Differential Repair of Platinum-DNA Adducts in Human Bladder and Testicular Tumor Continuous Cell Lines Philip Bedford, Anne Marie J. Fichtinger-Schepman, Sharon A. Shellard, et al. Cancer Res 1988;48:3019-3024. Published online June 1, 1988.
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[CANCER RESEARCH 48, 3019-3024, June 1, 1988)
Differential Repair of Platinum-DNA
Adducts in Human Bladder and Testicular
Tumor Continuous Cell Lines Philip Bedford, Anne Marie J. Fichtinger-Schepman, and Bridget T. Hill1
Sharon A. Shellard, M. Claire Walker, John R. W. Masters,
Laboratory of Cellular Chemotherapy, Imperial Cancer Research Fund Laboratories, Lincoln's Inn Fields, London WC2A 3PX[P. B., S. A. S., B. T. H.J, and Department of Histopathology, Institute of Urology, St. Paul's Hospital, 24, Endell Street, London WC2H 9AE [P. B., M. C. W., J. R. W. M., B. T. H.J, United Kingdom, and TNO Medical Biological Laboratory, Lange Kleiweg 139, 2280 AA Rijswijk, The Netherlands [A. M. J. F-SJ
ABSTRACT The formation and removal of four platinum-) )\A adducts were immunochemically quantitated in cultured cells derived from a human bladder carcinoma cell line (Rl 112) and from two lines derived from germ cell tumors of the testis (833K and SUSA), following exposure in vitro to 16.7 MM(5 fig/ml) cisplatin. Rl 112 cells were least sensitive to the drug and were proficient in the repair of all four adducts, whereas SUSA cells, which were 5-fold more sensitive, were deficient in the repair of DNA-DNA intrastrand cross-links in the sequences pApG and pGpG. Despite expressing a similar sensitivity to SUSA cells, 833K cells were proficient in the repair of all four adducts, although less so than the RT112 bladder tumor cells. In addition, SUSA cells were unable to repair DNA-DNA interstrand cross-links whereas 50-85% of these lesions were removed in RT112 and 833K cells 24 h following drug exposure. It is possible that the inability of SuSa cells to repair platinatoti DNA may account for their hypersensitivity to cisplatin.
INTRODUCTION Certain histológica! types of human tumor, notably those derived from germ cells in the testis, respond well to chemo therapy (1-3) whereas others, such as those derived from tran sitional cells in the bladder, are seldom eradicated by chemo therapy and periods of remission are usually short (4-6). To investigate the biological basis for this difference, we collected a series of cell lines from human testicular and bladder tumors and determined their in vitro sensitivities to cytotoxic agents (7-9). From these studies it was established that testicular tumor cell lines are, in general, more sensitive to the cytotoxic effects of a range of antitumor drugs including cu-diamminedichloroplatinum(II) (cisplatin), Adriamycin (7), and cyclophosphamide (8) and also to 7 -irradiation (9) and X-irradiâtion (10). These findings suggest that the differential chemosensitivities of these two tumor types observed clinically might not be governed by humoral factors such as altered blood supply or im munogen icity but are modulated at the cellular and molecular levels. We have investigated possible mechanisms responsible for the differential sensitivities to cisplatin expressed by these con tinuous cell lines. Cisplatin has been used effectively in the treatment of germ cell tumors of the testis (1-3) and is one of the few agents with demonstrable activity against metastatic bladder carcinoma (4,5). The antineoplastic activity of cisplatin is almost certainly due to its bifunctional interaction with DNA (for a review, see Ref. 11). Until recently, the only DNA lesions which could be reliably quantitated in mammalian cells exposed to cytotoxically relevant concentrations of drug were ISC2 and Received 7/16/87; revised 11/17/87, 2/18/88; accepted 2/29/88. 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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at the Imperial Cancer Research Fund Laboratories, Lincoln's Inn Fields, London WC2A, United King dom. 1The abbreviations used are: ISC, DNA-DNA interstrand cross-links; ELISA, enzyme-linked immunosorbent assay; Pt-GMP, Pt-(NH)3)3dGMP; Pt-AG, c/t-Pt(NH3)2d(pApG); Pt-GG, c/s-Pt(NH.,)2d(pGpG); Pt-(GMP)2, cisPt(NH3),d(GMP)2.
DNA-protein cross-links (12-14). Several reports have corre lated the number of ISC induced by cisplatin with cytotoxicity, e.g., in murine leukemia LI210 cells (12), normal and trans formed human fibroblasts (15), human bladder carcinoma cells (16), and human squamous carcinoma cells (17). The decreased ability to remove ISC of repair-deficient Chinese hamster ovary cells (18) and cells derived from persons suffering from Fanconi's anemia (19), both hypersensitive to cisplatin, would support the hypothesis that ISC are important cytotoxic lesions. In contrast, however, similar kinetics for repair of ISC has been reported in sensitive and resistant sublines of L1210 (20) and also in differentially sensitive Walker 256 rodent carcinoma cells (21), which would imply that other DNA lesions are more relevant to cytotoxicity. ISC account for less than 1% of the total platination of DNA (22) and the remaining bifunctional adducts are between bases on the same DNA strand, so-called intrastrand cross-links (23-25). Recently it has been possible, with the aid of polyclonal antisera raised to specific synthetic platinum-1 >\ A adducts, to detect intrastrand cross-links in cells exposed to cytotoxically relevant concentrations of cisplatin (26, 27). In addition, an antiserum has been raised which recognizes monofunctionally platinated guanine (27) which may be a precursor to DNA intrastrand and interstrand cross links. In this study, the induction and repair of four platinumDNA adducts has been compared in one cell line derived from a transitional cell carcinoma of the bladder and two lines originating from germ cell tumors of the testis. In addition, the physicochemical technique of alkaline elution (28) has been used to compare the repair of ISC in the bladder and testicular tumor cell lines. MATERIALS
AND METHODS
Cell Culture and Drug Treatment. The cell line RT112 was derived from a transitional cell carcinoma of the bladder (29). The line 833K was derived from an abdominal metastasis from a germ cell tumor of the testis (30) whereas the SUSA line was derived from a primary testicular teratoma (31). RT112, SUSA, and 833K cells were grown in RPMI 1640 (Gibco, Paisley, United Kingdom) supplemented with 5% fetal bovine serum (Flow Laboratories, Irvine, United Kingdom). All cells were grown as monolayers at 36.5°Cin a humidified atmosphere of 5% CO2 in air. Sensitivity to cisplatin was determined after a 1-h exposure to a range of drug concentrations using an in situ colonyforming assay as described previously for human bladder carcinoma cell lines (32). The population-doubling times of all lines were between 20 and 24 h. For quantitation of Pt-DNA adducts, cells were exposed for l h to 16.7 ¿¿M (5 /ig/ml) cisplatin (Sigma Chemical Co., Poole, United Kingdom) dissolved in 0.9% sodium chloride solution immediately prior to use. After drug exposure cells were washed and harvested either immediately or 18 h after incubation in drug-free medium. To produce the required number of cells for DNA isolation and to negate possible variations in growth state, cells grown and treated on 2-3 separate occasions were pooled. Cell pellets were stored at —20°C. For uptake studies, cells were treated in suspension with ["SmPt]-
cisplatin (kindly supplied by Dr. H. L. Sharma, Department of Medical 3019
Downloaded from cancerres.aacrjournals.org on July 10, 2011 Copyright © 1988 American Association for Cancer Research
REPAIR OF PLATINUM-DNA
ADDUCTS IN HUMAN TUMOR CELL LINES
100
Biophysics, University of Manchester, Manchester, United Kingdom) as previously described (16). Quantitation of Platinum-DNA Adducts. DNA was isolated from 5 x 10MO" cells using a combined phenol and chloroform/isoamyl alcohol technique (27). Lysis of cells was carried out overnight at 37'C in the presence of 100 m\i ammonium bicarbonate solution to inactivate monofunctionally bound drug and of proteinase K to digest the protein in DNA-protein cross-links and to destroy nuclease activity. DNA was digested to nucleotides and platinum-containing oligonucleotides as described previously (24) except that sodium azide was omitted from the incubation mixture. The purity of DNA was verified by the ratio of absorbance measurements at 260 and 280 nm and the concentration calculated using an E260of 1.11 1 mol cm~' (23). Separation of platinated DNA digestion products was achieved by anión exchange chromatography (Mono-Q; Pharmacia, Sweden) (24). The elution positions of the unmodified nucleotides dTMP and dGMP (detected spectrophotometrically at 254 nm) were used to determine the fractions to be analyzed by competitive ELISA. The expected positions in the elution pattern were established by calibrating the column with a digest of highly platinated salmon sperm DNA (24). The competitive ELISA was performed as described elsewhere (26) with the exception that only SO p\ of the various solutions were added to the microtiter wells. The precautions necessary to prevent nonspecific inhibition of the ELISA assay were routinely observed (27). Inhibition curves at four different dilutions of column fractions or standard reference solution (assayed on the same microtiter plate) were computed by linear regression (IBM, PDP-11) allowing interpolation of dilutions producing 50% inhibition to be made from which the amounts of inhibitor giving 50% inhibition in ELISA were calculated. Antiserum W101 at a dilution of 1:17,500 was used to detect the adduct-derived platinum compounds Pt-GG and Pt-(GMP)2 (26). Antiserum 3/43 was used at a dilution of 1:4x10" to detect the product Pt-GMP (27). Antiserum 3/65 was used at a dilution of 1:10* to detect the product Pt-AG (27). Correction for Dilution by DNA Synthesis. Since the occurrence of Pt-DNA adducts was quantitated per g DNA isolated, correction for dilution by DNA synthesized during the 18-h posttreatment incubation period was carried out using parallel cell cultures. Cells were labeled for 24 h with 25 nCi/ml [2-14C]thymidine (56 mCi/mmol; Amersham International, Amersham, United Kingdom) followed by 4 h in isotopefree medium. Cells were then exposed for l h to 5 Mg/ml cisplatin, as above, and harvested either immediately or 18 h later. DNA was extracted by heating cell pellets at 70°Cfor l h in l N perchloric acid. Radioactivity was determined in 50-/J aliquots of supernatant and the DNA content of the remainder was estimated spectrophotometrically after reaction with diphenylamine (33). The dilution factor was calcu lated as Specific activity of DNA at 18 h Specific activity of DNA at 0 h Values quoted are the mean ±SE of three experiments in which duplicate cultures were evaluated. The apparent number of lesions at 18 h divided by the dilution factor gave a "true" number of Pt-DNA adducts. Measurement of ISC. ISC were measured in the RT112, 833K, and SUSA cells 0, 14, and 24 h after a 1-h exposure to cisplatin using the alkaline elution technique of Kohn et al. (28) with minor modifications (16).
RESULTS Sensitivity to Cisplatin. Survival curves fitted by eye are shown in Fig. 1, from which drug concentrations reducing colony formation to 50% of controls were interpolated (Table 1). Based on a comparison of these 50% colony formation-reducing drug concentration values, the cell lines expressed a 5-fold range of sensitivities to cisplatin. SUSA cells were approximately 5 times more sensitive to the drug than K l'I 12 cells whereas 833K cells expressed a low (1.5-fold) level of resistance com pared with the SUSA cells.
U e u
40
BO
;iM CISPLATIN Fig. 1. Survival curves for (A) SUSA, (O) 833K, and (•)RT112 cells exposed to cisplatin for 1 h. Values are mean ±SE (him} of 2-5 experiments in which triplicate cultures were treated. Table I Cisplatin sensitivity and uptake off Ã-9Ã-l"PtJcisplatin in testicular and bladder tumor cell lines following a 1-h exposure to drug Cell lineSUSA
O*M)3.04.7
(pumi cells)*19.9 10"
±1.4 833K 11.7 ±0.6 RT112IC« 14.7Uptake 5.5 ±0.3 " 1C.,,, concentration of drug reducing colony formation to 50% of controls, interpolated from survival curves presented in Fig. 1. * Mean ±SD of two experiments in which duplicate samples were evaluated.
Uptake of ["SmPt|Cisplatin. Drug uptake normalized for cell number is shown in Table 1. After a 1-h exposure to 16.7 ¿IM [l95mPt]cisplatin, uptake was 3.6-fold higher in SUSA cells than in the RT112 cells, which is of the same order as their differ ential drug sensitivities. Furthermore, 833K cells, which were 1.5-fold less sensitive than SUSA cells, accumulated 1.7 times less cisplatin. Dilution by DNA Synthesis. The specific activity of radiola beled DNA decreased in all three cell lines during the 18-h posttreatment incubation period to approximately 70-80% of the values immediately following treatment. The calculated dilution factors were: SUSA, 0.70 ±0.02 (SE); 833K, 0.80 ± 0.01; and RT112, 0.79 ±0.08. Given the approximate 24-h population-doubling time of the cells, a 40% fall in specific activity in untreated cells would have been expected and it can therefore be concluded that drug exposure had caused a slight inhibition in semiconservative DNA synthesis in each line. Induction and Repair of Platinum-DNA Adducts. The elution profile of a DNA digest from SUSA cells immediately after a 1-h exposure to 16.7 fiM cisplatin is shown in Fig. 2. Platinated
3020
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REPAIR OF PLATINUM-DNA
dTMP
ADDUCTS IN HUMAN TUMOR CELL LINES
32282420
dGMP [3|
16124nSUSArw-, 120
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