Functional detection of MDR1/P170 and MRP/P190-mediated multidrug resistance in tumour cells by flow cytometry

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

Theoretical and practical considerations for the measurement of P-glycoprotein function in acute myeloid leukemia

“…We have quantified the N/C ratio by fluorescence 833). 1,31 In the latter case, the cells cannot be loaded with dye in the presence of the modulator if subsequently the laser scan microscopy in MDR cell lines and shown that it correlates with resistance even in very low resistant tumor cell dye efflux is to be studied. 1 (6) Finally, one has to be aware that the protein concentration lines.…”

“…10 For the combination A double labelling procedure with R123 and FITC labels is impossible and the combination with PE label still requires R123/PSC833, it is short-term accumulation that provides Pgp specificity. 1,27 Since some MRP-overexpressing cell extensive compensation. 25 For that reason, the procedure of Leith et al 17 who used DiOC 2 (3) combined with PE-labelled lines respond to PSC833, its Pgp specificity is not absolute.…”

“…[1][2][3][4][5][6][7][8][9][10] Additional specific details are given when considered Introduction useful, in the Results section.…”

“…[1][2][3] About 0.3 × 10 6 KB cells or 0.6 × 10 6 leukemic uptake, efflux, steady-state accumulation or intracellular discells per ml were incubated with regular shaking. After incutribution.…”

“…After incutribution. Our experience is built upon extensive comparison bation the cells were washed twice with ice-cold phosphateof methods to determine Pgp MDR in tumor cell lines, also buffered saline (PBS) and cytocentrifuged for 5 min at 350 with low levels of Pgp/MDR1 expression, [1][2][3][4][5][6] experiments and r.p.m. on slides precoated with 0.1% bovine serum albumin model calculations intended to understand the kinetics of Pgpsolution.…”