Inhibition of membrane transport of 5-fluoro[6-3H]deoxyuridine into L5178Y mouse leukemia cells.

The influx of 1.0 muM 5-fluoro[6-3H]deoxyuridine (5F[6-3H]dUrd) into L5178Y mouse leukemia cells followed a linear function with time from 2 to 10 min. Ammonium 5-bromodeoxyuridine 5'-methylphosphonate (BrdUrd-OPO2Me) inhibited the membrane transport of 5F[6-3H]dUrd into L5178Y cells. Influx of 5F[6-3H]dUrd into inhibited cells was observed from zero to 3 min; after 3 min the net rate of 5F[6-3H]dUrd uptake into the cells treated with 18 muM BrdUrd-OPO2Me was almost zero. The cellular uptake of 2'-deoxy[6-3H]uridine or 5-bromo[6-3H]deoxyuridine was inhibited by BrdUrd-OPO2Me. The L5178Y cells were grown for 96 hr in a medium that contained tritium-labeled BruDur-OPO2Me. An analysis of the labeled products in the growth medium showed that the ester linkage is not cleaved to separate the [3H]methylphosphonate group and the nucleoside moiety of BrdUrd-OPO2[3H]Me. The activity of thymidine kinase in a cell-free preparation from L5178Y cells was demonstrated. Although 37 muM 5-bromo-2'deoxyuridine produced an inhibition of approximately 45% in kinase activity, BrdUrd-OPO2Me had no effect on enzyme activity. The results indicate that BrdUrd-OPO2Me is an inhibitor of the cell membrane transport of the 5-fluoro and 5-bromo derivatives of 2'-deoxy[6-3H]uridine.

Reversal agent inhibition of the multidrug resistance pump in human leukemic lymphoblasts.

Multidrug resistant cancer cells of the MDR-1 phenotype utilize an ATP-dependent pump to excrete toxic drugs. Rhodamine 123 (R123) is a fluorescent substrate of the MDR pump. An assay for the ATP-dependent initial efflux of R123 from CEM/VLB100 human leukemic lymphoblasts has been developed. The MDR-1 cells were treated with a reversal agent and preloaded with 40.0 nM R123 in buffer at 30 degrees C that contained sodium azide and 2-deoxyglucose. The cells were rinsed with cold buffer and resuspended in L-glutamine/glucose solution at 23 degrees C. The cell suspension was passed through a filter and R123 in the filtrate was detected at 2-s intervals by fluorescence. Efflux of R123 was inhibited by the reversal agents amiodarone, cyclosporin A, Ro11-2933 (DMDP), quinidine, and the optical isomers of propranolol. The MDR pump is stereospecific for the (R)-diastereomer quinidine; however, the (S)-diastereomer quinine is a relatively weak inhibitor of the pump. Cyclosporin A was the most potent inhibitor tested against the efflux of R123 by the MDR pump.

Inhibition of the multidrug resistance efflux pump.

An ATP-dependent efflux pump is found in the plasma membrane of certain multidrug resistant (MDR) cancer cells. Drug resistance is due to decreased intracellular drug levels that have been reduced to subcytotoxic concentrations. Inhibition of the MDR efflux pump with a reversal agent may 'trap' the cytotoxic drug inside the cell; thus, cellular drug resistance is reversed. Although many different lipophilic substances exhibit reversal activity, inhibition of the pump is stereospecific with respect to the chiral agent cinchonine. In this article, several methods for the estimation of reversal potency are reviewed. Furthermore, information on the transport characteristics of reversal agents is presented. The rate equations for ATP-dependent drug efflux, competitive inhibition of the MDR pump, and noncompetitive inhibition of the pump are derived. A method is presented that discriminates between competitive or noncompetitive inhibition of the pump. These studies show the potential contribution of fundamental inhibition studies to the design of clinical reversal protocols.

Cellular drug efflux and reversal therapy of cancer.

A prevalent form of multidrug resistance (MDR) in cancer cells is caused by an ATP-dependent drug efflux pump; this pump catalyzes the rapid exit of cytotoxic chemotherapy drugs from the cells. The Michaelis equation can be used to describe drug efflux through the MDR pump at a low drug substrate concentration [S]. The inhibition mechanism of an MDR reversal agent can be characterized when two different values of [S] are used to determine two values for the half-inhibition of efflux through the pump (I50). The reaction is noncompetitive when the two values of I50 are identical; the reaction is competitive when an increase in [S] produces a significant increase in the value of I50. The I50 has been determined for several different reversal agents with the substrate rhodamine 123. The inhibition potency observed is: cyclosporin A > DMDP > amiodarone > verapamil > quinidine > quinine > propranolol. Chemotherapy drugs that are potent inhibitors of the MDR pump could be used for the treatment of MDR neoplasia.

PSC833, cyclosporin A, and dexniguldipine effects on cellular calcein retention and inhibition of the multidrug resistance pump in human leukemic lymphoblasts.

A convenient functional assay of the multidrug resistance (MDR) pump is useful for the diagnosis of MDR-1 cancers and the quantitative determination of the potency of inhibitors of the pump. Calcein-AM, a substrate of the MDR pump, was used to determine the concentration of SDZ PSC833 needed to completely inhibit the pump in CEM/VLB100 drug-resistant cells. The initial rates (in percent) for calcein retention by these MDR-1 cells were used to calculate values for the percent initial efflux of calcein-AM through the MDR pump in the presence of the inhibitors PSC833, cyclosporinA, and dexniguldipine. The percent efflux values at 250 and 60 nM calcein-AM were used to calculate the required concentration of each inhibitor to produce half-inhibition (I50) of initial efflux through the pump. These results are consistent with a noncompetitive inhibition of the MDR pump by each of the three inhibitors.

Perfluorodecanoic acid inactivation of a channel for 2-aminopurine in the L5178Y cell membrane and recovery of the channel.

Treatment of L5178Y mouse lymphoma cells with perfluoro-n-decanoic acid (PFDA) in growth medium for 24 hr at 30 degrees C produces a dose-dependent inactivation of a channel in the cell membrane. Activity of the channel was estimated from the initial rate of efflux of a fluorescent purine, 2-aminopurine (AP). The L5178Y cells were preloaded with 100 microM AP and excess AP was removed. The preloaded cells were put in a flow system, and AP efflux was estimated continuously at 21 degrees C from the fluorescence emission of AP at 370 nm. The AP channel was markedly inactivated by a treatment with 150 micrograms/ml PFDA for 24 hr at 30 degrees C. There was no significant recovery of AP flux after 3 days at 30 degrees C in fresh growth medium; however, recovery was significant after 6 days. Recovery of activity of the AP channel occurs in 1 day at 37 degrees C. The initial rate of AP efflux for control cells increases with AP concentration; the reaction is not saturated at 1000 microM AP. The efflux of AP was inhibited by the presence of uric acid in the external buffer. An apparent inhibition constant value of 355 microM was determined for urate inhibition of AP efflux. These observations suggest the presence of a urate-sensitive channel for AP in the membrane of L5178Y cells. The channel was inactivated by PFDA under conditions that had no significant effect on cell viability.

Epoxide metabolite of quinine and inhibition of the multidrug resistance pump in human leukemic lymphoblasts.

Multidrug resistance (MDR) in neoplastic cells is usually due to decreased cellular retention of drugs such as vincristine or doxorubicin. An ATP-dependent drug efflux pump has been detected in MDR-1-phenotypic cells; inhibition of the MDR pump is probably the primary mechanism for reversal of MDR. Although quinine (SQ1) and quinidine are reversal agents and inhibitors of the MDR pump, the results from in vivo experiments and in vitro experiments with these diastereomers are contradictory. These observations suggest that an oxidized metabolite of SQ1 is a more potent inhibitor of the MDR pump than is the parent compound. The chemical synthesis of the epoxides of SQ1 and quinidine is reported. The epoxy compounds have been tested as inhibitors of the ATP-dependent MDR pump in human CEM/VLB100 cells. The procedure is based on preloading the cells with an inhibitor and a low concentration of a substrate, rhodamine 123 (R123). After several cold rinses, the cell suspension is passed through a filtration-flow apparatus and the R123 in the filtrate (determined by fluorescence measurements) reveals the initial efflux of R123 through the MDR pump. When tested as an inhibitor of the MDR pump, quinine-10,11-epoxide is approximately 8-fold more potent than SQ1.