The acridonecarboxamide GF120918 potently reverses P-glycoprotein-mediated resistance in human sarcoma MES-Dx5 cells

The doxorubicin-selected, P-glycoprotein (P-gp)-expressing human sarcoma cell line MES-Dx5 showed the following levels of resistance relative to the non-P-gp-expressing parental MES-SA cells in a 72 h exposure to cytotoxic drugs: etoposide twofold, doxorubicin ninefold, vinblastine tenfold, taxotere 19-fold and taxol 94-fold. GF120918 potently reversed resistance completely for all drugs. The EC50s of GF120918 to reverse resistance of MES-Dx5 cells were: etoposide 7 ± 2 nM, vinblastine 19 ± 3 nM, doxorubicin 21 ± 6 nM, taxotere 57 ± 14 nM and taxol 91 ± 23 nM. MES-Dx5 cells exhibited an accumulation deficit relative to the parental MES-SA cells of 35% for [3H]-vinblastine, 20% for [3H]-taxol and [14C]-doxorubicin. The EC50 of GF120918, to reverse the accumulation deficit in MES-Dx5 cells, ranged from 37 to 64 nM for all three radiolabelled cytotoxics. [3H]-vinblastine bound saturably to membranes from MES-Dx5 cells with a KD of 7.8 ± 1.4 nM and a Bmax of 5.2 ± 1.6 pmol mg–1 protein. Binding of [3H]-vinblastine to P-gp in MES-Dx5 membranes was inhibited by GF120918 (Ki = 5 ± 1 nM), verapamil (Ki = 660 ± 350 nM) and doxorubicin (Ki = 6940 ± 2100 nM). Taxol, an allosteric inhibitor of [3H]-vinblastine binding to P-gp, could only displace 40% of [3H]-vinblastine (Ki = 400 ± 140 nM). The novel acridonecarboxamide derivative GF120918 potently overcomes P-gp-mediated multidrug resistance in the human sarcoma cell line MES-Dx5. Detailed analysis revealed that five times higher GF120918 concentrations were needed to reverse drug resistance to taxol in the cytotoxicity assay compared to doxorubicin, vinblastine and etoposide. An explanation for this phenomenon had not been found. © 1999 Cancer Research Campaign

(Paisley, UK). MES-SA and MES-Dx5 were obtained from the American Type Tissue Culture Collection (Rockville, MD, USA) and MCF-7/ADR cells from the European Collection of Cell Culture (Salisbury, UK). [ 3 H]-vinblastine (11.4-13.5 Ci mmol -1 and [ 14 C]-doxorubicin (60 mCi mmol -1 ) were purchased from Amersham (Little Chalfont, UK). [ 3 H]-taxol (19.3 Ci mmol -1 ) was obtained from the National Cancer Institute (Bethesda, MD, USA). [ 3 H]-taxol and [ 14 C]-doxorubicin were aliquoted and not frozen after thawing. Doxorubicin, etoposide, taxol, vinblastine and cyclosporin A were supplied by Sigma (Poole, UK). Taxotere was a gift from Rhône-Poulenc Rorer (Paris, France). Verapamil and GF120918 (Figure 1) were supplied by GlaxoWellcome (USA). CP100356 was a gift from Dr Kajiji, Pfizer (Groton, CT, USA), Dexniguldepine from Raine Boer, Byk-Gulden Lomberg GmBH (Konstanz, Germany), CGP41251 from Professor Gescher, MRC Toxicology Unit (Leicester, UK) and PSC833 from Sandoz (Basel, Switzerland). Stock solutions of drugs were made up in dimethyl sulphoxide (DMSO) at a concentration of 10 mM and stored at -20°C. Materials used for Western blotting were: monoclonal antibody NCL-JSB-1 from Novocastra Laboratories Ltd (Peterborough, UK), mouse peroxidase IgG from Sigma (Poole, UK) and enhanced chemiluminescence (ECL) protein detection reaction from Amersham (Little Chalfont, UK). Protein concentrations were measured with the Bio-Rad Protein Assay, Bio-Rad Laboratories Ltd (Hemel Hempstead, UK). All other reagents were of the highest available purity obtained from commercial sources. (Harker et al, 1983;Harker and Sikic, 1985) were grown as a monolayer in McCoy's 5A medium supplemented with 10% fetal calf serum (FCS), 20 000 U penicillin and 20 mg streptomycin per litre. The P-gp expressing MCF-7/ADR cells, derived from a human breast cancer cell line were grown as a monolayer in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 20 000 U penicillin and 20 mg streptomycin per litre as previously described (Ferry et al, 1995). Fifty per cent FCS was employed in experiments assessing the effect of protein binding. The cells were maintained in a humidified atmosphere containing 5% carbon dioxide at 37°C. All cell lines were mycoplasma-negative.

Cytotoxicity assay
The tetrazolium dye based MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Mosmann, 1983) was used to assess cytotoxicity. MES-SA or MES-Dx5 cells were seeded into flat-bottom 96-well plates at 2000 cells per well and grown for 24 h in drug-free medium. Cells were incubated for 72 h with cytotoxic agents ± GF120918. A total of 20 µl of MTT dye (5 mg ml -1 phosphate-buffered saline (PBS)) was added and incubated for 4 h at 37°C. All medium was removed and the formazan crystals dissolved in 100 µl DMSO. The absorption was measured in a spectrophotometer at a wavelength of 550 nm.

Radiolabelled drug accumulation assay
Drug accumulation assays were performed as previously described (Ferry et al, 1995). Briefly, 80 × 10 4 MES-SA or MES-Dx5 cells ml -1 were plated into each well of a 12-well plate and incubated for 24 h. McCoy's 5A medium was replaced with 1 ml uptake medium per well (McCoy's 5A medium without supplements and added 5 mM glucose and 5 mM magnesium chloride (MgCl 2 )). The cells were incubated in a shaking waterbath at 37°C in near darkness. GF120918 (1 µM) in 10 µl of DMSO or 10 µl DMSO for the control were added to each well. [ 3 H]-vinblastine (≈ 1 nM), [ 3 H]-taxol (≈ 0.5 nM) or [ 14 C]-doxorubicin (≈ 20 nM) were added at a volume of 10 µl per well which commenced the assay and incubated for 30 min to 4 h at 37°C. The reaction was stopped by removal of the uptake medium and addition of 1 ml liquid scintillant. Radioactivity was quantitated by liquid scintillation counting.

Preparation of cell surface membranes
MES-SA and MES-Dx5 cell surface membranes were prepared by a method as previously described (Ferry et al, 1992). Ten 80 cm 2 flasks of cells were grown to confluence (equivalent to 5 × 10 8 cells). After removal of medium and addition of ice-cold buffer A (50 mM Tris-HCl, pH 7.4, 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 0.1 mM EDTA) the cells were harvested with a rubber policeman. The cell suspension was centrifuged (3000 g, 4°C for 3 min) and the supernatant discarded. The packed cell volume was measured and 1 ml pellet was resuspended in 9 ml ice-cold buffer A. Cells were homogenized with a polytron (6 × 30 s, setting 6). The crude homogenate was centrifuged for 10 min (3500 g at 4°C) in a Sorvall centrifuge and the supernatant was centrifuged for 30 min at 40 000 g at 4°C. The resulting membrane pellet was resuspended in 3 ml ice-cold buffer B (50 mM Tris-HCl, pH 7.4 and 0.1 mM PMSF). Aliquots of membranes were stored at -80°C and they will retain their saturable [ 3 H]-vinblastine binding activity for at least a year (Ferry et al, 1995). Protein concentrations were measured with a Bio-Rad kit using bovine serum albumin as a standard.

Gel electrophoresis and Western blotting
A 6% sodium dodecyl sulphate (SDS) gel (Sambrook et al, 1989) was loaded with 10-100 µg membrane protein after denaturation with gel sample buffer and run at 12 mA overnight. The protein was transferred over 4 h onto a nitro-cellulose blotting membrane. Non-specific binding was blocked with 5% milk powder in PBS and Tween. Incubation with the primary monoclonal antibody NCL-JSB-1 (1:500 in 5% milk in PBS/Tween) lasted 16 h at 4°C. The secondary antibody, mouse peroxidase IgG (1:1000 in 5% milk in PBS/Tween), was added for 1 h and the protein was visualized using the ECL protein detection reaction.

Membrane binding assay
Binding of [ 3 H]-vinblastine to membrane preparations was performed as previously described (Ferry et al, 1992). Briefly, 50 µl buffer B (50 mM Tris-HCl, pH 7.4 and 0.1 mM PMSF), 50 µl drug diluted in buffer B, 50 µl [ 3 H]-vinblastine (5-7 nM) were incubated with 100 µl membrane preparation (protein concentration 50-60 µg per assay) for 3 h in near darkness at 21°C. The total assay volume was 0.25 ml. The assay was stopped by adding 3.5 ml ice-cold washbuffer (20 mM Tris-HCl, pH 7.4 and 20 mM MgCl 2 ) followed by rapid filtration through a Whatman GF/C filter (pre-wetted with 50 mM Tris-HCl and 0.1% albumin) to separate bound from free radioactivity. The filter was washed twice with 5 ml washbuffer to remove free radioactivity, dried and added to scintillation vials filled with 3 ml scintillant. Retained, bound radioactivity was measured with a liquid scintillation counter.

Rhodamine dye accumulation and efflux assay measured by FACS analysis
The rhodamine (Rh123) dye accumulation assay was performed as previously described (Davies et al, 1996). Briefly, 1 × 10 6 MES-SA or MES-Dx5 cells were seeded into 55-mm Petri dishes and allowed to become adherent overnight at 37°C. The medium was replaced with 5 ml additive free medium and incubated with 0.4 µM Rh123 for 20 min at 37°C in darkness. The cells were washed with PBS (37°C). Control cells were harvested before the dye could efflux. Additive free medium was added to the other samples and the cells were incubated for 1.5 h at 37°C. Cells were harvested with trypsin-EDTA at 4°C. Following centrifugation (3000 g, 4°C for 3 min) the cell pellet was washed in ice-cold PBS and resuspended in 1 ml ice-cold PBS and propidium iodate (5 µg ml -1 ), incubated for 10 min on ice followed by a wash in ice-cold PBS and centrifugation (3000 g, 4°C for 3 min). The cells were fixed for 20 min in 1 ml 1% paraformaldehyde (in PBS).
Flow cytometric analysis of the paraformaldehyde fixed cells was carried out by FACScan flow cytometer with an excitation wavelength of 488 nm. R123 and propidium iodate fluorescence were determined for 15 000 cells, considering only viable cells (PI-excluding cells). Fluorescence data were shown on a threedecade log scale.

Data analysis
All cytotoxicity data were modelled by non-linear regression using Kaleidagraph (Ablebeck Software, USA). Dose-response curves for cytotoxicity of drugs in absence or presence of the P-gp inhibitor GF120918 were analysed by non-linear curve fitting, using measured absorption of formazan produced by viable cells at 550 nm wavelength. Data were modelled with the general dose-response equation (DeLean et al, 1981): where Y is the absorption of formazan salts produced by viable cells at a molar concentration of cytotoxic drug X. The maximum of the curve is a, the minimum is d, the slope factor is b and C the drug concentration which inhibits the cell proliferation by 50%. For most curves a, b, C and d were the modelled parameters.
The effect of GF120918 to sensitize MES-Dx5 cells to cytotoxic drugs was expressed as % maximal shift of resistance, using the following formula: EC 50 RϬEC 50 (R+I max conc ) with the EC 50 , the concentration of the cytotoxic drug required to inhibit cell proliferation by 50% for R the resistant cell line MES-Dx5, S the sensitive cell line MES-SA and I the P-gp inhibitor GF120918 at various concentrations. Ligand binding experiments were analysed by non-linear curve fitting as previously described (Malkhandi et al, 1995). Means from independent experiments (n) are given with standard errors of mean, s.e.m. Statistical significance was analysed with the t-test.

Reversal of MES-Dx5 resistance to cytotoxics by GF120918
The wild-type MES-SA cells were subjected to a 3-day exposure to various cytotoxic agents and cell viability was assessed with the formazan-based MTT assay. The MES-SA cells were sensitive to vinblastine, taxotere and taxol with an EC 50 of 3 ± 1 nM (n = 4), 5 ± 1 nM (n = 2) and 5 ± 1 nM (n = 5) respectively, less sensitive to doxorubicin with an EC 50 of 58 ± 14 nM (n = 5) and etoposide with an EC 50 of 3003 ± 1006 nM (n = 5) ( Table 1 and Figure 2 A-E).
In MES-Dx5 cells a low slope was observed for the doseresponse curve of all tubulin inhibitors (vinblastine < taxol < taxotere) (Table 1) compared to (i) doxorubicin and etoposide or (ii) the dose-response curves in MES-SA cells. The dose-response curves for taxanes in particular had slope factors of < 1.0 (P < 0.0001), implying that proportional increases in the doses of taxanes have less effect than for drugs with steep dose-response curves such as doxorubicin.
The addition of the P-gp inhibitor GF120918 (100 nM) for 72 h did not affect sensitivity of the parental MES-SA cells to any of the five cytotoxics tested (results not shown). GF120918 alone did not produce a cytotoxic effect during the 72-h exposure in the MTT assay. An example of the sensitizing effect of GF120918 for taxol in MES-Dx5 cells is shown in Figure 3A. GF120918 concentrations of greater than 10 nM steepened the dose-response curves for taxol, vinblastine and taxotere. GF120918 was able to fully reverse resistance of the MES-Dx5 cells to the level of drug sensitivity shown by the parental MES-SA cells for all tested cytotoxic drugs. Based upon the analysis undertaken, the EC 50 of GF120918 to sensitize MES-Dx5 cells to taxol was 91 ± 23 nM (n = 8) but 7 ± 2 nM (n = 3) for etoposide (Table 2 GF120918 concentration needed to restore 50% of sensitivity (r = 0.9, P = 0.03). To test if this observation is due to P-gp in MES-Dx5 cells or the effect of GF120918, we have performed reversal experiments with another P-gp inhibitor, verapamil, in MES-Dx5 cells which modulated resistance to taxol, doxorubicin, vinblastine and etoposide equipotently with an EC 50 of ~3 µM ( Figure 3D).

[ 3 H]-vinblastine, [ 3 H]-taxol and [ 14 C]-doxorubicin accumulation assays
The   A dose-dependent effect of GF120918 existed for all three radiolabelled cytotoxic drugs (Figure 4). The EC 50 for the reversal of the accumulation deficit by GF120918 in MES-Dx5 cells ranged from 37-64 nM (Table 2).

Gel electrophoresis and Western blotting of membrane preparations
The monoclonal antibody NCL-JSB-1 could detect P-gp in Western blots of membranes prepared from MES-Dx5 cells (limit of detectability 10 µg membrane protein) but did not in MES-SA membranes. The calculated molecular weight for P-gp in MES-Dx5 cells was 156 kDa (data not shown).

FACS analysis of P-gp expression
FACS analysis of rhodamine accumulation and its efflux was used to determine if the MES-Dx5 cells consisted of more than one population. MES-Dx5 cells were found to consist of a single-cell population with regard to rhodamine efflux ( Figure 5).

Membrane binding assay
Saturation analysis of [ 3 H]-vinblastine binding in MES-Dx5 cell membrane preparations revealed a B max of 5.2 ± 1.6 pmol mg -1 protein and a K D of 6.8 ± 1.4 nM. The addition of ATP and MgCl 2 had only a minor effect on [ 3 H]-vinblastine binding ( Figure 6).
[ 3 H]-vinblastine binding to membrane preparations of MES-SA was not displaceable by cyclosporin A (Figure 7A), a well described, competitive inhibitor of [ 3 H]-vinblastine binding to P-gp (Ferry et al, 1992). Cyclosporin A displaceable [ 3 H]-vinblastine binding to P-gp in MES-Dx5 membrane preparations was proportional to the amount of protein used (up to 300 µg). Cyclosporin A inhibited [ 3 H]-vinblastine binding to P-gp to the level of non-specific binding in MES-SA cells.

MES-Dx5 cells grew poorly in medium supplemented with 50%
FCS. Therefore MCF-7/ADR cells and their drug-sensitive wildtype MCF-7 cells were used to explore the effect of a high protein concentration on the potency of GF120918 (MTT assay). The cells were incubated with the cytotoxic drug taxol in the presence of a series of potent P-gp inhibitors and either 10% or 50% FCS. The data are shown in Table 3. Although all P-gp inhibitors were less potent in 50% FCS, this effect was significantly smaller (P < 0.05, t-test) for GF120918 (2.6 ± 1.5-fold) compared to CP100356 (88 ± 22-fold), dexniguldipine (37 ± 9-fold), CGP 412251 (30 ± 12-fold) or PSC833 (19 ± 6-fold).

DISCUSSION
MES-Dx5 cells were derived from a human uterine sarcoma cell line, MES-SA after long-term selection in 500 nM doxorubicin (Harker et al, 1983;Harker and Sikic, 1985). The resistance factors to doxorubicin, vinblastine and etoposide in the 72-h MTT cytotoxicity assay we report were lower than those previously reported for the 1-h drug exposure in a colony-forming assay (Harker and Sikic, 1985). This is not surprising for tubulin-binding drugs and etoposide which show marked cell-cycle/phase dependency. After a 72-h exposure of MES-Dx5 cells to cytotoxic drugs, a resistance pattern similar to the one we have observed has been described (Gosland et al, 1989). MES-Dx5 cells expressed P-gp as assessed by Western blotting and [ 3 H]-vinblastine binding, which allowed quantification of the number of P-gp molecules per mg protein. In the drug sensitive clone MES-SA P-gp could not be detected by either method. The density of [ 3 H]-vinblastine binding sites in MES-Dx5 membrane preparations was about 25% that of MCF-7/ADR cells (Ferry et al, 1995). This correlates with the higher resistance of MCF-7/ADR cells to e.g. doxorubicin (200-fold) and taxol (1000-fold) (MA Russell and DR Ferry, unpublished data).  One striking feature of the dose-response curves of the cytotoxic drugs in MES-Dx5 cells was the variation in their slopes. Thus the slopes of the dose-response curves for taxanes and vinblastine were significantly lower than for doxorubicin. When the P-gp inhibitor GF120918 (Hyafil et al, 1993), an acridonecarboxamide, was added to these cells, the slopes of the dose-response curves for taxanes and vinblastine became steeper, reaching the same level as those observed in the parental MES-SA cells.
One possible explanation for this phenomenon could be, that MES-Dx5 cells consisted of two or more populations of cells expressing different amounts of P-gp. However, in a rhodamine FACS analysis (using the method described by Davies et al (1996)), a single population of MES-Dx5 cells was detected. Other explanations needed to be further considered, including heterogeneity of tubulin expression due to mutations in microtubules. Mutations in tubulin subunits were described in a series of mutant Chinese hamster ovary cell lines which rendered the cells resistant to taxol (Schibler and Cabral, 1986). Assuming the level of resistance to taxol in MES-Dx5 cells was due to the combined effects of P-gp and mutated microtubulin, the inhibition of P-gp should only lead to a shift of the dose-response curve to the left, e.g. concentrations of cytotoxic drugs achieve higher level of cytotoxicity but not to an alteration in the steepness of the dose-response curve. This was clearly not the case since the P-gp inhibitor GF120918 fully sensitized MES-Dx5 and additionally increased the slope of the dose-response curve for all tubulin binding drugs, taxol, taxotere and vinblastine.
MES-Dx5 cells were fully sensitized to a variety of natural product cytotoxics with nM concentrations of the recently described potent P-gp inhibitor GF120918, achieving the same sensitivity as the parental MES-SA cells. GF120918 acted potently on P-gp with concentrations as low as 1 nM, exhibiting a measurable reversal of resistance in the MTT assay. However, fivefold higher concentrations of GF120918 were required to reverse resistance to taxol by 50% compared to doxorubicin and vinblastine. Similarly an eightfold higher concentration of GF120918 was required to reverse resistance to taxotere by 50% compared to etoposide. If GF120918 bound to a common site, 'plugging' P-gp, the EC 50 to reverse resistance expressed in the cytotoxicity assay and the EC 50 to increase the cellular accumulation of drugs, should be equal. A number of possible explanations for this phenomenon are discussed below.
The potency of GF120918 to reverse resistance relates to the affinity of cytotoxics to bind to P-gp We know that [ 3 H]-GF120918 can bind saturably and with a 0.8 nM K D to human P-gp (Ferry et al, 1996). Theoretically the explanation may lie in the relative affinities of the cytotoxic drugs for P-gp. Thus, if drug A (e.g. taxol or vinblastine) has a very high affinity for P-gp relative to drug B (e.g. doxorubicin or etoposide) and both are present at equal concentrations, more GF120918 will be needed to inhibit the binding of drug A by 50%. However, there was no clear relationship between the potency to modulate a given cytotoxic drug and the affinity to bind to P-gp. Furthermore, since the EC 50 for GF120918 to reverse drug resistance was higher for taxol than for vinblastine this explanation must be flawed. These observations therefore imply that P-gp cannot be regarded as a simple receptor at which drugs compete.
The potency of GF120918 to reverse resistance relates to binding affinities for different P-gp drug acceptor sites. Accepting that taxol and GF120918 both bind to P-gp, one possibility is that GF120918 has a lower affinity for a taxaneselective binding domain of P-gp. This would be revealed by higher concentrations of GF120918 required to reverse the [ 3 H]-taxol accumulation deficit. However the EC 50 of GF120918 to reverse the [ 3 H]-taxol accumulation deficit was 37 ± 3 nM and not significantly higher than for the other drugs.
One reason for the failure of the above model to explain why GF120918 is less potent in modulating resistance to taxol and taxotere compared to the other cytotoxic drugs may be that the assumptions underlying the models are incorrect. The mechanisms of permeation of P-gp by cytotoxics is poorly characterized. The initial rate of transport and turnover numbers of P-gp are not known for any substrate.
The model assumes that GF120918, taxol and the other drugs bind to a common drug acceptor site of P-gp. Whilst previous data was interpreted as suggesting that P-gp has a unitary drug acceptor site (Raviv et al, 1990), more recent data suggest that P-gp has more than one drug acceptor site which may even be allosterically coupled (Spoelstra et al, 1992;Ferry et al, 1994;Boer et al, 1996;Day et al, 1997).
Thus if GF120918 has a lower affinity for the taxol drug acceptor site versus that for vinblastine, the potency to modulate resistance would be lower. This remains to be tested using [ 3 H]-GF120918 as a radioligand in binding studies.
The effect of other membrane drug transporters Resistance to etoposide and doxorubicin can be conferred by the expression of the multidrug-resistance associated protein, MRP1 (Cole et al, 1992), a membrane transporter protein. Both, the wildtype MES-SA cells and the P-gp-positive clone MES-Dx5 express similar levels of MRP1 (Chen et al, 1994). Additionally, it was demonstrated that the lung cancer cell line COR-L23 R (Barrand et al, 1994), which expresses MRP1 but not P-gp, was not sensitized to doxorubicin by 1 µM GF120918 (HCL Traunecker and DR Ferry, unpublished data). Therefore, the effect of GF120918 is unlikely to be due to the inhibition of MRP1.
The major vault protein, LRP, a transporter protein present in vesicular membranes has been implicated in resistance to doxorubicin (Scheper et al, 1993). Although LRP has been detected in parental MES-SA cells, no expression was detectable in the MES-Dx5 cells (Chen et al, 1994).
The resistance factor correlates with the concentration of GF120918 required to modulate the resistance The striking correlation between the EC 50 of GF120918 to reverse resistance in the cytotoxicity assay and the resistance factor of each cytotoxic agent (r = 0.9) suggests that the potency of GF120918 relates closely with the resistance factor of the cytotoxic drug. This implies, that a given concentration of GF120918 induces a given fractional modulation of resistance. It is difficult to envisage how this could occur at the molecular level.
Although there is no direct evidence, it is mechanistically plausible that GF120918 is a more potent modulator of resistance to vinblastine, doxorubicin and etoposide than the taxanes probably as a consequence of the pattern of binding to P-gp and subsequent inhibition of substrate transport by P-gp in MES-Dx5 cells. Experiments aimed at measuring direct binding of [ 3 H]-taxol and [ 3 H]-GF120918 to P-gp may cast some light and give some clues as to the molecular mechanisms underlying the phenomenon observed in the cytotoxicity assays.
GF120918 is the most potent P-gp inhibitor described (Hyafil et al, 1993) and retains most of its activity at high protein concentrations (50% FCS). All other tested P-gp inhibitors, i.e. CP100356, dexniguldipine, CGP 412251 and PSC833 were significantly less potent in the presence of 50% FCS. This finding is important for clinical studies in view of the high protein concentrations present in whole blood. In this manuscript detailed pharmacological data regarding the reversal of resistance to vinblastine, doxorubicin, etoposide, taxotere and taxol by GF120918 have been documented for the first time. The results have significant implications on a number of levels. Functional studies of P-gp, particularly binding and transporting of cytotoxic agents and P-gp inhibitors are not yet sophisticated enough to estimate initial rates of transport or turnover rates for any substrate. The relatively low expression of P-gp in cells was a major problem which some groups recently overcame by purifying P-gp (Shapiro et al, 1995;Callaghan et al, 1997). This may allow to re-address these fundamental questions in the future.
GF120918 has entered phase I clinical trials in combination with doxorubicin (Ferry et al, 1998;Planting et al, 1998). The serum levels achieved are known to reverse the P-gp mediated efflux of rhodamine in CD56 cells (natural killer cells) in normal volunteers  and the full trial data will be presented soon (Ferry et al, in preparation).
In terms of the future clinical application, it may prove important that the resistance to doxorubicin could be modulated at lower concentrations of GF120918 compared to the taxanes, taxol and taxotere but remains to be tested.