No abstract

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

Summary. P-glycoprotein (PGP),All the patients received standard induction and consolidation treatments containing MDR-related (idarubicin, mitoxantrone, etoposide) and other (arabinosyl cytosine) drugs. Multivariate analysis showed that PGP overexpression was significantly associated with a poor response to treatment, both in terms of primary resistance or shorter survival. Other independent prognostic factors were age and cytogenetics. LRP overexpression did not reach statistical significance, although for LRP þ cases the trend was unfavourable. Due to small numbers, no conclusion could be made regarding MRP overexpression, but 5/8 cases showed unfavourable karyotypic abnormalities, 8/8 had a defective IDA and 6/8 failed to achieve remission. This study showed that both PGP and LRP overexpression are common features in de novo ANLL at onset whereas MRP overexpression is more rare. It suggested that overexpression of one of the MDR related proteins was associated with a defective IDA, and confirmed that, in addition to age and cytogenetics, PGP retains an independent prognostic value. It also suggested that LRP did not affect clinical outcome when patients were treated with idarubicin or mitoxantrone and arabinosyl cytosine.

NHW-proximal MSD (Cole et al. 1992: Bakos et al. 1996: Loe et al. 1996a: Deelev and Cole. 1997: Stride et al. 1996. We haxe recently shoun bv site-directed mutag,enesis of N-glycosylation sites that. in MRP. this MSD contains an odd number of transmembrane helices and. in contrast to P-glycoprotein. the amino-terminus of MRP is extracytosolic .Whereas both NRP and P-glycoprotein confer resistance to chemotherapeutic agents such as doxorubicin and xincristine bv reducing cellular accumulation of these drugs. MRP has also been demonstrated to be a prmary active transporter of several structurally diverse conjugated organic anions. Known high-affinity

Summary Resistance to multiple antitumour drugs, mostly antibiotics or alkaloids, has been associated with a cellular plasma membrane P-glycoprotein (Pgp), causing energy-dependent transport of drugs out of cells. However, in many common chemotherapy resistant human cancers there is no overexpression of Pgp, which could explain drug resistance. In order to characterise early steps in multidrug resistance we have derived a series of P-glycoprotein-positive (Pgp/+) and P-glycoprotein-negative (Pgp/-) multidrug resistant cell lines, from a human non-small cell lung cancer cell line, SW-1573, by stepwise selection with increasing concentrations of doxorubicin. These cells were exposed to doxorubicin and its fluorescence in nucleus (N) and cytoplasm (C) was quantified with laserscan microscopy and image analysis. The fluorescence N/C ratio in parent cells was 3.8 and decreased both in Pgp/+ and Pgp/-cells with increasing selection pressure to