Acquisition of multidrug resistance by L1210 leukemia cells decreases their tumorigenicity and enhances their susceptibility to the host immune response

The use of antineoplastic drugs for cancer treatment is frequently associated with the acquisition of a multidrug-resistant (MDR) phenotype that renders tumoural cells insensitive to antineoplastics. It remains elusive whether the acquisition of the MDR phenotype alters immunological parameters that could influence the cell sensitivity to an eventual host immune response. We report that immunisation of syngeneic mice with -irradiated L1210S (parental line) and L1210R (MDR phenotype) cells results in a significant rejection of subsequently implanted L1210R-based tumours, but not of the L1210S ones. Notably, L1210R tumours display a twofold reduction in vivo proliferative capacity and are less aggressive in terms of mouse survival than their sensitive counterparts. Also, analysis of surface expression of molecules involved in antigen presentation and cytokine activity revealed a slight increase in IFN- receptor expression, a decrease of Fas molecule, and a fourfold up-regulation of MHC class I molecules in L1210R cells. Nonetheless, both cell lines were able to induce a cytotoxic response in syngeneic mice and were equally susceptible to cytotoxicity by splenic cells. Together, these findings indicate that acquisition of drug resistance by L1210 cells is accompanied by pleiotropic changes that result in reduced tumour proliferative capacity and tumorigenicity in syngeneic mice. Hence, immunological studies of MDR tumours may assist in the design of specific therapeutic strategies that complement current chemotherapy treatments.     

Overexpression of P-glycoprotein in L1210/VCR cells is associated with changes in several endoplasmic reticulum proteins that may be partially responsible for the lack of thapsigargin sensitivity

L1210/VCR cells, which express an abundant amount of P-glycoprotein (P-gp), were found to be resistant to thapsigargin--an inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA). In the current paper, we have studied the possible differences among L1210 and L1210/VCR cells in expression of endoplasmic reticulum proteins involved in the regulation of calcium homeostasis and calcium-dependent processes. Amounts of mRNA encoding both calcium release channels (ryanodine receptor channels--RyR and IP3-receptor channels--IP3R) were found to be at similar levels in sensitive and resistant cells. However, mRNAs encoding IP3R1 or 2 were decreased in resistant cells cultivated in the presence of VCR (1.08 micromol/l), while mRNA encoding RyR remained unchanged. The amount of mRNA for SERCA2 was decreased in resistant cells when compared with sensitive cells. This decrease was more pronounced when resistant cells were cultivated in the presence of vincristine (VCR). Calnexin was found to be less expressed at the protein level in resistant as in sensitive cells. The level of mRNA encoding calnexin was decreased only when resistant cells were cultivated in the presence of VCR. Calnexin was found to be associated with immature P-gp in resistant cells. Thus, differences exist between sensitive and resistant cells in the expression of endoplasmic reticulum proteins involved in the control of intracellular calcium homeostasis or calcium-dependent processes. These changes may be at least partially responsible for the lack of sensitivity of resistant cells to thapsigargin.     

Prolongation of pentoxifylline aliphatic side chain positively affects the reversal of P-glycoprotein-mediated multidrug resistance in L1210/VCR line cells

We reported previously that derivatives of pentoxifylline (PTX) reverse multidrug resistance (MDR) in P-glycoprotein (P-gp) positive L1210/VCR cells. Based on the results of a recent study using 25 N-alkylated methylxanthines with carbohydrate side-chains of various lengths, we formulated the following design criteria for a methylxanthine molecule to effectively reverse P-gp mediated MDR: i) a massive substituent at the N1 position is crucial for MDR reversal potency; ii) elongation of the substituents at the N3 and N7 positions (from methyl to propyl) increases the efficacy of a xanthine to reverse MDR; iii) elongation of the substituent at the C8 position (from H to propyl) decreases the efficacy of a xanthine to reverse MDR. Based on these criteria, we synthesized and tested for potency to reverse MDR a new PTX derivative, 1-(10-undecylenyl)-3-heptyl-7-methyl xanthine (PTX-UHM), with prolonged substituents at the N1 and N3 positions. The derivative was obtained by alkylation of 3-heptyl-7-methyl xanthine with 1-methylsulfonyloxy-10-undecylenyl. NMR and IR structural analyses proved the identity of the product. Cytotoxicity study showed that PTX-UHM is only slightly more toxic to L1210/VCR cells than PTX. We found that both PTX-UHM and PTX were able to reverse vincristine resistance of L1210/VCR cells, yet PTX-UHM was significantly more efficient in the reversal than PTX.     

Carbonyl group of aliphatic side chain of pentoxifylline does not play role for P-glycoprotein antagonizing effect of pentoxifylline

Previously we have found that pentoxifylline (PTX), but not caffeine, theophylline, or 1-methyl-3-isobutylxanthine, affects sensitivity of L1210/VCR cells, a line with multidrug resistance mediated by P-glycoprotein (P-gp) to vincristine (VCR) and doxorubicine. Comparison of chemical structure of PTX with other above xanthines has revealed only one marked difference. PTX contains extended aliphatic chain containing reactive electrophilic carbonyl group in the position N1. The investigation of possibility that this group is crucial for PTX-induced MDR reversal represents the aim of the current paper. To prove this hypothesis, we used the new synthesized PTX derivative in which the carbonyl group is modified by a substance containing amino-group and the product of reaction is the respective Schiff base (SB). Successful reaction was observed when PTX reacted with 3,5-diaminobenzenesulfonyl acid (DABS). The product of reaction of DABS with carbonyl group of aliphatic part of PTX was proved using NMR and IR spectroscopy. We found that the resulting PTX derivative PTX-SB revealed higher cytotoxicity on both sensitive L1210 and multidrug resistant L1210/VCR cells than PTX. Moreover, PTX-SB exerts more pronounced MDR reversal effect on L1210/VCR cells than PTX. These results indicate that electrophilic carbonyl group on aliphatic chain located in position N1 of PTX is not essential for MDR reversal effects of PTX.     

Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy

Multidrug resistance (MDR) phenotype of L1210/VCR cell line, acquired by selection for vincristine (VCR), is predominantly mediated by P-glycoprotein (Pgp). Calcein/AM (Cal) was recently described as a fluorescent substrate for Pgp and may be used for measuring of transport activity of Pgp. Expression of Pgp in the cells prevents them to be loaded with the fluorescent marker. To detect the activity of Pgp, verapamil (Ver) or cyclosporine A (CsA) has to be used as Pgp inhibitors. Multidrug resistance protein (MRP), another drug efflux pump, may be inhibited by probenecid (Pro), i.e, the inhibitor of a wide variety of anion transporters. Ver, but not Pro, is able to induce the loading of L1210/CR cells by Cal that is measurable by fluorescence-activated cell sorter (FACS). Another dye, fluo-3/AM (F-3), has a similar behaviour like Cal. Using confocal microscopy we have proved that L1210/VCR cells, in contrast to parental sensitive cells, are not loaded with F-3. Marking of cells with the dye can be achieved using inhibitors of Pgp like Ver or CsA but not by Pro. These results indicate that F-3 is usable for detection of Pgp function in various MDR tissue cells.     

Protein kinase C-alpha antagonizes apoptosis induction by histone deacetylase inhibitors in multidrug resistant leukaemia cells

Previous studies have documented that while several drug-resistant cells enter apoptosis upon treatment with histone deacetylase inhibitors (iHDACs), their drug-sensitive counterparts do not. In the present study, we have investigated at the molecular level why parental drug-sensitive tumor cells do not respond to Trichostatin A and suberoylanilide hydroxamic acid, two iHDACs that promote apoptosis in drug-resistant leukaemia cells. Taking murine leukaemia L1210 cells as a model, we have determined that: (i) PKC-alpha expression is more elevated in parental L1210 than in drug-resistant L1210/R cells, (ii) activation of PKC neutralizes iHDACs-mediated apoptosis in L1210/R cells, (iii) depletion of PKC in parental L1210 cells results in a positive response to iHDACs-mediated apoptosis, and (iv) transfection of a mutant constitutively active PKC-alpha form in L1210/R cells makes the cells refractory to apoptosis induction by iHDACs. These results allow us to conclude that activation/high expression of PKC-alpha protects parental drug-sensitive L1210 cells from iHDACs-mediated apoptosis. Thus, determination of PKC-alpha levels/activity in leukaemia seems to be relevant when choosing efficient chemotherapy protocols based on the use of apoptosis-inducing anticancer drugs.     

Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells that renders them unsusceptible to anti-neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of P-glycoprotein (MDR1, P-gp, ABCB1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In a study of MDR cell survival under cold stress conditions, it was found that resistant leukemic cells with P-gp over-expression, but not their sensitive counterparts, are hypersensitive to cold-induced cell death when exposed to temperatures below 4 °C. The transfection of parental cells with a P-gp-expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between P-gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase-3 at physiological temperature (37 °C) in MDR cells compared with their parental cell line. Treatment with a caspase-3 inhibitor partially rescues MDR leukemic cells from cold-induced apoptosis, which suggests that the cell death mechanism may require caspase-3 activity. Taken together, these findings demonstrate that P-gp expression plays a role in MDR cell survival, and is accompanied by a collateral sensitivity to death induced by cold stress. These findings may assist in the design of specific therapeutic strategies to complement current chemotherapy treatment against cancer.     

Differential expression of regulatory proteins in L1210/VCR cells with multidrug resistance mediated by P-glycoprotein.

Phosphorylation of P-glycoprotein (PGP) by some protein kinases may play an important role in the regulation of its drug transport activity, and may also be important for the development of multidrug resistance (MDR) phenotype. In the present study we investigated the expression of three groups of mitogen-activated protein kinases (MAPKs). The expression of ERKs, SAPK/JNKs and p38-MAPK was studied at the protein level in sensitive (L1210) and multidrug resistant (L1210/VCR) cells. The expression of ERKs in multidrug resistant cells did not differ from those observed in parental sensitive cells. On the other hand, the development of multidrug resistance phenotype in L1210/VCR cells was associated with increased expression of cytosolic p38-MAPK and also proteins of 90 and 130 kDa that react with antibody specific for SAPK/JNKs. The expression of the proteins mentioned was stimulated above all in conditions when vincristine was present in cultivation medium and the stimulation of transport activity of PGP was necessary for the cell survival. The development of multidrug resistance phenotype in L1210/VCR cells was not associated with significant changes in expression of several heat-shock proteins (hsp25, hsp60, hsp70, hsp90). The levels of these proteins were comparable in sensitive L1210 and resistant L1210/VCR cells, and vincristine did not influence the expression of heat-shock proteins in resistant cells.     

A mechanism for P-glycoprotein-mediated apoptosis as revealed by verapamil hypersensitivity

Selection of tumor cell lines with anticancer drugs has led to the appearance of multidrug-resistant (MDR) subclones with P-glycoprotein 1 (P-gp1) expression. These cells are cross-resistant to several structurally and functionally dissimilar drugs. Interestingly, in the process of gaining resistance, MDR cells become hypersensitive or collaterally sensitive to membrane-active agents, such as calcium channel blockers, steroids, and local anaesthetics. In this report, hypersensitivity to the calcium channel blocker, verapamil, was analyzed in sensitive and resistant CHO cell lines. Our results show that treatment with verapamil preferentially induced apoptosis in MDR cells compared to drug-sensitive cells. This effect was independent of p53 activity and could be inhibited by overexpression of the Bcl-2 gene. The induction of apoptosis by verapamil had a biphasic trend in which maximum cell death occurred at 10 microM, followed by improved cell survival at higher concentrations (50 microM). We correlated this effect to a similar biphasic trend in P-gp1 ATPase activation by verapamil in which low concentrations of verapamil (10 microM) activated ATPase, followed by inhibition at higher concentrations. To confirm the relationship between apoptosis and ATPase activity, we used two inhibitors of P-gp1 ATPase, PSC 833 and ivermectin. These ATPase inhibitors reduced hypersensitivity to verapamil in MDR cells. In addition, low concentrations of verapamil resulted in the production of reactive oxygen species (ROS) in MDR cells. Taken together, these results show that apoptosis was preferentially induced by P-gp1 expressing cells exposed to verapamil, an effect that was mediated by ROS, produced in response the high ATP demand by P-gp1.     

P-glycoprotein-mediated multidrug resistance phenotype of L1210/VCR cells is associated with decreases of oligo- and/or polysaccharide contents

Multidrug resistance of murine leukaemic cell line L1210/VCR (obtained by adaptation of parental drug-sensitive L1210 cells to vincristine) is associated with overexpression of mdr1 gene product P-glycoprotein (Pgp)-the ATP-dependent drug efflux pump. 31P-NMR spectra of L1210 and L1210/VCR cells (the latter in the presence of vincristine) revealed, besides the decrease of ATP level, a considerable lower level of UDP-saccharides in L1210/VCR cells. Histochemical staining of negatively charged cell surface binding sites (mostly sialic acid) by ruthenium red (RR) revealed a compact layer of RR bound to the external coat of sensitive cells. In resistant cells cultivated in the absence or presence of vincristine, the RR layer is either reduced or absent. Consistently, resistant cells were found to be less sensitive to Concanavalin A (ConA). Moreover, differences in the amount and spectrum of glycoproteins interacting with ConA-Sepharose were demonstrated between sensitive and resistant cells. Finally, the content of glycogen in resistant cells is lower than in sensitive cells. All the above facts indicate that multidrug resistance of L1210/VCR cells mediated predominantly by drug efflux activity of Pgp is accompanied by a considerable depression of oligo- and/or polysaccharides biosynthesis.     

PI3K/AKT 通路参与调控乳腺癌多药耐药和侵袭转移的研究

目的:探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(phosphatidylinositol 3 kinase/serine-threonine kinase,PI3K/AKT)信号通路与乳腺癌多药耐药和侵袭转移的相关性。方法:以乳腺癌细胞系MCF-7为母本,持续低浓度加药诱导建立阿霉素(Adriamycin,ADR)耐药系MCF-7/ADR’。细胞免疫荧光检测两细胞系中磷酸化AKT(phosphorylated AKT,P-AKT)、P-糖蛋白(P-Glycoprotein,P-gp)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)的表达。PI3K抑制剂LY294002作用两系前后,Western Blot检测P-AKT、MMP-2、P-gp的表达改变及qRT-PCR检测MMP-2、MDR1的表达改变。结果:P-AKT、P-gp(MDR1)、MMP-2在MCF-7中为低表达或不表达,MCF-7/ADR’中为高表达。LY294002作用两系后,P-AKT、P-gp(MDR1)、MMP-2在MCF-7/ADR’中的表达明显减低(P<0.05),MCF-7无明显改变。结论:抑制PI3K/AKT信号通路可有效降低MCF-7/ADR’耐药和侵袭转移能力,PI3K/AKT通路是调控乳腺癌多药耐药和侵袭转移的重要信号通路之一。     

Different modalities of intercellular membrane exchanges mediate cell-to-cell p-glycoprotein transfers in MCF-7 breast cancer cells

Multi-drug resistance (MDR) is a phenomenon by which tumor cells exhibit resistance to a variety of chemically unrelated chemotherapeutic drugs. The classical form of multidrug resistance is connected to overexpression of membrane P-glycoprotein (P-gp), which acts as an energy dependent drug efflux pump. P-glycoprotein expression is known to be controlled by genetic and epigenetic mechanisms. Until now processes of P-gp gene up-regulation and resistant cell selection were considered sufficient to explain the emergence of MDR phenotype within a cell population. Recently, however, "non-genetic" acquisitions of MDR by cell-to-cell P-gp transfers have been pointed out. In the present study we show that intercellular transfers of functional P-gp occur by two different but complementary modalities through donor-recipient cells interactions in the absence of drug selection pressure. P-glycoprotein and drug efflux activity transfers were followed over 7 days by confocal microscopy and flow cytometry in drug-sensitive parental MCF-7 breast cancer cells co-cultured with P-gp overexpressing resistant variants. An early process of remote transfer was established based on the release and binding of P-gp-containing microparticles. Microparticle-mediated transfers were detected after only 4 h of incubation. We also identify an alternative mode of transfer by contact, consisting of cell-to-cell P-gp trafficking by tunneling nanotubes bridging neighboring cells. Our findings supply new mechanistic evidences for the extragenetic emergence of MDR in cancer cells and indicate that new treatment strategies designed to overcome MDR may include inhibition of both microparticles and Tunneling nanotube-mediated intercellular P-gp transfers.     

P-gp localization in mitochondria and its functional characterization in multiple drug-resistant cell lines

Multidrug resistance (MDR) phenotype is characterized by the over-expression of P-glycoprotein (P-gp) on cell plasma membranes that extrudes several drugs out of cells. Cells that express the MDR phenotype are resistant to the mitochondrial related apoptosis and to several anticancer drugs. This study assessed the presence of P-gp in mitochondria and its role in parental drug-sensitive (P5) and in P5-derived MDR1 cells P1(0.5) hepatocellular carcinoma (HCC) cell lines and in drug-sensitive (PSI-2) and mdr1-transfected (PN1A) NIH/3T3 cells. By using Western blot analysis, confocal laser microscopy, measurements of Rhodamine 123 transport across mitochondrial membranes, MDR1 small interfering RNA and flow cytometry analysis, experiments indicate that P-gp is expressed in mitochondria of P1(0.5) and PN1A cells and it is functionally active. Rho 123 accumulation was largely reduced in mitochondria of P1(0.5) cells as compared to those of P5 cells; the reduced uptake of fluorescence in mitochondria of MDR cells was due to P-gp-mediated Rho 123 efflux. In conclusion, these data demonstrate that functionally active P-gp is expressed in the mitochondrial membrane of MDR-positive cells and pumps out anticancer drugs from mitochondria into cytosol. Therefore, P-gp could be involved in the protection of mitochondrial DNA from damage due to antiproliferative drugs.     

Azidopine noncompetitively interacts with vinblastine and cyclosporin A binding to P-glycoprotein in multidrug resistant cells.

It is believed that P-glycoprotein (P-gp) is an energy-dependent drug efflux pump responsible for decreased drug accumulation in multidrug resistant (MDR) cells. In this study, we investigated whether azidopine, a photoactive dihydropyridine calcium channel blocker, is transported by P-gp in MDR Chinese hamster lung cells, DC-3F/VCRd-5L, and whether its binding site(s) on P-gp are distinct from those of Vinca alkaloids and cyclosporins. The efflux of azidopine from MDR cells was energy-dependent and inhibited by the cytotoxic agent vinblastine (VBL). Cyclosporin A (CsA), a modulator of MDR, also increased azidopine accumulation in MDR cells by decreasing the energy-dependent efflux of azidopine. P-gp in these cells was the only protein specifically bound to [3H]azidopine in photoaffinity experiments. The specific photoaffinity labeling of P-gp by [3H]azidopine was inhibited by CsA, SDZ 33-243, nonradioactive azidopine, and VBL with median concentrations (IC50) of 0.5, 0.62, 1.7, and 25 microM, respectively. The equilibrium binding of azidopine to plasma membranes of MDR variant DC-3F/VCRd-5L cells showed a single class of specific binding sites having a dissociation constant of 1.20 microM and a maximum binding capacity of 4.47 nmol/mg of protein. Kinetic analysis indicated that the inhibitory effect of VBL and CsA on azidopine binding to plasma membranes of MDR cells was noncompetitive, indicating that azidopine binds to P-gp at a binding site(s) different from the binding site(s) of these drugs.     

Synthesis and chemical characterization of 2-methoxy-N(10)-substituted acridones needed to reverse vinblastine resistance in multidrug resistant (MDR) cancer cells

In an attempt to find clinically useful modulators of multidrug resistance (MDR), a series of 19 N(10)-substituted-2-methoxyacridone analogues has been synthesized. 2-Methoxyacridone and its derivatives (1-19) were synthesized. Compound 1 was prepared by the Ullmann condensation of o-chlorobenzoic acid and p-anisidine followed by cyclization using polyphosphoric acid. This compound undergoes N-alkylation in the presence of phase transfer catalyst (PTC). Stirring of 2-methoxy acridone with 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane in a two-phase system consisting of organic phase (tetrahydrofuran) and 6N potassium hydroxide in the presence of tetrabutylammonium bromide leads to the formation of compounds 2 and 11 in good yield. N-(omega-Chloroalkyl) analogues were found to undergo iodide catalyzed nucleophilic substitution reaction with various secondary amines. Products were characterized by UV, IR, 1H and 13C NMR, mass-spectral data and elemental analysis. The lipophilicity expressed in log(10) P and pK(a) of compounds have been determined. All compounds were examined for their ability to increase the uptake of vinblastine (VLB) in MDR KBCh(R)-8-5 cells and the results showed that the compounds 7, 10, 12, and 15-19 at 100 microM caused a 1.05- to 1.7-fold greater accumulation of vinblastine than did a similar concentration of the standard modulator, verapamil (VRP). However, the effects on VLB uptake were specific because these derivatives had little effect in the parental drug sensitive line KB-3-1. Steady state accumulation of VLB, a substrate for P-glycoprotein (P-gp) mediated efflux, was studied in the MDR cell line KBCh(R)-8-5 in the presence and absence of novel MDR modulators. Results of the efflux experiment showed that VRP and each of the modulators (1-19) significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp. From among the compounds examined, 14 except 1, 2, 4, 8, and 11, exhibited greater efflux inhibiting activity than VRP. All the 19 compounds effectively compete with [(3)H] azidopine for binding to P-gp, pointed out this transport membrane protein as their likely site of action. Cytotoxicity has been determined and the IC(50) values lie in the range 8.00-18.50 microM for propyl and 4-15 microM for butyl derivatives against KBCh(R)-8-5 cells suggesting that the antiproliferative activity increases as chain length increases from 3 to 4 carbons at N(10)-position. Compounds at IC(10) were evaluated for their efficacy to modulate the cytotoxicity of VLB in KBCh(R)-8-5 cells and found that the modulators enhanced the cytotoxicity of VLB by 5- to 35-fold. Modulators 12, 14-16, and 19 like VRP, were able to completely reverse the 24-fold resistance of KBCh(R)-8-5 cells to VLB. Examination of the relationship between lipophilicity and antagonism of MDR showed a reasonable correlation suggesting that hydrophobicity is one of the determinants of potency for anti-MDR activity of 2-methoxyacridones.     

Calcium ion-dependent proliferation of L1210 cells in culture

Maximum growth of L1210 cells in culture required the presence of free extracellular calcium ions. Reducing the free extracellular calcium ion concentration with EGTA served to decrease the growth rate of the cells. The decrease in cell growth was not due to cell death but rather due to the "pile-up" of the L1210 cells in the GO/Gl phase of the cell cycle. With the readdition of excess calcium ions, there was a lag period of 3 to 6 hours before the L1210 cells initiated DNA synthesis or transited from the G0/G1 phase to S-phase. Cells enriched for S and G2/M phase by elutriation and which were incubated in EGTA-containing culture medium, continued through the cell cycle and were blocked in GO/Gl. These data indicate that the proliferation of L1210 cells in culture requires a calcium ion-dependent process to allow movement from the G0/G1 to S-phase of the cell cycle.     

Retroviral transfection of Madin-Darby canine kidney cells with human MDR1 results in a major increase in globotriaosylceramide and 10(5)- to 10(6)-fold increased cell sensitivity to verocytotoxin. Role of p-glycoprotein in glycolipid synthesis

Retroviral infection of the Madin-Darby canine kidney (MDCK) renal cell line with human MDR1 cDNA, encoding the P-glycoprotein (P-gp) multidrug resistance efflux pump, induces a major accumulation of the glycosphingolipid (GSL), globotriaosylceramide (Galalpha1-4Galbeta1-4glucosylceramide-Gb(3)), the receptor for the E. coli-derived verotoxin (VT), to effect a approximately million-fold increase in cell sensitivity to VT. The shorter chain fatty acid isoforms of Gb(3) (primarily C16 and C18) are elevated and VT is internalized to the endoplasmic reticulum/nuclear envelope as we have reported for other hypersensitive cell lines. P-gp (but not MRP) inhibitors, e.g. ketoconazole or cyclosporin A (CsA) prevented the increased Gb(3) and VT sensitivity, concomitant with increased vinblastine sensitivity. Gb(3) synthase was not significantly elevated in MDR1-MDCK cells and was not affected by CsA. In MDR1-MDCK cells, synthesis of fluorescent N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-aminocaproyl (NBD)-lactosylceramide (LacCer) and NBD-Gb(3) via NBD-glucosylceramide (GlcCer) from exogenous NBD-C(6)-ceramide, was prevented by CsA. We therefore propose that P-gp can mediate GlcCer translocation across the bilayer, from the cytosolic face of the Golgi to the lumen, to provide increased substrate for the lumenal synthesis of LacCer and subsequently Gb(3). These results provide a molecular mechanism for the observed increased sensitivity of multidrug-resistant tumors to VT and emphasize the potential of verotoxin as an antineoplastic. Two strains (I and II) of MDCK cells, which differ in their glycolipid profile, have been described. The original MDR1-MDCK parental cell was not specified, but the MDR1-MDCK GSL phenotype and glycolipid synthase activities indicate MDCK-I cells. However, the partial drug resistance of MDCK-I cells precludes their being the parental cell. We speculate that the retroviral transfection per se, or the subsequent selection for drug resistance, selected a subpopulation of MDCK-I cells in the parental MDCK-II cell culture and that drug resistance in MDR1-MDCK cells is thus a result of both MDR1 expression and a second, previously unrecognized, component, likely the high level of GlcCer synthesis in these cells.