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Effect of D,L-Buthionine-S,R-sulfoximine on Cytotoxicity and DNA Cross-Linking Induced by Bifunctional DNA-reactive Cytostatic Drugs in Human Melanoma Cells¹

Radiumhemmet, Karolinska Hospital; Department of Tumor Biology II, Karolinska Institute; and Karolinska Pharmacy, S-104 01 Stockholm, Sweden

The effects of D,L-buthionine-S,R-sulfoximine (BSO) on cytotoxicity and DNA cross-linking induced by bifunctional DNA-reactive cytostatic agents in a human melanoma cell line (RPMI 8322) were investigated. RPMI 8322 cells were exposed to 0.01 mM BSO for 24 h, which resulted in a decrease in cellular glutathione to 14% without any reduction of cell proliferation or plating efficiency. BSO pretreatment significantly enhanced cytotoxicity of melphalan with a dose modification factor (DMF) of 3.4 and nitrogen mustard (HN2) (DMF 3.3). The increased cytotoxicity was paralleled by similar increases in DNA cross-linking (melphalan: DMF 2.2, HN2: DNF 2.5). A small but significant potentiation by BSO of cis-diamminedichloroplatinum(II) toxicity was seen (DMF 1.5), with a corresponding minor but significant increase in DNA cross-linking (DMF 1.1). Similarly, the potentiation of bis-chloroethylnitrosurea toxicity was small but significant (DMF 1.1), with no significant increase in DNA cross-linking (DMF 1.0). No effect of BSO pretreatment on the rate of removal of HN2-induced DNA cross-links was observed. Thus, the observed sensitization of RPMI 8322 cells to melphalan, HN2, cis-diamminedichloroplatinum(II), and bis-chloroethylnitrosourea was correlated to similar changes in drug-induced DNA cross-linking. Despite the increased cytotoxicity and DNA cross-linking BSO did not significantly increase the intracellular concentration of intact melphalan. These findings support the hypothesis that the potentiation of the cytotoxicity of bifunctional alkylating agents by BSO is due to an increased DNA cross-linking caused by a reduced intracellular conjugation of drug with glutathione, which results in an increased binding of drug to DNA targets.

Drug resistance is a major problem in clinical cancer chemotherapy. Malignant melanoma is a tumor with a high degree of inherent resistance to all available cytostatic drugs. Treatment of patients with metastatic malignant melanoma with drugs such as melphalan, BCNU,³ or cis-DDP as single agents induces objective tumor responses in only 10–20% of patients (1). Moreover, those tumors which initially respond to chemotherapy rapidly become resistant, since the median duration of remission is only a few months. The reason for the resistance to chemotherapy is poorly understood.

Several factors are of importance for cellular resistance to bifunctional alkylating agents and other antineoplastic agents that bind bifunctionally to DNA (for reviews, see Refs. 2–4). In some (5–8), but not all (9, 10), cases resistance to melphalan has been related to a reduced cellular uptake of drug. In previous investigations we have found that a human melanoma cell line (RPMI 8322) is relatively resistant to melphalan (11) and cis-DDP,⁴ as compared to normal phytohemagglutinin-stimulated lymphocytes. The accumulation of melphalan (12) and platinum⁴ was similar in both cell types, but the cellular content of glutathione (GSH) was 1.8-fold higher in the melanoma cells (12). The tripeptide GSH (L--glutamyl-L-cysteinyl-glycine), the main intracellular thiol, is known to have important functions in many biological phenomena (for review, see Ref. 13). It has been known for two decades that murine tumor cells with acquired resistance to bifunctional alkylating agents such as nitrogen mustard and melphalan may have increased levels of GSH (14). More recently an increase in the levels of GSH or nonprotein thiols (most of which consist of GSH) has been found in human tumor cells (9, 15) including melanoma cells (10) with induced resistance to alkylating agents. In melphalan-resistant murine L1210 cells the increase in GSH has been correlated to an increased dechlorination of melphalan to the inactive derivative dihydroxy-melphalan (16). Further, it has been demonstrated that such L1210 cells can be sensitized to melphalan in vitro by reducing the concentration of L-cysteine in the growth medium (17) and in vivo by excluding L-cysteine and L-methionine from the diet of tumor-bearing mice (18). Both manipulations efficiently decrease the GSH concentration in the L1210 cells. BSO is a potent and specific inhibitor of -glutamylcysteine synthetase and thus inhibits GSH synthesis at low doses without causing significant cytotoxic effects (19, 20). It has been shown that drug-resistant tumor cells can be sensitized to alkylating agents such as melphalan by treatment with BSO (15, 21–26). In addition, some (15, 24) but not all (23) investigators have found a sensitization of tumor cells to cis-DDP by BSO. However, the mechanisms of sensitization of tumor cells to alkylating agents and cis-DDP by a decrease in GSH levels remain obscure.

The present investigation aims to increase our knowledge of the mechanisms of sensitization of malignant cells to bifunctional DNA-reactive cytostatic agents by BSO. We have investigated the effect of BSO on the cytotoxity of melphalan, HN2, cis-DDP, and BCNU on RPMI8322 human melanoma cells. The effect of BSO on the induction of DNA cross-links by the drugs has been studied with alkaline elution of DNA. In addition, the effect of BSO on the intracellular concentration of active drug following exposure to melphalan has been measured with liquid chromatography.

¹ This investigation was supported by King Gustaf V's Jubilee Foundation, the Swedish Cancer Society, and Robert Lundberg's Memorial Fund, Stockholm, Sweden.

³ The abbreviations used are: BCNU, bis-chloroethylnitrosourea; cis-DDP, cis-diamminedichloroplatinum(II); GSH, glutathione; BSO, D,L-buthionine-S,R-sulfoximine; HN2, nitrogen mustard; MEM, minimal essential medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; TEAH, tetraethylammoniumhydroxide; DMF, dose-modification factor.

⁴ J. Hansson, R. Lewensohn, and U. Ringborg. cis-Diamminedichloroplatinum(II) toxicity in human melanoma cells and lymphocytes as related to cellular platinum accumulation, DNA cross-linking, and inhibition of DNA synthesis, submitted for publication.

² To whom requests for reprints should be addressed.

Received 5/21/87. Revised 9/16/87. Accepted 9/28/87.