It has been suggested that the inappropriate sequestration of weak-base chemotherapeutic drugs in acidic vesicles by multidrug-resistance (MDR) cells contributes to the mechanisms of drug resistance. The function of the acidic lysosomes can be altered in MDR cells, and so we investigated the effects of lysosomotropic agents on the secretion of lysosomal enzymes and on the intracellular distribution of the weak-base anthracycline daunomycin in drug-resistant renal proximal tubule PKSV-PR(col50) cells and their drug-sensitive PKSV-PR cell counterparts. Imaging studies using pH-dependent lysosomotropic dyes revealed that drug-sensitive and drug-resistant cells exhibited a similar acidic lysosomal pH (around 5.6-5.7), but that PKSV-PR(col50) cells contained more acidic lysosomes and secreted more of the lysosomal enzymes N -acetyl-beta-hexosaminidase and beta-glucuronidase than their parent PKSV-PR cells. Concanamycin A (CCM A), a potent inhibitor of the vacuolar H(+)-ATPase, but not the P-glycoprotein modulator verapamil, stimulated the secretion of N -acetyl-beta-hexosaminidase in both drug-sensitive and drug-resistant cells. Fluorescent studies and Percoll density gradient fractionation studies revealed that daunomycin accumulated predominantly in the lysosomes of PKSV-PR(col50) cells, whereas in PKSV-PR cells the drug was distributed evenly throughout the nucleo-cytoplasmic compartments. CCM A did not impair the cellular efflux of daunomycin, but induced the rapid nucleo-cytoplasmic redistribution of the drug in PKSV-PR(col50) cells. In addition, CCM A and bafilomycin A1 almost completely restored the sensitivity of these drug-resistant cells to daunomycin, doxorubicin and epirubicin. These findings indicate that lysosomotropic agents that impair the acidic-pH-dependent accumulation of weak-base chemotherapeutic drugs may reverse anthracycline resistance in MDR cells with an expanded acidic lysosomal compartment.

Dihydrotestosterone (DHT) binding studies and the effects of DHT on the expression of beta-glucuronidase (Gus) and kidney androgen-regulated protein (KAP) genes and cell growth were investigated in immortalized early PKSV-PCT and late PKSV-PR proximal tubule cells, derived from transgenic mice carrying the L-pyruvate kinase/SV40 hybrid gene. [3H]DHT binding studies indicated that both cell lines have conserved substantial amounts of androgen receptors. The levels of KAP and Gus transcripts in PKSV-PCT cells, and those of KAP transcripts in PKSV-PR cells, decreased when cells were shifted from a serum-supplemented to a steroid-free medium. The addition of 30 nM DHT to the steroid-free medium resulted in a slight increase in Gus and in a more marked increase in KAP transcripts in both cell lines. Dihydrotestosterone also affected the growth of PKSV-PCT and PKSV-PR cells, since this hormone added to the steroid-free medium stimulated the incorporation of [3H]thymidine in a dose-dependent manner and induced the formation of domes, which represent indicators of ionic transport processes. Thus, because these early and late mouse proximal tubule cells have conserved androgen receptors, they represent attractive cell systems to analyze the action of androgens on specific functions of the mouse proximal tubule.

Vinblastine (VBL) transport and efflux were studied in mouse proximal tubule PKSV-PR cells and in their multidrug-resistant derivatives PKSV-PRcol50 cells. The PKSV-PRcol50 cells produced more mdr1b transcripts and had higher resistance to various drugs. PKSV-PRcol50 cells had a predominantly basal-to-apical flux of [3H]VBL, 2.7 times larger than that in PKSV-PR cells. This flux was partially inhibited by verapamil (VRP) (10 microM) and cyclosporin A (CsA) (200 nM). [3H]VBL efflux was also greater in PKSV-PRcol50 than in PKSV-PR cells. Treatment with NH4Cl (30 mM), a lysosomotropic weak base, and concanamycin A (CCM A) (20 nM), an inhibitor of the vacuolar H+/ATPase, further increased [3H]VBL efflux from PKSV-PRcol50 cells. The cytoplasmic pH (pHcyt) of these drug-resistant cells transiently increased in the presence of NH4Cl deltapHcyt: +0.4). CCM A caused a moderate, delayed increase in pHcyt (deltapHcyt: +0.1) and made the acidic intralysosomal compartment more alkaline (deltapHlys: +1.3). VRP and CsA prevented the NH4Cl- and CCM A-induced [3H]VBL efflux from PKSV-PRcol50 cells. However, VRP (10 microM) did not significantly affect pHcyt of PKSV-PRcol50 cells, the NH4Cl-and CCM A-induced pHcyt responses, and the effect of CCMA on pHlys. Thus, lysosomotropic agents may affect the kinetics of [3H]VBL efflux. Our results also suggest that the inhibitory action of VRP on VBL efflux was not directly mediated by a pH-dependent process in these drug-resistant renal proximal tubule cells.

This review considers the mechanisms associated with the pleiotropic resistance of cancer cells to chemotherapeutic drugs, and more particularly those related to intracellular pH (pHi). The multidrug resistance (MDR) phenomenon responsible for the decreased accumulation and increased efflux of cytotoxic drugs is generally associated with excess levels of P-glycoproteins (Pgps) encoded by MDR genes and/or the multidrug resistance-associated protein (MRP). MDR cell lines, derived from normal or tumor cells, frequently exhibit abnormally elevated pHi and changes in the production of various proteins. Recent studies have suggested that, in addition to the impact of the ATP-dependent membrane transporters Pgp and MRP on drug transport, other mechanisms linked to pHi changes in MDR cells may play an important role in drug resistance. We have shown that alkalinization of the acidic compartments (endosomes and lysosomes) by lysosomotropic agents could stimulate the efflux of vinblastine from drug-resistant mouse renal proximal tubule cells. The fact that weak base chemotherapeutic drugs can be sequestered within the acidic organelles of MDR cells sheds new light on the cellular mechanisms of drug resistance.