[CANCER RESEARCH 49, 2988-2993, June 1, 1989]

Correlation of Multidrug Resistance with Decreased Drug Accumulation, AlteredSubcellular Drug Distribution, and Increased P-Glycoprotein Expression inCultured SW-1573 Human Lung Tumor Cells1

Hiskias G. Keizer,2 Gerrit J. Schuurhuis, Henk J. Broxterman, Jan Lankelma, Willem G. E. J. Schoonen,Johan van Rijn, Herbert M. Pinedo, and Hans Joenje3

Institute of Human Genetics [H. G. K., W. G. E. J. S., H. J.] and Departments of Medical Oncology [G. J. S., H. J. B., J. L., H. M. P.] and Radiotherapy [J. v. R.], FreeUniversity, Amsterdam, The Netherlands

ABSTRACT

Four mull¡drug-resistantvariants of the human squamous lung cancercell line SW-1573 with levels of doxorubicin resistance ranging from 10-to 2000-fold were characterized with respect to drug accumulation andefflux, subcellular drug distribution pattern, antioxidant defenses, and P-glycoprotein expression. For all these parameters except the antioxidantdefenses a correlation was observed with the level of doxorubicin resistance; with increasing drug resistance cellular drug accumulation capacity(as measured for doxorubicin) progressively decreased, initial drug effluxrates (as measured for daunorubicin) progressively increased, while thesubcellular doxorubicin distribution (as measured by fluorescence microscopy) gradually shifted from a "mainly nuclear" to a "mainly cyto-plasmic" pattern. Our data suggest that in the present set of cell lines

the same mechanism of resistance is operating at all levels of doxorubicinresistance.

INTRODUCTION

The development of drug resistance is one of the mainobstacles to effective cancer chemotherapy. MDR4 or pleio-

tropic drug resistance constitutes a particular type of resistancewhich has been studied extensively in vitro and in which cross-resistance is expressed to a series of drugs, many of which areof natural origin. MDR is thought to be mediated by a membrane-bound Mr 170,000-180,000 glycoprotein termed P-gly-coprotein (1-5).

To elucidate the various mechanistic aspects of MDR, usuallycell lines with high levels of resistance are examined. Suchsystems, however, do not necessarily mimic the clinical situation, where relatively low levels of resistance may prevail. Wetherefore monitored a number of cellular parameters currentlyconsidered to be important for MDR in a set of four sublinesof SW-1573 human squamous lung tumor cells. The parametersstudied include (a) cellular drug accumulation, (/>) drug effluxrate and P-glycoprotein expression, (e) subcellular drug distribution, and (¡I)major components of the cellular antioxidantdefense system, including glutathione-5-transferase.

MATERIALS AND METHODS

Chemicals and Drugs. Doxorubicin (Adriblastina, produced by Far-mitalia, Carlo Erba, Milan, Italy) was a gift from Bergel Nederland

Received 11/18/88; revised 2/24/89; accepted 3/7/89.1Financial support from The Netherlands Cancer Foundation (Queen Wilhel

mina Fund) by Grant IKA VU-84-14 to H. J.; G. J. S. and H. J. B. were supportedby Grants IKA VU-88-22 and IKA-VU-85-5.

2Present address: Department of Toxicology, Duphar B.V., P. O. Box 2, 1380

AA Weesp, The Netherlands.3To whom correspondence should be addressed, at the Institute of Human

Genetics, Free University, P. O. Box 7161, 1007 MC Amsterdam, The Netherlands.

' The abbreviations used are: MDR, multidrug resistance; SOD, Superoxide

disimilase: PCS, fetal calf serum; GSH, reduced glutathione; GSHPX, glutathioneperoxidase; GSSG, oxidized glutathione; GST, glutathione-5-transferase;HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; IC50, dose giving50% growth inhibition.

B.V., Heerhugowaard, The Netherlands); VP-16-213 (etoposide) wasfrom Bristol Myers, Bergisch Gladbach, Federal Republic of Germany;colchicine and menadione were from Sigma Chemical Company, St.Louis, MO; mitomycin C was from Kyowa Hakko Kyogo Co., Ltd.,Tokyo, Japan; bleomycin was from Lundbeck B.V., Amsterdam, TheNetherlands; cisplatin was from Bristol Myers S.A.E., Madrid, Spain;verapamil was from Knoll AG, Ludwigshafen, Federal Republic ofGermany; vincristine sulfate was from Sigma; and daunorubicin wasfrom Specia (Paris, France). All chemicals and drugs were dissolvedfreshly before use, except doxorubicin, which was stored in a stocksolution of 5 HIMin 0.9% NaCl solution at -20°Cin the dark. All drug

solutions were prepared as lux concentrated stock solutions in 0.9%NaCl solution, pH 7.4, and diluted in 10 volumes of complete culturemedium (see below). Cisplatin and VP-16-213 powders were firstdissolved in dimethyl sulfoxide and further diluted in 0.9% NaClsolution. Final concentrations of dimethyl sulfoxide in the culturemedium (less than 0.5% v/v) did not influence cell growth.

Cells and Culture Conditions. Before the present study was undertaken, the human lung tumor cell line SW-1573, originally isolated andcharacterized as a squamous cell carcinoma by Dr. A. Leibovitz (Scottand White Clinic, Temple, TX), had been cultured for several years inthe laboratory of Dr. J. van Rijn (Department of Radiotherapy, FreeUniversity Hospital). Monolayers of SW-1573 cells and the doxorubi-cin-resistant sublines derived from them were grown in 25-cm2 tissueculture flasks (Nunc, Roskilde, Denmark) containing 5 ml Ham's F-10

medium supplemented with 10% PCS (Flow Laboratories, Ltd., Irvine,Scotland) and 2 HIMglutamine (Flow). Cells were maintained at 37°C

under air/3% CO2 and were subcultured every 3-4 days. All experiments were carried out with Mycoplasma-iree cells, as tested by "infection" and Hoechst staining of a Afycop/owia-sensitive indicator strain

of monkey kidney cells.Doxorubicin-resistant Cell Strains. Three resistant sublines were se

lected that were 10-, 250-, and 2000-fold more resistant to doxorubicinthan SW-1573 parental cells. After reaching these levels of resistancethe cells were cultured for at least 400 population doublings in thecontinuous presence of doxorubicin at 50, 500, and 10,000 nM, respectively, before their use in the present study. These cell lines were termedSW-1573/50, SW-1573/500, and SW-1573/10000, respectively. A stable partially revertant subline was obtained by culturing SW-1573/500cells for more than 200 population doublings in drug-free medium.This cell line was termed SW-1573/500-0.

Cell Growth. Doubling times of all five cell lines were determined byseeding IO5cells in 25-cm2 culture flasks and following their growth by

counting triplicate flasks over a period of 5 days using a Coulter Counter(C. C. Electronics Ltd., Harpenden, Hertfordshire, England). Growthwas found to be logarithmic from day 1 to day 4. The percentage oftrypan blue excluding cells was typically >95% for all cell lines.

Plating Efficiency. Plating efficiency was determined in triplicateinoculations of 100 or 400 cells in 25-cm2 culture flasks containing 5ml Ham's F-10 medium supplemented with 10% PCS and 2 HIM

glutamine. Colonies were fixed after 7 to 10 days and counted afterstaining with Giemsa. Plating efficiency was calculated by dividing thenumber of colonies per flask by the number of cells seeded, x 100%.

Growth Inhibition Assay. Approximately 5 x IO4cells, grown for 1week in the absence of drugs, were seeded in each well of a 6-well plate.After 24 h drug was added in various concentrations and cells wereallowed to proliferate for 4 more days, after which cell numbers werequantified by counting in a Coulter Counter. ICso values represent drug

2988

Research. on September 29, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

MULTIDRUG RESISTANCE IN HUMAN LUNG TUMOR CELLS

concentrations that gave 50% growth inhibition in this assay. Relativeresistance of sublines was calculated by dividing the IC50value of thesubline by that of the parental SW-1573 cells. The modifying effect of

verapamil on doxorubicin sensitivity was tested by incubating the cellssimultaneously with 20 /<Mverapamil and doxorubicin; verapamil remained present during the entire assay period. Growth inhibition by 20/IM verapamil was usually less than 25%.

Oxygen Consumption. Cellular oxygen consumption was determinedusing a Clark type electrode (Yellow Springs Instruments) with aBeckman 0260 oxygen analyzer. The reaction chamber volume was0.89 ml; the medium used was Ham's F-10 supplemented with 2 mM

glutamine and 10% PCS, buffered with 25 mM HEPES, pH 7.4. Afterthe medium was allowed to equilibrate for 10 min at 37"C approximately 5 x IO6cells were added and oxygen consumption was recorded.

The rate of oxygen consumption was stable for at least 10 min. Standarddeviations shown are based on three independent measurements.

Adenylate Energy Charge. The adenylate energy charge was determined from cellular AMP, ADP, and ATP levels, which were assayedby using the 3M A.E.C, kit from Lumac (Landgraaf, The Netherlands).Adenylate energy charge was calculated as

ATP + 0.5 ADPATP + ADP + AMP

Antioxidant Defenses. GST was assayed according to the method ofMannervik and Guthenberg (6), GSHPX according to the method ofGünzleret al. (7), Superoxide dismutases according to that of Joenje etal. (8), catalase following DelRio (9), and total glutathione (GSH andGSSG) following the method of Tietze (10).

Doxorubicin Accumulation. Cellular doxorubicin uptake was determined essentially as described by Howell et al. (11). Subconfluentcultures in Ham's F-10 complete medium were exposed to various

concentrations of doxorubicin for 2 h, rinsed twice with ice-cold phosphate-buffered saline containing 10 mM NaN3 (pH 7.4), and disruptedby freezing and thawing. Three ml butanol were added and allowed toextract the doxorubicin during 2 h at 37°C.After extraction 1 ml water

was added, and the phases were mixed vigorously on a vortex mixerand separated by centrifugation (30 min, 5000 x g). Doxorubicinconcentrations were determined in the butanol phase by fluorescencespectrometry using 475 and 580 nm as excitation and emission wavelengths, respectively. A standard concentration curve was constructedby adding known amounts of doxorubicin to disrupted untreated cellsand measuring doxorubicin fluorescence after extraction with butanol.Each measurement consisted of at least three independent extractions.

Subcellular Doxorubicin Distribution. Subcellular doxorubicin distribution was visualized with a Zeiss fluorescence microscope using aband pass filter of 515-560 nm, a long wave pass filter of 590 nm, anda chromatic beam splitter of 580 nm. Cells were incubated at 37"C for

15 min in HEPES-buffered saline containing 10 mM HEPES (pH 7.2),140 mM NaCl, 5 mM KC1, 1 mM CaCl2, 1 mM MgCl2, and 10 mMglucose, in the presence of 10-50 ^M doxorubicin. When testing theeffect of verapamil this drug was added 30 min before doxorubicin.Subcellular distribution patterns of doxorubicin were found not to bedependent on the extracellular doxorubicin concentration (up to 250MM);only the intensity (and not distribution pattern) of fluorescencechanged with the concentration of doxorubicin. Subcellular distributionof doxorubicin was similar when cells were incubated in completeculture medium (Ham's F-10 supplemented with 2 mM glutamine and

10% FCS) instead of supplemented saline, as defined above. Only twotypes of drug distribution were clearly distinguishable: mainly nuclearand mainly cytoplasmic. The first type was defined by clear-cut bordersof fluorescence exactly matching the form and place of the nucleus.The second type was defined as diffuse fluorescence, in which nucleiwere virtually free of fluorescence. Cases in which either cytoplasm ornucleus was not virtually free of fluorescence were scored as intermediate.

Drug Efflux. Initial daunorubicin efflux was monitored by a methodbased on the flow-through system recently described by Lankelma etal. (12). Cells attached to a glass plate, placed in a small chamber ofthe flow-through system, were placed in the light beam of a KontronUvikon 722 LC spectrophotometer at 37"C. This system allows the

continuous flow of medium over the cell layer, while the cellular drugcontent can be monitored spectrophotometrically. Cells were loadedwith daunorubicin by a 3-min exposure to growth medium containinga high concentration pulse of daunorubicin, with a maximum concentration of 1 mM. Initial drug efflux was recorded during elution withdrug-free growth medium at 37°C,measuring light absorption at a fixed

wavelength of 480 nm. After a first rapid decline representing a looselybound daunorubicin fraction the half-life of cellular daunorubicin wascalculated. The residual light absorption at 30 min after the pulse wastaken as the reference level.

P-Glycoprotein Expression. P-glycoprotein expression was assayedby using the monoclonal antibodies against P-glycoprotein MRK-16and JSB-I (13,14) and a control antibody (II6A3) directed against classI major histocompatibility complex antigens. P-glycoprotein expressionwas calculated as the absorption signal in enzyme-linked immunosor-bent assay using anti-P-glycoprotein monoclonals minus the absorptionsignal in enzyme-linked immunosorbent assay using control antibodiesdivided by cellular protein contents in the assay. P-glycoprotein expression in the most resistant cells was taken as a reference and set at 100arbitrary units.

RESULTS

Isolation of Doxorubicin-resistant Cell Lines. At the start ofthe selection procedure SW-1573 cells were grown in the continuous presence of 15 nM doxorubicin, which is the IC5oof theparental cell line. Cells were subcultured in the continuouspresence of 15 nM doxorubicin until the population-doublingtime was similar to that of control cultures. This procedure wasrepeated using 2- to 5-fold increments in doxorubicin concentration until, after a period of 2 years, the cells tolerated thecontinuous presence of 10 ^M doxorubicin. Cells were subcultured at this concentration for more than 100 population doublings. Cells that were adapted to growth at 50 and 500 nMduring this procedure were subcultured separately for at least100 passages at 50 and 500 nM, respectively, after which anydoxorubicin-induced growth inhibition had disappeared. Prolonged cultivation in the presence of these drug concentrationsapparently did not result in a further increase in the level ofdrug resistance. The sublines selected in this way were termedSW-1573/50, SW-1573/500, and SW-1573/10000, indicatingthe doxorubicin concentrations (nM) at which they were able togrow without a detectable inhibition.

Stability of Resistance. When at some point during the selection procedure the cells had reached an IC5o of 2000 nM, partof the cells were subcultured in drug-free medium. After 90days of culturing under drug-free conditions the cells had gradually lost part of their resistance and finally reached an ICso of650 nM. Further cultivation in drug-free medium for more than1 year did not lead to any further decrease in ICso values. Thissubline, termed SW-1573/500-0, was included in further studies.

General Characteristics. As shown in Table 1, SW-1573 andits doxorubicin-resistant sublines are rapidly dividing cells withdoubling times ranging from 16.0 to 22.7 h. Plating efficienciesof the cell lines were similar and ranged between 60 and 87%,except for the most resistant line which exhibited a platingefficiency of 35%. The adenylate energy charge was very similarin all cell lines and ranged from 0.68 to 0.72. Respiratoryactivity, which was found to be approximately 40% less thanthat reported for HeLa cells (8), was very similar in all sublines,except SW-1573/50 cells which respired at a somewhat suppressed rate.

Pattern of Cross-Resistance and Reversal by Verapamil. Wetested the cross-resistance to several drugs with various mechanisms of action. High levels of cross-resistance were observed

2989

Research. on September 29, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

MULTIDRUG RESISTANCE IN HUMAN LUNG TUMOR CELLS

Table 1 General characteristics ofSW-1573 cells and doxorubicin-resistant sublines

Population-doubling time (h)Plating efficiency*Oxygen consumption'

Adenylate energy chargeSW-1,57317.0

±0.4°

87 ±32.1 ±0.1

0.72 ±0.02SW-

1573/5018.6

±0.660 ±101.5 ±0.3

0.71 ±0.02S

W-1573/500-016.0

±0.675 ±2

2.1 ±0.10.68 ±0.03SW-

1573/50018.6

±0.278 ±2

2.2 ±0.10.69 ±0.02SW-1573/10,00022.7

±0.435 ±6

2.3 ±0.20.70 ±0.01"

Mean ±SD, calculated for three independent experiments.* Percentage of seeded cells able to form a colony of >50 cells.c fmol Oj/cell/min.

Table 2 Patterns of cross-resistanceResistancefactor*DoxonibicinDaunorubieinVP-16-213VincristineColchicineMitomycinMenadioneBleomycinCisplatinDoxorubicin

+ verapamil (20 ¡iM)1C»(SW-1,573)7.55670.37.53.818,0002301703.5SW-1

573/50103.6235.5232.01.40.50.69CSW-1573/500-04020S3695.02.01.11.00.47SW-1573/50025070270540624.01.10.40.2522SW-1573/10,0002,000ND750ND2207.02.60.80.793

°Cells were exposed continuously to the drugs indicated and 1C»values were determined in ng/ml.* Resistance factors were determined by dividing 1C»values of resistant variants by the 1C»value of SW-1573 control cells. ND, not determined.' Resistance factor with respect to verapamil-treated SW-1573 cells.

16-

8-

01 5 10Doxorubicin concentration,;uM

Fig. 1. Cellular doxorubicin accumulation. Cells were exposed for 2 h at 37'C

to the indicated concentrations of doxorubicin, after which cellular drug accumulation was determined as described in "Materials and Methods." Bars, SD for

at least three independent experiments.

_

4-

2-

0.5

i\

1 \

10 40 250 2000Doxorubicin resistance,-fold

Fig. 2. Cellular daunorubicin half-life as a function of drug resistance level.Burs, SD of 3-5 independent experiments.

Table 3 Effect of verapamil on doxorubicin accumulation'

CelltypeSW-1573

SW-1 573/50SW-1573/500-0SW-1573/500SW-1573/10000Doxorubicin

exposure0.5

0.70.72.05.0Doxorubicin

accumulation(nmol/106cells)-verapamil0.31

±0.01*

0.30 ±0.020.39 ±0.020.32 ±0.010.31 ±0.01+verapamil0.30

±0.010.26 ±0.020.40 ±0.020.35 ±0.020.33 ±0.01

"Cells were exposed for l h at the indicated doxorubicin concentrations,leading to similar drug accumulation in the various resistant cell lines. Columns3 and 4 compare doxorubicin accumulation after l h exposure in the absence orpresence of 20 »IMverapamil, respectively.

* Mean ±SD for three independent experiments carried out on the same day.

Similar experiments carried out on 2 other days gave essentially similar results.

for daunorubicin, VP-16-213, colchicine, and vincristine; a lowlevel of cross-resistance to mitomycin C and menadione; andno cross-resistance, i.e., a tendency for collateral sensitivity, tobleomycin and cisplatin (Table 2). A similar pattern of cross-

resistance has been reported by others in cell lines selected forresistance to doxorubicin (15-17), indicating that a commonform of MDR is present in our set of SW-1573 cell lines.Verapamil, an agent known to decrease the level of drug resistance in many doxorubicin-resistant cell lines, reversed doxorubicin resistance also in our cell lines. Continuous incubation with20 MMverapamil increased the sensitivity to doxorubicin by2.1-, 2.4-, 12-, 24-, and 46-fold in SW-1573, SW-1573/50, SW-

2990

Research. on September 29, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

MULTIDRUG RESISTANCE IN HUMAN LUNG TUMOR CELLS

Table 4 Antioxidanl levels in SW-I573 and multidrug-resistant variants of SW-1573"

Copper- and zinc-containing SODManganese-containing SODCatalaseGSHPXGSTGlutathioneSW-1573208

±78.3 ±0.1135 ±49.8 ±0.638 ±3

19.2 ±1.1SW-1573/50224

±166.8 ±0.7163 ±56.5 ±0.144 ±3

16.8 ±1.0SW-

1573/500-081

±36.6±0.2

270 ±55.8 ±0.323 ±5

16.0 ±0.2SW-

1573/500234

±93.5 ±0.2103 ±67.9 ±0.536 ±4

16.8 ±0.1SW-1573/10,000240

±164.9 ±0.6195 ±95.7 ±0.275 ±6

6.4 ±0.6" Antioxidant factors in SW-1573 and its multidrug-resistant derivatives. Enzyme activities are expressed in units/mg protein, glutathione in nmol/mg protein.

Means ±SD for three independent experiments.

100-

80-

60-

ga.a 40c

a8 20H

0J

ITT '*- \yf A

10 40 250 2000Doxorubicin-resistance,-fold

Fig. 3. P-glycoprotein antigen expression as a function of drug resistance. P-glycoprotein was determined by enzyme-linked immunosorbent assay using themonoclonal antibodies JSB-I (A) and MRK-16 (O). The most resistant cells (SW-1573/ 10,000) were used as a reference, arbitrarily set at 100 units. Bars, SDdeviations for two independent experiments.

1573/500-0, SW-1573/500, and SW-1573/10000 cells, respectively.

Doxorubicin Accumulation: Effect of Verapamil. As shown inFig. 1, a clear drug accumulation defect can be observed in allresistant sublines. Interestingly, SW-1573/50 and SW-1573/500-0 cells exhibited very similar drug uptakes, even thoughSW-1573/50 cells were approximately 4 times more sensitiveto doxorubicin than SW-1 573/500-0 cells (Table 2). This suggests that in addition to drug accumulation other parameterscontribute to the drug-resistant phenotype. As shown in Table3, 20 /«Mverapamil did not significantly enhance drug uptakein any of our cell lines, even though this concentration increasedthe sensitivity to doxorubicin (Table 2). Possibly preincubationwith verapamil or longer incubation times are needed to detecta verapamil-mediated enhancement of doxorubicin accumulation in our cell lines, since the effect of verapamil on doxorubicin accumulation was measured during a 1-h exposure period,whereas the effect of verapamil on doxorubicin sensitivity wasinvestigated in the continuous presence of verapamil and doxorubicin during a period of 4 days.

Drug Efflux Rates. For experiments to determine drug effluxrates the anthracycline daunorubicin was used instead of doxorubicin since daunorubicin accumulates to a higher extentthan doxorubicin in all our cell lines. Since daunorubicin anddoxorubicin are closely related drugs, and both drugs seem tobe substrates for the drug efflux pump postulated in multidrugresistant cells, it was considered valid to use daunorubicin effluxas a measure for drug pump activity in doxorubicin-selected

multidrug-resistant cell lines. As shown in Fig. 2, drug effluxactivity increased in the order of 15-fold over the whole range

or drug resistance, which might explain the decrease in drugaccumulation observed in the resistant variants.

Antioxidant Defenses. Since doxorubicin might be cytotoxicdue to biological activation and free radical formation we havetested whether a correlation exists between antioxidant defensesand the level of doxorubicin resistance. In this study we haveincluded copper- and zinc-containing SOD, manganese-con

taining SOD, catalase, GSHPX, GST, and total glutathione.The levels of Superoxide dismutase, catalase, and GSHPXactivity as well as total glutathione in SW-1573 parental cells

are within the range established for human neoplastic cell lines(18). GSSG was always less than 1% of total glutathione. Thedata indicate that there is no consistent correlation betweenany of the antioxidant defenses and drug resistance in our fivecell lines (Table 4).

P-Glycoprotein Expression. P-glycoprotein expression, asquantified with the monoclonal antibodies MRK-16 and .ISBI, increased at increasing levels of resistance to doxorubicin,while its expression was hardly detectable in control cells (Fig.3). In the low-level-resistance variant SW-1573/50 only JSB-Iwas able to detect P-glycoprotein antigen. In all other cell linesP-glycoprotein expression agreed for both monoclonal antibod

ies.Subcellular Distribution of Doxorubicin. As shown in Fig. 4

fluorescence of doxorubicin was mainly confined to the nucleararea in SW-1573 parental cells, whereas it was predominantlyseen in the cytoplasm in SW-1573/10000 cells. At physiologicalpH (7.4) the low level resistance variant SW-1573/50 cells

exhibited a similar subcellular distribution of doxorubicin ascontrol SW-1573 cells, whereas SW-1573/500 cells had a distribution pattern similar to that of SW-1573/10000 cells; inSW-1573/500-0 cells an intermediate pattern was observed

(Table 5). At pH 7.2 it was possible to discriminate the distribution pattern of SW-1573 from that of SW-1573/50 (Table

5). In addition, at both pH 7.8 and 8.5 it was possible todistinguish the distribution pattern of SW-1573/500 from thatof SW-1573/10000 cells. These data indicate that there is agradual shift from mainly nuclear to mainly cytoplasmic doxorubicin fluorescence as a function of drug resistance level.

Since it is known that verapamil has the capacity to redistribute anthracyclines in multidrug resistant cells to a patternsimilar to that observed in parental SW-1573 cells (19, 20)5 we

also studied the effect of verapamil on drug distribution in ourcell lines. Verapamil was able to redistribute doxorubicin fromthe cytoplasm to the nucleus in all cell lines, although theconcentrations of verapamil needed for this effect were higherfor the more resistant cell types.

*G. J. Schuurhuis, H. J. Broxterman, H. M. Pinedo, and J. Lankelma.Accumulation defects can quantitatively account for doxorubicin resistance in 2multidrug resistant cell lines, submitted for publication.

2991

Research. on September 29, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

MULTIDRUG RESISTANCE IN HUMAN LUNG TUMOR CELLS

Fig. 4. Subcellular doxorubicin distributionpatterns in SW-1573 and SW-1573/10000 cells.SW-1573 and SW-1573/10000 cells were exposed for 15 min in complete medium containing10 and 40 ¿iMdoxorubicin, respectively. Underthese conditions total fluorescence was similarin both cell lines. A, SW-1573 cells, phase contrast microscopy; B, SW-1573 cells, fluorescencemicroscopy; C, SW-1573/10000 cells, phasecontrast microscopy; D, SW-1573/10000 cells,fluorescence microscopy. For both types of microscopy a magnification of x 1000 was used.

Table 5 Subcellular doxorubicin distribution in SW-1573 and multidrug-resistant sublines of SW-1573 as a function of extracellular pH"

pH7.2

7.47.88.5SW-

1573N

NNNSW-

1573/50IN

NNSW-1573/500-0CINNSW-1573/500CCINsw-1573/10,000CC

CI

" Subcellular drug distribution was studied using doxorubicin concentrations

between 10 and 40 JIM to compensate for differences in total fluorescence thatoccurred in some cases. Fluorescence was mainly nuclear (N), mainly cytoplasmic(C), or intermediate (I). Incubation for 2 h at pH 7.2 or 8.5 was completelynontoxic, as determined in a growth inhibition assay.

DISCUSSION

In an attempt to find out whether the same mechanism ofresistance is active in cells with low and high levels of resistance,we studied a number of parameters as a function of resistancelevel in four multidrug-resistant sublines of the human lungcancer cell line SW-1573. The pattern of cross-resistance tounrelated drugs (Table 2) and the partial reversal of doxorubicinresistance by verapamil suggest a "classical" form of MDR (15-

17) in these cell lines.Drug accumulation in all resistant sublines was lower than

in control cells, roughly in proportion to the level of doxorubicin resistance. Also the capacity to extrude daunorubicin appeared to increase proportionally to the level of drug resistance.Since doxorubicin and daunorubicin are thought to behavesimilarly during drug efflux (21), increased drug efflux mightexplain the difference in doxorubicin accumulation between ourcell lines.

P-glycoprotein expression is a common feature of almost all

multidrug resistant cell lines described so far and is thought tobe involved in drug efflux. The amount of mRNA coding forP-glycoprotein has been correlated with the level of MDR inseveral human tumor cell lines (17, 22) including one sublineof SW-1573 cells (22). Here we tested P-glycoprotein expression at the protein level in all available sublines of SW-1573cells using the monoclonal antibodies MRK-16 (13) and JSB-I(14). As shown in Fig. 3, there was a reasonable positivecorrelation between P-glycoprotein expression and the level ofdoxorubicin resistance.

According to several authors an increased antioxidant defensesystem or increased levels of GST may contribute to cellulardoxorubicin resistance (23, 24). However, in the present set ofSW-1573 cell strains, none of the antioxidant defenses investi

gated (superoxide dismutases, catalase, GSHPX, GSH, andGST) exhibited a progressive expansion in proportion to thelevel of drug resistance, although some variation in activityamong the various cell lines was observed. This observation isnot quite unexpected, since we have recently shown in Chinesehamster ovary cells that increased levels of copper- and zinc-containing SOD, manganese-containing SOD, catalase,

GSHPX, and total glutathione do not afford protection againstdoxorubicin-induced clonogenic cell death (18). Apparently theantioxidant defenses are not always limiting in the developmentof drug resistance.

A MDR-related parameter which has thus far received rela

tively little attention in the literature on doxorubicin resistanceis the pattern of subcellular drug distribution. Nevertheless, inmany multidrug-resistant cell lines including variants from

2992

Research. on September 29, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

MULTIDRUG RESISTANCE IN HUMAN LUNG TUMOR CELLS

AuxBl,5 KB (19), HL-60 (20), and A27805 an altered anthra-cycline distribution very similar to that observed in our SW-1573 sublines has been described. This suggests that a differencein drug distribution between sensitive and resistant cells mightbe a common feature of multidrug-resistant cells. Althoughonly two types of subcellular doxorubicin distribution canclearly be defined ("mainly nuclear" and "mainly cytoplasmic"),

using different external pH values during drug exposure it waspossible to show that there is a gradual change in subcellulardrug distribution that is correlated with the level of doxorubicinresistance. Differences in intracellular drug distribution werenot caused by an increase in doxorubicin uptake at higher pHsince drug distribution was apparently not dependent upon theextracellular doxorubicin concentration. It was more likely dueto a change in intracellular pH, since monensine (10 MM),aNa/H+ ionophore which increases intracellular pH, redistribu

ted doxorubicin from cytoplasm to nucleus in resistant cells,whereas nigericin (10 /¿M),a K+/H+ ionophore decreasing pH¡,had the opposite effect.6

In view of the observed progressive changes in correlationwith the level of MDR we conclude that (a) altered subcellulardrug distribution, (b) decreased drug accumulation, and (c)increased P-glycoprotein expression are determinants of bothlow and high levels of MDR in SW-1573 human lung tumorcells. It remains to be established to what extent clinical mul-tidrug resistance may exhibit similar characteristics. This iscurrently one of the subjects of investigation in our laboratories.

ACKNOWLEDGMENTS

We thank Wiebout Guikema, Karin Kuiper, Ingrid Gruijs, and HenkDekker for expert technical assistance and Dr. T. Tsuruo for providingMRK-16.

REFERENCES

1. Gros, P., Neriah, Y. B., Croop, J. M . and Housman, D. E. Isolation andexpression of a complementary DNA that confers multidrug resistance.Nature (Lond.), 323:728-731, 1986.

2. Horio, M., Gottesman, M. M., and Pastan, I. ATP-dependent transport ofvinblastine in vesicles from human multidrug-resistant cells. Proc. Nati.Acad. Sci. USA, «5:3580-3584, 1988.

3. Gerlach, J. H., Endicott, J. A., Juranka, P. F., Henderson, G., Sarangi, F.,Deuchars, K. L., and Ling, V. Homology between P-glycoprotein and abacterial haemolysin transport protein suggests a model for multidrug resistance. Nature (Lond.), 324: 485-489, 1986.

4. Bradley, G., luranka, P. F., and Ling, V. Mechanism of multidrug resistance.Biochim. Biophys. Acta, 948: 87-128, 1988.

5. Broxterman, H. J., Pinedo, H. M., Kuiper, C. M., Kaptein, L. C. M.,

1H. G. Keizer, unpublished results.

Schuurhuis, G. J., and Lankelma, J. Induction by verapamil of a rapidincrease in ATP consumption in multidrug-resistant tumor cells. FASEB J.,2: 2278-2282, 1988.

6. Mannervik, B., and Guthenberg, C. Glutathione transferase. Methods Inzymol., 77:231-232, 1981.

7. Günzler,W. A., Kremers, H., Flohe, L. An improved coupled test procedurefor glutathione peroxidase. Z. Klin. Chem. Klin. Biochem., 12: 444-448,1974.

8. Joenje, H., Gille, J. J. P., Oostra, A. B., and Van der Valk, P. Somecharacteristics of hyperoxia-adapted I lei .a cells. A tissue culture model forcellular oxygen tolerance. Lab. Invest., 52:420-428, 1985.

9. IVIK UP,L. A more sensitive modification of the catalase assay with the Clarkoxygen electrode. Anal. Biochem., 80: 409-415, 1977.

10. Tietze, F. Enzymic method for quantitative determination of nanogramamounts of total and oxidized glutathione: applications to mammalian bloodand other tissues. Anal. Biochem., 27: 502-522, 1969.

11. Howell, N., Belli, T. A., Zaczkiewicz, L. T., and Belli, J. A. High-level,unstable Adriamycin resistance in a Chinese hamster mutant cell line withdouble minute chromosomes. Cancer Res., 44:4023-4029, 1984.

12. Lankelma, J., Koopman, G., Van Grondelle, R., and Pinedo, H. M. In beamlight absorption measurement of anthracyclines in cells with a flow throughsystem. Biochim. Biophys. Acta, 964: 200-206, 1988.

13. Hamada, H., and Tsuruo, T. Functional role for the 170- to 180-kDaglycoprotein specific to drug-resistant tumor cells as revealed by monoclonalantibodies. Proc. Nati. Acad. Sci. USA, 83: 7785-7789, 1986.

14. Scheper, R. J., Bulle, J. W. M., Brakkee, J. G. P., Quak, J. J., Van derSchool, E., Balm, A. J. M., Meijer, C. J. L. M., Broxterman, H. J., Kuiper,C. M., Lankelma, J., and Pinedo, H. M. Monoclonal antibody JSB-I deteclsa highly conserved epitope on the P-glycoprotein associated with multidrugresistance. Int. J. Cancer, 42: 389-394, 1988.

15. Harker, W. B., and Sikic, B. I. Multidrug (pleiotropic) resistance in doxorub-icin-selected variants of the human sarcoma cell line MES-SA. Cancer Res.,45:4091-4096, 1985.

16. Beck, W. T., < ¡nain,M. C., Look, A. T., and Ashmun, R. A. Reversal ofVinca alkaloid resistance but not multiple drug resistance in human leukemiccells by verapamil. Cancer Res., 46: 778-784, 1986.

17. Fairchild, C. K., Ivy, S. P., Kao-Shan, C. S., Wang-Peng, J., Rosen, N.,Israel, M. A., Melera, P. W., Cowan, K. H., and Goldsmith, M. E. Isolationof amplified and overexpressed DNA sequences from Adriamycin-resistanthuman breast cancer cells. Cancer Res., 47: 5141-5148, 1987.

18. Keizer, H. G., Van Rijn, J., Pinedo, H. M., and Joenje, H. Effect ofendogenous glutathione, Superoxide dismutases, catalase and glutathioneperoxidase on Adriamycin tolerance of Chinese hamster ovary cells. CancerRes., 48:4493-4497, 1988.

19. Willingham, M. C., Cornwell, M. M., Cardarelli, C. O., Gottesman, M. M.,and Pastan, I. Single cell analysis of daunomycin uptake and efflux inmultidrug-resistant and -sensitive KB cells: effect of verapamil and otherdrugs. Cancer Res., 46: 5941-5946, 1986.

20. Hindenburg, A. A., Baker, M. A., Gleyzer, E., Stewart, V. J., Case, N., andTaub, R. N. Effect of verapamil and other agents on the distribution ofanthracyclines and on reversal of drug resistance. Cancer Res., 47: 1421-1425, 1987.

21. Peterson, C., Baurain, R., and Trouet, A. The mechanism for cellular uptake,storage and release of daunorubicin. Biochem. Pharmacol., 29: 1687-1692,1980.

22. Van der Bliek, A. M., Baas, F., Van der Velde-Koerts, T., Biedler, J. L.,Meyers, M. B., Ozols, R. F., Hamilton, T. C., Joenje, H., and Borst, P.Genes amplified and overexpressed in human multidrug resistant cell lines.Cancer Res., 48: 5927-5932, 1988.

23. Batist, G., Tulpule, A., Sinha, B. K., Katki, A. G., Meyers, C. H., and Cowan,K. H. Overexpression of a novel anionic glutathione transferase in multidrugresistant human breast cancer cells. J. Biol. Chem., 261:15544-15549,1986.

24. Kramer, R. A., Zakher, J., and Kim, G. Role of the glutathione redox cyclein acquired and de novo multidrug resistance. Science (Wash. DC), 241:694-696, 1988.

2993

Research. on September 29, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from

1989;49:2988-2993. Cancer Res   Hiskias G. Keizer, Gerrit J. Schuurhuis, Henk J. Broxterman, et al.   Human Lung Tumor CellsIncreased P-Glycoprotein Expression in Cultured SW-1573Accumulation, Altered Subcellular Drug Distribution, and Correlation of Multidrug Resistance with Decreased Drug

  Updated version

  http://cancerres.aacrjournals.org/content/49/11/2988

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/49/11/2988To request permission to re-use all or part of this article, use this link

Research. on September 29, 2020. © 1989 American Association for Cancercancerres.aacrjournals.org Downloaded from