Depletion of Cellular Glutathione by Exogenous Spermine in V79 Cells: Implications for Spermine-Induced Hyperthermic Sensitization

[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 37Â°Cor 43Â°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 37Â°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 37Â°Cor 43Â°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 37Â°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 43Â°C Â±0.05Â°C (SD). Control flasks were handled in the mammalian cells, results in thermosensitization at temperatures same manner but were maintained at 37Â°C. <43Â°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 37Â°C acid, centrifugad, and the supernatant was assayed for total GSH content GSH by the GSH reducÃase 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 37Â°C and 43Â°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 37Â°Cor 4-carboxylate (stimulates GSH synthesis to 200% of con trol prior to addition of spermine) 43Â°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 37Â°C,and in addition the elevation in GSH levels associated cium chloride addition, however, did not prevent either the cell with 43Â°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 37Â°Cwas used since the GSH depletion was similar for 37Â°C synthesis in the form of glutamine also failed to prevent GSH or 43Â°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. Addition of catalase, a well-known GSH depletion. Increasing the concentration of AG, a diamine oxidase inhibitor (24), to 10 3 M and increasing the cell density scavenger of extracellular H2O2, or calcium chloride failed to prevent spermine-induced GSH depletion. Calcium chloride was both resulted in less GSH depletion, to about 40% of control added because it was observed that the spermine treatment rather than the 5% level seen normally with spermine treatment. often resulted in cells becoming detached from the flask surface. No agent studied completely prevented the spermine-induced GSH depletion. If GSH levels were an indicator of spermine- induced thermal sensitization, perhaps increasing AG concentra tions or increasing cell density might reduce spermine sensiti zation. Survival of cells treated with 1 ITIMspermine at 43Â°Cwith two AG concentrations is shown in Chart 2. Thermal sensitization was observed for spermine addition, and the extent of sensiti zation was the same for both 10~5 and 10~3 M AG additions. *â€”*ofeeo A-A ^ GSH levels at 43Â°Cwere the same for both 10~3 and 10~5 M AG ""*/â€¢/â€¢V â€¢ additions, 45 and 49% of control, respectively. Therefore increas ing the AG concentration for heated spermine-treated cells af Oo#z forded no protection from thermal sensitization or GSH depletion. In contrast variation of the cell density at the time of hyperthermic and spermine exposure had pronounced effects on cell survival and GSH levels as shown in Chart 3. Chart 3A shows the survival of cells exposed to 43Â°C for 90 min with or without 1 mw Â« spermine. For higher cell densities there was less spermine- o240220200180160140120100806040 induced thermal sensitization. In like manner for higher cell ,,..Y>\^+sG densities the GSH depletion by spermine and hyperthermia was 20 less as shown in Chart 38. Therefore there appeared to be a relationship between the extent of spermine-induced thermal Oâ€¢â€¢â€¢43Â° sensitization and GSH depletion levels as the cell number was O 1 2 3 4 5 6 varied. Time (hr) Chart 1. Cellular GSH levels (expressed as a percentage of control values) for DISCUSSION cells exposed to 43Â°C(â€¢,A),37Â°C+ 1 mw spermine (S) (O, D, V, O), or 43Â°C+ 1 mw spermine (â€¢.â€¢)asa function of time. Different symbols represent replicate We have shown previously that intracellular levels of glutathi- experiments. one are elevated by exposing cells to hyperthermic stress (19-

21). Additionally we have shown that interference with this rise agents or conditions that have shown potential to thermosensi- in GSH by either diethylmaleate or BSO results in thermal sen- tize are of interest to us. Since polyamines are exquisite thermal sitization for temperatures <43Â°C (19-21 ). Recently Freeman et sensitizing agents (3-13), we reasoned that polyamines because al. (26) demonstrated that acid pH conditions that sensitize cells they are thermal sensitizers may have some interaction with to heat also results in reduction in cellular GSH levels. Therefore intracellular GSH. Furthermore this interaction with the cellular redox state may in part explain or offer an alternative mechanism for thermal sensitization by polyamines. Our data clearly show 43' that exogenously applied spermine is a potent GSH depleter both at 37Â°Cand 43Â°C.This observation is particularly attractive 10C from a biochemical point of view because the catabolism of polyamines results in the generation of hydrogen peroxide, al dehydes, and the a-ÃŸunsaturated carbonyl acrolein as shown in the proposed catabolic pathway in Chart 4. GSH can detoxify _i hydrogen peroxide and acrolein in conjunction with glutathione 10 O peroxidase (27) and glutathione^S-transferase (28), respectively. Â«Â« The mechanism of GSH depletion could result from interaction o of GSH enzymatic catalysis of and subsequent export of either GSSG or the acrolein-GSH adduct from the cell. There are .2 u. 10 several enzymes capable of such catalysis including diamine oxidase, found in fetal calf serum (14, 29) and various spermine oxidases (15). Aminoguanidine, a diamine oxidase inhibitor (24) failed to prevent spermine-induced sensitization when concen 10J -^Spartitine trations were increased (see Chart 2). There is precedent for GSSG export after detoxification of benzyl amine in in vivo systems (30). Moreover the adduct of acrolein and GSH would be exported from the cell. Hence, given that the catabolic path 10' ways are operative, it is apparently straightforward to account for GSH depletion by polyamines. Yet it is not clear that this is the only means by which GSH might be depleted. If the poly amine catabolic products were to interact with GSH anabolic enzymes GSH depletion would result. This is the case for BSO, and BSO results in thermal sensitization (19-21). We attempted to in 60 80 100 crease the available intermediates for GSH synthesis by supply ing cysteine via the intracellular cysteine delivery system OTZ (23, 31) or glutamine. Neither of these potential substrates for Time (min) GSH synthesis could sustain intracellular levels of GSH in the Chart 2. Survival of V79 ceils heated at 43Â°C.A, 43Â°Cwith no drugs; O, 43"C + 1u"3 M AG; D, 43Â°C+ 1(T5 M AG; â€¢,43Â°C+ 1 mm spermine + 1(T3 M AG; B, presence of spermine. We have not ruled out the possibility that 43Â°C+ 1 muÂ»spermine + 10"5 M AG. depletion of intracellular GSH by spermine is more complex than

10" 120

2 10- -No S p B r m i n t C100 V o o â€¢ 10- o 80 O Chart 3. Left, cell survival for cells heated at 43Â°Cfor 90 min plus 1 mm spermine and 10"s M AG (â€¢,A, â€¢)or heat alone (U. A, O) as a eo function of cell density; right, GSH values for 10' cells heated at 43Â°C with 1 mu spermine + 10~5 M AG as a function of cell density. Cell CO Â«O 40 densities are indicated on each curve. a 10" -^Sptrnlnt 20

(1.3 x 10*) (6.7 x IO4) (1.3 x IO4) 1 2 CÂ«ll Density (/cm2) mi thÂ« TimÂ« at 43Â°(hr) TimÂ« of Heating

CANCER RESEARCH VOL. 45 OCTOBER 1985 4912

202 + 2H20 Spannine Enzyme(s)

CHO-(CH2)2 NH-(CH2)4 NhHCH2)f CHO + 2H202 DiaWehyde Hydrogen Peroxide

Chart 4. Proposed mechanism(s) of spermine-induced GSH depletion. BIM, biologically im portant molecule; BIM' or Membrane', dam Dialdehyde r BIM aged or altered; GPO. glutathione peroxidase; GR, glutathione reducÃase. or Membrane

BIM* GSSG or .CHâ€”CH2 Membrane* CHO' Acrolein CHO. GSH-Transferase GSH GSH Adduct simple detoxification of catabolic polyamine products. Previously may be important in thermal sensitization by spermine. It also it has been shown in bacteria (32) that GSH can combine through shows for the first time that there is an interaction of exogenously an amide bond with polyamines. The assay system that we used applied polyamine and intracellular sulfhydryl groups. The exact in this study (25) is specific for GSH and GSSG; hence we would implications of such biochemical interactions are not known at not have discerned this particular metabolite had it been formed this time. We are currently undertaking studies to understand and cannot rule out this possibility. Catatase also failed to prevent these interactions better and to determine the role that endoge GSH depletion and thermosensitization (data not shown) for 1 nous polyamines impose on the intracellular redox state. mu spermine. This result is not surprising since exogenously applied catatase does not enter into the intracellular space and REFERENCES therefore would not be expected to protect the intracellular system from intracellularly generated hydrogen peroxide. Re 1. Dewey, W. C., Freeman, M. L., Raaphorst, G. P., Clark, E. P., Wong, R. S. L., cently it has been shown that the cytotoxicity of 0.01 HIM HkjhfiekJ, D. P., Spiro, I. J., Tomasovic, S. P., Denman, D. L, and Coss, R. A. Cell biology of hyperthermia. In: R.E. Meyn and H. R. Withers (eds.), Radiation spermine can be reversed by catatase addition.3 The discrepancy Biology in Cancer Research, pp. 589-621, New York: Raven Press, 1980. in these findings may be related to cell density and/or concentra 2. Hahn, G. M. Hyperthermia and Cancer. New York: Renum Press, 1982. 3. Gerner, E. W., and Russell, D. H. The relationship between polyamine accu tion differences in the spermine used. mulation and DNA replication in synchronized Chinese hamster ovary cells Increasing the cell numbers that are exposed to spermine after heat shock. Cancer Res., 37: 482-489,1977. 4. Ben-Hur, E., Prager, A., and Riklis, E. Enhancement of thermal killing by results in less thermosensitization and greater levels of intracel polyamines. I. Survival of Chinese hamster cells. Int. J. Cancer, 22: 602-606, lular GSH (Chart 3). There seems to be a correlation between 1978. the cell density, the level of GSH, and the thermosensitization 5. Gemer, E. W., Cross, A. E., Stickney, D. G., Holmes, D. K., and Culver, P. S. Factors regulating membrane permeability after thermal resistance. Ann. NY induced by spermine. This observation is consistent with that of Acad. Sci., 335:215-230,1980. Ben-Hur et al. (13) who showed less spermine-induced thermo 6. Ben-Hur, E., and Riklis, E. Enhancement of thermal killing by polyamines. II. Uptake and metabolism of exogenous polyamines in hyperthermic Chinese sensitization for cells treated as multicellular spheroids versus hamster cells. Int. J. Cancer, 22: 607-610,1978. monolayer cultures. When a greater number of cells are exposed 7. Ben-Hur, E., and Riklis, E. Enhancement of thermal killing by polyamines. III. Synergism between spermine and gamma-irradiation in hyperthermic Chinese to spermine there is a larger quantity of metabolic machinery to hamster cells. RadiÃ¢t.Res., 78:321-328,1979. detoxify spermine without resulting in drastic reductions in GSH. 8. Ben-Hur, E., and Riklis, E. Enhancement of thermal killing by polyamines. IV. If GSH levels are an index of the amount of cellular stress that Effect of heat and spermine on protein synthesis and ornithine decarboxylase activity. Cancer Btochem. Btophys., 4: 25-31, 1979. is being imposed, it is not surprising that there is greater relative 9. Fuller, D. J. M., and Gemer, E. W. Delayed sensitization to heat by inhibitors survival associated with the use of larger numbers of cells. of polyamine-biosynthetic enzymes. Cancer Res., 42: 5046-5049,1982. 10. Fuller, D. J. M., Stickney, D. G., and Gerner, E. W. Structural aspects of heat This study does support in part the premise that oxidative sensitization by the polyamines. Nati. Cancer Inst. Monogr., 67: 93-94,1982. stress as reflected in perturbation of intracellular levels of GSH 11. Gemer, E. W., Stickney, D. G. Herman, T. S., and Fuller, D. J. M. Polyamines and polyamine biosynthesis in cells exposed to hyperthermia. RadiÃ¢t.Res., 3 R. Issels, personal communication. 93/340-352,1983.

12. Fuller, D. J. M., and Gemer, E. W. Serialization to heat by the polyaminesand derivatives. Bull. Chem. Soc. Jpn., 37: 242-244,1964. their analogs. RadiÃ¢t.Res.,95:124-129,1983. 23. Russo, A., and Mitchell, J. B. Radiation response of Chinese hamster cells 13. Ben-Hur, E , Shaw, J. J., and Bleehen. N. M. Enhancementof thermal killing after elevation of intracellular glutathione levels. Int. J. RadiÃ¢t.Oncol. Biol. by polyamines.V. The responseof EMT6 multicelular tumour spheroidsversus Phys., 10:1243-1247,1984. monolayercells. Br. J. Cancer, 47:51-55,1983. 24. Shore, P. A., and Cohn, V. H., Jr. Comparativeeffects of monoamineoxidase 14. Cohen, S. S. Introduction to the polyamines. Englewood, NJ: Prentice-Hall, inhibitor on monoamineoxidase and diamine oxidase. Biochem. Pharmacol., Inc., 1971. 5:91-95,1960. 15. Tabor, C. W., and Tabor, H. Polyamines.Annu. Rev. Biochem., 53: 749-790, 25. Tietze. F.Enzymicmethodfor quantitativedeterminationof nanogramamounts 1984. of total and oxidized glutathione: applicationsto mammalianblood and other 16. Dethmers, J. Â«..andMeister, A. Glutathioneexport by human lymphoid cells: tissues. Anal. Biochem., 27: 502-522, 1969. depletion of glutathione by inhibition of its synthesis decreases export and 26. Freeman, M. L. Malcolm, A. W., and Meredith, M. J. Decreasedintracellular increasessensitivity to irradiation. Proc. Nati. Acad. Soi. USA, 78:7492-7496, glutathione concentration and increased hyperthermic cytotoxicity in an acid environment.Cancer Res., 45: 504-508,1985. 1981. 17. Griffith, O. W., and Meister, A. Potent and specific inhibition of glutathione 27. Paglia, D. E., and Valentine, W. N. Studies on the quantitaive and qualitative synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J. characterization of erythrocyte peroxidase. J. Lab. Clin. Med., 70: 158-169, Btol. Chem., 254; 7558-7560,1979. 1967. 18. Meister, A., and Anderson, M. A. Glutathione. Annu. Rev. Biochem., 52; 711- 28. Jakoby. W. B., and Habig, W. H. Glutathione tranferases In: W. B. Jakoby (ed.), Enzymatic Basis of Detoxification, pp. 63-94. New York: Academic 760,1983. Press, Inc., 1980. 19. Mitchell, J. B., and Russo, A. Thiols, thiol depletion, and thermosensitivity. 29. Hegre, O. D., Marshall, S., and Hickey, G. E. Spermidine cytoxicity in vitro: RadiÃ¢t.Res.,95: 471-485,1983. effect of serum and oxygen tension. In Vitro (Rockviile).20:198-204,1984. 20. Mitchell,J. B., Russo, A., Â«insella.T.J., and Glatstein, E. Glutathioneelevation 30. SÃes,H.,and Wondel. A. (eds.).Functions of Glutathione in Liver and Kidney. during thermototeranceinduction and thermosensitization by glutathione de New York: Springer-VerlagNew York, Inc., 1978. pletion. Cancer Res., 43: 987-991,1983. 31. Williamson,J. M., Boettcher, B., and Meister, A. Intracellulardelivery system 21. Russo, A., Mitchell, J. B.. and McPherson, S. The effects of glutathione that protects against toxicity by promoting glutathione synthesis. Proc. Nat). depletion on thermototerance and heat. Stress protein synthesis. Brit. J. Acad. Sci. USA, 79: 6246-6249,1982. Cancer, 49: 753-758,1984. 32. Tabor, H., and Tabor, C. W. Glutathionylspermidine.In: H. Tabor and C. W. 22. Kaneko,T., Shirmokobe, T., Yoshimoto, 0., Yoyokama,E., Inui, T., and Shiba, Tabor (eds.), Methods in Enzymology, Vol. 94, pp. 434-437. New York: T. Syntheses and properties of 2-oxothiazolidine-4-carboxylic acid and its Academic Press, Inc., 1983.

CANCER RESEARCH VOL. 45 OCTOBER 1985 4914 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1985 American Association for Cancer Research. Depletion of Cellular Glutathione by Exogenous Spermine in V79 Cells: Implications for Spermine-induced Hyperthermic Sensitization

Angelo Russo, James B. Mitchell, William DeGraff, et al.

Cancer Res 1985;45:4910-4914.

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