EXPERIMENTAL CELL RESEARCH 226, 133–139 (1996)ARTICLE NO. 0211

Cross-Resistance, Cisplatin Accumulation, and Platinum–DNA AdductFormation and Removal in Cisplatin-Sensitive and -Resistant

Human Hepatoma Cell Lines

S. W. JOHNSON,*,1 D.-W. SHEN,† I. PASTAN,‡ M. M. GOTTESMAN,† AND T. C. HAMILTON*

*Department of Medical Oncology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, Pennsylvania 19111;and †Laboratory of Cell Biology and ‡Laboratory of Molecular Biology, National Cancer Institute,

National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892

tion with other drugs in the treatment of a wide varietyThe BEL7404 human hepatoma cell line was selected of malignant solid tumors [1]. The success of platinum-

in vitro for primary cisplatin resistance. A panel of based regimens, however, is limited by the emergencefour cisplatin-resistant sublines were generated which of drug resistance which may occur through alteredexhibited resistance to cisplatin (up to 34-fold) but host pharmacokinetics or by the acquisition of drugwere not cross-resistant to adriamycin, taxol, etopo- resistance mechanisms by a subpopulation of tumorside, or mitomycin C. Further characterization of this cells [2]. The ability of cisplatin to form covalent intra-panel of cell lines revealed that increased cisplatin re- strand and interstrand crosslinks in genomic DNA issistance was associated with decreased cisplatin accu- believed to be responsible for its cytotoxic effects [3].mulation in the selected sublines (up to 14-fold) rela- Therefore, a cell’s ability to limit the formation of thesetive to the parental BEL7404 cell line. A significant DNA lesions or its capacity to repair or tolerate thesereduction in platinum–DNA adduct formation (9-fold) lesions once they are formed should influence survival.and ribosomal RNA gene-specific interstrand crosslink

Mechanisms of cellular cisplatin resistance which haveformation (12-fold) were also observed in the 7404-been identified thus far in various model systems in-CP20 cell line. No differences in the rate of platinumclude: (1) decreased cisplatin accumulation, (2) in-efflux from the BEL7404 and 7404-CP20 cell lines werecreased drug inactivation by protein and nonproteindetected following a 4-h loading period, and total plati-thiols, (3) enhanced platinum–DNA adduct repair, (4)num–DNA adduct and gene-specific interstrand cross-alteration in the types of platinum–DNA adductslink removal rates were similar in both cell lines.formed, and (5) increased platinum–DNA damage tol-There were approximately 3-fold more total platinum–erance. The underlying molecular bases for these resis-DNA adducts present in the BEL7404 cells relative to

the 7404-CP20 cells at equitoxic concentrations of cis- tance mechanisms are largely unknown.platin, suggesting that DNA damage tolerance also The use of in vitro-derived cisplatin resistance mod-contributes to the cisplatin resistance phenotype. els has been important in the identification of cisplatinOverall, these results indicate that decreased cisplatin resistance mechanisms. These systems offer severalaccumulation is the major cisplatin resistance mecha- advantages over the use of cell lines derived from tu-nism present in the in vitro-selected cell lines. This mors of cisplatin-refractory patients. For example, highmodel system of acquired cisplatin resistance may be levels of primary cisplatin resistance can be achievedvaluable in determining the molecular basis for de- by repeated cisplatin exposure of a relatively drug-sen-creased cisplatin uptake and be useful for the study of sitive cell line to cisplatin. Relative cisplatin resistancepotential resistance modulators. q 1996 Academic Press, Inc.

levels of up to 1000-fold have been reported in humanovarian cancer and human epidermoid carcinoma celllines [4, 5]. As a result, the individual resistance mech-

INTRODUCTION anisms are often present in increased magnitude rela-tive to those present in in vivo cisplatin resistance mod-

Cisplatin is a commonly used chemotherapeutic els. This may facilitate the identification of the genesagent which is effective as a single agent or in combina- responsible for resistance and provide a system for ex-

amining potential resistance modulators. Further-more, an in vitro-derived resistant cell line has a paren-1 To whom correspondence and reprint requests should be ad-

dressed. Fax: (215) 728-2741. tal drug-sensitive counterpart which can be of impor-

133 0014-4827/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

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134 JOHNSON ET AL.

ately or incubated for 0.5 to 4 h in fresh medium. Following thetance in efforts to identify potential resistance genesincubation period, the flasks were rinsed three times with PBS andusing techniques such as subtractive hybridization andthe cells were trypsinized, counted, and pelleted by centrifugation.

differential display. Certainly, there are potential The cell pellets were redissolved in 70% nitric acid and platinumdrawbacks to the use of in vitro models since resistance content was determined by AAS as described above.mechanisms which are identified might not be clini- DNA platination and interstrand crosslink formation. To mea-

sure platinum–DNA adduct formation, BEL7404 and 7404-CP20cally relevant and such models exclude the study ofcells were incubated for 4 h with 0–200 mM cisplatin, washed threealtered host pharmacokinetics. This report describestimes with PBS, and incubated overnight at 377C in lysis buffer (10the characterization of cisplatin resistance mecha-mM Tris–HCl, pH 7.4, containing 0.15 M NaCl, 10 mM EDTA, 0.1%

nisms in a panel of human hepatoma cell lines which (w/v) SDS, and 200 mg/ml proteinase K). High-molecular-weight DNAwere selected in vitro for primary cisplatin resistance. was isolated by the phenol/chloroform method, restriction-digested

with HindIII, and an aliquot of DNA (2 mg) was used for interstrandcrosslink determination by renaturing agarose gel electrophoresis

MATERIALS AND METHODS (RAGE) [8]. Briefly, DNA was incubated at 657C for 5 min in 0.2 NNaOH and placed on ice for 2 min. Samples were loaded onto a 0.5%

Chemicals and reagents. Cisplatin, etoposide, and mitomycin C agarose gel prepared in 90 mM Tris-borate buffer containing 2 mMwere obtained from Bristol Myers Squibb (Syracuse, NY). Adriamy- EDTA and electrophoresed for 4 h at 90 V. Following Southern blot-cin was obtained from Cetus Corp. (Emeryville, CA). Taxol for clinical ting, the membrane was placed in hybridization solution containinguse was provided by the Division of Cancer Treatment, National the ABEII probe (provided by Dr. J. E. Sylvester, University of Penn-Cancer Institute, and was resuspended at a concentration of 6 mg/ sylvania) which recognizes a 17-kb fragment of the 28S ribosomalml in 50% polyoxyethylated castor oil (Cremophor EL) and 50% dehy- RNA gene [9]. Histograms were generated for each lane using andrated alcohol. Cell culture reagents were obtained from GIBCO AMBIS radioanalytic imaging system (AMBIS Systems, San Diego,(Grand Island, NY). All other chemicals were obtained from Sigma CA), and the fraction of crosslinked strands was determined byChemical Co. (St. Louis, MO) unless otherwise indicated. weight analysis of the peaks. The average number of interstrand

crosslinks per fragment was calculated using the Poisson distribu-Cell culture. The BEL7404 human liver carcinoma cell line wastion equation: 0ln(1 0 Fc), where Fc is the fraction of DNA strandsselected for resistance to cisplatin as described [5]. The four cisplatin-containing crosslinks [8]. The remainder of the DNA samples wereresistant sublines 7404-CP1, 7404-CP2.5, 7404-CP7.5, and 7404-used for total platinum–DNA adduct determination. Samples wereCP20 generated by this selection procedure were used for the currentincubated at 957C in 10% HCl and platinum was measured by AASstudy. Cells were maintained at 377C in a humidified incubator con-as described [7]. Relative differences in platinum–DNA adduct for-taining 5% CO2 in Dulbecco’s modified Eagle’s medium with 4.5 g/mation were determined by comparing the slopes of the regressionliter glucose and supplemented with 12% (v/v) fetal calf serum, 100lines generated for each cell line in triplicate experiments.mg/ml streptomycin, 100 units/ml penicillin, 0.3 mg/ml glutamine,

and 0.3 units/ml insulin (porcine). Platinum–DNA adduct removal. Triplicate flasks (175 cm2) con-Cytotoxicity. The MTT assay was used to determine the relative taining BEL7404 and 7404-CP20 cells were incubated with 16 and

sensitivities of the BEL7404 cell line and its cisplatin-resistant sub- 150 mM cisplatin, respectively, for 4 h at 377C. Cells were eitherlines to cisplatin, adriamycin, taxol, etoposide, and mitomycin C [6]. harvested immediately or incubated in fresh medium for 8 h. DNACells were plated in 150 ml of medium per well in 96-well plates was isolated and analyzed for platinum content by AAS or for ribo-(Corning Co., Corning, NY). Following overnight incubation, cells somal RNA gene-specific interstrand crosslinks using RAGE as de-were exposed to various concentrations of drug which were added in scribed above.10-ml volumes. Following a 72-h incubation, 40 ml of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added

RESULTSper well. After 2 h at 377C, the cells were lysed by adding 100 ml of20% (w/v) sodium dodecyl sulfate, 50% (v/v) N,N-dimethylformamide,pH 4.7, and incubated overnight at 377C. The absorbance at 570 nm A series of cisplatin-resistant human hepatoma cellwas determined for each well using a Bio-Rad Model 3550 microplate

lines (7404-CP1, 7404-CP2.5, 7404-CP7.5, and 7404-reader. The reported IC50 values are the result of triplicate determi-nations on at least two separate occasions. CP20) were derived by exposure of the parental

Cisplatin accumulation. Duplicate 80-cm2 flasks of cells were BEL7404 cell line to stepwise, increasing cisplatin con-treated with cisplatin (0–200 mM ) for 4 h at 377C. Each flask was centrations (0.3 to 20 mg/ml) over a period of 24 monthsrinsed three times with Dulbecco’s phosphate-buffered saline, pH 7.1 [5]. The relative sensitivities of these five cell lines to(PBS), trypsinized, and counted using a hemacytometer. The cells

cisplatin, adriamycin, taxol, etoposide, and mitomycinwere aliquoted in triplicate into 1.5-ml microfuge tubes and pelletedC were measured using the MTT assay (Table 1, Fig.by centrifugation. Following an overnight incubation in 70% nitric

acid, the samples were incubated at 957C for 10 min and analyzed 1). The 7404-CP20 cell line was 34-fold resistant tofor platinum content by atomic absorption spectrometry (AAS) essen- cisplatin compared to the BEL7404 cell line (IC50 val-tially as described [7]. An additional three aliquots of the cells were ues 144 and 4.2 mM, respectively), while the 7404-CP1,pelleted and redissolved in 1 ml of 0.5 M Na2HPO4/NaH2PO4, pH

7404-CP2.5, and 7404-CP7.5 cell lines were 3.9-, 7.6-,8.5, containing 1% (w/v) SDS, 1 mM EDTA, and 4 M urea. Proteinand 13.6-fold resistant relative to the parental cell line.concentrations were measured for these samples using the bicincho-

ninic acid protein determination kit (Sigma). Relative drug accumu- Significant cross-resistance to adriamycin, taxol, etopo-lation differences were determined by comparing the slopes of the side, or mitomycin C was not observed (i.e., ú2-fold) inlines generated for each cell line. The experiment was repeated once any of the cisplatin-resistant sublines.for the BEL7404 cell line and twice for the 7404-CP20 cell line.

Cisplatin accumulation was measured in the five cellPlatinum efflux. Duplicate flasks (25 cm2) containing BEL7404lines following a 4-h exposure to a range of cisplatinand 7404-CP20 cells were incubated with cisplatin (50 and 500 mM,

respectively) for 4 h at 377C. The cells were either harvested immedi- concentrations and was linear up to 200 mM cisplatin

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135CISPLATIN RESISTANCE IN HUMAN HEPATOMA CELLS

TABLE 1

Sensitivity of BEL7404 and Cisplatin-Resistant Sublines to Various Drugs

Cisplatin Adriamycin Taxol Etoposide Mitomycin CCell line IC50 (mM)a IC50 (mM) IC50 (nM) IC50 (mM) IC50 (nM)

BEL7404 4.2 (1.0) 0.126 (1.0) 2.2 (1.0) 0.90 (1.0) 0.37 (1.0)7404-CP1 16.3 (3.9) 0.210 (1.7) 2.0 (0.9) 1.84 (2.0) 0.41 (1.1)7404-CP2.5 32.0 (7.6) 0.065 (0.5) 1.9 (0.9) 0.62 (0.7) 0.23 (0.6)7404-CP7.5 57.0 (13.6) 0.083 (0.7) 3.0 (1.4) 1.09 (1.2) 0.46 (1.2)7404-CP20 144.0 (34.3) 0.151 (1.2) 4.5 (2.0) 0.93 (1.0) 0.64 (1.7)

a The IC50 is the concentration of drug which kills 50% of the cells as measured by the MTT assay. The values shown are the averageof triplicate determinations measured on at least two separate occasions. Relative resistance to the BEL7404 cell line is indicated inparentheses.

(Fig. 2). There was a strong correlation (r Å 0.98) be- atomic absorption spectrometry following a 4-h expo-sure to a range of cisplatin concentrations (Fig. 4). Plat-tween cisplatin accumulation and relative cisplatin re-

sistance among the cell lines, with the parental inum–DNA adduct formation was linear with cisplatinconcentration (up to 200 mM ) and there was a 9-foldBEL7404 cell line containing the most cell-associated

platinum followed in decreasing order by the 7404-CP1, decrease in the adduct levels formed in the 7404-CP20cell line relative to the parental BEL7404 cell line. Cis-7404-CP2.5, 7404-CP7.5, and 7404-CP20 cell lines, re-

spectively. The magnitude of the accumulation differ- platin interstrand crosslink levels formed in the ribo-somal RNA gene, as measured by renaturing agaroseence between the most cisplatin-sensitive (BEL7404)

and -resistant (7404-CP20) cell lines was 14-fold. There gel electrophoresis (RAGE), were also 12-fold lower inthe 7404-CP20 cell line relative to the BEL7404 cellwas little change in the levels of cell-associated plati-

num between these two cell lines after treating cells line (Figs. 5 and 6). Using these data, it was calculatedthat 2 to 3% of the total platinum–DNA adducts werewith cisplatin for 4 h followed by a 4-h drug-free incuba-

tion period (Fig. 3). present as interstrand crosslinks in these cell lines.Platinum–DNA adduct removal was measured inThe formation of platinum–DNA adducts was mea-

sured in the BEL7404 and 7404-CP20 cell lines by

FIG. 1. Sensitivities of the 7404-CP1, 7404-CP2.5, 7404-CP7.5, FIG. 2. Cisplatin accumulation in the BEL7404 and cisplatin-resistant sublines. Duplicate flasks were incubated with 0–200 mMand 7404-CP20 cell lines to cisplatin, adriamycin, taxol, etoposide,

and mitomycin C as measured relative to the parental BEL7404 cell cisplatin for 4 h, harvested, and processed for atomic absorptionspectrometry as described under Materials and Methods.line.

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136 JOHNSON ET AL.

FIG. 5. Measurement of cisplatin interstrand crosslink forma-tion in the BEL7404 and 7404-CP20 cell lines using renaturing agar-ose gel electrophoresis (RAGE). DNA was denatured and electropho-resed on a 0.5% agarose gel. Following Southern transfer, the mem-brane was hybridized with 32P-labeled ABEII fragment which isspecific for the ribosomal RNA gene.

tively. Cisplatin interstrand crosslinks were also re-moved from the ribosomal RNA gene at a similar rateby the two cell lines.

DISCUSSION

Cisplatin resistance may be multifactorial, con-sisting of cellular mechanisms which limit the forma-FIG. 3. Platinum efflux in the BEL7404 and 7404-CP20 cell lines.

Following a 4-h exposure to cisplatin, the cells were incubated in tion of platinum–DNA damage and mechanisms whichdrug-free medium for various times up to 4 h, harvested, and plati- enable DNA lesions to be repaired or tolerated. Wenum content was measured by AAS. have characterized a panel of cisplatin-resistant hu-

man hepatoma cell lines which were derived by in vitrothe BEL7404 and 7404-CP20 cell lines (Table 2). Fol- exposure of the parental BEL7404 cell line to cisplatin.lowing an 8-h incubation in drug-free medium, the The BEL7404 cell line, which was established from aBEL7404 and 7404-CP20 cell lines removed 17 and21% of their total platinum–DNA adducts, respec-

FIG. 4. Formation of platinum–DNA adducts in BEL7404 and FIG. 6. Results of renaturing agarose gel electrophoresis (RAGE)for measuring the formation of cisplatin interstrand crosslinks in7404-CP20 following a 4-h exposure to cisplatin (0–200 mM ). DNA

was isolated and measured for platinum content as described under the ribosomal RNA gene of BEL7404 and 7404-CP20 following a 4-h exposure to cisplatin (0–200 mM ).Materials and Methods.

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137CISPLATIN RESISTANCE IN HUMAN HEPATOMA CELLS

TABLE 2 analogs [20–22]. Several observations have led investi-gators to conclude, however, that a carrier-mediatedRemoval of Total Platinum–DNA Adducts and Ribosomaltransport protein may also be involved in cisplatin up-RNA Gene-Specific Interstrand Crosslinks (ICL) in BEL7404

and 7404-CP20 Cells take. For example, cisplatin accumulation has beenshown to be partially energy-dependent, ouabain-in-

Repair Pt–DNA hibitable, sodium-dependent, and affected by mem-time adducts (pg

brane potential and cAMP levels [23–25].Cell line (h) Pt/mg DNA)a ICL/10 kba

Based on the current understanding of cisplatin pas-BEL7404 0 23 { 3 0.020 { .003 sive and facilitated transport, several approaches have

8 19 { 2 (17%) 0.017 { .002 (15%) been explored to circumvent decreased cisplatin accu-7404-CP20 0 42 { 1 0.022 { .003 mulation in cisplatin-resistant cells. The polyene mac-8 33 { 2 (21%) 0.019 { .001 (14%)

rolide antibiotic amphotericin B, which permeabilizescell membranes by associating with sterols resultinga Total platinum–DNA adduct levels were determined by AAS and

gene-specific interstrand crosslink levels were determined by RAGE. in the formation of pores or channels, has been shownThe percentage of platinum–DNA adduct removal is indicated in to selectively increase cisplatin accumulation in cis-parentheses. platin-resistant human non-small lung carcinoma and

ovarian carcinoma cell lines [26–29]. The levels of am-photericin B required to achieve a potentiating effectin vivo, however, are nephrotoxic. In another study,primary liver carcinoma of an untreated patient [10], is

intrinsically resistant to cisplatin [5]. In the present twocalmodulin antagonists were shown to increase cis-platin uptake in cisplatin-resistant human ovarianstudy, the cisplatin-selected sublines 7404-CP1, 7404-

CP2.5, 7404-CP7.5, and 7404-CP20 showed increased re- cancer cell lines, restoring their intracellular platinumlevels to that of the similarly treated parental cell linesistance to cisplatin (up to 34-fold) relative to the parental

cell line, but did not exhibit cross-resistance to adriamy- [30]. Although not selective for cisplatin-resistant cellsexhibiting the cisplatin accumulation defect, othercin, taxol, etoposide, or mitomycin C. Shen et al. [5] re-

ported previously, however, that the 7404-CP7.5 subline pharmacologic agents have been reported to increasecellular cisplatin accumulation such as forskolin and 3-is 13-, 23-, and 39-fold resistant to 6-mercaptopurine, 5-

fluorouracil, and methotrexate, respectively, compared to isobutyl-1-methylxanthine which increase cAMP levels[25], the nucleotide transport inhibitor dipyridamolethe BEL7404 cell line. The values reported for cisplatin

differ somewhat from the previously reported IC50 values [31], the mitotic inhibitor 1-propargyl-5-chloropyrimi-dine-2-one [32], and the fatty acid docosahexaenoic acidfor these cell lines using a different growth assay [5].

In order to determine the mechanism(s) responsible [33]. Alternatively, lipophilic platinum analogs, suchas the ammine/amine platinum (IV) dicarboxylates de-for the increased cisplatin resistance in the 7404-CP1,

7404-CP2.5, 7404-CP7.5, and 7404-CP20 cell lines, cis- scribed by Kelland et al. (18), have been shown to entercells more readily than cisplatin.platin accumulation and platinum–DNA adduct for-

mation and removal were measured. Reduced cisplatin Although no specific proteins have been directly im-plicated in the cisplatin accumulation defect often ob-accumulation (up to 14-fold in 7404-CP20) was ob-

served in the cisplatin-selected sublines and was asso- served in cisplatin-resistant cells, increased amountsof several proteins have been found to be associatedciated with decreased overall platinum–DNA adduct

formation, decreased interstrand crosslink formation, with this phenotype. Shen et al. [5] observed increasedlevels of a 50- and 52-kDa protein and decreasedand decreased cisplatin sensitivity. No differences in

platinum efflux rates were measured following a 4-h amounts of a 35-kDa protein in 7404-CP20 and KB-CP20 cells relative to the cisplatin-sensitive cells fromexposure to cisplatin, indicating that the decreased ac-

cumulation results from either rapid efflux immedi- which they were derived. The KB-CP20 cells also con-tained decreased levels of a 57-kDa protein. Subse-ately after cisplatin enters the cells or from reduced

drug uptake. This phenotype has frequently been ob- quent microsequencing and Western blot analysis indi-cated that the major 52-kDa protein was heat shockserved in other cell lines selected for primary cisplatin

resistance; however, the mechanism(s) by which de- protein HSP60. Elevated levels of HSP60 in cisplatin-resistant human ovarian cancer and human bladdercreased cellular cisplatin accumulation occurs is un-

known. [7, 11–18]. carcinoma cell lines have also been reported [34]. Anearlier study found the overexpression of a 200-kDaThe available data indicate that cisplatin enters cells

by passive diffusion and/or carrier-mediated transport membrane glycoprotein in a cisplatin-resistant murinelymphoma cell line which was associated with cisplatin(reviewed in 19). Evidence for cisplatin uptake by pas-

sive diffusion has been provided by studies in which resistance [35]. Despite these findings, it remains tobe determined whether these proteins have a role incisplatin uptake was shown to be nonsaturable, even

up to its solubility limit, and not inhibited by structural cisplatin resistance or whether their increased levels

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138 JOHNSON ET AL.

result from chronic cisplatin exposure during in vitro BEL7404 and 7404-CP20 cells (14- and 9-fold, respec-tively).selection. We have speculated that this chronic cis-

In conclusion, the BEL7404 cell line and its cisplatin-platin exposure could upregulate transcription factorsresistant sublines provide a valuable model system forwhich activate a range of genes, some relevant to resis-understanding the molecular basis of decreased cis-tance and others only by virtue of their similar mecha-platin accumulation, especially since the magnitude ofnism of transcriptional activation [36].the accumulation defect is relatively high (14-fold) com-A second cisplatin-resistance mechanism which wepared to that observed in other model systems [7, 11–observed in the 7404-CP20 cell line is increased plati-18]. If decreased cisplatin accumulation proves to be anum–DNA damage tolerance. This was determined byclinically relevant obstacle to the successful treatmentcalculating the amount of platinum–DNA damageof cisplatin-refractory cancer patients, then a thoroughpresent at equitoxic cisplatin doses for the BEL7404understanding of cisplatin uptake will be required forand 7404-CP20 cell lines. We found that at their respec-the development of effective modulation strategies.tive cisplatin IC50 values, the 7404-CP20 cell line con-

tained 48 pg Pt/mg DNA and the BEL7404 cell lineThis work was supported in part by CA51228 to T.C.H.contained 15 pg Pt/mg DNA, resulting in a 3-fold differ-

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Received December 28, 1995Revised version received April 8, 1996

AID ECR 3200 / 6i10$$$$$1 06-05-96 04:49:37 eca AP: Exp Cell