Abstract: ABC multidrug transporters are key players in cancer multidrug resistance and in general xenobiotic elimination, thus their functional assays provide important tools for research and diagnostic applications. It has been found that in cells expressing functional ABCG2, ABCB1, or ABCC1 transporters, cellular PG fluorescence is strongly reduced. The invention relates to methods and uses of fluorescein derivative ester compounds of formula Ia which are analogs of PG for assessing ABC transporter activity of ABC multidrug transporters. The present accumulation assay is a novel tool for the parallel determination of the function of the multidrug transporters, in particular ABCG2, ABCB1, and ABCC1. The assay is applicable for diagnostic purposes and also allows the selection, separation and culturing of selected cell populations expressing such transporters.

Abstract: The invention relates to methods for screening selective modulators of half transporter proteins of the ABCG family, more closely of ABCG1 and ABCG4. In particular the invention relates to methods for determining whether a substance is a selective activator, an inhibitor or a substrate of an ABCG1 or ABCG4 homodimer or of an ABCG1/ABCG4 heterodimer protein, methods for detection of ABCG1 protein in a biological sample, methods for modulating the function of said proteins, and methods for detecting the presence of and/or quantitating ABCG1/ABCG4 heterodimer activity in a biological sample. Moreover, the invention relates to isolated ABCG1/ABCG4 heterodimer proteins and antibodies selective for ABCG1 or ABCG4. The closely related human ABC half-transporters, ABCG1 and ABCG4, have been suggested to play an important role in cellular lipid/sterol regulation. ABCG1 and ABCG4 and mutants thereof have been expressed and studied by the present inventors in whole cells as well as isolated membrane preparations.

Abstract: The invention relates to a simple quantitative fluorescent assay method for determining the activity of transport proteins, more specifically multi-drug resistance associated proteins (MRPs). The method of the invention is performed on a well sealed culture of polarized cells expressing a transport protein of interest grown to confluency on a permeable support, said confluent cell culture forming well separated apical and basolateral compartments. The cells are contacted with a cell permeable non-fluorescent derivative of a fluorescent compound (e.g. calcein AM) in one compartment and, after a certain period of incubation, the fluorescence intensity detected in a sample taken from the opposite compartment is indicative of the activity of the transport protein of interest expressed in the cells.

Abstract: The invention relates to a simple quantitative fluorescent assay method for determining the activity of transport proteins, more specifically multi-drug resistance associated proteins (MRPs). The method of the invention is performed on a well sealed culture of polarized cells expressing a transport protein of interest grown to confluency on a permeable support, said confluent cell culture forming well separated apical and basolateral compartments. The cells are contacted with a cell permeable non-fluorescent derivative of a fluorescent compound (e.g. calcein AM) in one compartment and, after a certain period of incubation, the fluorescence intensity detected in a sample taken from the opposite compartment is indicative of the activity of the transport protein of interest expressed in the cells.

Abstract: Disclosed herein are methods and reagent kits for the quantitative in vitro determination of the functional determination of multi-drug resistance in cells, as well as for the clinical screening of potential modulators of multi-drug resistant transport activity in cells. The method of the invention is based on the measurement of the accumulation rate of free calcein within the cells of the specimen (advantageously by fluorescence measurement), after exposing the cells in vitro to a cell permeable form of calcein that is a good substrate for MDR proteins present in the sample. The cell permeable form of calcein is converted within the cell by intracellular enzymes to free calcein. Comparison of free calcein accumulation in the presence and absence of a potential inhibitor of transport activity permits the rapid screening of such inhibitors.

Abstract: Disclosed herein are methods and reagent kits for the quantitative in vitro determination of the functional determination of multi-drug resistance in cells, as well as for the clinical screening of potential modulators of multi-drug resistant transport activity in cells

Abstract: Disclosed herein are methods and reagents kits forth quantitative in vitro determination of the functional determination of multi-drug resistance in cells, as well as for the clinical screening of potential modulators of multi-drug resistant transport activity in cells. The method of the invention is based on the measurement of the accumulation rate of free calcein within the cells of the specimen (advantageously by flourescence measurement), after exposing the cells in vitro to a cell permeable form of calcein that is a good substrate for MDR proteins present in the sample. The cell permeable form of calcein is converted within the cell by intracellular enzymes to free calcein. Comparison of free calcein accumulation in the presence and absence of a potential inhibitor of transport activity permits the rapid screening of such inhibitors.

Abstract: The invention generally features a rapid, quantitative method of diagnosing multi-drug resistance in a patient. The method involves (a) exposing cells of a biological specimen to a calcein compound, the calcein compound becoming fluorescent in the cell; and (b) measuring calcein compound accumulating in the specimen cells relative to control cells, reduced calcein accumulation in specimen cells relative to control cells indicating the presence of multi-drug resistance in the biological specimen. The method is useful for diagnosing multi-drug resistance in patients undergoing drug therapy, e.g., chemotherapeutic or antibiotic therapy.