Cancer Letters, 37 (1987) 181- 187 Elsevier Scientific Publishers Ireland Ltd.

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THE EFFECT OF HYPERTHERMIA ON INTRACELLULAR K’ IN CHINESE HAMSTER OVARY CELLS

DIANA A. BATES and WILLIAM J. MacKILLOP+*

McGill Cancer Center, McGiU University, Montreal (Canadal

(Received 15 January 1987) (Revised version received 10 April 1987) (Accepted 13 April 1987)

SUMMARY

The effect of hyperthermia on intracellular K’ concentrations was studied in Chinese hamster ovary (CHOl cells in vitro, using a flame photometer. Intracellular K’ concentrations decreased with increasing exposure time at temperatures from 40°C to 45OC. The decrease in K’ concentrations preceded any loss of reproductive capability at 43OC and also occurred at the non-lethal temperature of 40°C. Prolonged exposure to 45OC resulted in an irreversible decrease in K’ concentrations. The decrease in K+ concentrations at elevated temperatures was not accounted for by changes in cell volume, loss of cells or failure of the Na’/K.’ pump.

INTRODUCTION

Although hyperthermia is now being used to treat cancer in the clinic, the mechanism by which heat kills mammalian cells remains unknown. The cell membrane has been suggested as the critical target for heat damage [15] and we have recently shown that the development of heat resistance in Chinese hamster ovary (CHOl cells is associated with an increase in membrane viscosity [3]. It has been shown that many membrane transport processes are very sensitive to changes in temperature and several studies have shown alterations in ion fluxes at elevated temperatures [2,5,7,9,12 - 14,171. There is general agreement that both potassium influx and potassium efflux

*Present address: Department de Chimie, Universite du Quebec a Montreal, Montreal, Quebec, Canada. **Address for correspondence: Dr. William J. Mackillop, Ontario Cancer Foundation, Kingston Regional Cancer Centre, King Street West, Kingston, Ontario K7L 2V7, Canada. Abbreviations: BSA, bovine serum albumin; CHO, Chinese hamster ovary: FBS, foetal bovine serum; HEPES, 4_(2-hydroxyethyl-l-piperazinelethanesulfonic acid; MEM Alpha, minimum essential medium Alpha; PBS, phosphate buffered saline.

0304-3835/87/$03.50 0 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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[2,5,12,13] are increased at elevated temperatures but there is little information about the net effect of these changes on intracellular ion concentrations and that which is available is contradictory. Some studies report a decrease in intracellular K’ levels after exposure to elevated temperatures [11,17] whereas others report no change [5,14]. We have therefore studied the effect of elevated temperatures on intracellular levels of K’ in CHO cells, and have correlated this with cell survival.

MATERIzdLS AND METHODS

Tissue culture The CHO cell line Aux Bl [lo] was grown in monolayer in 75 cm2 plastic

tissue culture flasks (Falcon, Becton-Dickinson Canada Inc., Mississauga. Ontario) at 37OC under 5% CO, in minimum essential medium Alpha (MEM Alpha) (Gibco Canada, Burlington, Ontario, Canada), supplemented with 10% foetal bovine serum (FBS) and 1% penicillin (50 units/ml)-streptomycin (50 rg/ml) both from Flow Laboratories, Mississauga, Ontario, Canada. Experiments were carried out using cells grown to confluence and incubated for 24 h at 37OC with fresh MEM Alpha plus loo,6 FBS. The cells were harvested with titrated phosphate buffered saline (0.14 M NaCI, 0.01 M Na Phosphate, 0.015 M Na Citrate), washed by centrifugation and resuspended in MEM Alpha, 100/o FBS and 20 mM 4-(&hydroxyethyl-l- piperazinelethanesulfonic acid (HEPES).

Determination of the effect of hyperthermia on cell volume Freshly harvested CHO cells were resuspended at 5 x lo6 cells/ml in a

total volume of 0.2 ml MEM Alpha plus 10% FBS plus 20 mM HEPES in glass tubes, and were incubated for varying times at several defined temperatures in a temperature controlled water bath (Haake D,, Saddlebrook, NJ). At different times 20-~1 aliquots of the cell suspension were diluted into 8 ml of phosphate buffered saline (PBS), containing 1% bovine serum albumin (BSAl and 10 mM glucose, prewarmed at the incubation temperatures. Relative measurements of mean cell volume were made using a Coulter counter attached to a pulse height analyser. The instrument was flushed with PBS prewarmed to the incubation temperature. The zero time point control at 37OC was arbitrarily assigned a value of 1.00 and other measurements were assigned a cell volume relative to the control point. Absolute cell volume was determined by measuring the diameters of 50 cells under a phase contrast microscope (Leitz Wetzlar, Germany) with a calibrated eyepiece microchrometer. The cells were assumed to be spherical.

Determination of intracellular K’ Freshly harvested l-ml aliquots containing lo7 CHO cells in MEM Alpha

plus loo/b FBS and 20 mM HEPES were heated in screw-capped glass tubes in a temperature controlled water bath (Haake D3, Saddle Brook, NJ) for

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varying times at temperatures between 37OC and 45OC at pH 7.4. Temperatures were monitored with a 24-gauge thermistor probe (Yellow Springs Instrument Co., Yellow Springs, Ohio) and the cell suspension reaches a temperature within O.lOC of the water bath temperature within 3 min. After the incubation, 3 ml of ice-cold choline chloride (0.14 Ml lOA M ouabain were added and the cells were centrifuged (1 min, 1000 x gl and washed once with 4 ml ice-cold choline chloride plus ouabain. The dry pellet of cells was lysed by boiling for 1 min with 30%1 nitric acid and neutralized with 1 M tris (hydroxymethyl) aminomethane. Lithium chloride was added at a final concentration of 15 mM and the sample volume made up to 3 ml with deionized distilled water. The K’ concentration was measured in the samples using a Beckman Klina flame photometer against a 15 mM lithium chloride internal standard. The flame photometer was calibrated using standard solutions of KCl.

Heat survival experiments Cell suspensions (lo6 cells/ml in MEM Alpha plus 10% FBS and 20 mM

HEPES) were divided into l-ml aliquots in screw-capped glass tubes (Kimax, U.S.A.). The tubes to be heated were placed in a temperature controlled water bath at a temperature of 43OC or 45OC. Tubes were removed from the water bath and placed on ice at different time intervals. The control tube was removed after the 3-min warm-up period (which is considered as time zero). The cells were carefully mixed before diluting to the appropriate concentration and plating in tissue culture coated petri dishes. The petri dishes were incubated at 37OC in an atmosphere of 5Oh CO, for 7 days. The plates were then washed with PBS, fixed with 95% ethanol, and stained with methylene blue before counting macroscopic colonies. Control plating efficiencies were greater than 80%. Percentage survival was expressed as the mean number of colonies obtained at a given time divided by the mean number of colonies obtained in the control. Two hundred cells per plate were seeded in the control, but for the higher temperatures with the longer periods of exposure larger numbers of cells were plated and the results were corrected accordingly. We. have demonstrated that in this system there is linearity between the number of cells plated and the colonies formed over the range of 10 - lo4 (data not presented).

RESULTS

Cell volumes were measured at 37OC and after exposure to 40°C, 43OC or 45OC for varying times up to 60 min and no measurable differences were detected. Intracellular ion concentrations were therefore calculated based on a mean cell volume of 1194 + 155 pg9 obtained in 7 separate experiments. Intracellular K’ concentrations are shown in Fig. 1 for CHO cells incubated for varying times up to 120 min at temperatures from 37OC to 45OC. At 37OC, the K’ concentration remained relatively constant within the normal

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0 20 40 60 80 100 120

Time (minutes)

Fig. 1. Intracellular K’ concentrations versus heating times at elevated temperatures. Means are given for triplicate estimations from 2 to 7 separate experiments after incubating 10’ CHO cells in 1 ml MEM Alpha plus 10% FBS and 20 mM HEPES for varying times at 37T (01, 40°C ( + ),43% (Al and 45% ( o ). Cells were heated for 60 min at 45°C and then returned to 37OC for up to 60 min ( x 1. SD. are not shown but were typically 2-3OA of the mean value.

range of 120- 160 mM for mammalian cells [B]. During the first 45 min heating period at 40 OC the K’ concentrations were similar to those in cells incubated at 37OC, however, by 60 min at 40°C the K’ concentration was measurably lower than in cells at 37OC and continued to decrease to approximately 85% of the control by 120 min. At 45OC there was a steady decrease in K’ concentrations which reached 50% of the initial concentration by 120 min. The reversibility of the loss of intracellular K’ at 45OC was determined by returning the cells to 37OC for periods varying from 15 min to 60 min after an initial incubation period of 60 min at 45OC. Figure 1 shows that the intracellular K’ concentration remained constant in the heated cells which were returned to 37OC while the cells that were maintained at 45OC continued to lose K’. The K’ concentration remained below the normal range after return to 37OC and did not return to control levels.

Aliquots of the cell suspension were counted after varying incubation times at temperatures from 37OC to 45OC to determine whether the decrease in intracellular K l concentrations at elevated temperatures could be accounted for by an absolute loss of cells. The cells were counted in the presence of trypan blue at 15-min intervals during 120-min incubations at

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0 30 60 SO 120 Tlmo (mln)

(b)

0 30 60 90 120 Time (mln)

Fig. 2. (a) Heat survival curves. Percent cell survival versus heating time in CHO cells at 42OC (ml and 45% (Al. Means are given for 2-4 estimations and lines of best fit and confidence intervals on the data were obtained using the method described in Ref. 4. These are vertical confidence intervals and their right boundary ends at the last data point. These data are typical of those obtained in 6 separate experiments. (bl Killing time frequency distributions are given for the data in Fig. 2a and were calculated using the method in Refer. 4.

37OC, 40°C, 43OC and 45OC. The absolute number of cells remained constant at all temperatures. Trypan blue positive cells remained at control levels of 2Orb throughout the incubation but increased slowly with increasing incubation time to 5% at 43OC and 6Ob at 45OC after 2 h.

Changes in intracellular K+ concentrations at elevated temperatures were correlated with changes in cell survival measured in a clonogenic assay. Figure 2a shows time courses for cell survival at 43OC and 45OC. There was no loss of cell viability after a 120-min incubation at either 37OC or 40°C (data not presented). At 43OC, the survival curve had a broad shoulder for approximately 30 min after which the slope of the curve increased. Cell survival decreased to approximately 10% after 60 min at 43OC and by llO- min cell survival was less than 0.1%. At 45OC the shoulder of the survival curve was much less than at 43OC and the subsequent slope was steeper. Cell survival decreased to 101 after 12.5 min at 45OC and by 20 min cell survival was lob and by 30 min cell survival was less than 0.1%. Figure 2b shows the killing time frequency distributions at these temperatures together with confidence intervals [4]. The mean killing time increased with increasing temperature. At 45OC, there is sharp peak at 9 min and at 43OC there is broad peak at 38 min.

DISCUSSION

We have observed a decrease in intracellular K’ levels at elevated temperatures from 40°C to 45OC in CHO cells in vitro. The decrease in intracellular K+ levels was not accounted for by a loss of cells since the total

number of cells remained constant during the incubation period. The small increase in the number of trypan blue positive cells at 43OC and 45OC shows that gross leakiness was not sufficient to account for the magnitude of the loss of intracellular K’. Furthermore, the decrease in intracellular K + levels during 60 min did not arise as a result of a change in cell volume since we measured no change in cell volume after heating cells for up to 60 min at temperatures from 40°C to 45OC. Our results are in apparent contradiction to those of 2 other groups who have reported no change in intracellular K’ levels at elevated temperatures [5,14]. This can, however, be explained by consideration of experimental conditions in these 2 studies. In one study, measurements of intracellular ion concentrations-were made on the adherent cells under conditions which select for the survivors of heat treatment [5]. In another study, limited measurements of intracellular ion concentrations were made only after a 30-min non-lethal heat treatment at 42OC [14]. The decrease in intracellular K’ levels at elevated temperatures which we have observed in CHO cells is, however, in agreement with several studies of murine tumour cell lines [11,16,17].

Previous studies report an increase in efflux of K’ from CHO cells at temperatures from 40°C to 45OC [2] and an increase in the efflux of K’ by active transport at elevated temperatures [2,3,5,13], but the net effect of these changes was unknown. It appears, however, that the observed increase in ouabain-sensitive K’ influx is not sufficient to maintain intracellular K’ levels in the face of a greater increase in K’ efflux.

We found here that the decrease in intracellular K’ levels after 60 min at 45OC was not reversed after return of these cells to 37OC for up to 60 min although the level of intracellular K’ then remained constant and showed no further decrease during a further 60-min incubation at 37OC. Other studies report that the decrease in intracellular K’ levels is irreversible when cells are returned to 37OC after heating at 43OC for 60 min [16] or 44OC for more than 20 min [ll]. Since we have shown that initial rates of K’ influx measured at 37OC are not affected by previous exposure to 45OC for 30 min [2] this suggests that the irreversible decrease in K’ concentration is due to an irreversible increase in the rate of efflux.

We have observed changes in intracellular K’ concentrations at the non- lethal temperatures of 40°C, and at 43OC the intracellular K’ concentration falls during the first 30 min, before any significant cell death occurs. These changes in intracellular K’ concentration therefore precede any loss of reproductive integrity and cannot therefore be regarded as a consequence of cell death. It is thus possible that changes in ionic concentrations at elevated temperatures may be either directly or indirectly involved in cell killing by hyperthermia.

ACKNOWLEDGEMENTS

This work was supported by a grant from the National Cancer Institute of Canada (W.J.M.1. D.A.B. is a research fellow of the Cancer Research Society.

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We thank Dr. R.M. Johnstone, Department of Biochemistry, McGill University for allowing us to use her flame photometer, Anoush Cotchikan and Claire Turbide for helpful assistance and Sandra Tirelli for assistance in preparation of the manuscript.

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