Treatment Patients, and Its Induction by Buthionine Sulfoximine RNA Expression in Peripheral Mononuclear Cells of Cancer -Glutamylcysteine Synthetase Messenger

The role of glutathione (GSH) in tumor cell resistance to alkylating agents and platinum compounds is suggested by a body of laboratory and clinical studies. The rate-limiting enzyme in GSH synthesis is y-glutamylcysteine synthetase (y-GCS), the expression of which is proportional both to GSH content and to the level of resistance in ovarian cancer cell lines. The role of this enzyme in regulating GSH levels is unclear, however. Reversal of resistance is achieved in vitro and in vivo with the use of buthionine sulfoximine (BSO), a potent inhibitor of y-GCS. In the course of a Phase I clinical trial of BSO and melphalan, we have measured GSH and expression of y-GCS mRNA in peripheral mononuclear cells before and at intervals after the initiation of treatment with BSO. Mean baseline GSH content was 6.89 nmol/mg protein. Treatment with BSO (10.5 to 17 g/m2 i.v. every 12 h for six doses) resulted in a mean nadir GSH decline to 19% of control values, most commonly on day 3. Baseline expression of y-GCS mRNA was measured by a reverse transcriptase polymerase chain reaction-based method. When described relative to that of 0-actin. the expression of y-GCS varied over 3-fold among individuals. Following GSH depletion by BSO, the level of y-GCS mRNA rose successively on days 3 and 5 to reach a mean increase of 2-fold on day 8. Differences were observed among patients in their capacity to respond to GSH depletion by increasing y-GCS steady-state mRNA levels (1.4to 3.1-fold). These re sults show that the expression of y-GCS is variable in the population and suggest that the cellular content of GSH may be involved in the regulation of its expression. Introduction A role for GSH1 in tumor cell resistance to alkylating agents and platinum compounds was proposed by Meister and Anderson (1), and is increasingly well established. Elevated GSH content was described in L1210 murine leukemia cells resistant to melphalan (2, 3). Manip ulation of thiol content by altering growth conditions or by depleting GSH with diethylmaleate resulted in sensitization of the cells to alkylatotinduced cytotoxicity (2. 3). Subsequent studies in tumor cell lines of human origin have demonstrated that elevated GSH levels are frequently associated with resistance in vitro (4, 5). Behrens et al. (6) have isolated a series of stably resistant human ovarian cancer cell lines by selection in escalating concentrations of cisplatin. The resis tance of these lines to cisplatin is directly correlated with their GSH .content (7). Additional evidence has been provided by the analysis of clinical specimens from patients who have relapsed from treatment. These specimens have higher GSH content than those obtained from Received 5/5/93; accepted 7/2/93. 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. 1Supported by NIH Grants CA 06972, C A 49820. CA 51175. and C A 52181 and an appropriation from the Commonwealth of Pennsylvania. 3 To whom requests for reprints should be addressed, at Fox Chase Cancer Center. 7701 Burholme Avenue. Philadelphia. PA 19111. ' The abbreviations used are: GSH, glutathione; -y-GCS. -y-glutamylcysteine syn thetase; BSO. buthionine sulfoximine; PMN. peripheral mononuclear cells; PCR, poly merase chain reaction; cDNA, complementary DNA. untreated patients (8). These results indicate that modulation of GSH metabolism may be an appropriate means of reversing drug resistance. The tripeptide GSH is synthesized intracellularly in two reactions. The initial conjugation of glutamate to cysteine is catalyzed by y-GCS and is the rate-limiting step in GSH synthesis (9). Addition of glycine by GSH synthetase concludes the reaction pathway (9). The cisplatinresistant ovarian carcinoma cell lines with elevated GSH content demonstrate markedly increased steady-state levels of y-GCS mRNA (7). The basis for this overexpression is not yet known; Southern analysis reveals no evidence of gene amplification (7). Current studies are directed to elucidating the regulation of GSH synthesis; the ex istence of a feedback mechanism is postulated but not established. The variability of y-GCS expression in the population has not been char acterized; the extent of this variability may have profound conse quences for the pharmacodynamics of antitumor drugs and other reactive compounds. In an ongoing clinical trial, we have administered the y-GCS in hibitor BSO to patients with advanced cancer (10). The synthesis of BSO by Meister and Griffith (11) provided a means of depleting cells of GSH. BSO treatment of cells in culture and tumor-bearing rodent models results in depletion of GSH in a doseand schedule-dependent manner (12). Patients entered in this Phase I trial received varying doses (ranging from 10.5 to 17 g/min these patients) of BSO by 30-min i.v. infusion every 12 h for a total of six doses. In a group of ten patients, we measured GSH and RNA expression of y-GCS in PMN before and at intervals following treatment. The results show that the basal level of y-GCS mRNA expressed varies by a factor of 3 among individuals. Following the institution of BSO treatment, the expression of y-GCS increased, with a time course that mirrored the fall in GSH. These findings suggest that GSH concentrations may influence the expression of the gene responsible for their maintenance. Materials and Methods Drugs, Chemicals, and Reagents. BSO for (his study was provided by (he Cancer Therapy Evaluation Program. Division of Cancer Treatment. National Cancer Institute, in glass vials containing 1 g. Separation of PMN was per formed using lymphocyte preparation medium (Organon Teknika; density. 1.080). [y-';P]CTP (3000 Ci/mmol) was purchased from Amersham. All other chemicals were obtained from Sigma except where stated and were reagent grade. Patients and Treatment. The patients entered on the clinical trial of BSO (10) were required to have a histological diagnosis of cancer and to have exhausted the standard therapeutic options for their disease. They were over 18 years of age, with an Eastern Cooperative Oncology Group performance status of 0 or 1. They had adequate bone marrow (WBC >4.000/mm\ granulocyte count >2.000/mnr\ platelets 5100,000/mm-'), liver [bilirubin <1.5 mg/dl. aspartate aminotransferase <4 times upper normal value] and kidney (serum creatinine <1.5 mg/dl or creatinine clearance >60 ml/min) function. All pa tients gave written informed consent in accordance with Federal. State, and Institutional guidelines. BSO was reconstituted in 50 to 250 ml of 0.9% sodium chloride, USP, and administered i.v. over 30 min at 12-h intervals for six doses. Immediately