Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level

Abstract

ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2-and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. V C 2016 International Society for Advancement of Cytometry Key terms functional transporter assay; multidrug transporters; ABCG2; ABCB1; DyeCycle Violet; Hoechts 33342; high content screening and analysis MULTIDRUG resistance is a major problem in chemotherapy of cancer or in prolonged viral infections. An important cause of the emergence of cellular multidrug resistance is the increased expression of ABC multidrug transporters (1,2). Three human ABC proteins, ABCB1, ABCG2, and ABCC1 are the key membrane proteins responsible for an increased outward drug transport in most multidrug-resistant cells. These transporters are members of the ancient ABC transporter protein family, performing primary, ATP-dependent active transport processes. The human multidrug transporters consist of conserved cytoplasmic ABC domains, responsible for the ATP binding and hydrolysis, and multipass membrane region(s) involved in drug substrate binding and outward drug transport (3,4). The above mentioned three human multidrug transporters show promiscuous drug binding characteristics and have partially overlapping substrate specificities. Prediction of interactions between ABC multidrug transporters and drugs is a challenging task, because the structure of these proteins is not known at an atomic level, and the drug-protein interaction site is probably a large and versatile interface (5).Both in vitro and in vivo studies clearly indicate that in multidrug-resistant cells the therapeutically effective drug concentrations strongly depend on the actual, func-

Efficient intracellular delivery makes cancer cells sensitive to nanoemulsive chemodrugs

“…The cellular uptake experiments were carried out with CLSM and flow cytometry. Nile red and Hoechst 33342 were selected as the fluorescence probesto stain the cytoplasm [ 33 ] or nuclei [ 34 ], respectively. Nile red not only had a similar chemical structure to EVO (see Figure 1D and 1E ), but also had the ability to dye the intracellular lipids with red fluorescence, while Hoechst 33342 could pass through the hydrophilic nuclear membrane and locate into the cell nucleus.…”

Characterization of new, efficient Mycobacterium tuberculosis topoisomerase-I inhibitors and their interaction with human ABC multidrug transporters

“…The transport assays were carried out in 96-well plates. 1x10 5 cells were incubated in the presence of 0.1 μM Calcein-AM or 1 μM DCV (Dye Cycler Violet) for the ABCB1 and for the ABCG2 assays, respectively [30, 31]. The assay was performed in HPMI medium (NaCl 120 mM, KCl 5 mM, MgCl 2 0.4 mM, CaCl 2 0.04 mM, HEPES 10 mM, NaHCO 3 10 mM, glucose 10 mM, Na 2 HPO 4 5 mM; pH 7.4).…”

Identification of novel cell-impermeant fluorescent substrates for testing the function and drug interaction of Organic Anion-Transporting Polypeptides, OATP1B1/1B3 and 2B1

“…Calcein-AM, originally developed as a viability dye, was discovered to be a high affinity substrate of several pharmacologically relevant ABC transporters 27 – 29 . Similarly, Hoechst 33342 and DyeCycle Violet, two nucleotide/DNA binding dyes, are ABCG2 and ABCB1 substrates that can be used to characterize transporter function 30 , 31 . Screening assays based on the OATP1B1/3-mediated uptake of fluorescein, fluorescein-methotrexate or various fluorescein derivatives have also been developed 32 – 34 .…”