed by cyclosporine analogues or verapamil (ver). We investigated the in vitro cross-resistance to AraC in a doxorubicin-resistant HL60 cell line, with an elevated expression of the MDR-1 gene. The resistant clone showed an eightfold increased resistance to AraC and a two- to fourfold resistance to the other analogues, as measured by cytotoxicity test. There was no significant increase in the activity of 5'-nucleotidase or in the amount of deoxyribonucleotide pools between cell lines. We could, however, detect a reduction in deoxycytidine kinase (dCK) activity (30%, P = 0.021, using deoxycytidine as substrate) and the level of AraC triphosphates was significantly reduced in the resistant cells (70%, P = 0.009). When the cells were exposed to cyclosporin A (CsA) or the cyclosporine analogue PSC 833 (PSC) in combination with AraC, there was more extensive apoptosis, as measured by formation of oligonucleosomal DNA fragmentation and caspase-3-like activity, than with exposure to AraC alone. We also found an increased retention of AraC in the resistant cells when incubated with AraC in combination with CsA. Ver in combination with AraC, failed to increase apoptosis for the resistant cell line. Our data suggests that the resistance to AraC for the P-gp-expressing cells is a result of a reduction of dCK activity and an increase in efflux, the latter possibly depending on P-gp. A combination of CsA or PSC with AraC may improve the effect of AraC in vivo.
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PMID:Cross-resistance to cytosine arabinoside in a multidrug-resistant human promyelocytic cell line selected for resistance to doxorubicin: implications for combination chemotherapy. 1155 80

Drug resistance eventually occurs in most hematologic malignancies treated with chemotherapy. The mechanisms responsible for drug resistance include expression of transporters of xenobiotics of the adenosine triphosphate-binding cassette protein superfamily (P-glycoprotein, multidrug resistance associated proteins, breast cancer resistance protein), modifications of enzymes like deoxycytidine kinase, and defects in chemotherapy-induced apoptosis. The efforts to overcome this drug resistance have been focused, thus far, on modulation of P-glycoprotein. Several compounds were manufactured for this purpose, and phase III trials of PSC833, one of the most potent P-glycoprotein inhibitors, are completed. The emergence of modulators with several adenosine triphosphate-binding cassette protein targets, like GG120918 (inhibiting P-glycoprotein and breast cancer resistance protein) and VX710 (inhibiting P-glycoprotein and multidrug resistance associated protein 1), are of clinical interest in malignancies often expressing several efflux pumps simultaneously. Another approach is the use of "furtive" drugs like liposomal or nanoparticular anthracyclines.
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PMID:Drug resistance in hematologic malignancies. 1167 86


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