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[CANCER RESEARCH 52. 3029-3034, June I. 1992]

Multidrug-resistant Phenotype of Disease-oriented Panels of Human Tumor Cell

Lines Used for Anticancer Drug Screening

Lin Wu, Anne M. Smythe, Sherman F. Stinson, Leslie A. Mullendore, Anne Monks, Dominic A. Scudiero,Kenneth D. Paull, Antonis D. Koutsoukos, Lawrence V. Rubinstein, Michael R. Boyd, and Robert H. Shoemaker1

Laboratory of Drug Discovery Research and Development [L. W., A. M. S., S. F. S., L. M., M. R. B., R. H. S.J, and Information Technology Branch Â¡K.D. P.],Developmental Therapeutics Program, and Biometrie Research Branch [A. D. K., L. V. R.], Cancer Therapy Evaluation Program, Division of Cancer Treatment, NationalCancer Institute; and Program Resources Incorporated Â¡A.M., D. A. S.J, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick,Maryland 21702

ABSTRACT

Disease-oriented panels of human tumor cell lines used by the NationalCancer Institute for large-scale in vitro anticancer drug screening wereevaluated for multidrug-resistant phenotype at the functional (in vitrodrug sensitivity) and molecular levels. The cell line panels manifested abroad range of sensitivities to drugs typically associated with multidrugresistance (MDR) as well as to drugs not associated with MDR. Individual cell lines displayed unique and characteristic profiles of response.Patterns of correlated response were observed among, but not between,MDR and non-MDR drugs. Strong evidence of correlated response waslimited to drugs sharing an intracellular mechanism of action. Severaltumor cell lines exhibited a high degree of resistance to MDR drugs andrelative sensitivity to non-MDR drugs, contained high levels of MDR-1uiKN'A, and expressed cell surface P-glycoprotein detectable with one or

more monoclonal antibodies. Parallel expression of all of these featuresrepresenting the classic MDR phenotype was observed among membersof the colon and renal tumor panels. Certain individual cell lines amongother panels (lung, ovarian, melanoma, and central nervous system) alsomanifested some aspects of the MDR phenotype to various extents.Identification of MDR cell lines used for large-scale in vitro anticancerdrug screening will facilitate interpretation of data in a way which mayallow identification of new drug leads of potential value in treatment ofMDR tumor cell populations.

INTRODUCTION

MDR2 has been described as a phenomenon wherein a cell

population selected in vitro for resistance to a single agentsubsequently exhibited broad cross-resistance to other chemically unrelated agents to which the cells had never been exposed(1-3). This phenomenon has been extensively studied at thecellular and molecular levels, and several recent reviews of thesubject are available (4-9). The central feature of MDR is amembrane P-glycoprotein (P-170) which serves as a transmembrane pump of broad substrate specificity in extruding a varietyof relatively large polycyclic compounds from the cell, reducingintracellular accumulation of drugs and thereby reducing theireffects on cells. In addition to this "classic" form of MDR,

atypical or variant forms also have been described whereincross-resistance is not associated with operation of the efflux

Received 10/25/91; accepted 3/20/92.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.

' To whom requests for reprints should be addressed, at Laboratory of Drug

Discovery Research and Development, Developmental Therapeutics Program.Division of Cancer Treatment, National Cancer Institute-Frederick Cancer Research and Development Center, Building 1052. Room 121, Frederick, MD21702-1201.

2The abbreviations used are: MDR. multidrug resistance; VP-16. etoposide:VM-26, teniposide; NCI. National Cancer Institute; TCA. trichloroacetic acid:T,, time of drug addition: (â€¢!.,,.interpolated drug concentration resulting in apercentage of growth inhibition of 50 (50% less growth in net cell protein whencompared to the untreated control cell protein at the end of the incubation period;Ref. 28); PCR, polymerase chain reaction; AMSA, amsacrine; CNS, centralnervous system; BCNU, l,3-bis(2-chloroethyl)-l-nitrosourea; CCNU, l-(2-chlo-roethyl)-3-cyclohexyl-l-nitrosourea; CBDCA, carboplatin; 5-FUDR. floxuridine.

pump (10, 11). Alterations in DNA topoisomerase II have beenidentified in a variety of human tumor cell lines which wereselected in vitro for resistance to VP-16 or VM-26 (10-14).These variant MDR cell populations typically do not showcross-resistance to Vinca alkaloids and do not overexpressMDR-1 or P-glycoprotein.

Molecular studies have resulted in cloning and sequencing ofthe MDR-1 gene and demonstration that it codes for themembrane glycoprotein (15-17). This, together with development of antibodies to P-glycoprotein (18-20), has facilitateddemonstration of the multidrug-resistant phenotype amonghuman tumor cell populations. Occurrence of MDR is nowthought to represent one of the greatest challenges to development of curative chemotherapy regimens for many of the common adult solid tumors (21, 22).

For several years, the NCI has been working toward development of a large-scale, in vitro anticancer drug screen, basedon the use of disease-oriented panels of human tumor cell lines.The rationale for this screening model and methods used aredescribed in detail elsewhere (23-25). The primary logic usedfor selection of drug leads from this screen has been to prioritizesynthetic compounds or natural product samples showing selective growth inhibition and/or cell killing of particular tumortypes for further evaluation. In order to allow detection of drugleads with special potential for treatment of MDR cell populations, we undertook to identify MDR cell lines in the currentdisease-oriented drug-screening panels.

The objective of the present study was to determine the extentto which cell lines included in the screening panels displayMDR. This was approached by evaluating the response of thecell lines to drugs associated with MDR, by assessing levels ofMDR-1 mRNA, and by evaluating expression of P-glycoproteinusing immunocytochemical techniques and monoclonal antibodies to various epitopes of the glycoprotein.

MATERIALS AND METHODS

Cell Lines. Cell lines were obtained and processed as describedpreviously (23, 26). In brief, this entailed in vitro expansion andcryopreservation of a "master stock," followed by detailed characteriza

tion which included evaluation for the presence of adventitious agentsand mouse or human pathogens, karyology, isoenzyme analysis, tu-morigenicity testing, and morphological analysis at the light and electron microscopic levels.

In Vitro Assay Method. Cellular response to drugs was evaluatedusing a sulforhodamine B assay as previously described (25, 27). Briefly,the human tumor cell lines comprising the screening panels wereroutinely grown in RPMI 1640 medium containing 5% fetal bovineserum and 2 mM L-glutamine. Cells were inoculated into 96-wellmit-miiUT plates in 100 ni of complete medium at densities ranging

from 5,000 to 40,000 cells/well depending on the protein content andgrowth rate of the individual cell line (25). The microtiter platescontaining cells were incubated for approximately 24 h prior to additionof drugs. After 24 h, two plates of each cell line were fixed in situ with

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MDR PHENOTYPE OF TUMOR CELL LINE PANELS

TCA, to represent a measure of cell population for each cell line at T:.Experimental drugs were solubili/ed initially in dimethyl sulfoxide

at 400-fold the desired final maximum test concentration, with theexception of c/s-platinum which was diluted into medium immediatelyprior to addition of cells. The dimethyl sulfoxide "stock" concentrateswere made into multiple aliquots and stored at â€”¿�70Â°Cto provide

uniform samples for initial and subsequent tests. At the time of drugaddition, an aliquot of frozen concentrate was thawed at room temperature and then diluted with complete medium containing 50 Mg/mlgentamicin to twice the desired final maximum test concentration. Anadditional four, 10-fold serial dilutions were made to provide a total offive concentrations, spanning a 4-log concentration range. Aliquots(100 iA)of these intermediate dilutions were immediately added to theappropriate microtiter wells already containing 100 n\ of medium,resulting in the required five concentration levels.

Following the addition of agents to microtiter plates, the plates wereincubated for an additional 48 h at 37Â°C,5% CO2, and 100% humidity.

After this time, adherent cells were fixed in situ by gently adding 50 ^1of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubatingfor 60 min at 4Â°C.The supernatant was discarded, and the plates were

washed five times with deionized water and air dried. SulforhodamineB solution (100 M!)(Sigma Chemical Co., St. Louis, MO) at 0.4% (w/v) in 1% acetic acid was added to each microtiter well, and incubationwas continued for 10 min at room temperature. After the plates werestained for 10 min, excess unbound dye was removed by washing fivetimes with 1% acetic acid, and then the plates were air dried. Boundstain was solubilized with Tris buffer, and the absorbances were readon an automated plate reader at a wavelength of 515 nm. For suspensioncell cultures, i.e., the leukemias, the method was the same except that,at the end of the drug incubation period, the settled cells were fixed tothe bottom of the microculture well by gently adding 50 n\ of 80% TCA(final concentration, 16% TCA).

Using the seven absorbance measurements made [time zero (7"7),

control growth (C), and test growth in the presence of drug at the fiveconcentration levels (7",;i = !.. .5)], we calculated the percentage growth

inhibition at each of the five concentration levels. Percentage growthinhibition was calculated to be

TÂ¡- T,x 100

C- T:

for concentrations for which 7",> T: and

r, -T,

x 100

for concentrations for which 7",< T.. We also calculated the GI50value

(28).Evaluation of MDR-1 mRNA Levels. Levels of MDR-1 mRNA were

estimated relative to /3-actin mRNA using a modification of the RNAPCR method of Fuqua et al. (29). Total RNA was prepared by extraction of exponentially growing cells with guanidinium thiocyanate followed by ultracentrifugation over a cesium chloride cushion (30). Afterisolation, RNA was extracted with phenol/chloroform and precipitatedwith ethanol. RNA was then examined by agarose gel electrophoresisfor intact ribosomal RNA bands. RNA concentration was estimatedspectrophotometrically prior to use in the PCR assay.

Reverse transcription was carried out with total cellular RNA asfollows: a "master mix" was prepared for 60 reactions and aliquoted

into Eppendorf microcentrifuge tubes. Each tube contained a totalvolume of 100 n\ composed of 10 mM Tris-HCl (pH 8.3), 50 miviKC1,3 mM MgCl2, 0.01% gelatin, 800 JJMeach of the four deoxyribonucleo-tide triphosphates, and 1 MMof either MDR-1 primer 1 and primer 2or /3-actin primer 1 and 2 (29). Primers were used as specified by Fuquaet al. (29) and were prepared by the Nucleic Acid and Protein SynthesisLaboratory, Program Resources Inc., NCI-Frederick Cancer Researchand Development Center. The sense MDR-1 primer 1 (5'-ATAT-CAGCAGCCCACATCAT-3') corresponds to MDR-1 cDNA 3007-3026 (31). The MDR-1 primer 2 (5'-GAAGCACTGGGATGTCCGGT-3') corresponds to the antisense strand of MDR-1 cDNA se

quence 3141-3160. The sense /3-actin primer 1 (5'-ATCATGTTTGA-GACCTTCAA-3') corresponds to /3-actin cDNA 1854-1873 and 0-actin primer 2 (5'-CATCTCTTGCTCGAAGTCCA-3') represents the

antisense strand of the cDNA sequence 2151-2170 (32). One ^g oftotal cellular RNA derived from each cell line was added to individualreaction tubes. Tubes were heated to 94Â°Cfor 2 min to denature the

RNA and then cooled rapidly on ice. Two units of avian myeloblastosisvirus reverse transcriptase was added to each tube, and the reaction wasallowed to proceed at 42Â°Cfor 45 min. Thermostable DNA polymerase

(Taq-1 ; Perkin-Elmer/Cetus, Norwalk, CT) was added to each tube (2.5units), and MDR-1 or /3-actin cDNA products, if present, were amplified by PCR. Each cycle of amplification consisted of a 1-min denatu-ration at 94Â°Cfollowed by 2-min annealing at 58Â°Cand 3-min extensionat 72Â°C.Amplification was carried out for 35 cycles with the extensiontime for the final cycle extended to 7 min at 72Â°C.

PCR products obtained by amplification of /3-actin or MDR-1 sequences were combined, in equal proportions, heated to 94Â°Cfor 10

min, and then loaded onto 2% agarose gels. Electrophoresis was performed in buffer (0.045 M Tris-borate, 0.00l M EDTA) at 100 V for 2h. Gels were stained with ethidium bromide, examined on a UVtransilluminator, and photographed.

Following electrophoresis, PCR products were depurinated, denatured, and transferred to nylon hybridization membranes using a modification of the method of Southern (33). After transfer, DNA was UVcross-linked to the membranes at 0.3 J/cm2 using a Bioslink 312

apparatus (Bios, New Haven, CT).The membranes were hybridized with a mixture of two biotinylated

probes. The MDR-1 probe is a Hindl\\/Pst\ 1.3-kilobase product fromthe pMDR2000XS clone (16). The /3-actin probe is a BamHl 2-kilobaseproduct from the pHF/3a-l clone (34). Fragments were labeled by nicktranslation using a kit from Oncor (Gaithersburg, MD), and the extentof biotin incorporation was quantitated by titration against a standardprovided in the kit.

Probing of membranes and colorimetrie detection was carried outunder the conditions specified in a kit which was also obtained fromOncor. Briefly, this entailed prehybridization at 42Â°Cfor 30 min andhybridization overnight at 42Â°Cwith each probe at a final concentration

of 12.5 ng/ml, followed by washing and color detection using streptav-idin/alkaline phosphatase and 5-bromo-4-chloro-3-indolyl phosphateand nitroblue tetra/oliimi. Precipitated nitroblue tetrazolium producesa blue color at sites on the membrane corresponding to the locationsof the hybridized probes for MDR-1 and /3-actin DNA sequences.Following color development, membranes were baked at 80Â°Cin a

vacuum oven for 30 min and then scanned on a flat-bed scannerinterfaced to a Macintosh llfx computer. Densitometry was performedusing the "Image" program.3 MDR-1 mRNA levels for each cell line

were expressed relative to mRNA levels for /3-actin.Immunocytochemical Characterization of P-Glycoprotein Expression.

Cells were harvested when 70-90% confluent by scraping and appliedto Teflon-coated glass slides with multiple, uncoated assay wells for

immunoperoxidase staining with a panel of three monoclonal antibodies reactive with P-glycoprotein. Slides were air dried and assayed usingthe avidin-biotin complex technique (Vectastain Elite kit; Vector Laboratories, Burlingame, CA). Optimal antibody concentrations weredetermined by titration against known positive and negative cell lines.Dilutions used were 1:10 for C219 (Centocor Diagnostics, Malvern,PA), 1:50 for JSB-1 (Sanbio bv, Portland, ME), and 1:250 for MRK-16 (T. Tsuruo, Tokyo, Japan). Slides were evaluated microscopically atx400 magnification, and 100 cells were counted in each of threeseparate fields. The percentage of positive cells was scored, and therelative staining intensity was qualitatively assessed on a scale of 1 (+)to 3 (+++). A semiquantitative "staining index" was calculated as the

product of the percentage of positive cells and the relative stainingintensity. A minimum of five assays at different passage levels for eachcell line was used for calculation of average staining index.

3A public domain image analysis program obtained from Wayne Rasband,NIH, Bethesda, MD.

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RESULTS

Identification of MDR Cell Lines Based on In Vitro DrugSensitivity. A functional analysis of MDR was conducted basedon relative sensitivities of cell lines to drugs typically associatedwith MDR and those not associated with MDR. We selected agroup of MDR drugs as shown in Table 1. These are relativelylarge molecules and, with the exception of AMSA and thesemisynthetic VP-16, are derived from natural sources. Thenon-MDR drugs selected for study were comprised of relativelylow molecular weight alkylating agents and antimetabolites anda single antitumor antibiotic (bleomycin). Each drug was testedagainst the entire panel of 62 cell lines on at least three differentdays.

For each drug, relative cell line sensitivities were measuredby ranking the log(GIso) values (35) (averaged over 3 or morerepeated tests) for the 62 cell lines. For each drug, the rank of1 was assigned to the cell line with the lowest average log(GI?0)value and, thus, the greatest sensitivity to that particular drug.After the relative sensitivities of the cell lines were ranked foreach drug, the relative sensitivity of each of the cell lines to theMDR drugs versus the non-MDR drugs was measured as follows. For each cell line, the rankings associated with the 7MDR drugs were averaged, as were the rankings associatedwith the 15 non-MDR drugs. These two averages are given, foreach cell line, in Table 2. Also given in Table 2 are the differences, mean MDR rank minus mean non-MDR rank. Themagnitude of this difference is a measure of the degree to whichthe cell line displays relative drug sensitivities consistent witha classic MDR phenotype (i.e., showing significantly greatersensitivity to the non-MDR versus the MDR drugs). We takethis difference as an "MDR phenotype score" and list the cell

lines in Table 2 in decreasing order. We also compared, foreach cell line, the relative rankings associated with the MDRdrugs with those associated with the non-MDR drugs by meansof the Wilcoxon test, in order to test the statistical significanceof the MDR phenotype suggested by the highest differencescores. The 9 cell lines for which significant or suggestive Pvalues were obtained (P < 0.10, two-sided) are listed in Table3. It can be seen that the cell lines with the highest 6 differencescores all demonstrate the MDR phenotype at the P < 0.05significance level.

Patterns of cell line sensitivities were compared, across MDRand non-MDR drugs, by performing a Spearman rank ordercorrelation analysis on the cell sensitivity rankings for theindividual drugs. Table 4 gives a complete listing of the correlations exceeding 0.80. It can be seen that two drugs must beeither both MDR or both non-MDR to have such a high cell

line sensitivity correlation.Relative Expression of M DR-1 mRNA among Cell Lines.

RNA extracted from the tumor cell lines was subjected to twoindependent PCR procedures and evaluated as described in"Materials and Methods." To facilitate comparison with other

parameters, results are included in Table 2 as the average ofthe MDR-1 to /3-actin mRNA ratios obtained from these reac

tions for the various tumor cell lines.Immunocytochemical Characterization of P-Glycoprotein

Expression. Results of immunocytochemical staining of tumorcell lines derived from solid tumors for P-glycoprotein usingthree different monoclonal antibodies are also listed in Table2. Leukemic cell lines were not included in this analysis becauseof difficulties in scoring the peroxidase staining in these cellswhich contain only rather small amounts of cytoplasm.

Table 1 Classification of drugs utilized for analysis

Drugs associated withMDRActinomycin

DVinblastinesulfateVincristinesulfateDaunomycinAdriamycinVP-16AMSADrugs

not associated wtihMDRThioguanine6-Mercaptopurine5-Fluorouracil5-FUDRHydroxyurea1

-/3-D-ArabinofuranosylcystosineBusulfanNitrogen

mustardChlorambucilMelphalanCCNUr/i-platinumCBDCABCNUBleomycin

DISCUSSION

The cell lines included in these disease-oriented panels displayed a broad range of in vitro sensitivities to individual drugs.To facilitate recognition of multidrug-resistant cell lines, thedata in Table 2 are arranged in order of decreasing differencebetween the average rank for MDR and non-MDR drugs, i.e.,

the MDR phenotype score. Thus, the cell lines listed at the topof the table show an in vitro drug response pattern mostconsistent with the multidrug-resistant phenotype. Non-MDRdrugs have been described to exhibit a lack of cross-resistancewith MDR drugs or to demonstrate "collateral sensitivity" in

drug-selected MDR cells relative to wild-type populations (1).As indicated in Table 3, a formal analysis for cell lines showingthis sort of phenomenon identified nine cell lines showingsignificantly greater relative resistance to MDR drugs. Theseincluded the MCF-7/ADR breast cancer cell line and two CNS,two colon, one melanoma, one ovarian, and two renal tumorlines.

Comparison of the rank order pattern of cell line sensitivitiesto the MDR and non-MDR drugs yielded a number of interesting correlations. As indicated in Table 4, strong correlationswere observed among some MDR and some non-MDR drugs.The strongest correlations were obtained for drugs which shareintracellular mechanisms of action. For the seven MDR drugs,five correlation coefficients >0.80 were observed and all relatedto other MDR drugs. Strong correlations were noted amongagents thought to affect DNA topoisomerase II as part of theirintracellular mechanism of action (Adriamycin, VP-16, andAMSA). Such correlations between relative cellular sensitivitiesand intracellular mechanism of action were recently used as thebasis for selection of halichondrin B and homohalichondrin Bfor examination as tubulin-binding compounds (36). The extentof the correlations observed among MDR drugs may reflect themultifactorial nature of cellular responses to drugs: the intracellular drug concentration, as modulated by MDR-1 geneexpression, undoubtedly acts in concert with the quantity/quality of target molecule(s) and other factors to define drugsensitivity phenotype.

In the case of non-MDR drugs, strong correlations wereobserved between the alkylating agents BCNU and CCNU,melphalan and chlorambucil, and cisplatin and CBDCA. In thecase of antimetabolites, only thioguanine and 6-mercaptopurineshowed a correlation coefficient >0.80.

MDR-1 mRNA levels estimated by PCR varied broadly. Asshown in Table 2, the relative amount of MDR-1 mRNA in theMCF-7/ADR cell line, which was selected in vitro for resistanceto Adriamycin (37), was quite high (0.8). The HCT-15 colonand ACHN renal cell lines showed even higher levels of MDR-

3031




Table 2 Cellular parameters relevant to multidrug resistance

TumortypeBreastOvaryCNSRenalColonRenalRenalColonOvaryMelanomaRenalMelanomaLungOvaryCNSOvaryLeukemiaColonCNSLungRenalCNSRenalMelanomaRenalLungCNSLeukemiaLungMelanomaLungOvaryLeukemiaLungLeukemiaSCLCColonOvarySCLCLungMelanomaColonLungColonMelanomaMelanomaRenalRenalLeukemiaLungBreastMelanomaColonMelanomaCNSCNSColonColonLeukemiaLungCNSLungCelllineMCF-7/ADROVCAR-4SF-268UO-31HCT-15RXF-393CAKI-1DLD-1OVCAR-5MI4ACHNUACC-62HOP-

18IGROV1SF-539OVCAR-3HL-60HCC-2998SF-295HOP-92786-0SNB-78RXF-631UACC-257TK-10NCI-H23SNB-75CEMHOP-62SK-MEL-28EKVXSK-OV-3MOLT-4NCI-H460SRDMS-114HCT-116OVCAR-8DMS-273NCI-H522LOXKM20L2A549COLO-205MALME-3MM19-MELA498SN12CK-562NCI-H322MMCF-7SK-MEL-5HT29SK-MEL-2SNB-19U251KM12SW-620RPMI-8226LXFL-529LXF-498NCI-H226MDR

rank"60.8656.7133.0053.7152.0043.7130.2949.7151.5734.2929.7120.0050.4342.2921.4342.7115.8641.8626.4332.2920.1450.4334.6443.2954.0725.2934.5713.1426.2946.8656.5744.148.438.714.8618.7125.2930.7110.0720.4310.0036.4320.5734.5029.1436.0041.0022.8626.8646.4311.7122.7132.7141.1428.0014.7135.1418.7114.5727.5713.4323.29Non-MDRrank36.4039.2716.1337.5336.4030.5318.4738.8741.7325.3320.9311.3342.9335.0014.8736.8010.3336.5321.6727.6016.2046.6731.0739.8051.6723.2032.7312.1325.5346.4056.6044.709.7310.076.8721.2728.5034.0014.2024.6014.2040.9325.1339.3334.2741.2046.4728.8733.2753.0019.0730.7341.4050.6738.8027.0048.2033.2729.4046.3333.4743.40MDR-1mRNA*0.8000.0000.0450.6500.9600.3650.150NO1*0.000ND0.9300.4150.0000.0400.0000.0000.0400.0000.1870.0000.425NDND0.0000.4500.0000.0750.0900.1250.2500.6500.1150.000NDND0.0000.1850.1720.1300.0000.0000.2250.0000.0350.0000.1100.5250.085ND0.0000.0000.3650.0000.2200.0000.2520.0000.4350.000NDND0.045Staining

indexcMRK-161.800.020.000.010.920.000.170.060.000.000.000.000.000.000.000.00ND0.000.000.000.000.000.000.000.000.000.00ND0.000.000.170.00ND0.00ND0.000.000.000.000.000.000.000.000.310.000.000.000.00ND0.000.000.000.000.000.000.000.000.08ND0.000.000.00JSB-11.000.220.000.181.010.000.470.590.000.000.450.000.000.000.000.00ND0.000.000.000.090.000.000.000.000.000.00ND0.000.000.400.00ND0.00ND0.000.150.070.000.000.000.000.000.300.000.000.000.00ND0.000.000.000.000.000.000.000.000.00ND0.000.000.62C2190.250.000.000.000.630.000.340.320.000.000.000.000.000.000.000.00ND0.000.000.000.000.000.000.000.000.000.00ND0.000.000.450.00ND0.00ND0.000.000.000.000.000.000.000.000.000.000.000.000.00ND0.000.000.000.000.000.000.000.000.00ND0.000.000.24Rank

difference24.4617.4416.8716.1815.6013.1811.8210.849.848.968.788.677.507.296.565.915.535.334.764.693.943.763.573.492.402.091.841.010.760.46-0.03-0.56-1.30-1.36-2.01-2.56-3.21-3.29-4.13-4.17-4.20-4.50-4.56-4.83-5.13-5.20-5.47-6.01-6.41-6.57-7.36-8.02-8.69-9.53-10.80-12.29-13.06-14.56-14.83-18.76-20.04-20.11

"The rank order sensitivity of each cell line to each of the drugs shown in Table 1 was established, and the mean rank for the MDR and non-MDR drugs wascalculated. Mean ranks for each category of drug as well as the difference between the MDR and non-MDR mean ranks are tabulated. Cell lines are listed in order ofdecreasing order of difference between the ranks.

* Relative levels of MDR-1 and /3-actin mRNA were estimated by densitometric analysis of Southern transfers prepared from titrations of the PCR products asdescribed in "Materials and Methods." The MDR-1 to /3-actin ratios are listed. Cell lines listed as 0.00 showed no MDR-1 PCR product measurable within the linearrange of the assay for measurement of the fi-actin PCR product for this cell line.

c Staining index was calculated as the product of the percentage of positive cells and the staining intensity as described in "Materials and Methods." Cell lines

listed as 0.00 showed no detectable P-glycoprotein under these assay conditions.d ND. not done.

1 mRNA (0.96 and 0.93, respectively), while the EKVX lungand UO-31 renal tumor cell lines were also high (0.65). Twenty-one of the cell lines showed no MDR-1 PCR product in thelinear range of the assay for measurement of /3-actin PCR

product. For the purposes of the correlation analysis, and listingin the Table 2, these were considered to be zero values. How

ever, this is not to say that MDR-1 mRNA was entirely absentfrom the cells, since in many cases, a signal could be detectedwhen very large amounts of PCR products were loaded ontothe gels. MDR-1 to /3-actin ratios for the remainder of the cell

lines fell between 0.035 and 0.525. We note that three of thefive lines which show the highest MDR-1 mRNA levels are

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Table 3 Cell lines showing differential sensitivity to non-MDR versus MDR drugs

TumortypeBreastOvaryCNSRenalColonMelanomaRenalColonCNSCelllineMCF-7/ADROVCAR-4SF-268UO-31HCT-15UACC-62RXF-393DLD-1SF-539Wilcoxonscore3.68643.00952.85812.71772.36482.01301.9411.76831.6595Pvalue0.00020.00260.00430.00660.01800.04410.0522Â°0.0770"0.0970"

" P values of borderline significance.

also among the five lines which have the greatest MDR phe-notype score (mean MDR rank minus mean non-MDR rank)and which demonstrate most significantly (P< 0.01, one-sided)the classic MDR drug sensitivity pattern (in Table 3). However,the overall Spearman rank correlation between MDR pheno-type score and MDR-1 to /3-actin ratios is only 0.178.

Immunocytochemical evaluation revealed only a limitednumber of cell lines expressing detectable P-glycoprotein. Table2 lists results obtained for these lines using three monoclonalantibodies which recognize different P-glycoprotein epitopes.While the methods used here are best considered semiquantitative, some heterogeneity in degree of expression of the variousepitopes was apparent. The distribution of cell lines positivefor P-glycoprotein and/or MDR-1 mRNA across tumor typesshows considerable analogy to reported patterns of P-glycoprotein expression in normal tissues (33-40), i.e., cell lines derived

from tumors of the kidney and colon were most frequentlypositive. However, P-glycoprotein was also detected in somenon-small cell lung and ovarian carcinoma cell lines.

The P-glycoprotein staining indices obtained using the threemonoclonal antibodies correlated well, using Spearman rankcorrelation analysis. The correlation coefficients were 0.721,0.678, and 0.717 for MRK-16 versus JSB-1, MRK-16 versusC219, and JSB-1 versus C219, respectively. However, MDRphenotype scores yielded Spearman rank correlation coefficients of only 0.319, 0.329, and 0.243 with MRK-16, JSB-1,and C219 staining indices, respectively. MDR-1 to /3-actinratios yielded Spearman rank correlation coefficients of only0.368, 0.432, and 0.349 with MRK-16, JSB-1, and C219 staining indices, respectively.

The relationships among the results of the functional in vitrodrug sensitivity assays, the MDR-1 mRNA levels, and P-glycoprotein expression are complex. Numerous individual celllines manifested one or more features of MDR. Only three linesdisplayed all of the features examined in this study: a highdegree of resistance to MDR drugs and relative sensitivity tonon-MDR drugs, high levels of MDR-1 mRNA, and immuno-cytochemically detectable P-glycoprotein. These may be considered classic MDR lines and include the MCF-7/ADR breastcancer line, the colon tumor line HCT-15, and the renal tumorline UO-31. The experimentally derived MCF-7/ADR cell lineand the wild-type MCF-7 line were initially included in a pilotversion of the in vitro drug-screening panels (23) and served asuseful positive and negative controls for the present study.However, they are not included among the 60-cell line panelnow being used by NCI for routine primary drug screening (25).The cell lines included in the current panel were not selectedon the basis of reported or measured in vitro drug sensitivitybut rather for retention of differentiated features characteristicof the various tumor types and acceptable growth characteristicsunder the conditions required for the drug-screening assay.Among these cell populations, two cell lines have been shown

here to display a classic MDR phenotype very similar to MCF-7/ADR. It is interesting to note that the UO-31 renal and HCT-15 colon tumor lines displaying this phenotype were derivedfrom untreated patients. The OVCAR-4, TK-10, and EKVXcell lines demonstrated a high degree of resistance to MDRdrugs and at least one of the features of the MDR phenotypeevaluated in this study. The latter two lines did not showincreased sensitivity to the non-MDR drugs but rather a pan-resistant phenotype. Examination of the detailed drug sensitivity data (not shown) suggests that the NCI-H322M cell linemay represent an atypical MDR cell line. This line showed ahigh degree of resistance to most of the MDR drugs but didnot show MDR-1 mRNA or P-glycoprotein and was relativelysensitive to the Vinca alkaloids tested. This cell line may wellhave altered DNA topoisomerase activity as part of the basisfor the observed resistance.

The results presented here illustrate the complexity of MDRand support the notion described elsewhere that MDR can bemanifested in the absence of the selective pressure of drugtreatment (41, 42). Our data indicate that MDR is not an allor-none phenomenon but rather represents a continuous gradation of phenotype. The genetic background of the tumor cellpopulation appears to modulate expression of MDR-1. In somecell lines, notably several of the melanomas, relatively highlevels of MDR-1 mRNA were found in the absence of detectibleP-glycoprotein. This may, in part, be explained by phenomena

such as epitope masking related to posttranscriptional modification (sialylation) of P-glycoprotein which has recently beendescribed in leukemic cell populations (43). Conversely, one ofthe ovarian carcinoma cell lines (OVCAR-4) was reactive with2 of 3 P-glycoprotein antibodies, yet no MDR-1 mRNA wasmeasurable in our assay. This may reflect altered stability ofMDR-1 mRNA and/or P-glycoprotein in these cells. Differences in reactivity of the monoclonal antibodies may also resultfrom differences in cross-reactivity with other cellular epitopes.In screening the cell line panels, we established a single protocolfor cellular preparation, fixation, and staining. Somewhat different results, and perhaps better sensitivity for individual antibodies, might be obtained using other protocols. Furthercomplexity is layered onto this situation, since factors otherthan MDR-1 gene expression, such as tubulin, topoisomerase,and DNA repair phenotype, are also likely to be important indefining cellular response to MDR drugs.

The disease-oriented cell line panels examined in this studywere assembled for use in large-scale in vitro anticancer drugscreening. While the primary intent of this screen has been toidentify drugs with selective growth inhibitory or cell-killingactivity toward particular tumor types, we have found that thedata derived from the screen can also convey useful informationrelating to mechanism of drug action (35). In this report, wehave identified tumor cell lines which express a classic MDRphenotype as well as others displaying MDR to lesser extentsor which may represent variant or atypical forms of MDR.

Table 4 Drug response correlations

Drugs Correlation coefficient

Adriamycin and daunomycinAdriamycin and VP-16Daunomycin and VP-16Daunomycin and AMSAVP-16 and AMSABCNU and CCNUMelphalan and chlorambucilc/s-Platinum and CBDCAThioguanine and 6-mercaptopurine

0.900.880.840.820.860.830.920.880.82

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Drug screening with these cell lines is currently in progress atthe rate of approximately 20,000 samples per year (25). Theinformation presented here will facilitate the retrospective andcontinuing surveillance of screening data for detection of newdrug development candidates which may have special potentialfor treatment of MDR cell populations.

ACKNOWLEDGMENTS

We thank Dr. Michael Gottesman and Dr. Larry Kedes for providingthe MDR-1 pMDR2000XS and 0-actin pHF/Ã•A-1clones and Dr.Takashi Tsuruo for supplying the MRK-16 monoclonal antibody. Angela Pittman provided expert technical assistance with immunocyto-chemistry procedures. In addition, we express our appreciation to themany colleagues who have contributed human tumor cell lines for usein the in vitro drug-screening project.

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1992;52:3029-3034. Cancer Res Lin Wu, Anne M. Smythe, Sherman F. Stinson, et al. Human Tumor Cell Lines Used for Anticancer Drug ScreeningMultidrug-resistant Phenotype of Disease-oriented Panels of

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