Interaction of bioactive hydrophobic peptides ... - The FASEB Journal

RESEARCH COMMUNICATIONS

Interaction

of bioactive

human

multidrug

BALAZS URSULA

hydrophobic

peptides

with

the

transporter

SARKADI,’ 1 MARIANNA MULLER,’ LASZLO A GERMANN,1 MICHAEL M GOTTESMAN,

IIOMOLYA,’ ZSOLT HOLLO,’ JANOS SEPR6DI;t ELMER M PRICE,S AND R C BOUCHER

‘National Institute of Haematology, Blood Transfusion and Immunology Budapest, Hungary; l5t Institute of Biochemistry Semmelweis Medical University, Budapest Hungary tNational Cancer Institute National Institutes of Health, Bethesda, Maryland 20892, USA; and SThe University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

ABSTRACT In this report we demonstrate that various biologically active hydrophobic peptide derivatives, e.g., proteinase inhibitors, chemoattractants, ionophores, enkephalins, and immunosuppressants, stimulate a membrane ATPase activity associated with the human multidrug transporter (MDR1). The stimulation of the MDR1ATPase by these agents does not correlate with their known biochemical or pharmacological activities but rather with their hydrophobicity. The peptides that show high-affinity interaction with the MDR1-ATPase also interfere strongly with fluorescent dye extrusion catalyzed by the multidrug transporter in intact cells and some have been shown to reverse drug resistance in cultured cells. These data suggest that several hydrophobic peptides behave as substrates of the multidrug transporter and may be used to modulate the chemotherapy resistance of tumor cells.-Sarkadi, B., Muller, M., Homolya, L., Holl#{243},Z., Seprdi, J., Germann, U. A., Gottesman, M. M., Price, E. M., Boucher, R. C. Interaction of bioactive hydrophobic peptides with the human multidrug transporter. FASEB J. 8: 766-770; 1994. Key

porter

IN

Words: MDRJ . P-glycoprotein . peptides . ATPase activity fluorescent dye extrusion

THE

CURRENT

significant multiple

problem resistance

CHEMOTHERAPY

is the against

OF

appearance a wide range

multidrug

human or development of anticancer

trans-

cancer

One major cause of such multidrug resistance is the appearance of a protein in the plasma membrane of the tumor cells. This so-called multidrug transporter, MDRI,2 or P-glycoprotein, catalyzes an ATldependent extrusion of various cytotoxic drugs, e.g., Vinca alkaloids, colchicin, anthracyclines, as well as other natural antibiotics, and maintains their cellular level at a subtoxic concentration (1-5). The multidrug transporter has also been shown to interact with a wide range of chemicals, including calcium channel blockers (e.g., Verapamil or nifedipine), calmodulin inhibitors (trifluoperazine), or steroid receptor ligands (progesterone, methylprednisolone, tamoxifen). This relative nonspecificity of the MDR1 allows the reversal of multidrug resistance by a great variety of compounds that compete with cytotoxic drugs for this extrusion pump (5, 6). The physiological role of the MDRI protein is most probably the extrusion of toxic materials and waste products, as the MDR1 protein has a high level in tissues responsible for such function (e.g., lung, kidney, liver, etc.). Another physiological function of MDR1 maybe the secretion of hydrophobic compounds from exocrine or endocrine glands.

766

a

of a drugs.

The human segments and

MDRI protein contains two nucleotide-binding

resembling

various

the

prokaryote

and

12 transmembrane domains, strongly eukaryote

members

of the so-called ABC (ATP binding cassette) transporters or traffic ATPases (3, 6), some of which are involved in peptide transport. The Steril 6 (STE6) protein of yeast is necessary for secretion of the a factor mating pheromone peptide, whereas in mammalian cells the TAPI +TAP2 complex is involved in the import of peptides from the cytosol into the lumen of the endoplasmic reticulum, a transport required for the MHC class-I-dependent antigen presentation (see refs 3, 4, 7, 8). Moreover, in yeast cells that lack the STE6 transporter, a factor secretion could be restored by transfection with a mouse MDR cDNA (9). Sharma et al. (10) reported that exposing cultured mammalian cells to cytotoxic proteinase inhibitor peptides, such as N-acetyl-leucyl-leucylnorleucinal (ALLN) and N-acetyl-leucyl-leucyl-methioninal (ALLM), selected for expression of the human MDR1 protein, and these peptides were apparently extruded from the cells by the multidrug transporter. There have also been observations indicating that cyclic peptides such as cyclosporins (11, 12) and peptide ionophores, e.g., valinomycin or gramicidin (13, 14), may act as substrates of the MDRI pump. In the present work we have applied two in vitro assay systems to allow rapid identification of peptides that interact with MDR1. As we have reported earlier (15), the in vitro overexpressed human MDRI in isolated insect (Sf9) cell membranes

shows

is significantly

a vanadate-sensitive

stimulated

by

substrates

ATPase

activity

of this

that

transporter

but not by nonsubstrate molecules. The relative concentrations of various MDR1 substrates in stimulating, and at higher concentrations inhibiting, the MDR1-ATPase correlated well with their extrusion by the transporter (15). Thus

we

used

this

assay

as

a basic

screening

method

for

studying peptide interactions with the MDR1 protein. controls, we have also examined the effects of the peptides native and CFTR expressing Sf9 cell membranes.

As on

‘To whom correspondence should be addressed, at: National stitute of Haematology, Blood Transfusion and Immunology, Budapest, Dar#{243}cziu. 24, Hungary.

In1113

2Abbreviations:

ALLN,

N-acetyl-leucyl-leucyl-norleucinal;

ALLM,

N-acetyl-leucyl-leucyl-methioninal; AM, acetoxymethylester; CsA, cyclosporin A; CFTR, cystic fibrosis transmembrane conductance regulator; DAGO enkephalin, tyrosyl-D-alanyl-glycil-N-methyl-

phenylalanyl-glycinol; DMEM, Dulbecco’s modified Eagle’s medium; FMLP, formyl-methionyl-leucyl-phenylalanine; FMLROMe, formylmethionyl-leucyl-phenylalanine-methylester;

drug-resistance

protein;

STE6,

MDR1,

Steril 6 transporter

0892-6638/94/0008-0766/$0l

human multiof yeast.

.50. © FASEB

RESEARCH COMMUNICATIONS A second short-term assay for determining peptideMDR1 interactions was to follow MDR1-specific dye extrusion from intact cells. In this assay we measured the uptake of Calcein-AM, which as a hydrophobic molecule, rapidly penetrates most cell membranes and becomes trapped intracellularly upon conversion into the fluorescent Calcein (free acid) by nonspecific cytoplasmic esterases. Calcein is a fluorescein derivative with a high molar emission coefficient, with no apparent cytotoxicity, and is widely used in cell

viability and proliferation assays. It is well retained by the cells, with a fluorescence essentially insensitive to changes in pH, as well as Ca2 or Mg2 concentrations. We have shown that in cells expressing MDR1, Calcein-AM is extruded by the

multidrug

transporter

before

its intracellular

conversion

to the non-MDR1 substrate free Calcein (16, 17). When in the MDR1-expressing cells Calcein-AM extrusion is inhibited by agents that interfere with the MDR1 pump, the fluorescent dye rapidly accumulates, similarly to that in the control cells. This highly sensitive fluorescence assay allows the relative affinity of the transport system for different compounds to be examined. By using the above assay systems, we found that many hydrophobic peptides showed a strong interaction with the human MDR1 protein. As the steric structures of these peptides and peptide derivatives are relatively well characterized, these establishing

molecules may the structural

interactions of molecules

branes were isolated and the vanadate-sensitive ATPase activity was measured. This ATPase activity reflects the ATPdependent functioning of the multidrug transporter as this activity is not present in uninfected Sf9 cells or in cells infected with a 13-galactosidase baculovirus (15, 21). Figure 1 shows the effects of Verapamil, a well-known substrate of the multidrug

transporter

and

of several

biologically

active

pep-

tides on the Sf9 membrane MDR1-ATPase activity. As demonstrated in Fig. IA, the protease inhibitor peptide derivatives, acetyl-leucyl-leucyl-norleucinal (ALLN) and acetyl-leucyl-leucyl-methioninal (ALLM), were found to be strong activators of the MDR1-ATPase, producing a similar maximum ATPase activity as Verapamil, with a halfmaximum stimulation (Ka) at peptide concentrations of about 150-200 cM. This result is in accordance with the findings of Sharma et al. (7), who showed the MDR1dependent extrusion of these peptides tions. When the amino acids leucine

at

similar

and

concentra-

norleucine,

and

100

80

serve as a basis for further studies requirements of the MDR1-drug

and may be used as tools for a rational to modulate clinical drug resistance.

design

60

40

METHODS ATPase

measurements

20

Recombinant baculovirus carrying the human MDR1 gene was generated and the Sf9 (Spodopterafrugiperda) cells were infected and cultured according to the procedures described previously (18). CFTR cDNA was engineered into baculovirus vector and expressed in Sf9 cells, and fI-galactosidase carrying baculovirus was used as described in ref 19. The virus-infected Sf9 cells were harvested and their membranes were isolated and stored as in ref 15; ATPase activity of the isolated 519 cell membranes was estimated by measuring inorganic phosphate liberation (15). The data points in the figures show the means of triplicate determinations in a representative experiment. The differences between the ATPase activities measured in the absence and presence of vanadate (100 IsM) are plotted.

Fluorescence

--4

0

‘I

0-

I-i

0.1

0

100

1000

(MM)

100

0

measurements 0

rapid stirring in a Hitachi F-4000 fluorescence 493 nm, emission: 515 nm). The bioactive peptides and peptide derivatives

spectrophotometer were

purchased

80

60

40

(cxc: from

20

Sigma (St. Louis, Mo.). Tracheal antibacterial peptide and synthetic yeast a factor (farnesylated and nonfarnesylated forms) were kind gifts of Dr. Gill Diamond, The Children’s Hospital, Philadelphia, and of Dr. Jeffrey M. Becker, University of Tennessee, Knoxville, respectively.

0

,

II

0

MDR1-ATPase

10

Concentration

NIH 3T3 mouse fibroblasts were cultured under standard conditions in D-MEM containing 10% fetal calf serum, 5 mM glutamine, 100 units/mI penicillin, and 100 g/ml streptomycin. MDR1-transfected fibroblasts (NIH-MDRI-Gl85) were prepared and characterized for their drug resistance as described previously (20). Before each experiment the 3T3 cells were trypsinized, then washed and stored in D-MEM at 25#{176}C. Dye uptake was measured by incubating 0.5-I x 106 cells/ml in HPMI medium (16) containing 0.25 sM Calcein AM. Fluorescence was measured at 37#{176}C with

RESULTS

1

AND

0.1 Concentration

DISCUSSION measurements

The human MDRI protein was expressed in Sf9 cells by infection with a recombinant baculovirus, containing an MDR1 cDNA (18), and 3 days later the insect cell mem-

PEPTIDE SUBSTRATES OF THE MUI.TIDRUG

10

TRANSPORTER

Figure

1. Stimulation

activity

in

100

1000

(MM)

of the vanadate-sensitive MDR1-ATPase membranes of Sf9 cells by Verapamil and by peptide Additions: A) Verapamil (#{149}); ALLN (0); ALLM (U); Calpeptin (Li); Leupeptin (V). B) FMLP (0); FMLP-OMe (S); Valinomycin (0); gramicidin D (V); DAGOenkephalin (V).

isolated derivatives.

767

RESEARCH COMMUNICATIONS dipeptides or leucyl-leucine, leucyl-norleucine, stimulatory

tripeptides such as leucyl-leucine, acetylleucyl-leucyl-norleucine, or acetyl-leucylwere examined they had no or very little

effect

on

the

ATPase

activity

(data

not

be a good cells and reversing

shown).

In contrast, Calpeptin (benzoyloxycarbonyl-leucyl-norleucinal) stimulated the MDR1 ATPase to a maximum level and at much lower half-maximum activating concentrations (about 0.5-1 /LM) than ALLN or ALLM. This Ka value was similar to that of verapamil and an inhibition of the MDRI-ATPase at higher concentrations (above 100 tM) occurred both with Verapamil and Calpeptin. Thus a more hydrophobic, aromatic blocking group at the NH2 terminal seems to greatly increase the affinity of the transporter for the peptide derivatives. We have also examined several other commercially available proteinase inhibitors and it is interesting that pepstatin (isovaleryl-valyl-valyl-statinyl-alanyl-statine), a pentapeptide pepsin inhibitor of microbial origin, activated the MDRI-ATPase with a Ka of about 5 M, whereas chymostatin

(phenylalanyl-capreomycidinyl-leucyl-phenylalanylal)

and bestatin

([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]

-

had practically no effect. Isolated membranes of uninfected Sf9 cells and the membranes of Sf9 cells expressing large amounts of f3-galactosidase or CFTR (19) exhibited no peptide-stimulated ATPase activity. These data raise the issue of what are the key molecular features of these MDR1-stimulating agents: do they have to contain the COOH-terminal aldehyde groups, as most protease inhibitors do, or is it only the absence of charged residues that allows peptide interaction with the multidrug transporter? The absence of ATPase stimulation by leupeptin (acetyl-leucyl-leucyl-argininal, Fig. IA) suggests that the presence of blocking groups at both the NH2 and COOH terminals, as well as a COOH-terminal aldehyde group, is not enough to promote MDRI-peptide interaction, a charged arginine residue seems to eliminate such an effect. The data in Fig. lB further support the notion that hydrophobicity is a key feature of the MDRI-reactive peptides. Formyl-methionyl-leucyl-phenylalanine (FM LP), a chemoattractant, was found to be a poor substrate for the MDR1 ATPase, whereas the COOH-terminal methylester derivative of the same molecule (FMLROMe) produced a half-maximum activation between 20 and 50 ILM. In this case a COOH-terminal aldehyde group is absent, and thus clearly is not required for the MDR1-peptide interaction. From various biologically active peptide derivatives we demonstrate the effects of two ionophore compounds, valinomycin (a K carrier cyclododecadepsipeptide) and gramicidin D (a cation channel forming linear polypeptide complex), on the MDR1 ATPase (Fig. 1B). As seen, although they are both hydrophobic peptides, valinomycin stimulated the ATPase activity to a maximum level with a Ka of about 0.5 jIM, whereas gramicidin D was only slightly stimulatory at 1-5 jM and inhibitory at higher concentrations. We have also tested several enkephalin analogs on the MDR1-ATPase activity, and although enkephalins with an unblocked COOH-terminal behaved as poor substrates, the t opioid receptor-selective analog DAGO, which has a hydroxyl group at the COOH terminus (tyrosyl-D-alanylglycil-N-methyl-phenylalanyl-glycinol), was quite an effective stimulator of the MDR1-ATPase (Fig. 1B). Recently it has been reported (22) that synthetic and natural opiates are also substrates for P-glycoprotein. The foregoing results all suggest that hydrophobicity and molecular structure, not the known biological effects, are the major factors determining MDR1-peptide interactions. The cyclic undecapeptide cyclosporin-A (C5A) is known to leucine)

768

Vol. 8

July 1994

inhibitor has

been

of drug

extrusion

used

vitro

in

and

in multidrug-resistant in

clinical

trials

as

a

agent for multidrug resistance (8-12). CsA seems to interact directly with the MDR1 protein (12, 23), and recently an actual translocation of CsA by the MDR1 protein has been demonstrated (24), although with relatively high Km (about 8 tM) and low Vmax values. The experiments of Mintenig et al. (25) indicated that CsA acts differently from Verapamil or vinblastine on the functioning of MDR1dependent chloride channels: although CsA blocks the multidrug

transporter,

it does

not

interfere

with

the

putative

MDR1-related channel activity. As shown in Fig. 2, CsA had a biphasic effect on the MDRI ATPase. In concentrations between 50 and 100 nM, a slight activation of the ATPase was seen, whereas at higher concentrations (above 0.5 tM) a strong inhibition of the vanadate-sensitive ATPase was observed. Moreover, the action of Verapamil was also inhibited by higher concentrations of CsA: when the effects of different CsA concentrations were examined in the presence of maximum stimulating concentrations of Verapamil, this stimulation was half-maximally inhibited by about 0.3-0.5 iM CsA. Thus both the CsA activation of the ATPase and the ability of CsA to inhibit its Verapamil activation indicate a 10- to 20-fold greater affinity of MDRI for CsA than for Verapamil. In these ATPase assays we have also examined several natural peptides and peptide derivatives that may be candidate substrates of the multidrug transporter. These included the tracheal antibacterial peptide and the farnesylated and nonfarnesylated forms of the yeast a factor (the former being a substrate of the STE6 transporter). However, they had no effect on the MDR1 ATPase in a wide concentration range. Fluorescence

measurements

in intact

cells

In Fig. 3 the time-dependent accumulation of the fluorescent dye Calcein is shown, when control (Fig. 3A) or human MDR1-transfected (Fig. 3B) NIH 3T3 mouse fibroblasts

100 90 80 0

70 60 50

0

40

0

30

0

20 10 0

dv 0.001

0.01

Concentration

0.1

1

10

100

(,uM)

Figure 2. Modulation of the vanadate-sensitive MDR1-ATPase activity in isolated membranes of Sf9 cells by cyclosporin A (CsA) and/or Verapamil. Additions: Verapamil (0); CsA + 10 jsM Verapamil added at each CsA concentration examined (0); CsA + 50 sM Verapamil added at each CsA concentration examined (U).

The FASEB Journal

SARKADI

ET AL.

RESEARCH COMMUNICATIONS were incubated with the hydrophobic, nonfluorescent CalceinAM compound. The MDR1-expressing cells actively extrude Calcein-AM, greatly reducing cellular fluorescence associated with free Calcein trapping (see refs 16, 17). Figure 3 demonstrates that the addition of Verapamil (line 1) or Calpeptin (benzoyloxycarbonyl-leucyl-norleucinal, lines 2, 3), which exhibit high-affinity stimulation of the MDR1-ATPase (see Fig. IA), showed concentration-dependent inhibition of dye extrusion, manifested as a rapid increase in cellular fluorescence in the MDRI-cells (Fig. 3B) compared with no effect in the control fibroblasts (Fig. 3A). Because the halfmaximum MDR1-activation concentration for Calcein-AM was found to be about 0.5 tM (16), only agents with similar or smaller Ka values would be expected to inhibit the Calcein-AM extrusion in intact cells. Figure 4 reports a compilation of the data obtained in experiments similar to those shown in Fig. 3. Verapamil inhibited dye extrusion (thus increased Calcein accumulation) with a K1 of about 2-5 fLM. ALLN had only a slight effect on Calcein-AM transport by the MDR1 protein, whereas valinomycin and calpeptin had effects similar to Verapamil.

A. (Control)

1

2

V (50)

CP (50)

B. (MDRI) 3

2

if

if

if

1,2 I.. ..‘

80

N 5) 5)

‘ti

60

.4-4

0 .

40

.0

20

0.1

j 1

Concentration

10

100

1000

(SM)

Figure 4. Inhibition of MDR1-dependent Calcein exthision by Verapamil and peptide derivatives in NIH 3T3 fibroblasts stably transfected with the human MDR1 cDNA. Intact cells were incubated with 0.25 sM Calcein-AM as described in the Methods section. Verapamil or the peptides were added 5 mm after the addition of Calcein-AM and the rate of dye accumulation was estimated from the linear phase of uptake curve. Percent inhibition of Calcein-AM extrusion was calculated in each experiment by taking 100% the inhibition produced by 50 LM Verapamil. Means of triplicate measurements are plotted. Verapamil (U); ALLN (V); Valinomycin (0); Calpeptin (es); FMLP-OMe (#{149}); cyclosporin A (V).

i mm

/

V (50) CP (50)

100 0

ft

CP (2) V (50)

if CP (10)

ft V (50)

Figure 3. Effects of Verapamil and Calpeptin on Calcein accumulation in NIH 3T3 fibroblasts. Intact cells were incubated with 0.25 sM Calcein-AM as described in the Methods section. Verapamil (V) and Calpeptin (CP) in the concentrations indicated (fsM) were added at the times shown by the arrows. The ordinate represents fluorescence in arbitrary units. A) Control NIH 3T3 fibroblasts. B) NIH 3T3 fibroblasts stably transfected with the cDNA of the human multidrug transporter (MDR1) and grown previously in media containing 60 ng/ml coichicin.

PEPTIDE SUBSTRATES OF THE MULTIDRUG

TRANSPORTER

CsA was highly effective at submicromolar concentrations, again indicating a high-affinity interaction between CsA and MDR1. Of course, in this assay both competition with Calcein-AM transport and a direct inhibition of the transporter produce an inhibition of dye extrusion. FMLP-OMe was effective at higher concentrations (above 20 /LM), whereas leupeptin and FMLP had no measurable effect on Calcein-AM extrusion in the MDRI-transfected cells (not shown). None of these peptides exerted any effect on fluorescent dye accumulation in the control fibroblasts. All these data closely correlate with those obtained for the stimulation of the MDR1-ATPase by the peptide derivatives. Furthermore, the data presented in Fig. 4 were repeated in adriamycin-selected K562 cells, expressing large amounts of MDR1, and qualitatively similar results were obtained (data not shown here). The findings presented here, obtained by MDR1-ATPase measurements and MDR1-dependent dye extrusion experiments, collectively indicate that several hydrophobic peptides interact with the human multidrug transporter. These peptides activate the MDR1-ATPase and compete with the dye extrusion in intact cells. The relatively simple, sensitive, and short-term assays described in this paper allow the quantitative estimation of the affinity of the multidrug transporter to a given substrate, and thus may facilitate the selection of clinically applicable drug-resistance reversing agents. We have recently developed a series of nontoxic hydrophobic peptide derivatives that showed an extremely high-affinity interaction with MDR1 in these assays (with Km values around 20-40 nM) and these peptide derivatives proved to be effective multidrug resistance-reversing agents in various cytotoxicity tests (B. Sarkadi et al., patent pending, unpublished).

769

RESEARCH COMMUNICATIONS The authors are grateful to Drs. G. G#{225}rdos and M. Mag#{243}csi (NIHBTI, Budapest), and C. W. Davis (UNC, Chapel Hill) for their

valuable

Andrea

advice.

The

Siposs is gratefully

ported

by research

(195), the Hungary.

grants

technical

help

acknowledged. from

PHARE-ACCORD

the

CF

by

Anna

Thaly

and

This work has been supFoundation,

program,

and

from

OMFB

OTKA

(Mec),

REFERENCES Endicott,J. A., and Ling, V. (1989) The biochemistry of P.glycoproteinmediated multidrug resistance. Anne. Rev. Bioc/tern. 58, 137-171 2. Van der Bliek, A. M., and Borst, p. (1992) Multidrug resistance. Adv. Cancer Ret. 52, 165-203 3. Higgins, C. F. (1992) ABC transporters from microorganisms to man. I.

Annu.

Rev.

8, 67-113

Cell. Biol.

4. Gottesman, M. M., and Pastan, I. (1993) Biochemistry resistance mediated by the multidrug transporter. Anne.

of multidrug Rev. Biochern.

62, 385-427 5. Tsuruo, T., lida, H., Nojri, M., Tsukagoshi, S., and Sakurai, Y. (1983) Circumvention of vincristine and adriamycin resistance in vitro and in vivo by calcium influx blockers. Cancer Res. 43, 2905-2910 6. Chen, C. J., Chin, J. E., Ueda, K., Clark, D. P., Pastan, I., Gottesman, M. M., and Roninson, I. B. (1986) Internal duplication and homology with bacterial transport proteins in the mdrl (P-glycoprotein) gene from multidrug resistant human cells. Cell 47, 381-389 7. McGrath, J. P., and Warshavsky, A. (1989) The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P glycoprotein. Nature (London) 340, 400-404 8. Monaco, J. J. (1992) A molecular model of MHC class I-restricted antigen processing. Immunol. Today 13, 173-179 9. Raymond, M., Gros, P., Whiteway, M., and Thomas, D. Y. (1992) Functional complementation of yeast ste6 by a mammalian multidrug resistance mdr gene. Science 256, 232-234 10. Sharma, R. C., Inoue, S., Roitelman, J., Schimke, R., and Simoni, R. D. (1992) Peptide transport by the multidrug resistance pump.

J. Biol.

C/tern.267,

5731-5734

11. Slater, I. M., Sweet, P., Stupecky, M., and Gupta, 5. (1986) Cyclosporin A reverses vincristine and daunorubicin resistance in acute lymphatic leukemia in vitro. J. Clin. Invest. 77, 1405-1408 12. Twentyman, P. R. (1992) Cyclosporins as drug resistance modifiers. Biochern.

Pharmacol.

doxorubicin

resistance

in two human

tumor

cell lines. Cancer. Res. 48,

2 793-2797

14. Daoud, S. S., and Juliano, R. L. (1989) Modulation of doxorubicin resistance by valinomycin (NSC 122023) and liposomal valinomycin in Chinese hamster ovary cells. Cancer Res. 49, 266-271 15. Sarkadi, B., Price, E. M., Boucher, R. C., Germann, U. A., and Scarborough, G. A. (1992) Expression of the human multidrug resistance eDNA in insect cells generates a high activity drug-stimulated membrane ATPase. J. Biol. C/tern.267, 4854-4858 16. Homolya, L., Holl#{243}, Z., Germann, U. A., Pastan, I., Gottesman, M. M., and Sarkadi, B. (1993) Fluorescent cellular indicators are extruded by the multidrug resistance protein. j Bid. C/tern. 268, 21493-21496 17. Holl#{243}, Z., Homolya, L., Davis, C. W., and Sarkadi, B. (1993) Calcein accumulation as a fluorometric functional assay of the multidrug transporter. Biochirn. Biop/iys. Acta. In press 18. Germann, U. A., Willingham, M. C., Pastan, I., and Gottesman, M. M. (1990) Expression of the human multidrug transporter in insect cells by a recombinant baculovirus. Biochemistry 29, 2295-2303 19. Sarkadi, B., Bauzon, D., Huckle, W. R., Earp, H. S., Berry, A., Suchindran, H., Price, E. M., Olsen, J. C., Boucher, R. C., and Scarborough, G. A. (1992) Biochemical characterization of cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis epithelial cells. J. Biol. Chern.267, 2087-2096 20. Bruggemann, E. P., Currier, S. J., Gottesman, M. M., and Pastan, I. (1992) Characterization of the azidopine and vinblastine binding site of P-glycoprotein. J. Biol. C/tern. 267, 21020-21026 21. Ambudkar, S. V., Lelong, I. H., Zhang, J., Cardarelli, C. 0., Gottesman, M. M., and Pastan, I. (1992) Partial purification and reconstitution of the human multidrug resistance pump: characterization of the drug-stimulatable ATP hydrolysis. Proc. NatI. Acad. Sci. USA 89, 8472-8476 22. Callaghan, R., and Riordan, J. (1993) Synthetic and natural opiates interact with P-glycoprotein in multidrug-resistant cells. J. Biol. C/tern.

268, 16059-16064 23. Tamai, I., and Safa, A. R. (1990) Competitive interaction of cyclosporins with the Vinca alkaloid binding site of P-glycoprotein in multidrug-resistant cells. j Biol. Chern. 265, 16509-16513 24. Saeki, T, Ueda, K., Tanigawara, Y., Hori, R., and Komano, T. (1993) Human P-glycoprotein transports cyclosporin A and FK506. j Biol. C/tern. 268, 6077-6080 25. Mintenig, G. M., Valverde, M. A., Sepulveda, F. V., Gill, D. R., Hyde, S. C., Kirk, J., and Higgins, C. F. (1993) Specific inhibitors distinguish the chloride channel and drug transporter functions associated with the human multidrug resistance P-glycoprotein. Receptors Channels 1, 305-313

43, 109-117 Received for publication December 14, 1993. Accepted for publication March 9, 1994.

13. Slovak, M. L., Hoeltge, G. A., Dalton, W. S., and Trent, J. M. (1988) Pharmacological and biological evidence for differing mechanisms of

HYPOTHESES

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Journal publishes

hypotheses

to the following

guidelines.

that

A

valid hypothesis is a prediction, based on preliminary data or on a new approach to published information, that is well-defined, focused, novel, and testable. Scientific data and the published literature should be cited in such articles only to the extent necessary to support the major conjectures. The FASEB Journal welcomes such innovative articles as a way of stimulating the development of new experimental procedures and of new concepts. Hypotheses are subject to the usual refereeing procedures. Accepted manuscripts will be published as rapidly as possible. The article should conform to the style of the journal (see Information for Authors in each January issue) and be prefaced by an abstract of 100-200 words. Total length may not exceed 3,000 words (3 printed pages) or the equivalent, including illustrations, tabular matter, and bibliography. An original and four copies should be submitted to the Editor-in-Chief.

770

Vol. 8

July 1994

The FASEB Journal

SARKADI

ET AL.