(CANCER RESEARCH 47, 2594-2598, May 15, 1987]

Multidrug Resistance in a Human Small Cell Lung Cancer Cell Line Selectedin Adriamycin1

Shelagh E. L. Mirski, James H. Gerlach, and Susan P. C. Cole2

Ontario Cancer Treatment and Research Foundation, Kingston Regional Cancer Centre, Kingston, Ontario, Canada K7L 2V7 fS. P. C. C.J; Departments of OncologyfS. E. L. M., S. P. C. C.J and Microbiology and Immunology {S. P. C. C.J, Queen's University, Kingston, Ontario, Canada K7L 3N6; and Ontario Cancer Institute,

Princess Margaret Hospital, and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M4X1K9 [J. H. G.J

ABSTRACT

A multidrug resistant variant (H69AR) of the human small cell lungcancer cell line NCI-H69 was obtained by culturing these cells in gradually increasing doses of Adriamycin up to 0.8 *tMafter a total of 14months. H69AR expresses the multidrug resistant phenotype because itis cross-resistant to anthracycline analogues including daunomycin, epi-rubicin, menogaril, and mitoxantrone as well as to acivicin, etoposide,gramicidin D, colchicine, and the Vinca alkaloids, vincristine and vin-blastine. H69AR is also similar to other multidrug resistant cell lines inthat it displays little or no cross-resistance to bleomycin, S-fluorouracil,and carboplatin. It has a slight collateral sensitivity to 1-dehydrotestos-terone and lidocaine. H69AR has increased cell-cell adhesiveness compared to H69, but a similar growth rate in vitro and tumorigenicity innude mice. When cultured in the absence of Adriamycin, there is a 40%decrease in resistance by 35 days of culture, compared to cells incontinuous culture in drug, but no further decrease in resistance up to181 days. Monoclonal antibodies to P-glycoprotein have no detectablereactivity with H69AR cells as determined by enzyme-linked immuno-sorbent assay and immunoblotting techniques. Thus, unlike most multi-drug resistant cell lines, H69AR does not appear to express enhancedlevels of P-glycoprotein. H69AR will provide a useful model for the studyof multidrug resistance in human small cell lung cancer.

INTRODUCTION

Lung cancer is the leading cause of cancer death in Americanmen aged 35 or older and is increasing in incidence in womenin whom it is predicted that it will surpass breast cancer as theleading cause of cancer death during the 1980s (1). Twenty-five% of autopsied lung cancer patients have the histológica! typedesignated "small cell." The histológica! distinction betweenS( à <̄" and nonsmall cell lung cancer is of major clinical

importance because of the different responses to therapy ofthese two tumor types. Non-SCLC is treated with surgery and/or radiotherapy. SCLC does not respond well to surgery orradiotherapy alone but regimens of combination chemotherapyor chemotherapy and radiotherapy have resulted in increases inmedian survival to 1 year compared to 7 weeks with supportivecare only. However subsequent relapse is common with a 2-year survival of only 10% (2). The high rate of relapse andfailure of chemotherapy is believed to be due to a large degreeto drug resistant cells either existing prior to or arising duringtreatment.

A MDR phenotype has been observed in a variety of mammalian cell lines which provides a model for the study of thisclinical problem (3). Even though selected by a single agent,

Received 9/29/86; revised 12/18/86; accepted 2/19/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by grants to S. P. C. C. from the Medical Research Council ofCanada (MA 9355) and the Clare Nelson Bequest of Kingston General Hospital.

2To whom requests for reprints should be addressed.3The abbreviations used are: SCLC, small cell lung cancer; MAb, monoclonal

antibody; PBS, phosphate-buffered saline; ADM, Adriamycin; MDR, multidrugresistant(ce); FBS, fetal bovine serum; CHO, Chinese hamster ovary; MTT, 3-|4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; ELISA, enzyme-linked immunosorbent assay; ID»,mean drug dose that inhibits growth by 50%.

these cell lines are resistant to a wide range of chemically andfunctionally unrelated drugs. In most cases, this cross-resistancehas been closely associated with the increased expression of P-glycoprotein, a M, 170,000 plasma membrane glycoprotein (3,4). Because of the clinical importance of MDR in SCLC, wehave derived a MDR variant (designated H69AR) of the humanSCLC cell line NCI-H69 using doxorubicin (ADM) as theselecting drug. In this paper we describe the preliminary characterization of this cell line and show that, although it exhibitsthe MDR phenotype, enhanced expression of P-glycoprotein isnot detectable.

MATERIALS AND METHODS

Drugs. Gramicidin D, 1-dehydrotestosterone, lidocaine, 5/3-pregnan-3a-ol-20-one, 5j3-pregnan-3j3-ol-20-one, deoxycortisone, dexametha-sone, dibucaine, tetracaine, and procaine were obtained from SigmaChemical Co. Acivicin was the generous gift of Dr. R. A. Whitney,Department of Chemistry, Queen's University. The remaining drugs

were obtained from the pharmacy at the Kingston Regional CancerCentre.

Cell Culture. The human SCLC cell line NCI-H69 (H69) was kindlyprovided by J. Minna (National Cancer Institute, Bethesda, MD). Itwas routinely cultured in RPMI 1640 medium (GIBCO) supplementedwith 5 or 10% heat-inactivated FBS, 4 HIM L-glutamine, 50 nM 2-mercaptoethanol, and 1 HIMsodium pyruvate. CHO cell lines AuxBland CHRCS (S) were cultured in a-minimal essential medium supplemented with 10% FBS, 4 HIML-glutamine, 50 MM2-mercaptoethanol,and 1 mM sodium pyruvate. Cultures were checked monthly for My-coplasma contamination using the 4',6-diamidino-2-pheny!indole

DNA-binding assay (6) and found to be negative.A multidrug resistant subline of H69 was obtained by culturing the

cells in gradually increasing doses of ADM. After 8 months, cells whichgrew in 0.4 ¡MIADM were obtained. After a further 6 months, cellswhich grew in 0.8 ^M ADM were obtained. This cell line has beendesignated H69AR and has been maintained by alternate feedings withdrug-free medium or medium containing 0.8 «¿MADM.

The stability of the resistant phenotype was determined by culturingcontinuously in medium with either 0.8 ¿>MADM or no drug andassessing relative resistance after various periods of time up to 5months.

Attempts to derive multidrug resistant variants of other SCLC linesincluding MAR (a generous gift of Prof. A. Neville, Ludwig Institutefor Cancer Research, London, United Kingdom), NCI-H209, NCI-HI 28 (generous gifts of J. Minna, National Cancer Institute), and QUAD (7) in liquid culture or in soft agar have been unsuccessful to date.

Growth Curve. The growth curves of H69AR and H69 were determined by seeding 1 x 10s cells/ml in triplicate wells in a 24-well plate(Costar). Cell counts were done on days 3, 4, and 7 using a hemocytom-eter with trypan blue exclusion as an indicator of viability.

Tumorigenicity in Athymic Mice. The tumorigenicity of H69 andH69AR was determined by s.c. injection of 1.3 x IO7 viable cells ofeach type in a volume of 0.2 ml PBS into the left flank of 5- to 6-week-old male BALB/c nu/nu mice. Tumor size was measured in twodimensions, and the area was estimated. Tumors that developed afterinjection of H69AR were excised, placed into culture, and tested forsensitivity to ADM after 4 weeks. The experiment was performed threetimes with 3-4 mice in each experimental group.

Chemosensitivity Testing. The resistance of H69AR to other antineo-

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MULTIDRUG RESISTANT SMALL CELL LUNG CANCER

plastic agents and its collateral sensitivity to a number of anestheticsand steroids were tested using the MTT assay (8). In brief, H69 andH69AR cells were harvested by centrifugation 48-72 h after feedingand plated at 2.5 x 10*cells/well in 96-well plates. Preliminary exper

iments showed that cultures initiated at this cell density continue togrow exponentially for at least 7 days. After incubation at 37°Cfor 2-4 h, drugs were added and the plate incubated at 37°Cfor 7 days. Three

h before the end of drug exposure time, 25 n\ of MTT (Sigma; 2 mg/ml in PBS) were added and the plate incubated at 37°Cfor an additional

3 h. Isopropanol:! N HC1 (25:1) was added to solubilize the forma/ancrystals, and then the absorbance at 570 nm was determined using aDynatech MR600 microtitre plate reader. Within each experiment,determinations were done in quadruplicate, and each drug was testedin at least two separate experiments in most cases. Controls includedwells with cells but no drugs (base line) and wells with medium and thehighest drug concentration but no cells. The percentage of viability wasexpressed as a percentage of the base-line absorbance at 570 nm. Therelative resistance of H69AR compared to H69 is expressed as the ratioof drug concentrations which decrease the base-line absorbance by 50%.The H69AR cell line was cultured in drug-free medium at least 48 h

before testing.Cell ELISA. The expression of P-glycoprotein as detected by the

MAb C219 (9) was assessed in a cell ELISA (10). H69, H69AR, AuxB 1,and CHRC5 were washed with PBS and fixed in 70% methanol at-20°C for 5 min. The cell concentration was adjusted such that 5 x10" cells in 50 ^1 PBS were layered per well in a 96-well plate (Falcon3912). The plates were dried overnight in a 37'C warm room. The cells

were rehydrated in PBS and nonspecific binding was blocked with 1%bovine serum albumin/5% normal goat serum. MAb C219, an irrelevantMAb 1-7-1 (11), or medium were added to the wells and binding ofantibody detected using horseradish peroxidase conjugated goat anti-mouse IgG plus IgA plus IgM affinity purified F(ab'): fragments

(Cappel No. 23470) with o-phenylenediamine and hydrogen peroxideused as substrates. Color development was measured by scanning at490 nm on a Dynatek MR600 microtitre plate reader.

P-GIycoprotein Detection by Western Blotting. Radioiodinated MAbC219 and C494 were used to detect P-glycoprotein in crude membranepreparations of H69 and H69AR as described previously (9). Membranepreparations of AuxBl and CHRC5 were included as controls in allsteps of the procedure. Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis was performed using a modification (12) of the procedure of Fairbanks et al. (13) and the gel was replica blotted ontonitrocellulose paper essentially by the method of Towbin et al. (14).Nonspecific binding of antibody was blocked with 10% bovine serumalbumin in PBS and 15 HIMsodium azide for 2 h at 37°Cor at 4'C

overnight with shaking. The blot was incubated with approximately 5x 10* cpm/ml of I25l-labeled MAb for 16 h at 4'C with shaking. The

blot was washed extensively in PBS, dried, and exposed with intensifying screens on Kodak X-AR5 film at -70°C.

RESULTS

A multidrug resistant variant of H69 was obtained by cultur-ing these cells in gradually increasing doses of ADM up to 0.8UM after a total of 14 months.

Relative Resistance to ADM. The relative resistance to ADMof H69AR compared to H69 was determined using the MTTassay (Fig. 1). In this experiment the ratio of the ID50 for eachcell line indicates a 32-fold relative resistance to ADM ofH69AR compared to H69. The results from additional experiments are presented in Tables 2 and 3.

Growth of H69 and H69AR in Vitro. When grown in RPMI/10% FBS in the absence of ADM and at a starting cell densityof 1 x 10* cells/ml, H69 and H69AR have the same rate of

growth with a doubling time of about 24 h (Fig. 2).Tumorigenicity. The tumorigenicity of H69 and H69AR was

assessed by measuring tumor size in BALB/c nu/nu mice afters.c. inoculation of 1.3 x IO7 cells (Fig. 3). In three separate

100

-00 -4.7 -4

LOG DRUG CONC (;jM)Fig. 1. Resistance to Adriamycin of H69, H69AR, and H69AR which had

been passed as a solid tumor in BALB/c nu/nu mice and subsequently culturedI'Mvitro (H69AR passed in mouse). D, 116'»;O, H69AR passed in mouse; •,

H69AR. Points, mean of quadruplicate determinations. SEs were less than 10%of the mean values and have been omitted for clarity. CONC, concentration.

Table 1 Stability ofH69AR resistance to ADM

Time (days)35

49IOS140171181ID«,'H69AR

offADM*0.49

1.891.360.890.911.20(MM)H69AR

onADM'0.78

2.612.513.981.472.00

" Assessed in quadruplicate using the MTT assay.* Cells were cultured in the absence of ADM from day 0.c Cells were cultured in the presence of 0.8 ^M from day 0.

experiments, using a total of 11 mice given injections of H69and 12 mice given injections of H69AR, there was no significantdifference in the rate of tumor growth.

Stability of the Resistant Phenotype. The drug resistancephenotype of H69AR was stable when these cells were passagedin BALB/c nu/nu mice as solid tumors and returned to culturefor 4 weeks in the absence of ADM before testing for drugresistance (Fig. 1).

The resistance of H69AR which had been grown in theabsence of drug was compared to H69AR grown continuouslyin 0.8 MMADM using the MTT assay (Table 1). By 35 days ofculture without'ADM, the ID50 of H69AR cells had decreased

to about 60% of that of H69AR cultured continuously in thepresence of 0.8 /¿MADM. Although there was some variation,the resistance of cells cultured without ADM remained at about60% of those grown continuously in ADM up to 181 days inculture.

Multidrug Resistance of H69AR. To determine whetherH69AR exhibited the MDR phenotype, the relative resistanceof H69AR compared to H69 was assessed with a panel of drugs(Table 2). These results show that H69AR expresses the MDRphenotype as it is cross-resistant to anthracycline analoguesincluding daunomycin, epirubicin, menogaril, mitoxantrone, aswell as to acivicin, etoposide, gramicidin D, colchicine, and theVinca alkaloids, vincristine and vinblastine.

Although H69AR was resistant to colchicine and vincristine,the dose/response curves to these drugs were unusual. H69 cellswere very sensitive to these drugs (ID50 < 10~5 n\i). With

H69AR cells, the viability decreased to a certain level (usuallyless than 50%) and then did not decrease any further with anincrease in drug dose. For example, in one experiment, viabilityremained at 35% of control values from 0.001-100 UMcolchi-

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Table 2 Relative resistance ofH69AR compared to H69ID»"(MM)DrugAdriamycinDaunomycinEpirubicinMenogari!MitoxanlroncColchicineVincristineH69AR3.16

0.8251.993.98

0.3480.3160.619

0.4680.6310.448

0.4220.39810.0

0.0150.006

>1.000.00075.6

x IO-2

7.95.5 x IO-1H690.0316

0.0200.0250.047

<0.0010.0005<0.001

0.00036.3 x10-'0.050

0.0080.0040.631

«0.01<0.001

<0.000110-'5x

IO-4

3.2<1 x 10-'Relative

resistance*100.041.3

79.684.7

>348.0632.0>619.0

1,560.010,000.09.052.8

99.515.8

»1.5>6.0

>104

7,000112.0

2.5>55.0

Vinblastine

Etoposide

5-Fluorouracil

4.5 x IO-4

6.313.2

24.0

2.82 x IO"5

0.390.22

13.5

15.8

16.259.0

1.8

Bleomycin (milli-units/ml)CarboplatinAcivicinGramicidin

D1.6

1.62.010.0

8.028.20.010

0.25.63

1.62.00.47

0.71.78«0.003

1.I2X 10-'2.5

1.01.021.3

11.415.9»3.322,430.0

°Assessed in quadruplicate by the MTT assay. Each line is the result from one

experiment.* Ratio of ID»H69AR/ID„H69.

Table 3 H69AR tested for collateral sensitivity to various drugs

Experiment"123DrugAdriamycin1

-Dehydrotestosterone50-Pregnan-3a-ol-20-one5/3-Pregnan-3/3-ol-20-oneDeoxycortisoneDexamethasoneAdriamycinLidocaineDibucaineTetracaineAdriamycinProcaineID»H69AR1.5856.032.028.038.0100.01.01260.040.0178.00.381580.0,'O.M)H690.015879.032.028.038.0100.00.12000.040.0178.00.0061580.0Relative

resistance'100.00.711.01.01.01.010.00.631.01.063.01.0

" Adriamycin is included in each experiment as a positive control, demonstrat

ing the relative resistance of H69AR to this drug.* Assessed in quadruplicate by MTT assay.' Ratio of ID5oH69AR/ID»H69.

cine. Similarly, in another experiment, H69AR cultures remained 30% viable over the range of 0.0001-60 /¿Mvincristine.The ID50 for both H69 and H69AR exposed to vinblastine waslower than the lowest drug concentration tested (10~3-10~6 ¿¿M

in three experiments) but only with H69AR did the viabilityplateau. This phenomenon was not due to an artifact of the

LUOD

02468

TIME (DAYS)

Fig. 2. Growth of H69 and H69AR in vitro in the absence of ADM. Cultureswere set up in triplicate wells at 1 x 10*cells/ml on day 0. Results are from one

experiment and similar results were obtained in a second experiment. O, H69AR,mean ±SE (hur\): •.H69, mean ±SE.

500

400

300

LuM

200

CCO

100

10 20TIME (DAYS)

30 40

Fig. 3. Tumorigenicity of H69 and H69AR cells in nude mice. Each mousereceived a s.c. innoculation of 1.3 x IO7cells in 0.2 ml PBS on day 0. •,H69; O,

H69AR. Curves, tumor growth in an individual mouse. Results are from oneexperiment. Similar results were obtained in two additional experiments.

MTT assay because it was also observed in experiments whereviable cells were counted using a hemocytometer and trypanblue as an indicator of viability.

Collateral Sensitivity. To determine whether H69AR exhibited collateral sensitivity, the effect of a panel of steroids andanesthetics on cell viability was assessed (Table 3). H69ARshowed a slightly enhanced sensitivity to 1-dehydrotestosteroneand to lidocaine compared to H69 (relative resistance 0.71 and0.63, respectively). H69 and H69AR were equally sensitive to5/8-pregnan-3/3-ol-20-one, 5/î-pregnan-3a-ol-20-one, deoxycor-tisone, dexamethasone, dibucaine, tetracaine, and procaine.

P-Glycoprotein. P-Glycoprotein is recognized in Chinese andSyrian hamster, mouse, and human MDR cell lines by M AhC219, and in Chinese and Syrian hamster and human cell linesby MAb C494 (9). To determine whether H69AR expressed P-glycoprotein, H69AR and H69 were tested in a cell ELISAusing MAb C219 (Fig. 4). AuxBl was negative and CHRC5 waspositive for P-glycoprotein as expected. Expression of P-gly-

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MULTIDRUG RESISTANT SMALL CELL LUNG CANCER

0.3

0.2

NO)

0.1

m o o01<DX

eiIOX

No 1st Ab 1-7-1 C 219Fig. 4. Immimologie;!I detection of P-glycoprotein with MAb C219 in a cell

ELISA. Bars, mean absórbante of tests performed in duplicate. Negative controlsincluded wells with an irrelevant MAb (1-7-1) or no first antibody.

coprotein was not detectable above background levels in eitherH69 or H69AR.

H69 and H69AR were also tested for P-glycoprotein byimmunoblotting. Crude membrane fractions of AuxBl,CHRC5, H69, and H69AR were made, separated on electro-

phoretic gels, and replica blotted on to nitrocellulose paper.The blot was incubated with '"I-labeled C219 or C494. Auto-radiographs showed a band corresponding to P-glycoprotein inextracts of CHRC5 as expected, but not in AuxBl, H69, or

H69AR (results not shown).

DISCUSSION

By continuous culture of the human SCLC cell line H69 ingradually increasing doses of ADM we have obtained over aperiod of 14 months a cell line, H69AR, which will grow in 0.8iiM ADM. When compared to its parent line, H69AR is 10- to100-fold more resistant to ADM. H69AR is also resistant to apanel of anthracycline analogues, Vinca alkaloids, colchicine,acivicin, and etoposide (Table 2) and thus possesses the MDRphenotype which has been described in numerous rodent andhuman cell lines (3, 4, 15-26).

Variation in the level of resistance of H69AR (but not thepattern of resistance) was observed with all drugs tested. Suchvariation has been noted by others (22). We have no explanationfor this phenomenon but note that it is composed of variationsin the fix,, for both the parent (H69) and resistant line(H69AR). The unusual dose response curves with the Vincaalkaloids in which total cell kill is not achieved even at highdrug concentrations have been observed by others. For example,Hill and Bellamy (27) have described a HN-1 human squamouscell carcinoma line and its VP 16-213 resistant variant VPR,which exhibited a plateau survival fraction after exposure tovincristine. The VPR cells had a decreased slope in the exponential region and a higher plateau level of survival, indicatingcross-resistance to vincristine when cell survival was assessedby colony formation in soft agar.

H69AR is similar to other human MDR lines in that itdisplays little or no cross-resistance to bleomycin, 5-fluoroura-cil, and platinum-containing drugs (3, 4, 16, 21, 22) (Table 2).In the CHO derived MDR cell lines, a collateral sensitivity hasbeen observed to some local anesthetics and hormones (28). Inapparent contrast to the CHO cells, H69AR has only a slightcollateral sensitivity to l-de hydro testosterone and to lidocaine

and is equally as sensitive as H69 to the remaining steroids andanesthetics tested (Table 3).

Multidrug resistant Chinese hamster lung cells described byBiedler et al. (29) displayed an altered cell morphology andpatterns of growth including increased cell-substrate and cell-cell adhesiveness and weak tumorigenicity /'// vivo compared to

sensitive cells. H69AR also displayed increased cell-cell adhesiveness, growing in spheroids rather than loose aggregates asdid the parent H69. However, in contrast to the results ofBiedler et al. (29), there was no significant difference betweenH69 and H69AR growth rates in vitro or tumorigenicity in vivo.Similar in vitro growth characteristics for paired sensitive andresistant human leukemia cell lines have been observed previously (17, 19). In contrast, the human sarcoma MDR cell line,MES-SA, described by Harker and Sikic (21), and the SCLCMDR cell line, H69/LX4, described by Twentyman et al. (22)grew at about 70% of the rate of the parent line in vitro. Whetherthe altered growth characteristics observed in these cells reflectcell membrane changes which may be involved in the mechanism of MDR is unknown and remains to be determined.

The experiments aimed at determining the stability of mul-tidrug resistance suggest that the drug resistant phenotype ofH69AR may be complex and possibly has two components.One component is lost within 35 days of culture in the absenceof ADM, resulting in a 40% decrease in resistance comparedto cells in continuous culture in drug. The second componentis relatively stable, so that cells cultured in drug free mediumfor up to 181 days are still 60% as resistant as those culturedwith ADM. Similarly, Twentyman et al. (22) found a partialloss of resistance in H69/LX4 within 3 weeks, but no furtherloss up to 9 weeks in drug-free medium.

The MDR phenotype has been associated with changes incellular protein composition and gene amplification (30, 31).In particular, the expression of P-glycoprotein, a M, 170,000plasma membrane-associated glycoprotein, is elevated in MDRcell lines (4,16, 21, 25, 26, 32-34). In addition, P-glycoproteinoverexpression has been detected in tumor samples from patients with ovarian carcinoma who were resistant to multidrugtherapy (35). Although the exact function of P-glycoprotein isunknown, it is the molecular alteration found to be mostconsistently associated with the MDR phenotype (4). The levelof P-glycoprotein expression has been correlated with the degree of resistance (32), and the transfer of genomic DNA froman MDR line to a sensitive line resulted in the acquisition ofboth the MDR phenotype and P-glycoprotein (12). Recently, acomplementary DNA has been isolated which confers multi-drug resistance in a drug-sensitive cell, clearly demonstrating afunctional role for P-glycoprotein in the expression of the MDRphenotype (36). To our knowledge, H69AR is the first MDRcell line in which P-glycoprotein is not detectable using imnninological detection methods. The possibility exists that H69ARexpresses a form of P-glycoprotein that is not detected by MAbsC219 and C494. However, Southern and RNA blot analysiswith the pCHPl probe (31) for P-glycoprotein indicates thatthe gene for P-glycoprotein is not amplified, rearranged, oroverexpressed in H69AR cells (within the boundaries recognized by this probe) (37). Taken together, these results appear

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to suggest that cellular changes other than enhanced expressionof P-glycoprotein are responsible for the M DR phenotypeobserved in H69AR cells.

The development of drug resistance has been associated withspecific chromosomal alterations: homogeneously staining regions or double minute chromosomes (38, 39). Karyotypicanalysis of H69 and H69AR showed a marked increase in thenumber of double minute chromosomes per cell in the drugresistant cells compared to the parental cells, with the numberof double minute chromosomes per cell returning to near parental levels in cells cultured in the absence of drug (37). Therelationship of this observation to the multidrug resistance ofH69AR is unknown at the present time.

We have recently produced a panel of murine Mabs specificfor H69AR cells. These MAbs will be useful in the isolationand characterization of membrane components involved in theM DR phenotype in H69AR SCLC cells. Furthermore, theseMAbs may prove to be valuable aids in the detection of drug-resistant cells in SCLC patients.

ACKNOWLEDGMENTSWe wish to thank Norbert Kartner for his generous provision of

MAbs C219 and C494 and Dr. Victor Ling and Dr. Jeff Trent forhelpful discussions. The excellent technical assistance of Ingrid Louw-man, Elisabeth Vreeken, and Deanna Evernden-Porelle is gratefullyacknowledged. This paper is dedicated to the memory of Ingrid Louw-

man.

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1987;47:2594-2598. Cancer Res   Shelagh E. L. Mirski, James H. Gerlach and Susan P. C. Cole  Line Selected in AdriamycinMultidrug Resistance in a Human Small Cell Lung Cancer Cell

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