Relation between MDR1 mRNA levels, resistance factor, and the efficiency of P-glycoprotein-mediated efflux of pirarubicin in multidrug-resistant K562 sublines

2002 ◽  
Vol 80 (11) ◽  
pp. 1054-1063 ◽  
Author(s):  
Jintana Meesungnoen ◽  
Jean-Paul Jay-Gerin ◽  
Samlee Mankhetkorn
Keyword(s):  
Prolonged Exposure ◽  
Multidrug Resistant ◽  
Resistance Factor ◽  
Cytotoxic Agents ◽  
Hill Coefficient ◽  
Mrna Levels ◽  
Experimental Conditions ◽  
P Glycoprotein ◽  
Mdr1 Mrna ◽  
Acidic Organelles

In this work, we sought to investigate the relation existing between MDR1 mRNA levels, the resistance factor (RF), and the efficiency of efflux of pirarubicin (THP) mediated by P-glycoprotein (P-gp) in multidrug-resistant (MDR) K562 sublines. The MDR K562 sublines were selected from K562/adr cells by exposure to different adriamycin concentrations: 300 nM (K562/300), 1000 nM (K562/1000), and 10 000 nM (K562/10000), yielding RF values of 23.2, 26.5, and 39.6, respectively. The analysis of the P-gp encoding MDR1 gene overexpression by reverse transcriptase – polymerase chain reaction provided evidence of increased MDR1 mRNA levels when the adriamycin concentration used for the MDR cell selection increased. We used spectrofluorometric methods to determine the kinetics of the uptake and P-gp-mediated efflux of THP in the different selected MDR K562 sublines. Our data showed that (i) the maximal rate of P-gp-mediated efflux of THP, Vmax, increased with increasing RF; (ii) the observed Michaelis constant, Km, had the same value for all selected sublines, thus leading to an overall increase in the ratio Vmax/Km (5.1 × 10–3, 6.2 × 10–3, 6.8 × 10–3, and 9.3 × 10–3 s–1 for K562/adr, K562/300, K562/1000, and K562/10000 cells, respectively), and (iii) the determination of the Hill coefficient (nH) gave values close to 2, which suggested a positive cooperative transport of THP with the expelling of two molecules of THP per turnover of P-gp. This study demonstrated that, in the K562/adr sublines used in our experiments, P-gp played a major role in conferring the MDR phenotype. Moreover, under our experimental conditions, intracellular acidic organelles were shown to contribute to decreased drug–target interaction and, thereby, decreased cytotoxicity. The variation of the concentrations of THP accumulated in the acidic organelles as a function of the total THP concentration added to the cells was the same, within the limits of experimental errors, whatever the degree of resistance of the studied MDR K562 sublines. Finally, this study suggested that, in the selected MDR K562 sublines, the K+/H+ antiporter exchanger could be activated by the pirarubicin transport, leading to a probable acidification of intracellular pH. The P-gp-mediated efflux of THP and an accumulation of THP in acidic organelles confer an advantage for MDR cells in surviving prolonged exposure to cytotoxic agents and giving rise to high degrees of resistance. Key words: multidrug resistance, P-glycoprotein, pirarubicin, acidic organelles, MDR1 mRNA levels, fluorescence spectroscopy, kinetic parameters.

Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells

1994 ◽  
Vol 14 (8) ◽  
pp. 5202-5211
Author(s):  
M T Kuo ◽  
R C Vyas ◽  
L X Jiang ◽  
W N Hittelman
Keyword(s):  
Gene Amplification ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Fragile Site ◽  
Dna Amplification ◽  
Chromosome Breakage ◽  
Multidrug Resistant ◽  
Cytotoxic Agents ◽  
Drug Resistant ◽  
P Glycoprotein

Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification.

Immunosuppressants FK506 and rapamycin function as reversal agents of the multidrug resistance phenotype

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1528-1536 ◽  
Author(s):  
RJ Arceci ◽  
K Stieglitz ◽  
BE Bierer
Keyword(s):  
Efflux Pump ◽  
Multidrug Resistant ◽  
Chemotherapeutic Agents ◽  
Cytotoxic Drug ◽  
Cytotoxic Agents ◽  
Cross Resistance ◽  
Clinical Role ◽  
Reversal Agents ◽  
P Glycoprotein ◽  
Mdr Reversal

Abstract The multidrug-resistant (MDR) phenotype is characterized in vitro by the resistance displayed by cell lines to a broad spectrum of natural product cytotoxic agents. This high level of cross-resistance is due to the increased expression of a membrane glycoprotein termed P- glycoprotein. Encoded in humans by the mdr1 gene, P-glycoprotein functions as an energy-dependent efflux pump of these cytotoxic agents. In this report, we demonstrate that the newly characterized immunosuppressant FK506 and its structural analogue, rapamycin, are capable of functioning as MDR reversal agents. FK506 and rapamycin increase both intracellular, cytotoxic drug (daunomycin) accumulation, and the cytotoxicity of chemotherapeutic agents in multidrug-resistant cells. The increase in cytotoxic drug accumulation is observed at concentrations of FK506 and rapamycin 1,000-fold greater than the concentrations required for FK506 and rapamycin to inhibit T-lymphocyte activation and similar to those shown to be effective for other MDR reversal agents such as cyclosporine A (CsA) and verapamil. The effect of FK506 or rapamycin on both intracellular accumulation and cytotoxicity of daunomycin is additive. This is supported by the ability of FK506 and rapamycin to directly compete the binding of the photoaffinity analogue 125I-iodoaryl azidoprazosin to the P- glycoprotein. The data demonstrate that FK506 and rapamycin represent a new class of structurally distinct molecules that can function as MDR reversal agents and suggest a previously unidentified, potential clinical role for these compounds.

scholarly journals Role of the highly structured 5′-end region of MDR1 mRNA in P-glycoprotein expression

2007 ◽  
Vol 406 (3) ◽  
pp. 445-455 ◽  
Author(s):  
Rebecca A. Randle ◽  
Selina Raguz ◽  
Christopher F. Higgins ◽  
Ernesto Yagüe
Keyword(s):  
Multidrug Resistance ◽  
Drug Exposure ◽  
Cytotoxic Drugs ◽  
Initiation Factor ◽  
Mrna Levels ◽  
Translational Repressor ◽  
P Glycoprotein ◽  
Mdr1 Mrna ◽  
Cytotoxic Stress

Overexpression of P-glycoprotein, encoded by the MDR1 (multidrug resistance 1) gene, is often responsible for multidrug resistance in acute myeloid leukaemia. We have shown previously that MDR1 (P-glycoprotein) mRNA levels in K562 leukaemic cells exposed to cytotoxic drugs are up-regulated but P-glycoprotein expression is translationally blocked. In the present study we show that cytotoxic drugs down-regulate the Akt signalling pathway, leading to hypophosphorylation of the translational repressor 4E-BP [eIF (eukaryotic initiation factor) 4E-binding protein] and decreased eIF4E availability. The 5′-end of MDR1 mRNA adopts a highly-structured fold. Fusion of this structured 5′-region upstream of a reporter gene impeded its efficient translation, specifically under cytotoxic stress, by reducing its competitive ability for the translational machinery. The effect of cytotoxic stress could be mimicked in vivo by blocking the phosphorylation of 4E-BP by mTOR (mammalian target of rapamycin) using rapamycin or eIF4E siRNA (small interfering RNA), and relieved by overexpression of either eIF4E or constitutively-active Akt. Upon drug exposure MDR1 mRNA was up-regulated, apparently stochastically, in a small proportion of cells. Only in these cells could MDR1 mRNA compete successfully for the reduced amounts of eIF4E and translate P-glycoprotein. Consequent drug efflux and restoration of eIF4E availability results in a feed-forward relief from stress-induced translational repression and to the acquisition of drug resistance.

scholarly journals Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells.

1994 ◽  
Vol 14 (8) ◽  
pp. 5202-5211 ◽  
Author(s):  
M T Kuo ◽  
R C Vyas ◽  
L X Jiang ◽  
W N Hittelman
Keyword(s):  
Gene Amplification ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Fragile Site ◽  
Dna Amplification ◽  
Chromosome Breakage ◽  
Multidrug Resistant ◽  
Cytotoxic Agents ◽  
Drug Resistant ◽  
P Glycoprotein

Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification.

scholarly journals Expression of P-glycoprotein in adult T-cell leukemia cells

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2065-2071 ◽  
Author(s):  
Y Kuwazuru ◽  
S Hanada ◽  
T Furukawa ◽  
A Yoshimura ◽  
T Sumizawa ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cell ◽  
Multidrug Resistant ◽  
Leukemia Cells ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Glycoprotein P ◽  
Adult T Cell Leukemia ◽  
P Glycoprotein ◽  
Mdr1 Mrna

We have examined the expression of P-glycoprotein (P-gp) in adult T- cell leukemia (ATL) samples from 25 patients. Based on immunoblotting with a monoclonal antibody against P-gp, C219, 8 of 20 ATL patients were P-gp positive at the initial presentation. All 6 patients at the relapsed stage were P-gp positive, and refractory to chemotherapy. The expression of MDR1 mRNA in P-gp-positive ATL cells was increased at the relapsed stage of one patient. P-gp of this patient was photolabeled with [3H]azidopine and the labeling was inhibited with nimodipine, vinblastine and progesterone. These results suggest that P-gp expressed in ATL cells from patients at relapsed stage has the same binding site(s) for the drugs as that in multidrug resistant cells, and is correlated with the refractory nature of the cells to chemotherapy.

scholarly journals Expression of P-glycoprotein in adult T-cell leukemia cells

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2065-2071 ◽  
Author(s):  
Y Kuwazuru ◽  
S Hanada ◽  
T Furukawa ◽  
A Yoshimura ◽  
T Sumizawa ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cell ◽  
Multidrug Resistant ◽  
Leukemia Cells ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Glycoprotein P ◽  
Adult T Cell Leukemia ◽  
P Glycoprotein ◽  
Mdr1 Mrna

Abstract We have examined the expression of P-glycoprotein (P-gp) in adult T- cell leukemia (ATL) samples from 25 patients. Based on immunoblotting with a monoclonal antibody against P-gp, C219, 8 of 20 ATL patients were P-gp positive at the initial presentation. All 6 patients at the relapsed stage were P-gp positive, and refractory to chemotherapy. The expression of MDR1 mRNA in P-gp-positive ATL cells was increased at the relapsed stage of one patient. P-gp of this patient was photolabeled with [3H]azidopine and the labeling was inhibited with nimodipine, vinblastine and progesterone. These results suggest that P-gp expressed in ATL cells from patients at relapsed stage has the same binding site(s) for the drugs as that in multidrug resistant cells, and is correlated with the refractory nature of the cells to chemotherapy.

scholarly journals Mechanisms of P-Glycoprotein Modulation by Semen Strychni Combined with Radix Paeoniae Alba

2017 ◽  
Vol 2017 ◽  
pp. 1-8
Author(s):  
Li-Li Liu ◽  
Yong-Mei Guan ◽  
Xue-Ping Lu ◽  
Xin-Li Liang ◽  
Li-Hua Chen
Keyword(s):  
Atpase Activity ◽  
Cell Model ◽  
Chinese Herb ◽  
Therapeutic Window ◽  
Therapeutic Effects ◽  
Mrna Levels ◽  
P Glycoprotein ◽  
Mdr1 Mrna ◽  
Radix Paeoniae Alba ◽  
Semen Strychni

Semen Strychni has been extensively used as a Chinese herb, but its therapeutic window is narrowed by the strong toxicity of the compound, which limits its effectiveness. Radix Paeoniae Alba has been reported to reduce the toxic effects and increase the therapeutic effects of Semen Strychni, but the underlying mechanism remains unknown. This research aimed to explore the mechanism through which P-glycoprotein (P-gp) is modulated by Semen Strychni combined with Radix Paeoniae Alba in vitro. An MTT assay was used to study cytotoxicity in an MDCK-MDR1 cell model. Rh123 efflux and accumulation were measured to assess P-gp function. The expression levels of MDR1 mRNA and P-gp protein in MDCK-MDR1 cells were investigated. A P-gp ATPase activity assay kit was applied to detect the effect on P-gp ATPase activity. Semen Strychni combined with Radix Paeoniae Alba could induce P-gp-mediated drug transport by inhibiting brucine and strychnine transport in MDCK-MDR1 cells, enhancing the P-gp efflux function, upregulating the P-gp expression and MDR1 mRNA levels, and stimulating P-gp ATPase activity.

scholarly journals Immunosuppressants FK506 and rapamycin function as reversal agents of the multidrug resistance phenotype

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1528-1536
Author(s):  
RJ Arceci ◽  
K Stieglitz ◽  
BE Bierer
Keyword(s):  
Efflux Pump ◽  
Multidrug Resistant ◽  
Chemotherapeutic Agents ◽  
Cytotoxic Drug ◽  
Cytotoxic Agents ◽  
Cross Resistance ◽  
Clinical Role ◽  
Reversal Agents ◽  
P Glycoprotein ◽  
Mdr Reversal

The multidrug-resistant (MDR) phenotype is characterized in vitro by the resistance displayed by cell lines to a broad spectrum of natural product cytotoxic agents. This high level of cross-resistance is due to the increased expression of a membrane glycoprotein termed P- glycoprotein. Encoded in humans by the mdr1 gene, P-glycoprotein functions as an energy-dependent efflux pump of these cytotoxic agents. In this report, we demonstrate that the newly characterized immunosuppressant FK506 and its structural analogue, rapamycin, are capable of functioning as MDR reversal agents. FK506 and rapamycin increase both intracellular, cytotoxic drug (daunomycin) accumulation, and the cytotoxicity of chemotherapeutic agents in multidrug-resistant cells. The increase in cytotoxic drug accumulation is observed at concentrations of FK506 and rapamycin 1,000-fold greater than the concentrations required for FK506 and rapamycin to inhibit T-lymphocyte activation and similar to those shown to be effective for other MDR reversal agents such as cyclosporine A (CsA) and verapamil. The effect of FK506 or rapamycin on both intracellular accumulation and cytotoxicity of daunomycin is additive. This is supported by the ability of FK506 and rapamycin to directly compete the binding of the photoaffinity analogue 125I-iodoaryl azidoprazosin to the P- glycoprotein. The data demonstrate that FK506 and rapamycin represent a new class of structurally distinct molecules that can function as MDR reversal agents and suggest a previously unidentified, potential clinical role for these compounds.

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