[CANCER RESEARCH 45,4910-4914,1985] Depletion of Cellular Glutathione by Exogenous Spermine in V79 Cells: Implications for Spermine-induced Hyperthermic Sensitization Angelo Russo,1 James B. Mitchell, William DeGraff, Norman Friedman, and Janet Gamson Radiobiology Section, Radiation Oncology Branch, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, MD 20205 ABSTRACT one is responsible in part for maintenance of the intracellular redox state (18) and since lowering intracellular levels of gluta The relationship between spermine-induced thermosensitiza- thione results in thermal sensitization, hyperthermia may be tion and modulation in the cellular redox state as measured by imposing an oxidative stress (19-21). As part of our general glutathione levels was studied using Chinese hamster V79 cells. interest in hyperthermia and the role that oxidative stress may Marked cellular glutathione depletion was observed for cells have on hyperthermic sensitization, the present study addresses treated with exogenous 1 mw spermine at 37°Cor 43°C. Glu the possible role of exogenously applied polyamines on the tathione depletion and thermal sensitization by spermine were cellular redox state. Our data show that exogenous polyamines found to be cell density dependent with maximum depletion and may impose an oxidative stress on cells partly reflected through sensitization observed at low cell densities. These findings are modulation of intracellular glutathione levels. discussed in the context that treatment of cells with exogenous polyamines such as spermine can result in cellular oxidative stress which may in part contribute to spermine-induced thermal MATERIALS AND METHODS sensitization. Cell Culture. Stock cultures of exponentially growing Chinese hamster V79 cells were grown in F12 medium supplemented with 10% fetal calf serum, penicillin, and streptomycin. Cell cultures were maintained at INTRODUCTION 37°Cin an atmosphere of 5% C02:95% air at pH 7.3. Plating efficiencies The cytotoxicity of hyperthermia is well documented (1, 2). consistently ranged from 75-95%. However, the mechanism of hyperthermic cell death remains Drug Exposure. Spermine and spermidme were purchased from Calbiochem-Behring, San Diego, CA. Catatase, calcium chloride, AG2, obscure. Initial perturbation of the cellular membrane with sec and glutamine were purchased from Sigma Chemical Co., St. Louis, MO. ondary biochemical ramifications is considered the most likely OTZ was synthesized according to the procedures of Kaneko ef al. (22). primary effect (2). Over the last decade effects of polyamines on Characterization of the OTZ has been published elsewhere (23). Solu thermosensitization has been extensively studied (3-13). These tions of these drugs were always made up just prior to each experiment. studies strongly suggest that exogenously applied positively Cells were removed from stock cultures by trypsin, rinsed, counted, charged diamino-organic compounds have a profound thermo and plated in 75-cm2 plastic flasks at 5 x 10* cells/flask 15-18 h prior sensitization effect. How polyamines function in thermosensiti to each experiment. For some experiments the initial number of cells per zation is not known. It is particularly difficult to define a definite flask was varied so as to allow for cell density studies. Following the 15- mechanism by which polyamines sensitize to heat since the 18-h incubation the medium was removed and replaced with fresh exact role that polyamines have in a normally functioning cell is medium containing 1 mw of either spermine or spermidme. Unless otherwise stated AG was always added to the medium to a final concen not established. What is known about polyamines is that they tration of 10~5 M (24). The pH of the medium was maintained at 7.3. The are ubiquitous, their levels fluctuate as a function of cell cycle, cells were then incubated at 37°Cor 43°Cfor varying time periods. Cell their concentrations are increased in rapidly reproducing mam survival and GSH levels (described below) were assessed at each time malian cells, and they are associated with the anionic biomolecule point. Treated monolayers were rinsed twice with PBS, trypsinized, DMA (14,15). Moreover exogenously applied polyamines cause counted, diluted, and plated using appropriate numbers of cells to yield fluctuation in the concentrations of naturally occurring intracel- 50-75 macroscopic colonies/dish. Plating for each point was always in lular polyamines (14,15). These fluctuations in poly amine levels triplicate. Extended exposure times often resulted in cells dislodging from are thought to result from inhibition of S-adenosylmethionine the monolayer. When this occurred the medium was collected and pooled decarboxylase and omithine decarboxylase activity, two en with the cells that were trypsinized and then rinsed twice with PBS and handled as described above. After plating, cells were incubated 7-9 days zymes involved in polyamine biosynthesis (14,15). at 37°Cfor macroscopic colony formation. The plates were then fixed Catabolism of polyamines depends in part on amine oxidases using methanolracetic acid (3:1) and stained with crystal violet. Various (14, 15). These amine oxidases are enzymes that are oxygen experiments were conducted at least 2-3 times. Shown in the charts dependent and are known to produce hydrogen peroxide (14). are representative single self-contained experiments. Previously we have shown that modulation of the cellular redox Hyperthermia Exposure. Immediately following the addition of drugs state by applying diethylmaleate, a compound that binds sulfhy- the flasks were gassed with 5% CO2:95% air and sealed with paraffin dryls, or BSO, a compound that inhibits the synthesis of gluta wax. The flasks were then submerged in precision controlled water baths thione (16-18), the major nonprotein sulfhydryl compound in maintained at 43°C ±0.05°C (SD). Control flasks were handled in the mammalian cells, results in thermosensitization at temperatures same manner but were maintained at 37°C. <43°C (19-21). Furthermore, we postulated that since glutathi- GSH Assay. GSM levels in treated and control cells were assayed in parallel for each experiment. The cells were collected from the flasks and 1To whom requests for reprints should be addressed, at Radiation Oncology Branch, National Cancer Institute, Building 10, Room B3-B69. Bethesda, MD 2 The abbreviations used are: AG, aminoguanidine; GSH, glutathione; OTZ, 2- 20205. oxothiazolidine~4-carboxylate; GSSG, glutathione disulfide; BSO, buthionine sulfox- Received 3/13/85; revised 6/19/85; accepted 6/26/85. imine; PBS, phosphate-buffered saline. CANCER RESEARCH VOL. 45 OCTOBER 1985 4910 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1985 American Association for Cancer Research. SPERMINE-INDUCED GSH DEPLETION rinsed with cold PBS. Triplicate samples of each condition containing Table 1 106 cells/sample were then resuspended in 2 ml of 0.6% sulfosalicylic Various factors used to modify spermine-induced GSH depletion at 37°C acid, centrifugad, and the supernatant was assayed for total GSH content GSH by the GSH reducíase procedure (25). Control GSH values ranged from (% of 0.8-1.2 ^g/106 cells. Condition control) 4-h exposure, 1 RIMspermine + 10~5 M AG <5 4-h exposure, 1 mu spermine + 1fT5 M AG + catalase, <5 200 ng/mi (scavenger of extracellular H2O2) RESULTS 4-h exposure, 1 mm spermine + 10~* M AG + calcium chic- <5 ride 10 mw (to rule out spermine cationic chelation) The effects of exogenous spermine exposure on GSH levels 4-h exposure, 1 mm spermine + AG 10~3 M (diamine oxidase 40 for cells maintained at 37°C and 43°C are shown in Chart 1. inhibitor) 4-h exposure, 1 mw spermine + 10~5 M AG + glutamine, 10~3 12 Hyperthermic exposure alone resulted in a rapid elevation in M and 10"2 M (provides substrate energy for GSH synthe- 6 GSH to a near plateau level of 160-180% of control. Similar heat sis) 4-h exposure, 1 mw spermine + 10~5 M AG + oxothiazolidine- <5 induced GSH elevation has been observed previously (19-21). When cells were exposed to 1 mw spermine at either 37°Cor 4-carboxylate (stimulates GSH synthesis to 200% of con trol prior to addition of spermine) 43°Cthere was a steady decline in cellular GSH values. By 2-3 4-ti exposure, 1 mm spermine + 10"5 M AG + cell density at 37 1.3 x 10* cells/cm2, 6.7 x 104 cells/cm2, and 1.3 x 104 18 h of exposure at either temperature GSH values were <5% of cells/cm2 2 controls and thus lower than the detection limits of the assay. Similar results were found using 1 mM spermidine (data not shown). It was concluded from these experiments that exoge A possible reason for this might have been due to spermine nous spermine was exerting marked effects on GSH levels even cationic chelation for ions important in cell adherence. The cal at 37°C,and in addition the elevation in GSH levels associated cium chloride addition, however, did not prevent either the cell with 43°Cheating alone was not only totally inhibited but was detachment or GSH depletion. The GSH synthetic cycle was markedly reduced when spermine was present. stimulated and thus the GSH levels were increased by pretreat A series of experiments was next conducted to identify pos ment of cells for 2 h with OTZ (23). Despite GSH levels being sible approaches to stop or modify the spermine-induced GSH 200% of control, when spermine was added, GSH depletion was depletion. For these studies only exposure of cells to spermine still observed. Providing additional substrate energy for GSH at 37°Cwas used since the GSH depletion was similar for 37°C synthesis in the form of glutamine also failed to prevent GSH or 43°C. A 4-h exposure time was selected which completely depletion. There were only two parameters that had any appre depletes cellular GSH (see Chart 1). The data from these exper ciable affect toward partially preventing the spermine-induced iments are shown in Table 1.