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Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters.

Lebedeva IV, Pande P, Patton WF - PLoS ONE (2011)

Bottom Line: ABC transporters also determine the general fate and effect of pharmaceutical agents in the body.Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results.The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments.

View Article: PubMed Central - PubMed

Affiliation: ENZO Life Sciences, Inc., Farmingdale, New York, United States of America. ilebedeva@enzolifesciences.com

ABSTRACT
An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC(2)(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments.

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eFluxx-ID® MDR probes detect all three major types of ABC transporters in a similar manner as doxorubicin and mitoxantrone probes, but are significantly brighter, providing much higher sensitivity compared with the other dyes.Model cell lines (CHO K1, panel A, and A549, panel B) were trypsinized, washed with PBS, aliquoted at 5×105 cells/sample, and treated in triplicates with different inhibitors (5 µM of cyclosporin A, 20 µM of verapamil, 50 µM of MK-571, or 0.05 µM of novobiocin) or left untreated. Tested probes (eFluxx-ID® Green, eFluxx-ID® Gold dyes, doxorubicin or mitoxantrone) were added to every sample. The cells were incubated with the dye(s) in the presence or absence of inhibitors for 30 min at 37°C. Then cells were immediately analyzed by flow cytometry. Population comparison was performed using Kolmogorov-Smirnov statistics [26]. Clear histograms represent sample fluorescence in the presence of the inhibitor, shaded – without the inhibitor. The numbers indicate average D-values for each sample from at least three independent experiments, with SD not exceeding 10% for each value.
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pone-0022429-g002: eFluxx-ID® MDR probes detect all three major types of ABC transporters in a similar manner as doxorubicin and mitoxantrone probes, but are significantly brighter, providing much higher sensitivity compared with the other dyes.Model cell lines (CHO K1, panel A, and A549, panel B) were trypsinized, washed with PBS, aliquoted at 5×105 cells/sample, and treated in triplicates with different inhibitors (5 µM of cyclosporin A, 20 µM of verapamil, 50 µM of MK-571, or 0.05 µM of novobiocin) or left untreated. Tested probes (eFluxx-ID® Green, eFluxx-ID® Gold dyes, doxorubicin or mitoxantrone) were added to every sample. The cells were incubated with the dye(s) in the presence or absence of inhibitors for 30 min at 37°C. Then cells were immediately analyzed by flow cytometry. Population comparison was performed using Kolmogorov-Smirnov statistics [26]. Clear histograms represent sample fluorescence in the presence of the inhibitor, shaded – without the inhibitor. The numbers indicate average D-values for each sample from at least three independent experiments, with SD not exceeding 10% for each value.

Mentions: Sensitivity and specificity of eFluxx-ID® Green and Gold probes, mitoxantrone and doxorubicin were analyzed and compared using three model cell lines (CHO K1, A549 and HeLa). Cells were grown in tissue culture dishes and on the day of assay were treated according to a dye uptake protocol and analyzed by flow cytometry, as described in Materials and Methods. The results of the assay are presented in Figure 2 (representative experiment) where average D-values from three independent experiments are displayed. All four probes were capable of detecting all three major types of ABC transporters, however the eFluxx-ID® Green and Gold probes generate substantially higher fluorescence intensity (as shown by D-values), demonstrating they are capable of providing more sensitive detection of MDR.


Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters.

Lebedeva IV, Pande P, Patton WF - PLoS ONE (2011)

eFluxx-ID® MDR probes detect all three major types of ABC transporters in a similar manner as doxorubicin and mitoxantrone probes, but are significantly brighter, providing much higher sensitivity compared with the other dyes.Model cell lines (CHO K1, panel A, and A549, panel B) were trypsinized, washed with PBS, aliquoted at 5×105 cells/sample, and treated in triplicates with different inhibitors (5 µM of cyclosporin A, 20 µM of verapamil, 50 µM of MK-571, or 0.05 µM of novobiocin) or left untreated. Tested probes (eFluxx-ID® Green, eFluxx-ID® Gold dyes, doxorubicin or mitoxantrone) were added to every sample. The cells were incubated with the dye(s) in the presence or absence of inhibitors for 30 min at 37°C. Then cells were immediately analyzed by flow cytometry. Population comparison was performed using Kolmogorov-Smirnov statistics [26]. Clear histograms represent sample fluorescence in the presence of the inhibitor, shaded – without the inhibitor. The numbers indicate average D-values for each sample from at least three independent experiments, with SD not exceeding 10% for each value.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3142157&req=5

pone-0022429-g002: eFluxx-ID® MDR probes detect all three major types of ABC transporters in a similar manner as doxorubicin and mitoxantrone probes, but are significantly brighter, providing much higher sensitivity compared with the other dyes.Model cell lines (CHO K1, panel A, and A549, panel B) were trypsinized, washed with PBS, aliquoted at 5×105 cells/sample, and treated in triplicates with different inhibitors (5 µM of cyclosporin A, 20 µM of verapamil, 50 µM of MK-571, or 0.05 µM of novobiocin) or left untreated. Tested probes (eFluxx-ID® Green, eFluxx-ID® Gold dyes, doxorubicin or mitoxantrone) were added to every sample. The cells were incubated with the dye(s) in the presence or absence of inhibitors for 30 min at 37°C. Then cells were immediately analyzed by flow cytometry. Population comparison was performed using Kolmogorov-Smirnov statistics [26]. Clear histograms represent sample fluorescence in the presence of the inhibitor, shaded – without the inhibitor. The numbers indicate average D-values for each sample from at least three independent experiments, with SD not exceeding 10% for each value.
Mentions: Sensitivity and specificity of eFluxx-ID® Green and Gold probes, mitoxantrone and doxorubicin were analyzed and compared using three model cell lines (CHO K1, A549 and HeLa). Cells were grown in tissue culture dishes and on the day of assay were treated according to a dye uptake protocol and analyzed by flow cytometry, as described in Materials and Methods. The results of the assay are presented in Figure 2 (representative experiment) where average D-values from three independent experiments are displayed. All four probes were capable of detecting all three major types of ABC transporters, however the eFluxx-ID® Green and Gold probes generate substantially higher fluorescence intensity (as shown by D-values), demonstrating they are capable of providing more sensitive detection of MDR.

Bottom Line: ABC transporters also determine the general fate and effect of pharmaceutical agents in the body.Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results.The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments.

View Article: PubMed Central - PubMed

Affiliation: ENZO Life Sciences, Inc., Farmingdale, New York, United States of America. ilebedeva@enzolifesciences.com

ABSTRACT
An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC(2)(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments.

Show MeSH
Related in: MedlinePlus