Cytotoxic Effects of Hyperthermia and Adriamycin on Chinese Hamster Cells 1, 2 George M. Hahn and Douglas P. Strande 3,4 ABSTRACT-Chinese hamster cells, exposed to simultaneous hyperthermia (43 C) and adriamycin (ADM), were initially sensitized to the cytotoxic activity of ADM. If the duration of the combined treatment exceeded about 30 minutes, however, the cells became refractory to additional killing by ADM. If heat was applied before ADM exposure, the cells also could be rendered insensitive; this state persisted so that 24 hours after a 50-minute exposure to 430 C, the cells still showed considerable resistance to ADM. The most reasonable explanation for the findings was that cell membrane permeability to ADM was initially increased by hyperthermia, but prolonged (>30 min) heat exposure reversed this situation and inhibited additional ADM from penetrating to sensitive sites. However, the data yield no hints as to the precise mechanisms involved nor are they sufficiently precise to exclude other explanations. The results do point to precautions that must be observed if the combination of ADM and hyperthermia is to be used clinically.--J Natl Cancer Inst 57: 1063-1067, 1976. 0
In vitro or in vivo treatment of mouse mammary tumor cells (EMT-6) (1) with a combination of elevated temperature and ADM was shown to have a synergistic cytotoxic effect (2). This effect was irreversible in that the increased cell killing was seen irrespective of the duration of the combined treatment. The working hypothesis that attempts to explain this phenomenon was that hyperthermia modified the permeability of the EMT-6 cells to ADM and permitted more of the ADM to enter the cells at 43° C than at 37° C. Fluorescence studies measuring cellular drug uptake were consistent with this hypothesis. We now report that, when Chinese hamster cells were similarly treated, they had a more complex response. Sensitization could be observed only if the duration of exposure to 43° C was limited sharply; beyond about I hour, heating at that temperature appeared to protect the cells against further action by the chemotherapeutic agent. Although our results do not give a clear-cut explanation of the phenomenon, they do point out that the kinetics of drug-hyperthermia interactions must be considered carefully before attempts are made to use such a combination clinically. Variations in response from one cell line to another might well be reflected clinically in differential tumor responses. MATERIALS AND METHODS
Cells.-Chinese hamster cells (HAl), free of mycoplasma contamination, were grown in monolayers as described (3, 4). We obtained exponentially growing cells by seeding 1-2 x 105 cells into 25-cm 2 tissue culture flasks. The cultures were used for experiments 2-3 days later at a density of 104_105 cells/em". In all experiments, cell survival was assayed by Puck's cloning technique. ADM and heat treatment.-Concentrated stock solutions of ADM in balanced salt solution were prepared and stored frozen for up to 7 days with no significant loss of activity during this period (data not shown). VOL. 57, NO.5, NOVEMBER 1976
ADM treatment was accomplished by dilution of the concentrated stock solution into 37° C medium (Eagle's minimum essental medium supplemented with 15% fetal bovine serum); then the culture medium was exchanged for the ADM-containing medium. The flask was flushed with a 5% CO 2-95% air mixture for a few seconds and incubated at the appropriate temperature (37 or 43° C). Controls were handled identically except that no ADM was added to the medium. Although it is generally desirable to perform drug exposures in serum-free media to eliminate variability problems associated with drug binding to serum components (5), we chose a serum-containing medium for one overriding reason: To keep the cell survival within measurable limits during the long 43° C exposures (up to 4 hr), a system with relatively low heat sensitivity is required. HAl cells are most resistant when exposed to heat in serum-containing medium and while in exponential growth (4). By carefully controlling the serum concentration and by using the same serum lot for the entire series of experiments, we were able to obtain reproducible results despite serious binding of ADM to serum components (data not shown). We conducted heat treatment by submerging the flask in a circulating water bath (Forma-Temp Jr.; Forma Scientific Inc., Marietta, Ohio) at 43° ± a:
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TEXT-FIGURE 2.-Dose-response curve of short exposures (20 min) of cells to graded doses of ADM at 37 and 43° C.
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TEXT-FIGURE I.-Time response of HAl cells exposed simultaneously to 43° C and ADM. (A) Survival values. (B) Surviving fraction ratios which were obtained by dividing, point by point, the values of the lowest curve in (A) by the 43° C control values. The curve is a' measure of the cytotoxic action of ADM at 43° C.
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NATL CANCER INST
ponential over the exposure time; the 43° C heat control data were consistent with those published earlier (6). The combined curve showed appreciable cell killing for short-term exposures: At 30 minutes, survival was about 4x 10- 2 • This contrasted with survival levels of 90% for the heat control and 60% for the ADM control. Hence, with independent action, a 54% level of survival would be predicted for the combined treatment. However, beyond the 30-minute point, the combined treatment curve approached the shape of the 43° C control; the former was essentially parallel to the latter, as illustrated in text-figure IB, which shows the effect of ADM at 43° C. This curve was obtained by normalizing the combined curve point by point to the heat control. Beyond about 30 minutes, ADM clearly no longer contributed to cell killing. In fact, there was some protection against the ADM by heat: The last two time points were higher in survival by a factor of 2 than the earlier points (though this difference was not statistically significant). These results were not due to thermal inactivation of ADM: Heating ADM to 43° C for up to 5 hours did not appreciably reduce the drug's ability to subsequently kill cells at 37° C (data not shown). The results shown in text-figures lA and B indicated that considerable synergism could be demonstrated if the exposure were kept short (30 min or less). For this reason, we constructed a dose-response curve with ADM at a fixed, 20-minute treatment time (text-fig. 2). At such a short exposure, there was essentially no toxicity due to heat alone (text-fig. lA). Hence, the fact that the two curves of text-figure 2 diverge over the concentration range investigated showed that, for short exposures, 43° C appreciably sensitized the cells to AD M . For VOL. 57, NO.5, NOVEMBER 1976
1065
EFFECTS OF HYPERTHERMIA AND ADRIAMYCIN ON CHINESE HAMSTER CELLS
A
example, 0.1 JLg ADM/ml at 43° C killed the same fraction of HAl cells as 0.4-0.5 JLg ADM/ml at 37° C. The sensitization seemed to be most pronounced at low drug levels. The effect of heating (43° C) cells for various lengths of time before exposure to ADM is shown in text-figure 3A. The results showed that, if the preheating time was kept below 50 minutes, the effect of drug killing was essentially constant and equal to that of the unheated control (O-min preheating). Beyond that time, the survival of the cells subjected to consecutive treatments or to heat only was the same; i.e., the heated cells became refractory to ADM. This is illustrated in text-figure 3B
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TEXT-FIGURE 6.-Cumulative histograms of fluorescence of ADMtreated cells. (A) l l-rnin exposure. (B) 50-min exposure.
These show that preheating clearly inhibited the cells' acquisition of fluorescence; however, an increase in the incubation period from 11 (text-fig. 6A) to 50 minutes (text-fig. 6B) allowed both the preheated cells and the controls to increase uptake. Therefore, if the third hypothesis is correct, the initial metabolic step (leading to VOL. 57, NO.5, NOVEMBER 1976
1067
EFFECTS OF HYPERTHERMIA AND ADRIAMYCIN ON CHINESE HAMSTER CELLS
the fluorescing product) clearly is not the one that is affected by heat. Our current working hypothesis is that the simplest explanation, modulation of permeability as in the first hypothesis, is the most likely to be correct. As pointed out above, the data do not exclude other possibilities. Whatever the mechanism of the observed phenomenon, the differential response of the two cell lines is of interest because of possible clinical applications. Although neither of the two cell lines examined is human, and only one the (EMT-6 murine mammary sarcoma) is frankly malignant, the possibility exists that different human tumors might show similar variations in their response. Fluorescence measurements to determine drug uptake might then be desirable on biopsy specimens. Alternatively, shorter heating times (~20-30 min) might be more efficient. Heating to 41°C rather than 43° C could preclude the resistance to ADM introduced by the longer exposures to 43° C. REFERENCES (1) ROCKWELL S, KALLMAN RF: Cellular radiosensitivity and tumor
radiation response in the EMT 6 tumor cell system, Radiat Res 53:281-294, 1973
VOL. 57, NO.5, NOVEMBER 1976
(2) HAHN GM, BRAUN], HAR-KEDAR I: Thermochemotherapy: Synergism between hyperthermia (42-43°) and adriamycin (or bleomycin) in mammalian cell inactivation. Proc Nat! Acad Sci USA 72:937-940, 1975 (3) YANG S-], HAHN GM, BAGSHAW MA: Chromosome aberrations induced by thymidine. Exp Cell Res 42:130-135, 1966 (4) HAHN GM, STEWART ]R, YANG S-], et al: Chinese hamster cell monolayer cultures. 1. Changes in cell dynamics and modifications of the cell cycle with the period of growth. Exp Cell Res 49:285-292, 1968 (5) HAHN GM, GORDON LF, KURKJIAN SD: Responses of cycling and non-cycling cells to 1,3-bis(2-chloroethyl)-I-nitrosourea and to bleomycin. Cancer Res 34:2373-2377, 1974 (6) HAHN GM: Metabolic aspects of the role of hyperthermia In mammalian cell inactivation and their possible relevance to cancer treatment. Cancer Res 34:3117-3123,1974 (7) GERNER EW, SCHNEIDER MS: Induced thermal resistance In HeLa cells. Nature 256:500-502, 1975 (8) HENLE K], LEEPER DB: Interaction of hyperthermia and radiation in CHO cells: Recovery kinetics. Radiat Res 66:505-518, 1976 (9) GERWECK LE, GILLETTE EL, DEWEY we: Effect of heat and radiation on synchronous Chinese hamster cells: Killing and repair. Radiat Res 64:611-623, 1975 (10) WESTRA A, DEWEY WC: Variation in sensitivity to heat shock during the cell-cycle of Chinese hamster cells in vitro. Int] Radiat Bioi 19:467-477, 1971 (11) BACHUR NR, CRADOCK ]C: Daunomycin metabolism in rat tissue slices.] Pharmacol Exp Ther 175:331-337, 1970
J
NATL CANCER INST
In vitro or in vivo treatment of mouse mammary tumor cells (EMT-6) (1) with a combination of elevated temperature and ADM was shown to have a synergistic cytotoxic effect (2). This effect was irreversible in that the increased cell killing was seen irrespective of the duration of the combined treatment. The working hypothesis that attempts to explain this phenomenon was that hyperthermia modified the permeability of the EMT-6 cells to ADM and permitted more of the ADM to enter the cells at 43° C than at 37° C. Fluorescence studies measuring cellular drug uptake were consistent with this hypothesis. We now report that, when Chinese hamster cells were similarly treated, they had a more complex response. Sensitization could be observed only if the duration of exposure to 43° C was limited sharply; beyond about I hour, heating at that temperature appeared to protect the cells against further action by the chemotherapeutic agent. Although our results do not give a clear-cut explanation of the phenomenon, they do point out that the kinetics of drug-hyperthermia interactions must be considered carefully before attempts are made to use such a combination clinically. Variations in response from one cell line to another might well be reflected clinically in differential tumor responses. MATERIALS AND METHODS
Cells.-Chinese hamster cells (HAl), free of mycoplasma contamination, were grown in monolayers as described (3, 4). We obtained exponentially growing cells by seeding 1-2 x 105 cells into 25-cm 2 tissue culture flasks. The cultures were used for experiments 2-3 days later at a density of 104_105 cells/em". In all experiments, cell survival was assayed by Puck's cloning technique. ADM and heat treatment.-Concentrated stock solutions of ADM in balanced salt solution were prepared and stored frozen for up to 7 days with no significant loss of activity during this period (data not shown). VOL. 57, NO.5, NOVEMBER 1976
ADM treatment was accomplished by dilution of the concentrated stock solution into 37° C medium (Eagle's minimum essental medium supplemented with 15% fetal bovine serum); then the culture medium was exchanged for the ADM-containing medium. The flask was flushed with a 5% CO 2-95% air mixture for a few seconds and incubated at the appropriate temperature (37 or 43° C). Controls were handled identically except that no ADM was added to the medium. Although it is generally desirable to perform drug exposures in serum-free media to eliminate variability problems associated with drug binding to serum components (5), we chose a serum-containing medium for one overriding reason: To keep the cell survival within measurable limits during the long 43° C exposures (up to 4 hr), a system with relatively low heat sensitivity is required. HAl cells are most resistant when exposed to heat in serum-containing medium and while in exponential growth (4). By carefully controlling the serum concentration and by using the same serum lot for the entire series of experiments, we were able to obtain reproducible results despite serious binding of ADM to serum components (data not shown). We conducted heat treatment by submerging the flask in a circulating water bath (Forma-Temp Jr.; Forma Scientific Inc., Marietta, Ohio) at 43° ± a:
A
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TEXT-FIGURE 2.-Dose-response curve of short exposures (20 min) of cells to graded doses of ADM at 37 and 43° C.
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TEXT-FIGURE I.-Time response of HAl cells exposed simultaneously to 43° C and ADM. (A) Survival values. (B) Surviving fraction ratios which were obtained by dividing, point by point, the values of the lowest curve in (A) by the 43° C control values. The curve is a' measure of the cytotoxic action of ADM at 43° C.
J
NATL CANCER INST
ponential over the exposure time; the 43° C heat control data were consistent with those published earlier (6). The combined curve showed appreciable cell killing for short-term exposures: At 30 minutes, survival was about 4x 10- 2 • This contrasted with survival levels of 90% for the heat control and 60% for the ADM control. Hence, with independent action, a 54% level of survival would be predicted for the combined treatment. However, beyond the 30-minute point, the combined treatment curve approached the shape of the 43° C control; the former was essentially parallel to the latter, as illustrated in text-figure IB, which shows the effect of ADM at 43° C. This curve was obtained by normalizing the combined curve point by point to the heat control. Beyond about 30 minutes, ADM clearly no longer contributed to cell killing. In fact, there was some protection against the ADM by heat: The last two time points were higher in survival by a factor of 2 than the earlier points (though this difference was not statistically significant). These results were not due to thermal inactivation of ADM: Heating ADM to 43° C for up to 5 hours did not appreciably reduce the drug's ability to subsequently kill cells at 37° C (data not shown). The results shown in text-figures lA and B indicated that considerable synergism could be demonstrated if the exposure were kept short (30 min or less). For this reason, we constructed a dose-response curve with ADM at a fixed, 20-minute treatment time (text-fig. 2). At such a short exposure, there was essentially no toxicity due to heat alone (text-fig. lA). Hence, the fact that the two curves of text-figure 2 diverge over the concentration range investigated showed that, for short exposures, 43° C appreciably sensitized the cells to AD M . For VOL. 57, NO.5, NOVEMBER 1976
1065
EFFECTS OF HYPERTHERMIA AND ADRIAMYCIN ON CHINESE HAMSTER CELLS
A
example, 0.1 JLg ADM/ml at 43° C killed the same fraction of HAl cells as 0.4-0.5 JLg ADM/ml at 37° C. The sensitization seemed to be most pronounced at low drug levels. The effect of heating (43° C) cells for various lengths of time before exposure to ADM is shown in text-figure 3A. The results showed that, if the preheating time was kept below 50 minutes, the effect of drug killing was essentially constant and equal to that of the unheated control (O-min preheating). Beyond that time, the survival of the cells subjected to consecutive treatments or to heat only was the same; i.e., the heated cells became refractory to ADM. This is illustrated in text-figure 3B
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TEXT-FIGURE 6.-Cumulative histograms of fluorescence of ADMtreated cells. (A) l l-rnin exposure. (B) 50-min exposure.
These show that preheating clearly inhibited the cells' acquisition of fluorescence; however, an increase in the incubation period from 11 (text-fig. 6A) to 50 minutes (text-fig. 6B) allowed both the preheated cells and the controls to increase uptake. Therefore, if the third hypothesis is correct, the initial metabolic step (leading to VOL. 57, NO.5, NOVEMBER 1976
1067
EFFECTS OF HYPERTHERMIA AND ADRIAMYCIN ON CHINESE HAMSTER CELLS
the fluorescing product) clearly is not the one that is affected by heat. Our current working hypothesis is that the simplest explanation, modulation of permeability as in the first hypothesis, is the most likely to be correct. As pointed out above, the data do not exclude other possibilities. Whatever the mechanism of the observed phenomenon, the differential response of the two cell lines is of interest because of possible clinical applications. Although neither of the two cell lines examined is human, and only one the (EMT-6 murine mammary sarcoma) is frankly malignant, the possibility exists that different human tumors might show similar variations in their response. Fluorescence measurements to determine drug uptake might then be desirable on biopsy specimens. Alternatively, shorter heating times (~20-30 min) might be more efficient. Heating to 41°C rather than 43° C could preclude the resistance to ADM introduced by the longer exposures to 43° C. REFERENCES (1) ROCKWELL S, KALLMAN RF: Cellular radiosensitivity and tumor
radiation response in the EMT 6 tumor cell system, Radiat Res 53:281-294, 1973
VOL. 57, NO.5, NOVEMBER 1976
(2) HAHN GM, BRAUN], HAR-KEDAR I: Thermochemotherapy: Synergism between hyperthermia (42-43°) and adriamycin (or bleomycin) in mammalian cell inactivation. Proc Nat! Acad Sci USA 72:937-940, 1975 (3) YANG S-], HAHN GM, BAGSHAW MA: Chromosome aberrations induced by thymidine. Exp Cell Res 42:130-135, 1966 (4) HAHN GM, STEWART ]R, YANG S-], et al: Chinese hamster cell monolayer cultures. 1. Changes in cell dynamics and modifications of the cell cycle with the period of growth. Exp Cell Res 49:285-292, 1968 (5) HAHN GM, GORDON LF, KURKJIAN SD: Responses of cycling and non-cycling cells to 1,3-bis(2-chloroethyl)-I-nitrosourea and to bleomycin. Cancer Res 34:2373-2377, 1974 (6) HAHN GM: Metabolic aspects of the role of hyperthermia In mammalian cell inactivation and their possible relevance to cancer treatment. Cancer Res 34:3117-3123,1974 (7) GERNER EW, SCHNEIDER MS: Induced thermal resistance In HeLa cells. Nature 256:500-502, 1975 (8) HENLE K], LEEPER DB: Interaction of hyperthermia and radiation in CHO cells: Recovery kinetics. Radiat Res 66:505-518, 1976 (9) GERWECK LE, GILLETTE EL, DEWEY we: Effect of heat and radiation on synchronous Chinese hamster cells: Killing and repair. Radiat Res 64:611-623, 1975 (10) WESTRA A, DEWEY WC: Variation in sensitivity to heat shock during the cell-cycle of Chinese hamster cells in vitro. Int] Radiat Bioi 19:467-477, 1971 (11) BACHUR NR, CRADOCK ]C: Daunomycin metabolism in rat tissue slices.] Pharmacol Exp Ther 175:331-337, 1970
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NATL CANCER INST
