622
BIOCHEMICAL SOCIETY TRANSACTIONS
Mechanisms of cisplatin cytotoxicity in human ovarian carcinoma cell lines SHARON SHELLARD,* LOUISE HOSKING,* ANNE M A N E FICHTINGER-SCHEPMANt and BRIDGET T. HILL* *Laboratory of Cellular Chemotherapy, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, U.K. and t TNO Medical Biological Laboratory, Rijswijk, The Netherlands Cisplatin (cis-diamminedichloroplatinum 11) is a widely used antitumour agent for the treatment of ovarian and testicular cancers. The clinical efficacy of this drug is limited, however, by the development of drug resistance [ 11. Since the antineoplastic activity of cisplatin is almost certainly due to its interactions with DNA, we have studied the mechanisms of cisplatin-induced DNA damage and repair in two human ovarian carcinoma cell lines exhibiting differential drug sensitivities. The two continuous cell lines SK-OV-3 and TR175 were derived from adenocarcinomas of the ovary from patients pretreated with chemotherapy, although neither had previously received cisplatin (cf. [2]).Clonogenic assay data for a 1 h cisplatin exposure, indicated a 26-fold differential sensitivity when comparing the more sensitive TR175 cells with the more resistant SK-OV-3 cells. In an attempt to define the mechanisms of cisplatin action in these two cell lines we have studied the following parameters: (i) drug uptake and DNA binding; (ii) drug inactivation involving glutathione metabolism; (iii) DNA repair and (iv) tolerance to the druginduced damage. A similar pattern of drug uptake and DNA binding was observed in both cell lines following a 1 h exposure to radiolabelled cisplatin. When standardized for differential cell volumes, approximately 4 pmollunit of drug were taken up by both cell lines, whereas DNA binding occurred to a level of approximately 6 pmol/unit of cisplatin per g of DNA. Several reports suggest [3, 41 that the interaction beween cisplatin and DNA may be reduced by the elevation of glutathione and related enzyme activities, in addition to the cytosolic protein metallothionein. Elevated levels of glutathione (2.9-fold), glutathione reductase (6.3-fold) and glutathione peroxidase ( 1.8-fold) were associated with the more resistant SK-OV-3 cell line. However, compared with three other human ovarian carcinoma lines, no significant. differences in these various parameters were observed to account for the hypersensitivity of the TR175 cells to cisplatin. With the aid of polyclonal antisera, the induction and removal of platinum-DNA (Pt-DNA) adducts have been quantitated using a competitive enzyme-linked immunosorbent assay (e.1.i.s.a.). DNA was isolated from cells either immediately after a 1 h exposure to 33.3 pwcisplatin or Abbreviations used: cisplatin, cis-diamminedichloroplatinum Pt-DNA adducts, platinum-DNA adducts; Pt-GMP, Pt(NH),dGMP Pt-AG, cis-Pt(NH,),d(pApG); Pt-GG, cisPt(NH,)2d(pGpC);Pt-(GMP)2,cis-Pt(NH,)zd(GMP)z.
18 h after the initial drug exposure to allow for DNA repair. DNA was digested to nucleotide and platinum-containing oligonucleotides [ 5 ]and digestion products were separated using anion-exchange chromatography. Fractions collected from the column were analysed by competitive e.1.i.s.a. Three antisera were used to quantify, four Pt-DNA adducts. Antiserum 3/43 was used to detect Pt-GMP, antiserum 3/65 to detect Pt-AG and antiserum WlOl to detect both Pt-GG and Pt-(GMP), adducts. In both cell lines the predominant lesion appeared to be the Pt-GG adduct accounting for approximately 50% of the total platination. The more sensitive TR175 cells appeared proficient in the removal of all four Pt-DNA adducts. In contrast, the more resistant SKOV-3 cells appeared deficient in the repair of the major adduct Pt-GG and the difunctional Pt-(GMP)2adduct. Due to the inability of the SK-OV-3 cells to remove the Pt-GG and Pt(GMP), adducts, these cells appeared to tolerate higher levels of platination at the 18 h time point. The antiserum W 10 1 detects the major adduct Pt-GG and the difunctional Pt-(GMP), including interstrand cross-links. We have therefore employed the technique of alkaline elution [6] to quantify the latter lesion. Alkaline elution studies revealed that more interstrand cross-linking at equimolar concentrations of cisplatin were induced in the more resistant SK-OV-3 cells than the TR175 cells. However, both cell lines appeared proficient in the removal of this lesion. In conclusion, the more resistant SK-OV-3 cells appeared to be deficient in the repair of two Pt-DNA adducts, Pt-GG and Pt(GMP),. In this cell line resistance to cisplatin may be associated with increased tolerance of the drug-induced damage which therefore permits replication on a damaged DNA template. However, the ability of the TR175 cells to remove all Pt-DNA adducts, in addition to interstrand crosslinks, does not account for their hypersensitivity to cisplatin. The lethal event in these TR175 cells remains to be identified. 1. Frei, E., Cucchi, C. A., Rosowski, A., Tantravaki, R.. Bernal, S., Ervin, T. J., Ruprecht, R. M. & Haseltine, W. A. ( 1 985) l'roc.
Natl. Acad. Sci. U.S.A. 82.2 158-2 162 2. Hill, B. T., Whelan, R. D. H., Gibby. E. M., Sheer, D.. Hosking, L. K., Shellard, S. A. & Rupniak. H. T. (1987) Int. J. Cancer 39, 219-225 3. Hromas, R. A., Barlogie. B., Meyn, R. E., Andrews, P. A. & Burns, C. P. ( 1985) Proc. A m . A s s w . Cancer Kes. 26,26 1-265 4 . Behrens, B. C., Hamilton, T. C., Masuda, H., Grotzinger, K. R., Whang-Peng, J., Louie, K. G., Knutsen, T., McKoy, W., Young, R.C.&Ozols,R.F.(1987)CancerRes.47,414-418 5. Fichtinger-Schepman, A. M. J., Baan, R. A,, Luiten-Schuite, A., van Dijk, M. & Lohman, P. H. M. (1985) C'hem. Biol. Interact. 55,275-288 6 . Bedford, P., Walker, M. C., Sharma, H. L., Perera, A,, McAuliffe, C. A., Masters, J. R. W. & Hill, B. T. (1987) C'hem. Biol. Inteacr. 61.1-15
(11);
Received 23 November I989
1990
BIOCHEMICAL SOCIETY TRANSACTIONS
Mechanisms of cisplatin cytotoxicity in human ovarian carcinoma cell lines SHARON SHELLARD,* LOUISE HOSKING,* ANNE M A N E FICHTINGER-SCHEPMANt and BRIDGET T. HILL* *Laboratory of Cellular Chemotherapy, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, U.K. and t TNO Medical Biological Laboratory, Rijswijk, The Netherlands Cisplatin (cis-diamminedichloroplatinum 11) is a widely used antitumour agent for the treatment of ovarian and testicular cancers. The clinical efficacy of this drug is limited, however, by the development of drug resistance [ 11. Since the antineoplastic activity of cisplatin is almost certainly due to its interactions with DNA, we have studied the mechanisms of cisplatin-induced DNA damage and repair in two human ovarian carcinoma cell lines exhibiting differential drug sensitivities. The two continuous cell lines SK-OV-3 and TR175 were derived from adenocarcinomas of the ovary from patients pretreated with chemotherapy, although neither had previously received cisplatin (cf. [2]).Clonogenic assay data for a 1 h cisplatin exposure, indicated a 26-fold differential sensitivity when comparing the more sensitive TR175 cells with the more resistant SK-OV-3 cells. In an attempt to define the mechanisms of cisplatin action in these two cell lines we have studied the following parameters: (i) drug uptake and DNA binding; (ii) drug inactivation involving glutathione metabolism; (iii) DNA repair and (iv) tolerance to the druginduced damage. A similar pattern of drug uptake and DNA binding was observed in both cell lines following a 1 h exposure to radiolabelled cisplatin. When standardized for differential cell volumes, approximately 4 pmollunit of drug were taken up by both cell lines, whereas DNA binding occurred to a level of approximately 6 pmol/unit of cisplatin per g of DNA. Several reports suggest [3, 41 that the interaction beween cisplatin and DNA may be reduced by the elevation of glutathione and related enzyme activities, in addition to the cytosolic protein metallothionein. Elevated levels of glutathione (2.9-fold), glutathione reductase (6.3-fold) and glutathione peroxidase ( 1.8-fold) were associated with the more resistant SK-OV-3 cell line. However, compared with three other human ovarian carcinoma lines, no significant. differences in these various parameters were observed to account for the hypersensitivity of the TR175 cells to cisplatin. With the aid of polyclonal antisera, the induction and removal of platinum-DNA (Pt-DNA) adducts have been quantitated using a competitive enzyme-linked immunosorbent assay (e.1.i.s.a.). DNA was isolated from cells either immediately after a 1 h exposure to 33.3 pwcisplatin or Abbreviations used: cisplatin, cis-diamminedichloroplatinum Pt-DNA adducts, platinum-DNA adducts; Pt-GMP, Pt(NH),dGMP Pt-AG, cis-Pt(NH,),d(pApG); Pt-GG, cisPt(NH,)2d(pGpC);Pt-(GMP)2,cis-Pt(NH,)zd(GMP)z.
18 h after the initial drug exposure to allow for DNA repair. DNA was digested to nucleotide and platinum-containing oligonucleotides [ 5 ]and digestion products were separated using anion-exchange chromatography. Fractions collected from the column were analysed by competitive e.1.i.s.a. Three antisera were used to quantify, four Pt-DNA adducts. Antiserum 3/43 was used to detect Pt-GMP, antiserum 3/65 to detect Pt-AG and antiserum WlOl to detect both Pt-GG and Pt-(GMP), adducts. In both cell lines the predominant lesion appeared to be the Pt-GG adduct accounting for approximately 50% of the total platination. The more sensitive TR175 cells appeared proficient in the removal of all four Pt-DNA adducts. In contrast, the more resistant SKOV-3 cells appeared deficient in the repair of the major adduct Pt-GG and the difunctional Pt-(GMP)2adduct. Due to the inability of the SK-OV-3 cells to remove the Pt-GG and Pt(GMP), adducts, these cells appeared to tolerate higher levels of platination at the 18 h time point. The antiserum W 10 1 detects the major adduct Pt-GG and the difunctional Pt-(GMP), including interstrand cross-links. We have therefore employed the technique of alkaline elution [6] to quantify the latter lesion. Alkaline elution studies revealed that more interstrand cross-linking at equimolar concentrations of cisplatin were induced in the more resistant SK-OV-3 cells than the TR175 cells. However, both cell lines appeared proficient in the removal of this lesion. In conclusion, the more resistant SK-OV-3 cells appeared to be deficient in the repair of two Pt-DNA adducts, Pt-GG and Pt(GMP),. In this cell line resistance to cisplatin may be associated with increased tolerance of the drug-induced damage which therefore permits replication on a damaged DNA template. However, the ability of the TR175 cells to remove all Pt-DNA adducts, in addition to interstrand crosslinks, does not account for their hypersensitivity to cisplatin. The lethal event in these TR175 cells remains to be identified. 1. Frei, E., Cucchi, C. A., Rosowski, A., Tantravaki, R.. Bernal, S., Ervin, T. J., Ruprecht, R. M. & Haseltine, W. A. ( 1 985) l'roc.
Natl. Acad. Sci. U.S.A. 82.2 158-2 162 2. Hill, B. T., Whelan, R. D. H., Gibby. E. M., Sheer, D.. Hosking, L. K., Shellard, S. A. & Rupniak. H. T. (1987) Int. J. Cancer 39, 219-225 3. Hromas, R. A., Barlogie. B., Meyn, R. E., Andrews, P. A. & Burns, C. P. ( 1985) Proc. A m . A s s w . Cancer Kes. 26,26 1-265 4 . Behrens, B. C., Hamilton, T. C., Masuda, H., Grotzinger, K. R., Whang-Peng, J., Louie, K. G., Knutsen, T., McKoy, W., Young, R.C.&Ozols,R.F.(1987)CancerRes.47,414-418 5. Fichtinger-Schepman, A. M. J., Baan, R. A,, Luiten-Schuite, A., van Dijk, M. & Lohman, P. H. M. (1985) C'hem. Biol. Interact. 55,275-288 6 . Bedford, P., Walker, M. C., Sharma, H. L., Perera, A,, McAuliffe, C. A., Masters, J. R. W. & Hill, B. T. (1987) C'hem. Biol. Inteacr. 61.1-15
(11);
Received 23 November I989
1990
