Jan Lankelma

Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export

The multidrug resistance proteins MRP1 and MRP2 are members of the same subfamily of ATP-binding cassette transporters. Besides organic molecules conjugated to negatively charged ligands, these proteins also transport cytotoxic drugs for which no negatively charged conjugates are known to exist. In polarized MDCKII cells, MRP1 routes to the lateral plasma membrane, and MRP2 to the apical plasma membrane. In these cells MRP1 transports daunorubicin, and MRP2 vinblastine; both transporters export reduced glutathione (GSH) into the medium. We demonstrate that glutathione transport in MDCKII-MRP1 cells is inhibited by the inhibitors of organic anion transporters sulfinpyrazone, indomethacin, probenecid and benzbromarone. In MDCKII-MRP2 cells, GSH export is stimulated by low concentrations of sulfinpyrazone or indomethacin, whereas export is inhibited down to control levels at high concentrations. We find that unmodified sulfinpyrazone is a substrate for MRP2, also at concentrations where GSH export is inhibited. We also show that GSH export in MDCKII-MRP2 cells increases in the presence of vinblastine, and that the stochiometry between drug and GSH exported is between two and three. Our data indicate that transport of sulfinpyrazone and vinblastine is associated with GSH export. However, at high sulfinpyrazone concentrations this compound is transported without GSH. Models of MRP action are discussed that could explain these results.

Principles behind the multifarious control of signal transduction. ERK phosphorylation and kinase/phosphatase control.

General and simple principles are identified that govern signal transduction. The effects of kinase and phosphatase inhibition on a MAP kinase pathway are first examined in silico. Quantitative measures for the control of signal amplitude, duration and integral strength are introduced. We then identify and prove new principles, such that total control on signal amplitude and on final signal strength must amount to zero, and total control on signal duration and on integral signal intensity must equal −1. Collectively, kinases control amplitudes more than duration, whereas phosphatases tend to control both. We illustrate and validate these principles experimentally: (a) a kinase inhibitor affects the amplitude of EGF‐induced ERK phosphorylation much more than its duration and (b) a phosphatase inhibitor influences both signal duration and signal amplitude, in particular long after EGF administration. Implications for the cellular decision between growth and differentiation are discussed.

Functional detection of MDR1/P170 and MRP/P190-mediated multidrug resistance in tumour cells by flow cytometry.

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma membrane drug transporter P-glycoprotein (P-gp) or the recently discovered multidrug resistance-associated protein (MRP). In this study we investigated the specificity and sensitivity of the fluorescent probes rhodamine 123 (R123), daunorubicin (DNR) and calcein acetoxymethyl ester (calcein-AM) in order to detect the function of the drug transporters P-gp and MRP, using flow cytometry. The effects of modulators on the accumulation and retention of these probes were compared in several pairs of sensitive and P-gp- as well as MRP-overexpressing cell lines. R123, in combination with the modulator PSC833, provided the most sensitive test for detecting P-gp-mediated resistance. Moreover, in a 60 min drug accumulation assay R123 can be regarded as a P-gp-specific probe, since R123 is not very efficiently effluxed by MRP. In contrast to R123, a 60 min DNR or calcein-AM accumulation test could be used to detect MRP-mediated resistance. The MRP-specific modulator genistein could be used in combination with DNR, but not with calcein-AM. Vincristine (VCR) can be used to increase the cellular uptake of calcein-AM in MDR cells, but is not specific for MRP. Thus, although the combination of DNR with genistein appeared to be as sensitive as the combination of calcein-AM with VCR, the former may be used to probe specific MRP activity whereas the latter provides a combined (P-gp + MRP) functional MDR parameter. With these functional assays the role and relative importance of P-gp and MRP can be studied in, for example, haematological malignancies.

Simvastatin Improves Disturbed Endothelial Barrier Function

Background—Recent clinical trials have established that inhibitors of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) reduce the risk of acute coronary events. These effects of statins cannot be fully explained by their lipid-lowering potential. Improved endothelial function may contribute to the positive effects of statin treatment. Methods and Results—In the present study, we report that simvastatin reduces endothelial barrier dysfunction, which is associated with the development of atherosclerosis. Treatment of human umbilical vein endothelial cells for 24 hours with 5 &mgr;mol/L simvastatin reduced the thrombin-induced endothelial barrier dysfunction in vitro by 55±3%, as assessed by the passage of peroxidase through human umbilical vein endothelial cell monolayers. Similar effects were found on the thrombin-induced passage of 125I-LDL through human aortic endothelial cell monolayers. This reduction in barrier dysfunction by simvastatin was both dose and time dependent and was accompanied by a reduction in the thrombin-induced formation of stress fibers and focal adhesions and membrane association of RhoA. Simvastatin treatment had no effect on intracellular cAMP levels. In Watanabe heritable hyperlipidemic rabbits, treatment for 1 month with 15 mg/kg simvastatin reduced vascular leakage in both the thoracic and abdominal part of the aorta, as evidenced by the Evans blue dye exclusion test. The decreased permeability was not accompanied by a reduction of oil red O–stainable atherosclerotic lesions. Conclusions—These data show that simvastatin, in a relatively high concentration, improves disturbed endothelial barrier function both in vitro and in vivo. The data also support the beneficial effects of simvastatin in acute coronary events by mechanisms other than its lipid-lowering effect .

Genistein modulates the decreased drug accumulation in non-P-glycoprotein mediated multidrug resistant tumour cells.

In tumour cells the pharmacological basis for multidrug resistance (MDR) often appears to be a reduced cellular cytostatic drug accumulation caused by the drug efflux protein, P-glycoprotein (Pgp MDR), or by other drug transporters (non-Pgp MDR). Here we report the reversal of the decreased daunorubicin (DNR) accumulation in five non-Pgp MDR cell lines (GLC4/ADR, SW-1573/2R120, HT1080/DR4, MCF7/Mitox and HL60/ADR) by genistein. Genistein inhibited the enhanced DNR efflux in the GLC4/ADR cells. In these cells the decreased VP-16 accumulation was also reversed by genistein. Three other (iso)flavonoids biochanin A, apigenin and quercetin also increased the DNR accumulation in the GLC4/ADR cells. In contrast to the effects on non-Pgp MDR cells, 200 microM genistein did not increase the reduced DNR accumulation in three Pgp MDR cell lines (SW-1573/2R160, MCF7/DOX40 and KB8-5) or in the parental cell lines. In conclusion the use of genistein provides a means to probe non-Pgp related drug accumulation defects.

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.

Paper-Based Analytical Device for Electrochemical Flow-Injection Analysis of Glucose in Urine

This article describes a new design for a paper-based electrochemical system for flow-injection analysis. Capillary wicking facilitates a gravity-driven flow of buffer solution continuously through paper and nitrocellulose, from a buffer reservoir at one end of the device to a sink at the other. A difference in height between the reservoir and the sink leads to a continuous and constant flow. The nitrocellulose lies horizontally on a working electrode, which consists of a thin platinum layer deposited on a solid support. The counter and reference electrodes are strategically positioned upstream in the buffer reservoir. A simple pipetting device was developed for reliable application of (sub)microliter volumes of sample without the need of commercial micropipets; this device did not damage the nitrocellulose membrane. Demonstration of the system for the determination of the concentration of glucose in urine resulted in a noninvasive, quantitative assay that could be used for diagnosis and monitoring of diabetes. This method does not require disposable test strips, with enzyme and electrodes, that are thrown away after each measurement. Because of its low cost, this system could be used in medical environments that are resource-limited.

Preferential Reactivation of Motivationally Relevant Information in the Ventral Striatum

Spontaneous “off-line” reactivation of neuronal activity patterns may contribute to the consolidation of memory traces. The ventral striatum exhibits reactivation and has been implicated in the processing of motivational information. It is unknown, however, whether reactivating neuronal ensembles specifically recapitulate information relating to rewards that were encountered during wakefulness. We demonstrate a prolonged reactivation in rat ventral striatum during quiet wakefulness and slow-wave but not rapid eye movement sleep. Reactivation of reward-related information processed in this structure was particularly prominent, and this was primarily attributable to spike trains temporally linked to reward sites. It was accounted for by small, strongly correlated subgroups in recorded cell assemblies and can thus be characterized as a sparse phenomenon. Our results indicate that reactivated memory traces may not only comprise feature- and context-specific information but also contain a value component.

Modulation by (iso) flavonoids of the ATPase activity of the multidrug resistance protein

The multidrug resistance protein (MRP) is an ATP‐dependent transport protein for organic anions, as well as neutral or positively charged anticancer agents. In this study we report that dinitrophenyl‐S‐glutathione increases ATPase activity in plasma membrane vesicles prepared from the MRP‐overexpressing cell line GLC4/ADR. This ATPase stimulation parallels the uptake of DNP‐SG in these vesicles. We also show that the (iso)flavonoids genistein, kaempferol and flavopiridol stimulate the ATPase activity of GLC4/ADR membranes, whereas genistin has no effect. The present data are consistent with the hypothesis that certain (iso)flavonoids affect MRP‐mediated transport of anticancer drugs by a direct interaction with MRP.

Competitive inhibition by genistein and ATP dependence of daunorubicin transport in intact MRP overexpressing human small cell lung cancer cells

In several multidrug resistant tumor cell lines without overexpression of P-glycoprotein (non-Pgp MDR), a decreased accumulation of drugs has been shown to contribute to resistance. We have recently reported that daunorubicin (DNR) accumulation was decreased in the multidrug resistance-associated protein overexpressing GLC4/ADR non-Pgp MDR small cell lung cancer cell line due to an enhanced energy-dependent efflux which could be inhibited by the isoflavonoid genistein. The purpose of this work was 2-fold: (i) to investigate the mechanism by which genistein inhibits the DNR efflux in the GLC4/ADR cells; and (ii) to characterize the dependence of DNR transport on ATP concentration in intact GLC4/ADR cells. The active transport of DNR in GLC4/ADR cells appeared to be a saturable process with an apparent Km of DNR of 1.4 +/- 0.4 microM. Genistein increased the apparent Km value of DNR, suggesting that this agent is a competitive inhibitor of DNR transport. These data provide additional evidence that energy-dependent DNR transport in GLC4/ADR cells is a protein-mediated process. In addition, genistein decreased cellular ATP concentration in a dose-dependent manner in sensitive as well as in resistant cells. Marked inhibition of DNR transport activity in intact GLC4/ADR cells was found when cellular ATP concentration was decreased below 2 mM by sodium azide or 2-deoxy-D-glucose. Thus, since DNR transport in intact GLC4/ADR is already inhibited at modest cellular ATP depletion, a limitation in ATP supply might open ways to make MDR cells more susceptible to drug toxicity.