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Biomedical subjects

H Dekker

Publications and source records attributed to H Dekker.

At least 19 recordsLinked to original sources
PubMed2000

A mathematical model of drug transport in human breast cancer.

A mathematical model of drug transport in tissue has been developed on the basis of a clinical study of patients with breast cancer, treated with the drug doxorubicin and of drug transport experiments using cultured human breast cancer cells. The clinical study revealed doxorubicin gradients in tumor islets of densely packed cancer cells. The mathematical model allows simultaneous drug transport through the cellular network (transcellular pathway), through the intercellular interstitium (paracellular pathway), and across the boundary between the two networks. The effective diffusion coefficient of the interstitial network is found to be much higher than that of the cellular network, in spite of the fact that the interstitium thickness is only 20-40 nm. The model simulations can be made to fit the results of the clinical study. A long-continued simulation (40 days) of drug transport into a spherical islet with a radius of 150 microm, after a bolus injection of doxorubicin, reveals that the maximum average drug concentration at the islet centre is only reached after 224 h, while it decreases by a factor 15 from the boundary to the centre of the islet. The area under the curve in a plot of the average drug concentration versus time only decreases by 10% from the boundary to the centre of the islet.

Antineoplastic Agents↗
PubMed2000

Evaluating limited specificity of drug pumps reduced relative resistance in human MDR phenotypes.

In the parallel paper, we developed a property to characterize drug efflux pumps, i.e. the reduced relative resistance (RRR). Using this RRR, we here investigate whether the observed diversity in human multidrug resistance (MDR) phenotypes might be due to variable levels of P-glycoprotein encoded by MDR1. We analyzed resistance phenotypes of various human cell lines in which either one, or both, classical human multidrug resistance genes, MDR1 and MDR3, are overexpressed. In addition, RRR values were calculated for MDR phenotypes presented in the literature. The results suggest that more than a single mechanism is required to account for the observed phenotypic diversity of classical multidrug resistance. This diversity is only partly due to differences in plasma membrane permeabilities between cell line families. It is discussed whether the alternative MDR phenotypes might be MDR1 phenotypes modified by other factors that do not themselves cause MDR. The method we here apply may also be useful for other nonspecific enzymes or pumps.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Doxorubicin gradients in human breast cancer.

Ten patients with locally advanced breast cancer were given doxorubicin i.v., and an incision biopsy was subsequently taken. Doxorubicin autofluorescence was examined using computerized laser scanning microscopy, and microvessels were immunostained in the same sections. Overlays of both pictures revealed doxorubicin gradients in tumor islets with high concentrations in the periphery and low concentrations in the center of the tumor islets. Gradients were most pronounced shortly after the injection, but they could still be detected 24 h later. No gradients were observed in connective tissue. This study demonstrates a serious risk of the drug not reaching all of the cancer cells in those cases in which the cancer cells are densely packed in islets. The efficacy of drug treatment will thus depend on the histology of the tumor tissue.

Antineoplastic Agents↗
PubMed1997

Further evidence for the presence of mitochondrially encoded subunits in cytochrome c oxidase of the trypanosomatid Crithidia fasciculata.

Mitochondrial mRNAs in trypanosomatids are edited by uridylate insertion and deletion. The respiratory chain complexes cytochrome c reductase, cytochrome c oxidase and F0F1-ATPase of the insect trypanosomatid Crithidia fasciculata have been isolated and analysed by peptide microsequencing, but so far, proteins encoded by edited (and unedited) mitochondrial mRNAs have not been found. In this paper, we provide evidence that the mitochondrial mRNAs encoding the three large subunits of cytochrome c oxidase are indeed translated. First, purified holo cytochrome c oxidase turned out to be cysteine-rich, in agreement with the high cysteine codon-content of the sequence of mitochondrial cox subunit mRNAs. Second, in mass spectrometry measurements of cytochrome c oxidase, a protein was detected with the predicted molecular weight of cytochrome c oxidase subunit 2. Finally, an antibody generated against a fusion protein produced in Escherichia coli from constructs containing a segment of cytochrome c oxidase subunit 2 cDNA, specifically recognised protein bands present in cytochrome c oxidase following SDS PAGE. However, these proteins were present in the high molecular weight region of the gel, suggesting that cytochrome c oxidase subunit 2 aggregates in the presence of SDS.

Animals↗
PubMed1996

The sequence of a small subunit of cytochrome c oxidase from Crithidia fasciculata which is homologous to mammalian subunit IV.

The sequence of subunit 8 of cytochrome c oxidase from Crithidia fasciculata was determined by sequencing cDNA and N-terminus of the mature protein (Mr = 15.7 kDA). The (inferred) protein is homologous to mammalian cox IV and the corresponding cox subunits from yeast, Neurospora crassa and Dictyostelium discoideum, which is reflected in a very similar hydropathy profile. Elements that are conserved in the C. fasciculata sequence include (i) an N-terminal (D/E)-(K/R)-X-K-(X2)-W-(X2)-(I/L) motif, (ii) a putative membrane-spanning region in the middle portion of the protein, and (iii) a C-terminal W-(X13)-(N/D)-P motif. The C. fasciculata protein is synthesized with a cleavable presequence.

Amino Acid Sequence↗
PubMed1996

Saturable P-glycoprotein kinetics assayed by fluorescence studies of drug efflux from suspended human KB8-5 cells.

This article describes a new and rapid method to determine the pumping rate of P-glycoprotein (P-gp) in intact cells. Multidrug resistant (MDR) human epidermoid carcinoma KB8-5 cells (containing P-gp) were loaded with daunorubicin (DNR) in the absence or in the presence of verapamil, sufficient to inhibit DNR pumping by P-gp. In either case, the cells were resuspended in medium devoid of DNR and the subsequent increase of the DNR fluorescence intensity was measured as a function of time. For cells loaded with the same amount of drug, the free cytosolic drug concentration (Ci(t)) was a unique function of the DNR medium concentration (Co(t)). The cellular drug content in the presence of verapamil decreased nonlinearly with decreasing extracellular drug concentration, indicating that the intracellular drug apparent distribution volume increased with decreasing cellular drug content. At each fluorescence intensity, we calculated the P-gp mediated (verapamil-inhibitable) DNR transport rate from the rate of increase of the DNR fluorescence intensity in the absence of verapamil minus the rate of increase of the DNR fluorescence intensity in the presence of verapamil. When plotted against the intracellular free drug concentration (as calculated from the total cellular drug content and a separately determined relation between the total cellular drug content and the intracellular free drug concentration: the apparent distribution volume), this P-gp mediated DNR transport rate showed saturation of P-gp at higher DNR concentrations. The results imply that P-gp mediated DNR transport is saturable (the value of Km is in the order of 1 microM).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Purification and characterization of cytochrome c oxidase from the insect trypanosomatid Crithidia fasciculata.

Cytochrome c oxidase was purified from the mitochondrial lysate of the insect trypanosomatid Crithidia fasciculata with the aid of a methyl hydrophobic interaction column in a rapid one-step procedure. The purified complex displayed all characteristics expected from a eukaryotic cytochrome c oxidase: the presence of CuA in electron paramagnetic resonance analysis, a characteristic 605 nm peak in reduced-minus-oxidized optical spectroscopy, and the capacity to efficiently oxidize homologous, but not heterologous, cytochrome c. Two-dimensional PAGE showed that C. fasciculata cytochrome c oxidase consists of at least 10 different subunits. N-terminal sequences were obtained from the six smallest subunits of the complex, one of them showing significant similarity to Neurospora crassa cytochrome c oxidase subunit V. The N-terminus of each of the four largest subunits was found to be blocked.

Amino Acid Sequence↗
PubMed1995

The P-glycoprotein-mediated relative decrease in cytosolic free drug concentration is similar for several anthracyclines with varying lipophilicity.

We have used a new methodology to measure the activity of P-glycoprotein (P-gp) in multidrug-resistant (MDR) tumor cells. This activity leads to a lower cytosolic concentration and a lower cytotoxicity of the classical anthracyclines, daunorubicin (DNR), and doxorubicin (DOX). It has been reported that the anthracycline idarubicin (IDA), which is more lipophilic, has a higher clinical efficacy in acute myeloid leukemias (AML) than DNR and DOX. In our study, the aim was to determine for a series of anthracyclines how variations in the passive drug influx rate as well as the P-gp-mediated drug pumping rate affect their cytosolic free drug concentrations and how these parameters are related to drug cytotoxicity. We selected six anthracyclines: DOX, DNR, epidoxorubicin (EPI), IDA, cyano-morpholino-doxorubicin (CMD), and carminomycin (CAR), ordered according to their increasing octanol/PBS buffer concentration ratios, respectively. To measure the passive permeation coefficient, the P-gp-mediated drug pumping rate, and the cytosolic free drug concentration, we used a flow-through system in which cells were exposed to a flowing medium containing drugs. We used the MDR P-gp-containing cell line KB8-5. It was shown that the passive drug permeation coefficient as well as the drug pumping rate of P-gp increased with increasing lipophilicity in this series of anthracyclines. The cytosolic free drug concentration was lowered by P-gp to a similar extent in KB8-5 cells for all drugs tested (40-50% of the extracellular drug concentration). CMD, IDA, and CAR had lower IC50 values and lower resistance factors in comparison to DOX, DNR, and EPI. Verapamil reversed the resistance for all anthracyclines tested. In conclusion, for several anthracyclines the activity of P-gp leads to a similar relative decrease in the cytosolic free drug concentration; consequently, the reported lower resistance factor of IDA compared to that of DNR is not due to the inability of P-gp to export IDA from cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein.

Multidrug-resistance-associated protein (MRP) is a plasma membrane glycoprotein that can confer multidrug resistance (MDR) by lowering intracellular drug concentration. Here we demonstrate that depletion of intracellular glutathione by DL-buthionine (S,R)-sulfoximine results in a complete reversal of resistance to doxorubicin, daunorubicin, vincristine, and VP-16 in lung carcinoma cells transfected with a MRP cDNA expression vector. Glutathione depletion had less effect on MDR in cells transfected with MDR1 cDNA encoding P-glycoprotein and did not increase the passive uptake of daunorubicin by cells, indicating that the decrease of MRP-mediated MDR was not due to nonspecific membrane damage. Glutathione depletion resulted in a decreased efflux of daunorubicin from MRP-transfected cells, but not from MDR1-transfected cells, suggesting that glutathione is specifically required for the export of drugs from cells by MRP. We also show that MRP increases the export of glutathione from the cell and this increased export is further elevated in the presence of arsenite. Our results support the hypothesis that MRP functions as a glutathione S-conjugate carrier.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Differential in situ hybridization for herpes simplex virus typing in routine skin biopsies.

A herpes simplex virus (HSV) type 2 specific recombinant plasmid probe designated pH2S3 was constructed from non-HSV-1 crossreactive regions of the HSV-2 genome. DNA in situ hybridization on in vitro reconstructed tissue samples of sheep collagen matrix impregnated with herpes virus-infected human cells was used to demonstrate absence of crossreactivity of the pH2S3 probe with HSV-1 and varicella zoster virus under stringent posthybridization washing conditions. It was demonstrated that DNA in situ hybridization with pH2S3 allows specific detection of HSV-2 in routine formalin-fixed, paraffin-embedded patient material.

Animals↗
PubMed1994

Daunorubicin efflux against a concentration gradient in non-P-glycoprotein multidrug-resistant lung-cancer cells.

Multidrug-resistant, human non-small-cell lung carcinoma SW-1573/2R120 (2R120) cells, not containing the drug efflux pump P-glycoprotein (Pgp), have reduced initial daunorubicin (DN) accumulation rates and decreased cellular steady-state drug concentrations. Previously we found indications of the presence of a plasma membrane "vacuum cleaner", pumping DN directly from the membrane, and reported evidence of active DN pumping using digitonin. Further evidence of active DN pumping is now provided via a different methodology and the active drug pump flux is estimated. Cells were exposed to a flowing medium containing the cytotoxic agent DN. After reaching a steady state, in which net DN uptake equals net DN efflux, high concentration pulses of vincristine (VCR) were injected into the flowing medium. A rapid increase in cellular DN content was observed, while only a minimal effect was seen in SW-1573 wild-type cells. After passage of the VCR pulse, the extra accumulated DN was effluxed against a concentration gradient. Upon increasing the VCR concentration, a maximum pump inhibition was reached which was similar to the effect of cellular energy depletion. Similar effects were observed for Pgp-containing SW-1573/2R160 (2R160) cells as well as non-Pgp MDR human small-cell lung carcinoma GLC4/ADR cells. With increasing extracellular DN concentrations, saturation of the VCR-induced DN influx was observed (DN medium concentration 2.5 microM at 1/2 Vmax). At an extracellular DN concentration of 5 microM, higher concentrations of VCR were needed to reach the maximum effect in 2R120 cells than at 0.5 microM DN. This is an indication of competitive interaction between DN and VCR for the putative DN efflux system. In summary, we found indications of inhibition of active DN efflux by VCR and DN efflux against a concentration gradient in non-Pgp MDR 2R120 and GLC4/ADR cells. These features are consistent with the presence of a multidrug transporter, different from Pgp, in the plasma membrane of these cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

The multidrug-resistance-reverser verapamil interferes with cellular P-glycoprotein-mediated pumping of daunorubicin as a non-competing substrate.

We examined P-glycoprotein-mediated verapamil transport, using two drug-sensitive and multi-drug resistant cell-line couples, i.e. A2780, 2780AD and SW-1573, SW-1573/1R500. The interaction of 3H-labeled verapamil with cells was measured using a flow-through system. The verapamil-containing medium was pumped over the cells and monitored on-line for radioactivity. In the P-glycoprotein-expressing cells, verapamil accumulation was increased by vinblastine and some known multidrug resistant (MDR) modifiers. Subsequent removal of these modifiers caused release of verapamil into the medium against a verapamil concentration gradient. In this manner, we obtained evidence that verapamil is actively transported by the MDR-related P-glycoprotein. Using the flow-through system, we also exposed the cells to flowing culture medium containing daunorubicin, and measured the inhibition of daunorubicin efflux by verapamil. We found that, although the active efflux of daunorubicin was maximally blocked by verapamil short-term, longer-term active efflux of daunorubicin resumed. At a daunorubicin concentration in the flowing medium of 5 microM, increasing the verapamil concentration resulted in the same short-term effects, but in a significantly longer period of a maximal inhibition of daunorubicin efflux from the cells. At a daunorubicin concentration of 20 microM, increasing the verapamil concentration affected neither the short-term nor the long-term effects. These and other observations are in agreement with a model in which daunorubicin and verapamil are non-competing substrates for P-glycoprotein. In conclusion, we obtained evidence that verapamil is actively transported by the MDR-related P-glycoprotein and that verapamil and daunorubicin are non-competing substrates for P-glycoprotein. Consequently, the effectiveness of verapamil as an MDR antagonist may be compromised because it is extruded by P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Cortisol is transported by the multidrug resistance gene product P-glycoprotein.

The physiology of the multidrug transporter P-glycoprotein (Pgp) is still poorly understood. We now show evidence that cell lines with a high expression of Pgp display a reduced accumulation of cortisol and an ATP-dependent outward transport of the hormone. Cortisol efflux from Pgp negative cells does not have such an active component. Further we show that the steroid hormones cortisol, testosterone, and progesterone cause an immediate, dose-dependent increase of daunorubicin accumulation in Pgp overexpressing cells. These effects are particularly apparent for the more lipophilic steroids. These results demonstrate that Pgp may function as a transporter for cortisol and suggest a physiological role of the protein in steroid handling by organs such as the adrenal.

ATP Binding Cassette Transporter, Subfamily B, Mem↗