biologically active mk-801 and skf-10 047 binding

Molecular Biology of the CellVol. 3, 613-619, June 1992

Biologically Active MK-801 and SKF-10 047 BindingSites Distinct from Those in Rat Brain are Expressedon Human Lung Cancer CellsRhoda Maneckjee*t* and John D. Minnat

*NCI-Navy Medical Oncology Branch, Division of Cancer Treatment, National Cancer Institute,Bethesda, Maryland 20889; and ttSimmons Cancer Center, University of Texas,Southwestern Medical Center, Dallas, Texas 75235

Submitted February 20, 1992; Accepted April 13, 1992

We have shown previously that cultured human lung cancer cells of different histologictypes express multiple opioid receptors that can regulate their growth. In this report, weshow that these cells also express specific, saturable, and high-affinity binding sites (Kd- 1 nM) for the non-opioid phencyclidine (PCP), {(+)-5-methyl-10,11-dihydro-5H-di-benzo[a,b]cyclohepten-5,10-imine hydrogen maleate} (MK-801) and a N-allylnormeta-zocine (SKF-10 047) receptor ligands. Characterization of these binding sites showed themto be protein in nature and sensitive to the guanine nucleotide GTP. Pharmacologicalstudies showed that (+) MK-801 and (+) SKF-10 047 competed with each other for theirbinding sites and also for the methadone binding site present in these cells. However, the,u and a opioid ligands did not compete for (+) MK-801 and (+) SKF-10 047 binding sites.In addition, these binding sites on lung cancer cells appear to be distinct from the N-methylD-aspartate/PCP receptor ionophore complex reported to be present in rat brain. MK-801and SKF-10 047, at nM concentrations, were found to inhibit the growth of these cells inculture within a few hours of exposure, and this effect was irreversible after 24 h. Thegrowth effects of these ligands could not be reversed by the opioid antagonist naloxone,suggesting involvement of nonopioid type receptors in the actions of these ligands. Theabundant expression of biologically active MK-801 and SKF-10 047 binding sites in thesecell lines, distinct from those in rat brain, suggests that these cell lines may prove to be avaluable source for further characterization and purification of these binding sites.

INTRODUCTION

Previous studies from our laboratory have demonstratedthe existence of multiple opioid receptor types (A, 6, andK) in human lung cancer cells, and these receptors wereshown to play a key role in mediating the growth in-hibitory effects of several opioid agonists and peptides(Maneckjee and Minna, 1990). In addition, we recentlyhave identified the presence of a different type of re-ceptor for the growth inhibitory actions of methadonein these cell lines (Maneckjee and Minna, 1992). Thedissociative anesthetic phencyclidine (PCP)l is a widely

abused drug with potent psychotomimetic properties.{(+)-5-methyl- 10,11 -dihydro-5H-dibenzo[a,b]cy-clohepten-5,10-imine hydrogen maleate} (MK-801) isa PCP-like compound that displays a higher affinitythan PCP for binding to the PCP binding site (Wong etal., 1986; Loo et al., 1987). The psychotomimetic andstimulatory effects of N-allylnormetazocine (SKF-10 047) and certain benzomorphan drugs have beenshown to be mediated through another class of recep-tor-the a receptor (Martin et al., 1976; Zukin and Zu-kin, 1981). PCP and a opioids were originally thought

t Corresponding author and present address.' Abbreviations used: DAGO, [D-Ala2, N MePhe4, Gly5-ol] enkeph-

alin; DPDPE, [D-Pen2, D-Pen5]-enkephalin; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; MK-801, {(+)-5-methyl-10,1 1-

dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate};MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide;NMDA, N-methyl D-aspartate; PCP, phencyclidine; SCLC, smallcell lung cancer; SKF-10 047, N-allylnormetazocine; Tris, tris-(hydroxymethyl)aminomethane.

©) 1992 by The American Society for Cell Biology 613

R. Maneckjee and J.D. Minna

to exert their effects through a common a receptor.However, recent studies have suggested that there aredistinct PCP and a receptors, neither of which are nal-oxone sensitive (Teal and Holtzman, 1980; Vaupel,1983; Vaupel et al., 1986). There is evidence that thehigh-affinity PCP specific binding site is functionallyand structurally coupled to the N-methyl D-aspartate(NMDA)-type glutamate receptor and that drugs activeat the PCP receptor noncompetitively block the actionsof NMDA. Thus, MK-801 has been shown to be a potentnoncompetitive antagonist for the NMDA receptor(Wong et al., 1986; Kemp et al., 1987; Loo et al., 1987;Olney et al., 1987). It is able to protect central nervoussystem neurons against the neurotoxic action of certainendogenous excitatory amino acids, such as glutamateand aspartate (Rothman and Olney, 1987; Choi et al.,1988).PCP and MK-801 have been shown to bind to specific

high-affinity receptors present in rat brain membranes(Zukin and Zukin, 1979; Vincent et al., 1980; Zukin etal., 1983). a receptors have been shown to occur in sev-eral normal tissues, including brain, liver, kidney, ad-renal, testis and ovary (Musacchio et al., 1988; Romanet al., 1988; Samovilova et al., 1988; Su et al., 1988;Weissman et al., 1988; Wolfe et al., 1989), and in severalhuman tumors, including renal and colon cancers andin one metastatic lung adenocarcinoma (Thomas et al.,1990; Bem et al., 1991). However, PCP binding was notdetected in many of these tissues. The Chinese hamsterX neuroblastoma brain hybrid cell line NCB-20 is theonly cultured cell line that has been shown by bindingstudies to have both a and PCP receptors (Kushner etal., 1988). As an extension of our previous work withopioid receptors in lung cancer (Maneckjee and Minna,1990), the present study was carried out to determinewhether PCP and a receptors are present in humanlung cancer cell lines; whether these receptors are similarto those reported for rat brain, NCB-20 cells, and othertissues; and what role these receptors play in the invitro growth of human lung cancer cells.

MATERIALS AND METHODS

Reagents(+) 3-[3H]MK-801 (17.8 Ci/mmol) was obtained from New EnglandNuclear Research Products (Boston, MA). Nonradioactive (+) and (-)isomers of MK-801 and SKF-10 047 were obtained from ResearchBiochemicals (Natick, MA). [D-Ala2, N MePhe4, Gly5-ol]enkephalin(DAGO) and [D-Pen2, D-Pen5]-enkephalin (DPDPE) were purchasedfrom Peninsula Laboratories (Belmont, CA); (±) methadone-HCl andnaloxone-HCl, nicotine di-tartarate, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were obtained from SigmaChemical (St. Louis, MO). U-50 488H and PCP were generously do-nated by the National Institute on Drug Abuse (Rockville, MD).

Cell Lines and Growth AssaysPreviously characterized small cell lung cancer (SCLC) and non-SCLCcell lines were grown in RPMI-1640 media (GIBCO, Grand Island,

NY) supplemented with 10% fetal calf serum, as described (Cameyet al., 1985; Brower et al., 1986). Cells were maintained at 37°C in ahumidified atmosphere of 5% CO2 in air. Cells were free of myco-plasma contamination based on a molecular hybridization assay usedaccording to the manufacturer's instructions (Gen-Probe, San Diego,CA). A semiautomated colorimetric assay (MTT), based on the abilityof live cells to reduce a tetrazolium-based compound, 3-phenyl tet-razolium bromide (MTT) to give a purple-colored product, which isquantitated spectrometrically, was used to measure cell growth in thepresence of different ligands. Under the conditions used, the viablecell number is directly proportional to MTT reduction. Appropriatenumbers of cells (1000-5000 per well) were seeded in 96-well tissueculture plates and cultured with and without various concentrationsof ligands in RPMI-1640 medium with 10% fetal calf serum at 37°C.Five days later, MTT assays were carried out as previously described(Denizot and Lang, 1986). Absorbance was measured at 540 nm usingan automated microplate reader, with a program for determining meanand standard deviation (Biotek Instruments, Winooski, VT).

Receptor Binding AssaysCells were harvested at confluency and processed as described pre-viously (Zukin et al., 1983; Reynolds et al., 1987; Kushner et al., 1988).For the [3H]MK-801 assay, the cell pellets were homogenized in ice-cold 5 mM tris(hydroxymethyl)aminomethane (Tris)/N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 7.4,containing 1 mM EDTA, using a polytron (Brinkman, Rexdale, Ontario,Canada), and centrifuged at 30 000 X g for 15 min at 4°C. Resus-pension and centrifugation of the pellets were repeated four times,and the pellets finally were resuspended in 50 volumes of the samebuffer. For the PCP and SKF-10 047 assays, 5 mM Tris-HCl buffer,pH 7.4, was used. Membrane protein concentrations were determinedusing a protein assay kit (Bio-Rad Labs, Richmond, CA). [3H]MK-801binding to intact membranes was measured as follows: in a final vol-ume of 1 ml, aliquots of membrane preparations (containing -200,ug of protein) inS mM Tris-HEPES/1 mM EDTA buffer, pH 7.4, wereincubated in triplicate for 2 h at 24°C with various concentrations of[3H]MK-801 with or without excess of nonradioactive MK-801 (1 ,uM)or other indicated ligands. For the [3H]PCP and [3H]SKF-10 047 assays,5 mM Tris-HCl buffer, pH 7.4, was used, and the incubation wascarried out at 4°C for 45 min. At the end of the incubation period,free ligand was separated from the membrane-bound ligand by fil-tration under reduced pressure through GF/B glass filters (WhatmanInternational, Maidstone, England), followed by three washes withice-cold assay buffer. For the PCP and SKF-10 047 assays, the filterswere presoaked in 0.3% polyethyleneimine. The filters were placedin Ultrafluor scintillation fluid (National Diagnostics, Manville, NJ)and counted in a scintillation counter (Beckman Instruments, Fullerton,CA). Specific binding was calculated as the difference between totalbinding and binding in the presence of excess nonradioactive ligand.Bmax and Kd values were calculated by Scatchard analysis usingcomputer-assisted linear-regression analysis. All experiments wererepeated three times.

cAMP AssaysCells were cultured for 4 d in 24-well plated in 1 ml of medium, andthe medium was changed the day before ligand treatment. The cellswere incubated with the various ligands (100 nM) for 20 min at 37°C.The extracts were prepared and the intracellular cAMP levels measuredby radiometric assay kits (Amersham, Arlington Heights, IL), accordingto the manufacturer's instructions.

RESULTS

[3H]MK-801 binding to intact membranes prepared fromthe SCLC cell line NCI-H187 and the non-SCLC cell

Molecular Biology of the Cell614

Sigma and PCP Receptors in Lung Cancer

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1.9 nM (Bmax, 674 fmols/mg protein), whereas thebinding data for the SCLC cell line NCI-H187 could beresolved into two linear components with Kd values of1.2 and 20 nM, respectively (Bmax, 341 and 1820 fmol/mg protein for the high- and low-affinity binding sites).The specific binding of [3H]MK-801 and [3H]SKF-10 047to lung cancer cell lines was of higher affinity than thebinding of these ligands to rat brain membranes (Kd,33 and 4 nM, respectively). This is in agreement withthe results of others in rat brain membranes and neu-roblastoma cells (Zukin and Zukin, 1979; Zukin et al.,1983; Kushner et al., 1988). For example, Zukin et al.found the Kd values for [3H]MK-801 and [3H]SKF-10 047 binding to rat brain membranes to be 33 and 7nM, respectively (Zukin and Zukin, 1979).

Using a series of specific ligands for the differentopioid and nonopioid receptor types, we determinedtheir ability to displace [3H]MK-801 binding to lungcancer cells (Table 1). The (+) isomer of MK-801 wasmore effective in displacing [3H]MK-801 binding to thecells than the (-) isomer. The a ligand (+) SKF-10 047also significantly competed for [3H]MK-801 binding tothese cells. One-micromolar concentrations of both U-50 488H (K agonist) and PCP were able to effectively

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Figure 1. Saturation binding and scatchard analysis of specificbinding of ['H]MK-801 to (A) the SCLC cell line NCI-H187 and (B)the non-SCLC cell line NCI-H157. Various concentrations of [3H]MK-801 were incubated with cell membranes with and without excess

nonradioactive MK-801 as described in MATERIALS AND METH-ODS. The experiment was repeated twice. B, fmol/mg protein; B/F,bound/free.

line NCI-H157 was studied as a function of radioligandconcentration. Saturation analyses revealed specifichigh-affinity saturable binding to both cell lines (Figure1, A and B). Half-maximal binding was achieved at an-5 nM concentration of [3HIMK-801. Scatchard plotsof the equilibrium binding of [ H]MK-801 to membranesfrom both cell lines could be resolved into two linearcomponents, with Kd values of the high-affinity sitebeing 1 nM for the SCLC cell line NCI-H187 and 2 nMfor the non-SCLC cell line NCI-H157. The Bmax valueswere 462 and 1011 fmol/mg protein, respectively, forthe high-affinity sites. Specific binding of [3H]SKF-10 047 to the SCLC cell line NCI-H187 and the non-

SCLC cell line NCI-H157 was saturable, with respectto ligand concentration (Figure 2, A and B). Half-max-imal binding occurred at -4 nM concentration of[3H]SKF-10 047 for the SCLC cell line NCI-H187 andat a 2 nM concentration for the non-SCLC cell line NCI-H157. Scatchard analysis of the [3H]SKF-10 047 bindingdata to the non-SCLC NCI-H157 revealed a single classof high-affinity binding sites with an apparent Kd of

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Figure 2. Saturation binding and scatchard analysis of specificbinding of ['H]SKF-10 047 to (A) the SCLC cell line NCI-H187 and(B) the non-SCLC cell line NCI-H157.

Vol. 3, June 1992

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Table 1. Cross-reactivity of various ligands for [3H]MK-801 bindingto human lung cancer cell lines

Specific binding

Kd Bmax(nM) (fmol/mg protein)

NCI cell lines H187 H157 H187 H157Ligands tested(+)MK-801 1 2 462 1 011(-) MK-801 14 20 11 028 6 240(+)SKF-10 047 9 17 665 967(±) Methadone 50 4 900 2 318U-50 488H (K) 38 33 3 692 213

No displacement of [3H]MK-801 binding was seen with 1 ,uM con-centrations of DAGO (A), DPDPE (6), and naloxone.

displace [3H]MK-801 binding to membranes prepara-tions of both types of lung cancer cell lines (H187 andH157). The opioid ligands, DAGO (,t agonist) andDPDPE (b agonist), and the opioid antagonist naloxonedid not compete for [3H]MK-801 binding to both typesof lung cancer cells. Interestingly, we find that, in con-trast to rat brain membranes (Table 2), in human lungcancer cells, methadone significantly competed for[3H]MK-801 binding (Table 1). Similarly, in human lungcancer cells, binding of [3H]SKF-10 047 was effectivelydisplaced by both MK-801 and methadone (Table 3).In contrast, we found that MK-801 and methadone didnot compete for [3H]SKF-10 047 binding to rat brainmembranes (Table 2).

Incubation of membranes from the human lungcancer cell line NCI-H187 with protein modifyingagents, such as heat (15 min at 60°C) or Proteinase K(10 ,ug/ml), at 30°C for 15 min completely inhibited[3H]MK-801 binding. Trypsin and N-ethylmaleimide(NEM) decreased specific [3H]MK-801 binding by-50% relative to control samples (162 fmol/mg pro-

tein). [3H]MK-801 and [3H]SKF-10 047 binding to thesecell membrane preparations was also inhibited by theguanine nucleotide GTP (100 AM reduced specificbinding by - 75% relative to control binding). In thehuman lung cancer cell line NCI-H187, (+) SKF-10 047, added to the culture medium at a 100 nM con-centration for 20 min at 37°C, significantly decreasedcAMP levels below the control levels (from 65 to 12pmol/mg total cellular protein). However, in thesecells, the intracellular cAMP levels were not affectedsignificantly on treatment with MK-801.We have shown previously that specific ligands for

the ,u, 6, and K opioid receptors caused a significant con-centration-dependent inhibition of growth of both typesof lung cancer cell lines using the MTT growth assayand that these inhibitory effects could be reversed bythe opioid antagonist naloxone. This growth inhibitoryeffect of opioids was also reversed in the presence ofnicotine (Maneckjee and Minna, 1990). We have shownrecently that methadone is a potent inhibitor of thegrowth of lung cancer cells and that this growth inhi-bition was mediated by distinct methadone receptors.The inhibitory action of methadone could be reversedby naloxone but not by nicotine (Maneckjee and Minna,1992). Similarly, we found that MK-801 and SKF-10 047were able to inhibit the growth of human lung cancercells of both the SCLC (NCI-H187) and the non-SCLC(NCI-H157) types in a dose-dependent manner, usingthe MTT growth assay (Figure 3, A and B). Significantgrowth inhibition occurred at - 1 ,uM concentration ofthese ligands. However, naloxone or nicotine, at dosesranging from 10 nM to 1 1.M, was unable to reverse thegrowth inhibitory effects in the cell lines tested.

In human lung cancer cells, we found that NMDA,in the micromolar to millimolar range, inhibited thegrowth of these cells. However, Figure 4 shows thatthis toxic effect of NMDA in lung cancer cells could notbe reversed by the prior incubation of these cells withvarious concentrations of MK-801 or PCP, as was ob-served for neuronal cells (Kemp et al., 1987; Olney et

Table 2. Comparison of cross-reactivity of various ligands for MK-801 and SKF-10 047binding sites in lung cancer cell lines and rat brain membranes

Rat brain Lung cancer cells

Ligands tested [3H]MK-801 [3H]SKF-10 047 [3H]MK-801 [3H]SKF-10 047

MK-801 + - + +Methadone - - + +SKF-10 047 - + + +U-50 488H + + + +DAGO - - -DPDPE - - - -Naloxone



Table 3. Cross-reactivity of various ligands for [3H]SKF-10 047binding to human lung cancer cell lines

Specific binding

BmaxKd (fmol/mg(nM) protein)

NCI cell lines H187 H157 H187 H157Ligands tested(+)SKF-10 047 1.25 1.9 341 674(-)MK-801 0.9 1.7 135 1076(±) Methadone 2 3.3 514 2007

No displacement of [3H]SKF-10 047 binding was seen with 1 /Mconcentrations of DAGO (,u) and DPDPE (6).

al., 1987; Rothman and Olney, 1987; Choi et al., 1988).Also, in contrast to rat brain membranes (Reynolds etal., 1987), the binding of [3H]MK-801 to lung cancercell membranes is reduced rather than enhanced by ei-ther glycine, L-glutamate, or D-serine, at concentrations(30-100,uM) that enhanced [3H]MK-801 binding in ratbrain. Furthermore, various concentrations (1 nM-1i M)of the calcium channel blocker, diltiazem, did not blockthe binding of [3H]MK-801 to its receptor nor did itreverse the growth inhibitory effects of MK-801 whenadded to the culture medium of lung cancer cells.

lationship between the effects of PCP and a- opioids,such as SKF-10 047 and cyclazocine (for review, seeZukin and Zukin, 1988).

In the rat brain, [3H]labeled MK-801 and PCP-likecompounds bind to a site that appears to be linked tothe NMDA-ionophore complex, and this binding ofPCP-like compounds is to the open activated NMDAreceptor channel complex regulated by the excitatoryamino acids, L-glutamate and glycine and also by D-serine (Reynolds et al., 1987). MK-801, administeredparenterally, has been shown to have a remarkableneuroprotective role (Kemp et al., 1987; Olney et al.,1987; Rothman and Olney, 1987; Choi et al., 1988). Itis highly effective in preventing loss of neurons afterneurotoxic doses of NMDA. However, unlike the casewith rat brain membranes, the PCP receptors found inlung cancer cells appear not to be associated with theNMDA/PCP receptor complex and NMDA toxicitycould not be reversed by prior incubation with MK-801or PCP. In some passages of NCB-20 cells, PCP bindingcould be detected even in the absence of a functionalNMDA channel (Kushner et al., 1988). L-glutamate,

A

DISCUSSION

This study demonstrates the presence of specific andhigh-affinity MK-801 and SKF-10 047 binding sites onboth SCLC and non-SCLC cell lines that were foundto be sensitive to heat and protein modifying agents, aswell as GTP. The binding appears to be stereospecificin these cells, as the affinities of the less active (-) iso-mers were found to be lower than the (+) isomers. Theseligands also can significantly inhibit the in vitro growthof lung cancer cells. The only other cultured cell linereported to have PCP receptors is the Chinese hamster/neuroblastoma brain hybrid cell line, NCB-20 (Kushneret al., 1988). The PCP binding site in the NCB-20 cellline was similar to that in the rat brain, but the affinitywas an order of magnitude lower than that reported forrat brain. In contrast, the binding of [3H]MK-801 and[3H]SKF-10 047 to lung cancer cells was 30- and 5-foldhigher, respectively, than those reported for rat brainreceptors. Pharmacological analysis showed that the li-gands for the a- and PCP receptors cross-reacted withone another for receptor binding in these cells. Bem etal. (1991) have also demonstrated that a binding in tu-mor tissues (kidney and colon) was at least two-foldhigher than the control nonmalignant tissues. Previousbiochemical and behavioral studies have shown a re-

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Figure 3. Effect of various concentrations of (A) MK-801 and (B)SKF-10 047 on the in vitro growth of the human lung cancer cell linesNCI-H187 (SCLC) and NCI-H157 (non-SCLC cell line), as detectedby the MTT growth assay. Each data point represents the mean ofeight culture wells.

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Figure 4. Effect of various concentrations of PCP or MK-801 onNMDA toxicity in the human lung cancer cell line NCI-H157, asdetected by the MTT growth assay. Each data point represents themean of eight culture wells.

glycine, and D-serine appeared not to enhance thebinding of [3H]MK-801 to the lung cancer cell mem-branes. In addition, the calcium channel blocker dilti-azem did not block the binding of [3H]MK-801 to itsreceptor nor did it reverse the growth inhibitory effectsof MK-801 added to the culture medium of lung cancercells. These findings indicate that the MK-801 bindingsites in lung cancer cells are not associated with an openNMDA receptor channel complex.

Binding studies suggest that af agonists like SKF-10 047 interact with at least two sites: a high-affinitysite called the a site and a low-affinity site called thePCP site (Choi and Viseskul, 1988). In contrast to ratbrain membranes, we found that the binding of [3H]MK-801 and [3H]SKF-10 047 to lung cancer cells could eachbe displaced by each other's unlabeled ligand and bymethadone with relatively similar affinities. However,these compounds do not appear to exhibit a similarcross-reactivity in rat brain membranes. Althoughmethadone, MK-801, and SKF-10 047 compete forbinding, only the binding of methadone is inhibited bynaloxone. At present, we do not know whether separatebinding sites exist for methadone, MK-801, and SKF-10 047, which share cross-reactivity with these ligands,or whether there is one common binding site, variousportions of which are seen by the ligands. These resultsprovide further substantiation for the earlier suggestionby Zukin and Zukin (1981) that the a receptor and thePCP binding site may be related. Together with themethadone binding sites, these sites could possibly rep-resent a unique class of binding sites in lung cancercells.

Binding to PCP and a sites on human lung cancercells appears to involve the guanine nucleotide bindingproteins (G proteins), which play a role in several signaltransduction pathways. The inhibition of receptorbinding by GTP seems to suggest the involvement ofan inhibitory type of guanine nucleotide binding pro-

tein. In contrast, we previously found that methadonebinding was not coupled to a pertussis toxin-sensitiveG protein (Maneckjee and Minna, 1992). In lung cancercells, a binding appears to involve a cAMP related sys-tem, as treatment of the cells with SKF-10 047 decreasedcAMP levels by -80%. However, MK-801 binding didnot significantly affect cAMP levels and hence could beregulating cellular effector systems other than adenylatecyclase. MK-801 and PCP were not found to be directlycoupled to calcium channels using the Quin-2 technique(Tsien et al., 1982).

In addition to the SCLC cell line NCI-H187 and thenon-SCLC cell line NCI-H157, we have found that avariety of other human lung cancer cell lines (NCI linesN417, H345, H146, H82, H23, H460, H322, and H1299)express varying amounts of membrane receptors foropioids, as well as for a and PCP-like compounds. Inaddition, the growth of these cell lines was significantlyinhibited by these compounds. In conclusion, these celllines are useful models for studies on the mechanismof action of opioids and related compounds and thesedrugs may give new insights into finding effective treat-ment for this malignancy. Abundant expression ofbinding sites for PCP and a ligands on these cell linesshould prove to be valuable for their further purificationand characterization.

ACKNOWLEDGMENTSWe thank the G. Harold and Leila Y. Mathers Charitable Foundationfor generous support and the National Institute of Drug Abuse (NIDA)for their gift of various drugs. We thank Dr. A. Gazdar for the cancercell lines and S. Stephenson, M. J. Englee-Miller, and E. Russell fortechnical assistance. We particularly thank Dr. P. Skolnick for adviceand review of the manuscript.

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