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H Dekker

Publications and source records attributed to H Dekker.

35 records · Page 2Linked to original sources
PubMed1992

Kinetics of daunorubicin transport by P-glycoprotein of intact cancer cells.

Drug permeation across the plasma membrane of multidrug-resistant cells depends on the kinetics of the P-glycoprotein-mediated pump activity as well as on the passive permeation of the drug. We here demonstrate a method to characterize kinetically the pump in intact cells. To this purpose, we examined the membrane-transport properties of daunorubicin in various sensitive cancer cell lines and in their multidrug resistant (MDR) counterparts. First, we determined the passive permeability coefficient for daunorubicin. Then, using a flow-through system, the drug flux into the cell was measured after inhibition of the P-glycoprotein-mediated efflux pump. Combining the two results allowed us to calculate the intracellular free concentration of the drug. In the steady-state, the pump rate must equal the net rate of passive diffusion of the drug and, therefore, the same experiments gave us the pumping rate of daunorubicin. These experiments were then repeated at various extracellular drug concentrations. By plotting the pumping rate versus the intracellular drug concentration, we then characterized the P-glycoprotein kinetically. Four independent methods were used to measure the passive permeability coefficient for the cell line A2780. Similar values were obtained. Maximal pump rates (Vmax) showed a good correlation with the amount of P-glycoprotein in the cell lines used. We obtained saturation curves for the variation of the pump rates with the intracellular daunorubicin concentrations. These curves were typical for positive cooperativity, which provides evidence that at least two binding sites for daunorubicin are present on the active transport system of daunorubicin. The apparent Km values for P-glycoprotein-mediated transport, the intracellular free cytosolic daunorubicin concentrations at half-maximal velocity for the cell lines used, were approximately 1.5 microM. Except for the cell lines with the highest amount of P-glycoprotein, the passive efflux rate of daunorubicin proved to be a substantial part of the total daunorubicin efflux rate for the cell lines used. In cell lines with relatively low levels of P-glycoprotein, passive daunorubicin efflux was even the main route of daunorubicin transport from the cells, determining the intracellular steady-state concentrations of daunorubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Probing daunorubicin accumulation defects in non-P-glycoprotein expressing multidrug-resistant cell lines using digitonin.

Multidrug resistance (MDR) in tumor cells is frequently associated with reduced cellular cytostatic drug accumulation, caused by the drug efflux protein, P-glycoprotein (Pgp). The action of Pgp in tumor cells can be detected by measuring the increase of daunorubicin accumulation upon blocking Pgp with drugs such as verapamil. A number of MDR cell lines have been described, characterized by decreased drug accumulation without Pgp being present. For such non-Pgp MDR cells no gene probes or functional assays are available to study this phenotype in clinical tumor specimens. We have worked out a method which enables the detection of drug-transport-related decreases in cellular daunorubicin accumulations without the need for the use of specific Pgp blockers. The cells used were SW-1573-, GLC4- and HT1080-sensitive cell lines, which accumulated (corrected for DNA content) 272%, 1,288% and 203% more daunorubicin than the non-Pgp MDR sublines SW-1573/2R120, GLC4/ADR and HT1080/DR4. When the plasma membranes of these MDR lines were permeabilized with 20 microM digitonin an increase to 282%, 1,260% and 239% of 14C-daunorubicin control accumulation was measured (at pH = 7.35). The intracellular pH measured with BCECF was the same in parent and corresponding MDR cells, excluding the role of pH differences in the measured effects. This method provides a tool allowing the detection of cellular mechanisms (including Pgp) which are related to active outward transport of daunorubicin.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

P-glycoprotein drug efflux pump involved in the mechanisms of intrinsic drug resistance in various colon cancer cell lines. Evidence for a saturation of active daunorubicin transport.

We studied the resistance of colon tumors to anticancer agents in vitro. Using daunorubicin (DN), a number of cellular parameters which normally indicate acquired or multidrug resistance (MDR), were compared for several human wild-type colon cell lines, i.e. HT29, SW1116 and COLO 320, and the murine colon cell line C-26. The sensitive/MDR human ovarian cancer cell line couple A2780/2780AD was used as a reference. The amount of P-glycoprotein (P-gp) was in the order HT29, A2780 less than or equal to SW1116 less than C26 less than or equal to COLO 320 less than 2780AD. The MDR modifiers verapamil, Cremophor EL, cyclosporin A and Ro 11-2933/001 had significant effects on DN cytotoxicity, total DN accumulation and efflux, only if P-gp was present. A flow-through system was used to study the mechanism of DN transport. For the first time, evidence for saturation of an active transport of DN from the cells is reported. We discussed the possible presence of cooperative activity between at least two binding sites on the protein responsible for DN efflux, likely to be P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Measurement of in vitro cellular pharmacokinetics of 5-fluorouracil in human and rat cancer cell lines and rat hepatocytes using a flow-through system.

A flow-through system was used to study the cellular pharmacokinetics of 5-fluorouracil (5-FU) in four human cell lines (squamous-cell carcinoma HEp-2, colon carcinoma WiDr, hepatoma Hep G2, and breast carcinoma MCF-7) as well as in the rat hepatoma H35 cell line and in freshly isolated rat hepatocytes. The system made it possible to restrict the decrease in the concentration of 5-FU in the medium, to keep the volume in which the metabolites accumulated relatively small, and to study the dynamics of a response during and after a change in the composition of the eluent. Clearance of 5-FU from the eluent was achieved predominantly (greater than 95%) by its catabolism to dihydrofluorouracil in the tumor cell lines and to 2-fluoro-beta-alanine in the hepatocytes. Not only rat hepatocytes but also HEp-2 cells showed relatively high clearance values. A concentration-dependent 5-FU elimination was observed, indicating saturation of 5-FU elimination according to Michaelis-Menten kinetics (Km 14-22 microM). The maximal velocity (Vmax) values ranged from 0.025 to 0.13 nmol 5-FU/10(6) cells per minute. For HEp-2 cells, high-concentration pulse injections of 5-FU, thymine, uridine, or uracil immediately led to a reduction in 5-FU conversion, followed by recovery within 5 min. The flow-through system proved to be adequate for the study of the non-linear pharmacokinetics of 5-FU in different intact cells and for the comparison of various manipulations of these pharmacokinetics.

Animals↗
PubMed1990

Evidence for daunomycin efflux from multidrug-resistant 2780AD human ovarian carcinoma cells against a concentration gradient.

Using a flow-through system human multidrug-resistant 2780AD ovarian carcinoma cells were exposed to flowing culture medium containing the anticancer agent daunomycin (5 microM). A pulse of medium containing verapamil caused increased cellular daunomycin accumulation, resulting in a dip in the fluorescence signal from daunomycin in the effluent. After passage of this pulse we observed an efflux of more than 90% of this extra accumulated daunomycin within 10 min. This daunomycin efflux against a concentration gradient provides evidence for drug efflux from these cells being an active process. After the addition of methylamine to the medium to increase intravesicular pH, the dip in the fluorescence signal was not decreased, indicating that vesicular transport was not an important component of this efflux.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1988

Rate enhancement of the internal electron transfer in cytochrome c oxidase by the formation of a peroxide complex; its implication on the reaction mechanism of cytochrome c oxidase.

The oxidation of reduced cytochrome c oxidase by hydrogen peroxide was investigated with stopped-flow methods. It was reported by us previously (A.C.F. Gorren, H. Dekker and R. Wever (1986) Biochim. Biophys. Acta 852, 81-92) that at low H2O2 concentrations cytochrome a is oxidised simultaneously with cytochrome a3, but that at higher H2O2 concentrations the oxidation of cytochrome a is slower than that of cytochrome a3. We now report that for high peroxide concentrations (10-45 mM) the oxidation rate of cytochrome a increased linearly with the concentration of H2O2 (k = 700 M-1.S-1). Upon extrapolation to zero H2O2 concentration an intercept with a value of 16 s-1 (at 20 degrees C and pH 7.4) was found. A reaction sequence is described to explain these results; according to this model the rate constant (16 S-1) at zero H2O2 concentration represents the true value of the rate of electron transfer from cytochrome a to cytochrome a3 when the a3-CuB site is oxidised and unligated. However, when a complex of hydrogen peroxide with oxidised cytochrome a3 is formed, this rate is strongly enhanced. The slope (700 M-1.S-1) would then represent the rate of cytochrome a3(3+)-H2O2 complex formation. From experiments in which the pH was varied, we conclude that the reaction of H2O2 with cytochrome a3(2+) is independent of pH, whereas the electron-transfer rate from cytochrome a to cytochrome a3 gradually decreases with increasing pH. From the temperature dependence we could calculate values of 23 kJ.mol-1 and 45 kJ.mol-1 for the activation energies of the oxidations by H2O2 of cytochrome a3(2+) and cytochrome a2+, respectively. The similarity of the values that were obtained for cytochrome a oxidation both with H2O2 and with O2 as the electron acceptor suggests that the reactions share the same mechanism. In 2H2O the reactions studied decreased in rate. For the reaction of 2H2O2 with reduced cytochrome a3 in 2H2O, a small effect was found (15% decrease in rate constant). However, the internal electron-transfer rate from cytochrome a to cytochrome a3 decreased by 50%, Our results suggest that the internal electron transfer is associated with proton translocation.

Animals↗
PubMed1986

Kinetic investigations of the reactions of cytochrome c oxidase with hydrogen peroxide.

The reaction of H2O2 with reduced cytochrome c oxidase was investigated with rapid-scan/stopped-flow techniques. The results show that the oxidation rate of cytochrome a3 was dependent upon the peroxide concentration (k = 2 X 10(4) M-1 X s-1). Cytochrome a and CuA were oxidised with a maximal rate of approx. 20 s-1, indicating that the rate of internal electron transfer was much slower with H2O2 as the electron acceptor than with O2 (k greater than or equal to 700 s-1). Although other explanations are possible, this result strongly suggests that in the catalytic cycle with oxygen as a substrate the internal electron-transfer rate is enhanced by the formation of a peroxo-intermediate at the cytochrome a3-CuB site. It is shown that H2O2 took up two electrons per molecule. The reaction of H2O2 with oxidised cytochrome c oxidase was also studied. It is shown that pulsed oxidase readily reacted with H2O2 (k approximately 700 M-1 X s-1). Peroxide binding is followed by an H2O2-independent conformational change (k = 0.9 s-1). Resting oxidase partially bound H2O2 with a rate similar to that of pulsed oxidase; after H2O2 binding the resting enzyme was converted into the pulsed conformation in a peroxide-independent step (k = 0.2 s-1). Within 5 min, 55% of the resting enzyme reacted in a slower process. We conclude from the results that oxygenated cytochrome c oxidase probably is an enzyme-peroxide complex.

Animals↗
PubMed1985

The oxidation of cytochrome c oxidase by hydrogen peroxide.

The reaction of H2O2 with mixed-valence and fully reduced cytochrome c oxidase was investigated by photolysis of fully reduced and mixed-valence carboxy-cytochrome c oxidase in the presence of H2O2 under anaerobic conditions. The results showed that H2O2 reacted rapidly (k = (2.5-3.1) X 10(4) M-1 X s-1) with both enzyme species. With the mixed-valence enzyme, the fully oxidised enzyme was reformed. On the time-scale of our experiments, no spectroscopically detectable intermediate was observed. This demonstrates that mixed-valence cytochrome c oxidase is able to use H2O2 as a two-electron acceptor, suggesting that cytochrome c oxidase may under suitable conditions act as a peroxidase. Upon reaction of H2O2 with the fully reduced enzyme, cytochrome a was oxidised before cytochrome a3. From this observation it was possible to estimate that the rate of electron transfer from cytochrome a to a3 is about 0.5-5 s-1.

Animals↗
PubMed1971

Asthma and mites.

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