ABSTRACT
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.
Subject(s)
Antineoplastic Agents/metabolism , Drug Resistance, Multiple , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Humans , Hydrogen-Ion Concentration , Intracellular Fluid , MiceABSTRACT
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.
Subject(s)
Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/metabolism , Vinblastine/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Ammonium Chloride/pharmacology , Animals , Base Sequence , Biological Transport, Active/drug effects , Cell Line , Cell Polarity , Concanavalin A/pharmacology , DNA Primers/genetics , Drug Resistance, Multiple/genetics , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Genes, MDR , Hydrogen-Ion Concentration , Intracellular Fluid/metabolism , Kidney Tubules, Proximal/cytology , Kinetics , Lysosomes/drug effects , MiceABSTRACT
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.
