The multidrug-resistance associated protein MRP is a 180-to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an expression vector containing MRP cDNA. MRP-overexpressing SW-1573 cells are resistant to doxorubicin, daunorubicin, vincristine, VP-16, colchicine, and rhodamine 123, but not to 4'-(9-acridinylamino)methanesulfon-manisidide or taxol. The intracellular accumulation of drug (daunorubicin, vincristine, (20) and pRc/RSV (Invitrogen). All cDNA fragments used for the assembly of the MRP cDNA were sequenced and the integrity of the MRP cDNA fragment in the resulting expression vectors, pJ3fl-MRP and pRc/RSV-MRP (Fig. 1) Abbreviations: MDR, multidrug resistance (resistant); Pgp, P-glycoprotein; SCLC, small-cell lung cancer; pH;, intracellular pH; m-AMSA, 4'-(9-acridinylamino)methanesulfon-m-anisidide. 8822The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

In this study we report that the multidrug resistance protein (MRP) transports calcein from the cytoplasmic compartment of tumor cells, in contrast to P-glycoprotein which transports calcein acetuxymethyl ester from the plasmamembrane. The transport of calcein by MRP is ATP-dependent and is inhibited by probenecid and vincristine. Intracellular glutathione (GSH) depletion which occurred when cells were exposed to buthionine sulfoximine had no effect on the efflux of calcein, whereas it reversed the daunorubicin accumulation deficit in MRP overexpressing tumor cells. In conclusion, ATP-dependent transport of calcein and possibly other organic anions by MRP is not inhibited by a large decrease of the intracellular GSH concentration, that inhibits daunorubicin efflux by MRP.

Summary:Apoptosis is the common cell death pathway which is initiated by a variety of different stimuli. The recognition of early apoptotic events would markedly improve reliability and convenience of apoptosis assays. In the present study the vital stain Syto R 16 in combination with the permeability marker 7-amino actinomycin D, (7-AAD) has been used to identify an early stage of apoptosis, not detected with trypan blue or 7-AAD alone or with conventional apoptosis tests and not consistently and only partly detected by the early apoptosis marker annexin V. The method was established using solid tumour cell lines treated with TNF. Subsequently we applied it to determine apoptotic populations in CD34 + peripheral blood progenitor cells obtained from growth factor and/or chemotherapy mobilised patients and frozen/thawed according to standard stem cell transplantation protocols.

Summary Multidrug resistance (MDR) in tumour cells is often caused bv the overexpression of the plasma membrane drug transporter P-glycuprutein (P-gp) or the recently discosered multidrug resistance-associated protein (MRP). In this studs we insvestigated the specificity and sensitivity of the fluorescent probes rhodamine 123 (R123). daunorubicin (DNR) and calcein acetoxymethvl ester (calcein-AM) in order to detect the function of the drug transporters P-gp and MRP. using flow cytometrv. The effects of modulators on the accumulation and retention of these probes were compared in seseral pairs of sensitise and P-gp-as w-ell as MRPoserexpressing cell lines. R123. in combination with the modulator PSC833. provided the most sensitise test for detecting P-gp-mediated resistance. Moreoser. in a 60 min drug accumulation assay RI 23 can be regarded as a P-p-specific probe. since R123 is not ver-efficiently effluxed bv MRP. In contrast to R123. a 60 mm DNR or calcein-AM accumulation test could be used to detect MRP-mediated resistance The MRP-specific modulator genistein could be used in combination with DNR. but not w-ith calcein-AM. Vincristine (VCR) can be used to increase the cellular uptake of calcein-AM in MDR cells. but is not specific for MRP Thus.although the combination of DNR with genistein appeared to be as sensitive as the combination of calcein-AM with VCR. the former mav be used to probe specific MRP activity wshereas the latter provides a combined (P-gp + MRP) functional MDR parameter. With these functional assavs the role and relatise importance of P-p and MRP can be studied in. for example. haematological malignancies