Cross-Resistance, Cisplatin Accumulation, and Platinum–DNA Adduct Formation and Removal in Cisplatin-Sensitive and -Resistant Human Hepatoma Cell Lines

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

226, 133–139 (1996)

0211

Cross-Resistance, Cisplatin Accumulation, and Platinum–DNA Adduct Formation 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

The BEL7404 human hepatoma cell line was selected in vitro for primary cisplatin resistance. A panel of four cisplatin-resistant sublines were generated which exhibited resistance to cisplatin (up to 34-fold) but were not cross-resistant to adriamycin, taxol, etoposide, or mitomycin C. Further characterization of this panel of cell lines revealed that increased cisplatin resistance was associated with decreased cisplatin accumulation in the selected sublines (up to 14-fold) relative to the parental BEL7404 cell line. A significant reduction in platinum–DNA adduct formation (9-fold) and ribosomal RNA gene-specific interstrand crosslink formation (12-fold) were also observed in the 7404CP20 cell line. No differences in the rate of platinum efflux from the BEL7404 and 7404-CP20 cell lines were detected following a 4-h loading period, and total platinum–DNA adduct and gene-specific interstrand crosslink removal rates were similar in both cell lines. There were approximately 3-fold more total platinum– DNA adducts present in the BEL7404 cells relative to the 7404-CP20 cells at equitoxic concentrations of cisplatin, suggesting that DNA damage tolerance also contributes to the cisplatin resistance phenotype. Overall, these results indicate that decreased cisplatin accumulation is the major cisplatin resistance mechanism present in the in vitro-selected cell lines. This model system of acquired cisplatin resistance may be valuable in determining the molecular basis for decreased cisplatin uptake and be useful for the study of potential resistance modulators. q 1996 Academic Press, Inc.

INTRODUCTION

Cisplatin is a commonly used chemotherapeutic agent which is effective as a single agent or in combina1 To whom correspondence and reprint requests should be addressed. Fax: (215) 728-2741.

tion with other drugs in the treatment of a wide variety of malignant solid tumors [1]. The success of platinumbased regimens, however, is limited by the emergence of drug resistance which may occur through altered host pharmacokinetics or by the acquisition of drug resistance mechanisms by a subpopulation of tumor cells [2]. The ability of cisplatin to form covalent intrastrand and interstrand crosslinks in genomic DNA is believed to be responsible for its cytotoxic effects [3]. Therefore, a cell’s ability to limit the formation of these DNA lesions or its capacity to repair or tolerate these lesions once they are formed should influence survival. Mechanisms of cellular cisplatin resistance which have been identified thus far in various model systems include: (1) decreased cisplatin accumulation, (2) increased drug inactivation by protein and nonprotein thiols, (3) enhanced platinum–DNA adduct repair, (4) alteration in the types of platinum–DNA adducts formed, and (5) increased platinum–DNA damage tolerance. The underlying molecular bases for these resistance mechanisms are largely unknown. The use of in vitro-derived cisplatin resistance models has been important in the identification of cisplatin resistance mechanisms. These systems offer several advantages over the use of cell lines derived from tumors of cisplatin-refractory patients. For example, high levels of primary cisplatin resistance can be achieved by repeated cisplatin exposure of a relatively drug-sensitive cell line to cisplatin. Relative cisplatin resistance levels of up to 1000-fold have been reported in human ovarian cancer and human epidermoid carcinoma cell lines [4, 5]. As a result, the individual resistance mechanisms are often present in increased magnitude relative to those present in in vivo cisplatin resistance models. This may facilitate the identification of the genes responsible for resistance and provide a system for examining potential resistance modulators. Furthermore, an in vitro-derived resistant cell line has a parental drug-sensitive counterpart which can be of impor-

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tance in efforts to identify potential resistance genes using techniques such as subtractive hybridization and differential display. Certainly, there are potential drawbacks to the use of in vitro models since resistance mechanisms which are identified might not be clinically relevant and such models exclude the study of altered host pharmacokinetics. This report describes the characterization of cisplatin resistance mechanisms in a panel of human hepatoma cell lines which were selected in vitro for primary cisplatin resistance. MATERIALS AND METHODS Chemicals and reagents. Cisplatin, etoposide, and mitomycin C were obtained from Bristol Myers Squibb (Syracuse, NY). Adriamycin was obtained from Cetus Corp. (Emeryville, CA). Taxol for clinical use was provided by the Division of Cancer Treatment, National Cancer Institute, and was resuspended at a concentration of 6 mg/ ml in 50% polyoxyethylated castor oil (Cremophor EL) and 50% dehydrated alcohol. Cell culture reagents were obtained from GIBCO (Grand Island, NY). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. Cell culture. The BEL7404 human liver carcinoma cell line was selected for resistance to cisplatin as described [5]. The four cisplatinresistant sublines 7404-CP1, 7404-CP2.5, 7404-CP7.5, and 7404CP20 generated by this selection procedure were used for the current study. Cells were maintained at 377C in a humidified incubator containing 5% CO2 in Dulbecco’s modified Eagle’s medium with 4.5 g/ liter glucose and supplemented with 12% (v/v) fetal calf serum, 100 mg/ml streptomycin, 100 units/ml penicillin, 0.3 mg/ml glutamine, and 0.3 units/ml insulin (porcine). Cytotoxicity. The MTT assay was used to determine the relative sensitivities of the BEL7404 cell line and its cisplatin-resistant sublines to cisplatin, adriamycin, taxol, etoposide, and mitomycin C [6]. Cells were plated in 150 ml of medium per well in 96-well plates (Corning Co., Corning, NY). Following overnight incubation, cells were exposed to various concentrations of drug which were added in 10-ml volumes. Following a 72-h incubation, 40 ml of 5 mg/ml 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added per well. After 2 h at 377C, the cells were lysed by adding 100 ml of 20% (w/v) sodium dodecyl sulfate, 50% (v/v) N,N-dimethylformamide, pH 4.7, and incubated overnight at 377C. The absorbance at 570 nm was determined for each well using a Bio-Rad Model 3550 microplate reader. The reported IC50 values are the result of triplicate determinations on at least two separate occasions. Cisplatin accumulation. Duplicate 80-cm2 flasks of cells were treated with cisplatin (0–200 mM) for 4 h at 377C. Each flask was rinsed three times with Dulbecco’s phosphate-buffered saline, pH 7.1 (PBS), trypsinized, and counted using a hemacytometer. The cells were aliquoted in triplicate into 1.5-ml microfuge tubes and pelleted by centrifugation. Following an overnight incubation in 70% nitric acid, the samples were incubated at 957C for 10 min and analyzed for platinum content by atomic absorption spectrometry (AAS) essentially as described [7]. An additional three aliquots of the cells were pelleted and redissolved in 1 ml of 0.5 M Na2HPO4/NaH2PO4, pH 8.5, containing 1% (w/v) SDS, 1 mM EDTA, and 4 M urea. Protein concentrations were measured for these samples using the bicinchoninic acid protein determination kit (Sigma). Relative drug accumulation differences were determined by comparing the slopes of the lines generated for each cell line. The experiment was repeated once for the BEL7404 cell line and twice for the 7404-CP20 cell line. Platinum efflux. Duplicate flasks (25 cm2) containing BEL7404 and 7404-CP20 cells were incubated with cisplatin (50 and 500 mM, respectively) for 4 h at 377C. The cells were either harvested immedi-

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ately or incubated for 0.5 to 4 h in fresh medium. Following the incubation period, the flasks were rinsed three times with PBS and the cells were trypsinized, counted, and pelleted by centrifugation. The cell pellets were redissolved in 70% nitric acid and platinum content was determined by AAS as described above. DNA platination and interstrand crosslink formation. To measure platinum–DNA adduct formation, BEL7404 and 7404-CP20 cells were incubated for 4 h with 0–200 mM cisplatin, washed three times with PBS, and incubated overnight at 377C in lysis buffer (10 mM Tris–HCl, pH 7.4, containing 0.15 M NaCl, 10 mM EDTA, 0.1% (w/v) SDS, and 200 mg/ml proteinase K). High-molecular-weight DNA was isolated by the phenol/chloroform method, restriction-digested with HindIII, and an aliquot of DNA (2 mg) was used for interstrand crosslink determination by renaturing agarose gel electrophoresis (RAGE) [8]. Briefly, DNA was incubated at 657C for 5 min in 0.2 N NaOH and placed on ice for 2 min. Samples were loaded onto a 0.5% agarose gel prepared in 90 mM Tris-borate buffer containing 2 mM EDTA and electrophoresed for 4 h at 90 V. Following Southern blotting, the membrane was placed in hybridization solution containing the ABEII probe (provided by Dr. J. E. Sylvester, University of Pennsylvania) which recognizes a 17-kb fragment of the 28S ribosomal RNA gene [9]. Histograms were generated for each lane using an AMBIS radioanalytic imaging system (AMBIS Systems, San Diego, CA), and the fraction of crosslinked strands was determined by weight analysis of the peaks. The average number of interstrand crosslinks per fragment was calculated using the Poisson distribution equation: 0ln(1 0 Fc), where Fc is the fraction of DNA strands containing crosslinks [8]. The remainder of the DNA samples were used for total platinum–DNA adduct determination. Samples were incubated at 957C in 10% HCl and platinum was measured by AAS as described [7]. Relative differences in platinum–DNA adduct formation were determined by comparing the slopes of the regression lines generated for each cell line in triplicate experiments. Platinum–DNA adduct removal. Triplicate flasks (175 cm2) containing BEL7404 and 7404-CP20 cells were incubated with 16 and 150 mM cisplatin, respectively, for 4 h at 377C. Cells were either harvested immediately or incubated in fresh medium for 8 h. DNA was isolated and analyzed for platinum content by AAS or for ribosomal RNA gene-specific interstrand crosslinks using RAGE as described above.

RESULTS

A series of cisplatin-resistant human hepatoma cell lines (7404-CP1, 7404-CP2.5, 7404-CP7.5, and 7404CP20) were derived by exposure of the parental BEL7404 cell line to stepwise, increasing cisplatin concentrations (0.3 to 20 mg/ml) over a period of 24 months [5]. The relative sensitivities of these five cell lines to cisplatin, adriamycin, taxol, etoposide, and mitomycin C were measured using the MTT assay (Table 1, Fig. 1). The 7404-CP20 cell line was 34-fold resistant to cisplatin compared to the BEL7404 cell line (IC50 values 144 and 4.2 mM, respectively), while the 7404-CP1, 7404-CP2.5, and 7404-CP7.5 cell lines were 3.9-, 7.6-, and 13.6-fold resistant relative to the parental cell line. Significant cross-resistance to adriamycin, taxol, etoposide, or mitomycin C was not observed (i.e., ú2-fold) in any of the cisplatin-resistant sublines. Cisplatin accumulation was measured in the five cell lines following a 4-h exposure to a range of cisplatin concentrations and was linear up to 200 mM cisplatin

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TABLE 1 Sensitivity of BEL7404 and Cisplatin-Resistant Sublines to Various Drugs Cisplatin IC50 (mM)a

Cell line BEL7404 7404-CP1 7404-CP2.5 7404-CP7.5 7404-CP20

4.2 16.3 32.0 57.0 144.0

(1.0) (3.9) (7.6) (13.6) (34.3)

Adriamycin IC50 (mM) 0.126 0.210 0.065 0.083 0.151

Taxol IC50 (nM)

(1.0) (1.7) (0.5) (0.7) (1.2)

2.2 2.0 1.9 3.0 4.5

(1.0) (0.9) (0.9) (1.4) (2.0)

Etoposide IC50 (mM) 0.90 1.84 0.62 1.09 0.93

(1.0) (2.0) (0.7) (1.2) (1.0)

Mitomycin C IC50 (nM) 0.37 0.41 0.23 0.46 0.64

(1.0) (1.1) (0.6) (1.2) (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 average of triplicate determinations measured on at least two separate occasions. Relative resistance to the BEL7404 cell line is indicated in parentheses.

(Fig. 2). There was a strong correlation (r Å 0.98) between cisplatin accumulation and relative cisplatin resistance among the cell lines, with the parental BEL7404 cell line containing the most cell-associated platinum followed in decreasing order by the 7404-CP1, 7404-CP2.5, 7404-CP7.5, and 7404-CP20 cell lines, respectively. The magnitude of the accumulation difference between the most cisplatin-sensitive (BEL7404) and -resistant (7404-CP20) cell lines was 14-fold. There was little change in the levels of cell-associated platinum between these two cell lines after treating cells with cisplatin for 4 h followed by a 4-h drug-free incubation period (Fig. 3). The formation of platinum–DNA adducts was measured in the BEL7404 and 7404-CP20 cell lines by

atomic absorption spectrometry following a 4-h exposure to a range of cisplatin concentrations (Fig. 4). Platinum–DNA adduct formation was linear with cisplatin concentration (up to 200 mM) and there was a 9-fold decrease in the adduct levels formed in the 7404-CP20 cell line relative to the parental BEL7404 cell line. Cisplatin interstrand crosslink levels formed in the ribosomal RNA gene, as measured by renaturing agarose gel electrophoresis (RAGE), were also 12-fold lower in the 7404-CP20 cell line relative to the BEL7404 cell line (Figs. 5 and 6). Using these data, it was calculated that 2 to 3% of the total platinum–DNA adducts were present as interstrand crosslinks in these cell lines. Platinum–DNA adduct removal was measured in

FIG. 1. Sensitivities of the 7404-CP1, 7404-CP2.5, 7404-CP7.5, and 7404-CP20 cell lines to cisplatin, adriamycin, taxol, etoposide, and mitomycin C as measured relative to the parental BEL7404 cell line.

FIG. 2. Cisplatin accumulation in the BEL7404 and cisplatinresistant sublines. Duplicate flasks were incubated with 0–200 mM cisplatin for 4 h, harvested, and processed for atomic absorption spectrometry as described under Materials and Methods.

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FIG. 5. Measurement of cisplatin interstrand crosslink formation in the BEL7404 and 7404-CP20 cell lines using renaturing agarose gel electrophoresis (RAGE). DNA was denatured and electrophoresed on a 0.5% agarose gel. Following Southern transfer, the membrane was hybridized with 32P-labeled ABEII fragment which is specific for the ribosomal RNA gene.

tively. Cisplatin interstrand crosslinks were also removed from the ribosomal RNA gene at a similar rate by the two cell lines. DISCUSSION

FIG. 3. Platinum efflux in the BEL7404 and 7404-CP20 cell lines. Following a 4-h exposure to cisplatin, the cells were incubated in drug-free medium for various times up to 4 h, harvested, and platinum content was measured by AAS.

the BEL7404 and 7404-CP20 cell lines (Table 2). Following an 8-h incubation in drug-free medium, the BEL7404 and 7404-CP20 cell lines removed 17 and 21% of their total platinum–DNA adducts, respec-

FIG. 4. Formation of platinum–DNA adducts in BEL7404 and 7404-CP20 following a 4-h exposure to cisplatin (0–200 mM). DNA was isolated and measured for platinum content as described under Materials and Methods.

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Cisplatin resistance may be multifactorial, consisting of cellular mechanisms which limit the formation of platinum–DNA damage and mechanisms which enable DNA lesions to be repaired or tolerated. We have characterized a panel of cisplatin-resistant human hepatoma cell lines which were derived by in vitro exposure of the parental BEL7404 cell line to cisplatin. The BEL7404 cell line, which was established from a

FIG. 6. Results of renaturing agarose gel electrophoresis (RAGE) for measuring the formation of cisplatin interstrand crosslinks in the ribosomal RNA gene of BEL7404 and 7404-CP20 following a 4h exposure to cisplatin (0–200 mM).

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TABLE 2 Removal of Total Platinum–DNA Adducts and Ribosomal RNA Gene-Specific Interstrand Crosslinks (ICL) in BEL7404 and 7404-CP20 Cells

Cell line BEL7404 7404-CP20

Repair time (h)

Pt–DNA adducts (pg Pt/mg DNA)a

0 8 0 8

23 19 42 33

{ { { {

3 2 (17%) 1 2 (21%)

ICL/10 kba 0.020 0.017 0.022 0.019

{ { { {

.003 .002 (15%) .003 .001 (14%)

a Total platinum–DNA adduct levels were determined by AAS and gene-specific interstrand crosslink levels were determined by RAGE. The percentage of platinum–DNA adduct removal is indicated in parentheses.

primary liver carcinoma of an untreated patient [10], is intrinsically resistant to cisplatin [5]. In the present study, the cisplatin-selected sublines 7404-CP1, 7404CP2.5, 7404-CP7.5, and 7404-CP20 showed increased resistance to cisplatin (up to 34-fold) relative to the parental cell line, but did not exhibit cross-resistance to adriamycin, taxol, etoposide, or mitomycin C. Shen et al. [5] reported previously, however, that the 7404-CP7.5 subline is 13-, 23-, and 39-fold resistant to 6-mercaptopurine, 5fluorouracil, and methotrexate, respectively, compared to the BEL7404 cell line. The values reported for cisplatin differ somewhat from the previously reported IC50 values for these cell lines using a different growth assay [5]. In order to determine the mechanism(s) responsible for the increased cisplatin resistance in the 7404-CP1, 7404-CP2.5, 7404-CP7.5, and 7404-CP20 cell lines, cisplatin accumulation and platinum–DNA adduct formation and removal were measured. Reduced cisplatin accumulation (up to 14-fold in 7404-CP20) was observed in the cisplatin-selected sublines and was associated with decreased overall platinum–DNA adduct formation, decreased interstrand crosslink formation, and decreased cisplatin sensitivity. No differences in platinum efflux rates were measured following a 4-h exposure to cisplatin, indicating that the decreased accumulation results from either rapid efflux immediately after cisplatin enters the cells or from reduced drug uptake. This phenotype has frequently been observed in other cell lines selected for primary cisplatin resistance; however, the mechanism(s) by which decreased cellular cisplatin accumulation occurs is unknown. [7, 11–18]. The available data indicate that cisplatin enters cells by passive diffusion and/or carrier-mediated transport (reviewed in 19). Evidence for cisplatin uptake by passive diffusion has been provided by studies in which cisplatin uptake was shown to be nonsaturable, even up to its solubility limit, and not inhibited by structural

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analogs [20–22]. Several observations have led investigators to conclude, however, that a carrier-mediated transport protein may also be involved in cisplatin uptake. For example, cisplatin accumulation has been shown to be partially energy-dependent, ouabain-inhibitable, sodium-dependent, and affected by membrane potential and cAMP levels [23–25]. Based on the current understanding of cisplatin passive and facilitated transport, several approaches have been explored to circumvent decreased cisplatin accumulation in cisplatin-resistant cells. The polyene macrolide antibiotic amphotericin B, which permeabilizes cell membranes by associating with sterols resulting in the formation of pores or channels, has been shown to selectively increase cisplatin accumulation in cisplatin-resistant human non-small lung carcinoma and ovarian carcinoma cell lines [26–29]. The levels of amphotericin B required to achieve a potentiating effect in vivo, however, are nephrotoxic. In another study, two calmodulin antagonists were shown to increase cisplatin uptake in cisplatin-resistant human ovarian cancer cell lines, restoring their intracellular platinum levels to that of the similarly treated parental cell line [30]. Although not selective for cisplatin-resistant cells exhibiting the cisplatin accumulation defect, other pharmacologic agents have been reported to increase cellular cisplatin accumulation such as forskolin and 3isobutyl-1-methylxanthine which increase cAMP levels [25], the nucleotide transport inhibitor dipyridamole [31], the mitotic inhibitor 1-propargyl-5-chloropyrimidine-2-one [32], and the fatty acid docosahexaenoic acid [33]. Alternatively, lipophilic platinum analogs, such as the ammine/amine platinum (IV) dicarboxylates described by Kelland et al. (18), have been shown to enter cells more readily than cisplatin. Although no specific proteins have been directly implicated in the cisplatin accumulation defect often observed in cisplatin-resistant cells, increased amounts of several proteins have been found to be associated with this phenotype. Shen et al. [5] observed increased levels of a 50- and 52-kDa protein and decreased amounts of a 35-kDa protein in 7404-CP20 and KBCP20 cells relative to the cisplatin-sensitive cells from which they were derived. The KB-CP20 cells also contained decreased levels of a 57-kDa protein. Subsequent microsequencing and Western blot analysis indicated that the major 52-kDa protein was heat shock protein HSP60. Elevated levels of HSP60 in cisplatinresistant human ovarian cancer and human bladder carcinoma cell lines have also been reported [34]. An earlier study found the overexpression of a 200-kDa membrane glycoprotein in a cisplatin-resistant murine lymphoma cell line which was associated with cisplatin resistance [35]. Despite these findings, it remains to be determined whether these proteins have a role in cisplatin resistance or whether their increased levels

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result from chronic cisplatin exposure during in vitro selection. We have speculated that this chronic cisplatin exposure could upregulate transcription factors which activate a range of genes, some relevant to resistance and others only by virtue of their similar mechanism of transcriptional activation [36]. A second cisplatin-resistance mechanism which we observed in the 7404-CP20 cell line is increased platinum–DNA damage tolerance. This was determined by calculating the amount of platinum–DNA damage present at equitoxic cisplatin doses for the BEL7404 and 7404-CP20 cell lines. We found that at their respective cisplatin IC50 values, the 7404-CP20 cell line contained 48 pg Pt/mg DNA and the BEL7404 cell line contained 15 pg Pt/mg DNA, resulting in a 3-fold difference. Decreased cisplatin sensitivity associated with increased platinum–DNA damage tolerance has also been observed in other model systems [37–39]; however, the underlying mechanism(s) for this remains to be determined. Enhanced replicative bypass of a DNA lesion is one way a cell can exhibit damage tolerance. This has been demonstrated in cisplatin-resistant murine leukemia cells in which a 3- to 4-fold increase in DNA synthesis past platinum–DNA adducts was observed [40]. It has also been shown that DNA polymerase b is capable of efficiently bypassing a single d(GpG)Pt adduct in vitro, which suggests that it may play a role in translesion DNA synthesis [41]. Alternatively, cisplatin-resistant cells may require higher lesion densities in order to undergo programmed cell death. Although this pathway has not been fully elucidated, it has been demonstrated that the inhibition of apoptosis results in reduced sensitivity to a variety of chemotherapeutic agents [42]. Other cisplatin resistance mechanisms were also considered in the panel of human hepatoma cell lines. Increased DNA repair has been shown to be associated with cisplatin resistance in several cell lines [7, 37–39, 43–46]; however, we did not observe any differences in the removal of total platinum–DNA adducts or interstrand crosslinks between the BEL7404 and 7404CP20 cells. We have demonstrated previously using a human ovarian cancer cell line (C200) that the individual platinum–DNA lesions are removed with varying efficiency (50 to 80% at 12 h) and it is unclear from the present study if the removal of one or more of the individual adducts is favored [39]. We did not observe an increase in the removal of cisplatin interstrand crosslinks relative to removal of total platinum–DNA adducts which has previously been reported in human ovarian cancer cell lines [7, 39, 46]. It is also unlikely that increased cisplatin inactivation contributes to cisplatin resistance in the 7404-CP20 cells since the relative differences in cisplatin accumulation and platinum–DNA adduct formation were similar in the

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BEL7404 and 7404-CP20 cells (14- and 9-fold, respectively). In conclusion, the BEL7404 cell line and its cisplatinresistant sublines provide a valuable model system for understanding the molecular basis of decreased cisplatin accumulation, especially since the magnitude of the accumulation defect is relatively high (14-fold) compared to that observed in other model systems [7, 11– 18]. If decreased cisplatin accumulation proves to be a clinically relevant obstacle to the successful treatment of cisplatin-refractory cancer patients, then a thorough understanding of cisplatin uptake will be required for the development of effective modulation strategies. This work was supported in part by CA51228 to T.C.H.

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

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