Zsolt Holló

Calcein accumulation as a fluorometric functional assay of the multidrug transporter

Acetoxymethyl ester (AM) derivatives of various fluorescent indicators (fura-2, fluo-3, indo-1, BCECF, calcein) are actively extruded by the multidrug transporter (MDR1, P-glycoprotein-Homolya, L. et al. (1993) J. Biol. Chem. 268, 21493-21496). In the present paper we show that the measurement of the accumulation of a fluorescent cell viability marker, calcein, can be effectively used as a rapid and sensitive fluorometric and flow cytometric assay for studying P-glycoprotein function. The rate of calcein accumulation in human MDR1-expressing cells is significantly lower than in the control cells, while various drug-resistance reversing agents (verapamil, vinblastine, oligomycin, cyclosporin A and UIC2 monoclonal antibody) greatly increase calcein trapping only in the MDR1-expressing cells. Since calcein-AM is not fluorescent and free calcein is not a substrate of the multidrug transporter, the assay is readily applicable for rapid kinetic studies of the MDR1 function. Calcein has a high fluorescence intensity in the visible range, thus changes in calcein uptake can be easily visualised and MDR1-expressing and control cells separated by conventional flow cytometry.

Membrane Topology and Glycosylation of the Human Multidrug Resistance-associated Protein

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.

Transport properties of the multidrug resistance‐associated protein (MRP) in human tumour cells

In this paper we demonstrate that the expression of the multidrug resistance‐associated protein (MRP) in a variety of intact human tumour cells results in the ATP‐dependent, mutually exclusive extrusion of both the acetoxymethyl ester and the free anion forms of the fluorescent dye calcein, as well as that of a fluorescent pyrenemaleimide‐glutathione conjugate. The MRP‐dependent transport of all these three model compounds closely correlates with the expression level of MRP and is cross‐inhibited by hydrophobic anticancer drugs, by reversing agents for MDR1, and also by compounds not influencing MDR1, such as hydrophobic anions, alkylating agents, and inhibitors of organic anion transporters. Cellular glutathione depletion affects neither the MRP‐dependent extrusion of calcein AM or free calcein, nor its modulation by most hydrophobic or anionic compounds, although eliminating the cross‐inhibitory effect of glutathione conjugates. These results suggest that the outward pumping of both hydrophobic uncharged and water‐soluble anionic compounds, including glutathione conjugates, is an inherent property of MRP, and offer sensitive methods for the functional diagnostics of this transport protein as well as for the rapid screening of drug‐resistance modulating agents.

Interaction of bioactive hydrophobic peptides with the human multidrug transporter.

In this report we demonstrate that various biologically active hydrophobic peptide derivatives, e.g., proteinase inhibitors, chemoattractants, ionophores, enkephalins, and immunosuppressants, stimulate a membrane ATPase activity associated with the human multidrug transporter (MDR1). The stimulation of the MDR1‐ATPase by these agents does not correlate with their known biochemical or pharmacological activities but rather with their hydrophobicity. The peptides that show high‐affinity interaction with the MDR1‐ATPase also interfere strongly with fluorescent dye extrusion catalyzed by the multidrug transporter in intact cells and some have been shown to reverse drug resistance in cultured cells. These data suggest that several hydrophobic peptides behave as substrates of the multidrug transporter and may be used to modulate the chemotherapy resistance of tumor cells.—Sarkadi, B., Müller, M., Homolya, L., Holló, Z., Seprödi, J., Germann, U. A., Gottesman, M. M., Price, E. M., Boucher, R. C. Interaction of bioactive hydrophobic peptides with the human multidrug transporter. FASEB J. 8: 766‐770; 1994.

Calcein assay for multidrug resistance reliably predicts therapy response and survival rate in acute myeloid leukaemia

In this study, we evaluated the suitability of the calcein assay as a routine clinical laboratory method for the identification of multidrug‐resistant phenotype in acute leukaemia. This study presents the results of the calcein tests obtained in two large haematological centres in Hungary. Assays were performed with blast cells of 93 de novo acute leukaemia patients, including 65 patients with acute myeloid leukaemia (AML). Results were expressed as multidrug resistance activity factor (MAF) values. AML patients were divided into responders and non‐responders and MAF values were calculated for each group. In both centres, responder patients displayed significantly lower MAF values than non‐responders (P = 0·0045 and P = 0·0454). Cut‐off values were established between the MAFR + SEM and MAFNR − SEM values. On the basis of these cut‐off levels, multidrug resistance (MDR) negativity showed a 72% predictive value for the response to chemotherapy, whereas MDR positivity was found to have an average predictive value of 69% for therapy failure. MDR activity was a prognostic factor for survival rate and the test was suitable for detecting patients at relapse. The calcein assay can be used as a quantitative, standardized, inexpensive screening test in a routine clinical laboratory setting. The assay detects both P‐glycoprotein and multidrug resistance‐associated protein activities, and identifies AML patients with unfavourable therapy responses.

Determination of reference values of MDR-ABC transporter activities in CD3+ lymphocytes of healthy volunteers using a flow cytometry based method: MDR-ABC TRANSPORTER ACTIVITY VALUES IN CD3+ LYMPHOCYTES

MDR transporters are important biomarkers of drug resistance in cancer and in autoimmune conditions. We determined the MDR1, MRP1 and BCRP activity in CD3+ lymphocytes using a flow cytometry based method from 120 healthy volunteers in order to describe normal reference values of the activity of these transporters. The effects of gender and age were also determined.

Fluorescence Assay for Studying P-Glycoprotein Function at Single Cell Level

Ineffectivity of tumor chemotherapy is often caused by the resistance of malignant cells to a wide range of hydrophobic cytostatic agents. The main characteristic of these multidrug-resistant (MDR) cells is an energy dependent outward transport of drugs produced by a membrane protein, P-glycoprotein (MDR1, multidrug transporter). The functional assessment of this protein is essential for planning the proper chemotherapy. It has been previously demonstrated that MDR1-expressing cells show a decreased uptake of certain fluorescent anthracyclines, as well as fluorescent dyes as rhodamine 123, or fluo-3, and these compounds have been used to discriminate between drug-resistant and sensitive cells. We have demonstrated that hydrophobic acetoxy-methylester (AM) derivatives of various fluorescent indicators are actively extruded from cells by MDR1 (1). On the basis of this finding a quantitative assay method was developed by using calcein AM (3). Calcein AM, a non-fluorescent hydrophobic molecule, rapidly penetrates through cell membranes and becomes trapped intracellularly upon conversion into the fluorescent calcein (free acid) by nonspecific cytoplasmic esterases. In the MDR1-expressing cells, calcein AM is extruded by the multidrug transporter before its intracellular conversion to the non-MDR1 substrate free calcein (1, 2). However, when calcein AM extrusion is blocked by an agent that interferes with the MDR1 pump (e. g. verapamil), fluorescent free calcein rapidly accumulates. This assay provides an efficient experimental method for the quantitative determination of the multidrug transporter activity.