Acknowledgments
The authors kindly thank the Fraunhofer Society and the Bavarian State ministry for economy and media, energy and technology (Az.:VI/3-6622/453/12) for financially supporting the work.
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Daniel F. Gilbert and Oliver Friedrich (eds.), Cell Viability Assays: Methods and Protocols, Methods in Molecular Biology, vol. 1601, DOI 10.1007/978-1-4939-6960-9_11, © Springer Science+Business Media LLC 2017
Chapter 11
Assays for Analyzing the Role of Transport Proteins in the Uptake and the Vectorial Transport of Substances Affecting Cell Viability
Emir Taghikhani, Martin F. Fromm, and Jửrg Kửnig
Abstract
Endogenous compounds, drugs, or other xenobiotics may affect cell viability. A prerequisite for intracel- lular cell damage is the uptake of such substances across the plasma membrane into cells. Furthermore, the subsequent transporter-mediated export out of cells may influence cell viability. Therefore, transport proteins mediating the uptake (uptake transporter) or export (export pumps) of substances in and out of cells are important determinants of cell viability. Uptake transporters mostly belong to the superfamily of solute carriers (SLC transporters), whereas export pumps are members of the ABC-transporter superfamily (ATP-binding cassette). Cell systems recombinantly overexpressing uptake transporters (single transfec- tants) or multiple-transfected cell models expressing simultaneously an uptake transporter together with an export pump (double transfectants) are important in vitro tools for analyzing protein-mediated transport of potentially cell toxic compounds.
Here we describe different in vitro transport assays for the functional analysis of transport proteins.
Using single-transfected HEK293 cells stably overexpressing an uptake transporter, substances can be tested as potential substrates (uptake assay) or potential transport inhibitors (inhibition assay) for the respective transport protein. Vectorial transport of substances with the uptake across the basolateral plasma membrane and the export across the apical membrane of polarized grown MDCKII cells can be analyzed using double-transfected cell models with the simultaneous overexpression of an uptake transport and an export pump in vectorial transport assays, thereby mimicking physiological transport processes, e.g., in liver or kidney.
Key words Uptake assay, Vectorial transport, Uptake transporter, Export pump, Drug transport
1 Introduction
Uptake transporters and efflux pumps determine plasma and tissue concentrations of a broad range of drugs, xenobiotics, or endoge- nous compounds. Furthermore, protein-mediated uptake is a pre- requisite for the subsequent intracellular action or metabolism of substances. Therefore, the intracellular concentration of potential cytotoxic compounds depends on uptake transporters in the plasma membrane of cells. In addition, also the transporter-mediated
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export of substances (e.g., drugs) or their metabolites may affect this intracellular concentration. One well-known drug affecting cell viability is cisplatin. Cisplatin is widely used for chemotherapy but this therapy often is limited by severe side effects such as neph- rotoxicity and ototoxicity [1]. In kidney, cisplatin is taken up into cells of the terminal proximal tubule and of the distal nephron where it causes apoptosis or necrosis. The uptake transporter OCT2 (gene symbol SLC22A2) mediates this cisplatin uptake, and it has been demonstrated that OCT2-mediated uptake plays a piv- otal role in the development of cisplatin-induced oto- and nephro- toxicity [2]. Therefore, transport proteins mediating the uptake or the export of substances are important for cell viability and it is important to characterize substances as transporter substrates or transport inhibitors.
Functionally, transport proteins can be classified into trans- porters mediating the uptake of substances (e.g., drugs) into cells and transporters mediating the export of substances (e.g., drug metabolites) out of cells [3]. Uptake transporters mostly belong to the superfamily of SLC transporters (SLC = solute carriers). Today, this superfamily consists of 52 SLC families with more than 400 identified transport proteins. Particularly important for the trans- port of drugs are the families SLC21/SLCO and SLC22. The SLC21/SLCO family encodes for OATP proteins (OATP = Organic Anion Transporting Polypeptides) and members of this family are expressed in nearly all human tissues and cells investi- gated. Important family members for drug treatment are OATP1B1 (gene symbol SLCO1B1) and OATP1B3 (SLCO1B3), both expressed in human liver mediating the uptake of endogenous sub- stances and drugs (e.g., statins or antibiotics) from blood into hepatocytes. The SLC22 family comprises organic cation trans- porters (OCTs) and organic anion transporters (OATs), both of which are important for drug and xenobiotic transport.
Export transporters mostly belong to the superfamily of ABC transporters (ABC = ATP-binding cassette) mediating the export of substances out of cells against a concentration gradient driven by ATP hydrolysis. The ABC superfamily comprises seven families (ABCA–ABCG) with members of the ABCB family (e.g., P-glycoprotein), the ABCC family (e.g., multidrug resistance associ- ated protein 2–MRP2), and the ABCG family (breast cancer resis- tance protein–BCRP) as important for the transport of drugs, drug conjugates, and xenobiotics. In addition to ABC transporters, also family members of the SLC47 family are important for mediating the export out of cells. MATE (multidrug and toxin extrusion pro- teins) proteins are members of this family and important for the excretion of drugs and drug metabolites into bile and urine [3].
Taken the known substrate spectrum of all transporters impor- tant for drug and xenobiotic transport into account, it is obvious that in vitro cell systems for the standardized analysis of uptake Emir Taghikhani et al.
transporters and export pumps are important tools for investigating substances potentially affecting cell viability. Uptake transporters are investigated routinely by so-called single-transfected cells recombi- nantly overexpressing the respective transporter. They can be used in normal uptake assays or in inhibition assays, in which the inves- tigated substance is added as possible transport modulator to an uptake assay using a prototypic substrate for the respective trans- port protein.
Because it is much more difficult analyzing export pumps in normal transport assays, double- and multiple-transfected cell lines with the simultaneous expression of an uptake transporter and an export pump are important in vitro tools for analyzing the vecto- rial transport of substances. Such cell models are mostly based on MDCKII cells that grow in a polarized fashion separating a baso- lateral and an apical compartment. Uptake transporters are local- ized in the basolateral membrane of polarized grown MDCKII cells mediating the uptake of substances from the basolateral compartment into cells. The export is mediated by proteins in the apical membrane of MDCKII cells transporting the substance into the apical compartment. In recent years several double- and multiple- transfected MDCKII cell models have been established expressing different combinations of uptake transporters and export proteins [4–10].
In this chapter, we describe three different experimental setups for the analysis of transport proteins. Based on single-transfected HEK293 cells overexpressing solely an uptake transporter, sub- stances can be identified as substrates in simple uptake assays (uptake assay). Prerequisite for such analyses are substances that are available in radiolabeled form or for which an LC/MS-MS analysis can be performed after the uptake assay. If this is not the case, substances can be added as potential transport inhibitors by using the single-transfected HEK293 cells with a known transport substrate (inhibition assay). In addition, vectorial transport studies using double-transfected MDCKII cells will be described (vecto- rial transport studies).
2 Materials