Katharina Klappe

DIFFERENTIAL EXPRESSION OF SPHINGOLIPIDS IN MRP1 OVEREXPRESSING HT29 CELLS

We have obtained a novel multidrug resistant cell line, derived from HT29 G+ human colon carcinoma cells, by selection with gradually increasing concentrations of the anti‐mitotic, microtubule‐disrupting agent colchicine. This HT29col cell line displayed a 25‐fold increase in colchicine resistance and exhibited cross‐resistance to doxorubicin, VP16, vincristine and taxol. Immunoblotting, combined with RT‐PCR showed that the multidrug resistance phenotype was conferred by specific overexpression of the multidrug resistance protein 1. Confocal scanning laser microscopy revealed that multidrug resistance protein 1 specifically localized in the plasma membrane of HT29col cells. In a functional assay, using the fluorescent multidrug resistance protein 1 substrate 5‐carboxyfluorescein, an increased efflux activity of HT29col cells was measured, as compared to the wild‐type HT29 G+ cells. MK571, a specific inhibitor of multidrug resistance protein 1, blocked the 5‐carboxyfluorescein efflux, but only partially reversed resistance to colchicine, indicating that additional multidrug resistance mechanisms operate in HT29col cells. In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchicine pressure, indicating that colchicine is not a P‐glycoprotein‐specific substrate. Colchicine‐induced overexpression of multidrug resistance protein 1 is accompanied by a changed sphingolipid composition, i.e., enhanced levels of glucosylceramide and galactosylceramide. In addition, ceramide, a lipid messenger molecule involved in apoptosis‐related signal transduction processes, was much more abundant in HT29col cells, which is indicative of a stress response. Int. J. Cancer 87:172–178, 2000.

Lipid dependence of ABC transporter localization and function

Lipid rafts have been implicated in many cellular functions, including protein and lipid transport and signal transduction. ATP-binding cassette (ABC) transporters have also been localized in these membrane domains. In this review the evidence for this specific localization will be evaluated and discussed in terms of relevance to ABC transporter function. We will focus on three ABC transporters of the A, B and C subfamily, respectively. Two of these transporters are relevant to multidrug resistance in tumor cells (Pgp/ABCB1 and MRP1/ABCC1), while the third (ABCA1) is extensively studied in relation to the reverse cholesterol pathway and cellular cholesterol homeostasis. We will attempt to derive a generalized model of lipid rafts to which they associate based on the use of various different lipid raft isolation procedures. In the context of lipid rafts, modulation of ABC transporter localization and function by two relevant lipid classes, i.e. sphingolipids and cholesterol, will be discussed.

The absence of functional glucosylceramide synthase does not sensitize melanoma cells for anticancer drugs

Conversion of ceramide, a putative mediator of anticancer drug‐induced apoptosis, into glucosylceramide, by the action of glucosylceramide synthase (GCS), has been implicated in drug resistance. Herein, we compared GM95 mouse melanoma cells deficient in GCS activity, with cells stably transfected with a vector encoding GCS (GM95/GCS). Enzymatic and metabolic analysis demonstrated that GM95/GCS cells expressed a fully functional enzyme, resulting in normal ceramide glycosylation. However, cytotoxicity assays, as well as caspase activation and cytochrome c release studies, did not reveal any difference between the two cell lines with respect to their sensitivity toward doxorubicin, vinblastine, paclitaxel, cytosine arabinoside, or short‐chain ceramide analogs. Administration of doxorubicin resulted in ceramide accumulation in both cell lines, with similar kinetics and amplitude. Although glucosylceramide formation was detected in doxorubicin‐treated GM95/GCS cells, metabolism of drug‐induced ceramide did not appear to be instrumental in cell survival. Furthermore, N‐(n‐butyl)deoxynojirimycin, a potent and non‐toxic GCS inhibitor, had no chemosensitizing effect on wild‐type melanoma cells. Altogether, both genetic and pharmacological alterations of the cellular ceramide glycosylation capacity failed to sensitize melanoma cells to anticancer drugs, therefore moderating the importance of ceramide glucosylation in drug‐resistance mechanisms.

FLUORESCENCE ASSAYS TO MONITOR FUSION OF ENVELOPED VIRUSES

Publisher Summary This chapter explains fluorescence assays to monitor fusion of enveloped viruses. Viral nucleocapsids contain all the necessary genetic information for replication. However, viruses lack the synthesizing machinery to do so, which is why these particles exploit host cells for this purpose. The introduction of the nucleocapsid into the cytoplasm of a host cell is, therefore, a prerequisite for viral replication. Many enveloped viruses have a relatively simple membrane composition, consisting of lipids derived from the host cell and some (which can be as few as one) virus-specific membrane glycoproteins. The ability of a virus to fuse is contained in these glycoproteins. The intracellular membrane fusion events, which are crucial for the biological life of a cell, are also mediated by proteins makes viral systems particularly attractive as a model for protein-induced fusion of biological membranes. The chapter explains the detection of virus fusion activity.

Fusion of Sendai virus with human HL-60 and CEM cells: different kinetics of fusion for two isolates

The kinetics of fusion of Sendai virus (Z strain) with the human promyelocytic leukemia cell line HL-60, and the human T lymphocytic leukemia cell line CEM was investigated. Fusion was monitored by fluorescence dequenching of octadecylrhodamine (R-18) incorporated in the viral membrane. For one virus isolate (Z/G), the overall rate of fusion (at 37 degrees C) increased as the pH was lowered, reaching a maximum at about pH 5, the lowest pH tested. For another isolate (Z/SF) the rate and extent of fusion were lower at pH 5 than at neutral pH. Lowering the pH from neutral to 5 after several minutes of incubation of either isolate with HL-60 cells resulted in an enhanced rate of fluorescence dequenching. Nevertheless, experiments utilizing NH4Cl indicated that fusion of the virus with cells was not enhanced by the mildly acidic pH of the endosome lumen. Analysis of the kinetics of fusion by means of a mass action model resulted in good simulation and predictions for the time-course of fusion. For the isolate which showed maximal fusogenic activity at pH 5, the rate constant of fusion (approx. 0.1 s-1) at neutral pH was in the range found previously for virus-liposome fusion, whereas the rate constant of adhesion was close to the upper limit for diffusion-controlled processes (1.4.10(10) M-1 s-1). However, for the other isolate (Z/SF) the rate constant of fusion at neutral pH was very small (less than 0.01 s-1), whereas the rate constant of adhesion was larger (greater than or equal to 2.10(10) M-1 s-1). Lowering the temperature decreased the fusion rate. Experiments involving competition with excess unlabeled virions indicated that not all binding sites for Sendai virus on HL-60 cells are fusion sites. The virus fusion activity towards HL-60 cells at neutral pH was not altered significantly by pre-incubation of the virus at pH 5 or 9, in contrast to earlier observations with liposomes and erythrocyte ghosts, or results based on erythrocyte hemolysis or cell-cell fusion.

Multidrug Resistance-Related Protein 1 (MRP1) Function and Localization Depend on Cortical Actin

MRP1 (ABCC1) is known to be localized in lipid rafts. Here we show in two different cell lines that localization of Mrp1/MRP1 (Abcc1/ABCC1) in lipid rafts and its function as an efflux pump are dependent on cortical actin. Latrunculin B disrupts both cortical actin and actin stress fibers. This results in partial loss of actin and Mrp1/MRP1 (Abcc1/ABCC1) from detergent-free lipid raft fractions, partial internalization of Mrp1/MRP1 (Abcc1/ABCC1), and reduction of Mrp1/MRP1 (Abcc1/ABCC1)-mediated efflux. Pretreatment with nocodazole prevents latrunculin B-induced loss of cortical actin and all effects of latrunculin B on Mrp1 (Abcc1) localization and activity. However, pretreatment with tyrphostin A23 does not prevent latrunculin B-induced loss of cortical actin, lipid raft association, and efflux activity, but it does prevent latrunculin B-induced internalization of Mrp1 (Abcc1). Cytochalasin D disrupts actin stress fibers but not cortical actin and this inhibitor much less affects Mrp1/MRP1 (Abcc1/ABCC1) localization in lipid rafts, internalization, and efflux activity. In conclusion, cortical actin disruption results in reduced Mrp1/MRP1 (Abcc1/ABCC1) activity concomitant with a partial shift of Mrp1/MRP1 (Abcc1/ABCC1) out of lipid raft fractions and partial internalization of the ABC transporter. The results suggest that reduced Mrp1 (Abcc1) function is correlated to the loss of lipid raft association but not internalization of Mrp1 (Abcc1).

Rafts as missing link between multidrug resistance and sphingolipid metabolism

Since their discovery, detergent-insoluble glycosphingolipid-enriched membrane domains have accounted for several cellular functions. Besides their role in protein and lipid transport in polarized cells, most of the attention focuses on their organizing role in signal transduction. Given that virtually all multidrug-resistant cells exhibit a deviating sphingolipid composition, most typically increased levels of glucosylceramide, a possible role of sphingolipids in multidrug-resistance has been investigated. An increased conversion of cytotoxic drug-induced ceramide into glucosylceramide, thereby escaping ceramide-induced apoptosis, appeared as a novel and independent multidrug resistance mechanism. In addition, multidrug-resistant cells were found to have abundant caveolae, which harbored a large fraction of the cellular drug-efflux pump, P-glycoprotein. Soon thereafter, other drug-efflux pumps were shown to be located in membrane domains. Interestingly, alterations in cellular sphingolipid composition associated with multidrug resistance cells could largely be accounted for by these membrane domains. In this review, we present an overview of the current understanding of the relation between multidrug resistance and sphingolipid metabolism and the important role membrane domains appear to play in this respect. Multidrug Resistance, Sphingolipids and ATP-binding Cassette Transporters

Use of a fluorescence assay to monitor the kinetics of fusion between erythrocyte-ghosts, as induced by sendai virus

The kinetics of the fusion process between erythrocyte ghosts, as induced by Sendal virus, were readily revealed by a simple fluorescence procedure previously employed to characterize the fusion of viruses with biological membranes. The method relies on the relief of fluorescence selfquenching of the membrane-inserted probe octadecyl Rhodamine B chloride (R18) as occurs when labeled membranes fuse with unlabeled counterparts. The kinetics of R18 insertion into ghost membranes, the non-exchangeable properties of the fluorophore and the kinetics, and some characteristics of Sendai virus-induced fusion of ghosts, are described. We propose that the experimental approach may be particularly advantageous to obtain insight into the efficiency and mechanism of a wide range of fusogens, capable of inducing fusion of erythrocyte membranes.

Gangliosides do not affect ABC transporter function in human neuroblastoma cells

Previous studies have indicated a role for glucosylceramide synthase (GCS) in multidrug resistance (MDR), either related to turnover of ceramide (Cer) or generation of gangliosides, which modulate apoptosis and/or the activity of ABC transporters. This study challenges the hypothesis that gangliosides modulate the activity of ABC transporters and was performed in two human neuroblastoma cell lines, expressing either functional P-glycoprotein (Pgp) or multidrug resistance-related protein 1 (MRP1). Two inhibitors of GCS, d,l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (t-PPPP) and N-butyldeoxynojirimycin (NB-dNJ), very efficiently depleted ganglioside content in two human neuroblastoma cell lines. This was established by three different assays: equilibrium radiolabeling, cholera toxin binding, and mass analysis. Fluorescence-activated cell sorting (FACS) analysis showed that ganglioside depletion only slightly and in the opposite direction affected Pgp- and MRP1-mediated efflux activity. Moreover, both effects were marginal compared with those of well-established inhibitors of either MRP1 (i.e., MK571) or Pgp (i.e., GF120918). t-PPPP slightly enhanced cellular sensitivity to vincristine, as determined by 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide analysis, in both neuroblastoma cell lines, whereas NB-dNJ was without effect. MRP1 expression and its localization in detergent-resistant membranes were not affected by ganglioside depletion. Together, these results show that gangliosides are not relevant to ABC transporter-mediated MDR in neuroblastoma cells.

Function of MRP1/ABCC1 is not dependent on cholesterol or cholesterol-stabilized lipid rafts

MRP1 (multidrug-resistance-related protein 1)/ABCC1 (ATP-binding cassette transporter C1) has been localized in cholesterol-enriched lipid rafts, which suggests a role for these lipid rafts and/or cholesterol in MRP1 function. In the present study, we have shown for the first time that nearly complete oxidation of free cholesterol in the plasma membrane of BHK-MRP1 (MRP1-expressing baby hamster kidney) cells did not affect MRP1 localization in lipid rafts or its efflux function, using 5-carboxyfluorescein diacetate as a substrate. Inhibition of cholesterol biosynthesis, using lovastatin in combination with RO 48-8071, an inhibitor of oxidosqualene cyclase, resulted in a shift of MRP1 out of lipid raft fractions, but did not affect MRP1-mediated efflux in Neuro-2a (neuroblastoma) cells. Short-term methyl-β-cyclodextrin treatment was equally effective in removing free cholesterol from Neuro-2a and BHK-MRP1 cells, but affected MRP1 function only in the latter. The kinetics of loss of both MRP1 efflux function and lipid raft association during long-term methyl-β-cyclodextrin treatment did not match the kinetics of free cholesterol removal in both cell lines. Moreover, MRP1 activity was measured in vesicles consisting of membranes isolated from BHK-MRP1 cells using the substrate cysteinyl leukotriene C4 and was not changed when the free cholesterol level of these membranes was either decreased or increased. In conclusion, MRP1 activity is not correlated with the level of free cholesterol or with localization in cholesterol-dependent lipid rafts.