Int. J . Cancer: 22, 602-606 (1978)
ENHANCEMENT OF THERMAL KILLING BY POLYAMTNES. I. SURVIVAL OF CHINESE HAMSTER CELLS E. BEN-HUR,A. PRAGER and E. RIKLIS Israel Atomic Energy Commission, Nuclear Research Center- Negev, P . 0. Box 9001, Beer-Sheva, Israel
Exposure of Chinese hamster cells t o polyamines at an elevated temperature (42'C) results i n synergistic cell killing. The effectiveness of polyamines i n potentiating thermal killing decreases in the following order: sperrnine:-sperrnidine> cadaverine>putrescine. The magnitude of t h e synergism increases w i t h exposure time. The survival curves, when plotted as a function o f polyamine concentration, display a shoulder during I h exposure at 42' C, followed by exponential cell killing. Longer exposure times eliminate t h e shoulder and result in steeper slopes of the survival curves. The effect is maximal when exposure t o polyamines and heat i s simultaneous. Separation i n t i m e between the t w o treatments causes a rapid disappearance of the synergism. The order o f application is of only minor importance in this regard. The results suggest that the intracellular level of spermine may be a major factor in determining heat sensitivity o f Chinese hamster cells.
Mammalian cells are sensitive to temperatures above 41" C (hyperthermia). Continued exposure to hyperthermia leads to cell death, measured by loss of proliferative ability. The quantitative biology of this response was studied by various laboratories for different cell lines. Heat sensitivity varies with the position of the cell in the cell cycle, S-phase cells being most heat-sensitive (Palzer and Heidelberger, 1973; Westra and Dewey, 1971). Environmental factors such as lowered PH (Gerweck and Rottinger, 1976) and nutritional deficiency (Hahn, 1974) increase heat sensitivity. In mammalian cells, heat interacts synergistically with radiation (Ben-Hur et al., 1972, 1974; Gerweck et a!., 1975) and drugs (Ben-Hur and Elkind, 1974; Hahn et al., 1975). The mechanisms of this synergism are not clear. In many cases (e.g. radiation) DNA is the target molecule inside the cell (Ben-Hur and Elkind, 1975; Ben-Hur et al., 1977). One of the effects of heat is the inhibition of the repair capacity of the cells (Ben-Hur et al., 1974). This could be due to effects on proteins associated with DNA (Ben-Hur, 1976) since proteins are considered the major target molecule for heat-inactivation (Dewey et al., 1977). Hyperthermia combined with radiation or drugs is now considered potentially useful in cancer therapy. The cellular aspects which underline the usefulness of the combined modality were reviewed (Ben-Hur et al., 1978; Leith et al., 1977). In the course of our studies we have found that polyamines cause a dramatic potentiation of heat-induced cell killing. Since this may help to explain some features of heat-inactivation, the phenomena were further explored for all the naturally-occurring
polyamines. The results suggest that spermine may play a role in determining heat-sensitivity of mammalian cells.
MATERIAL AND METHODS
Chinese hamster V79 fibroblasts were grown attached t o glass or plastic in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal calf serum. The cells doubled in number in about 9 h in 5 % CO, humidified atmosphere at 37" C. Colony-forming ability in vitro was used as a
\
I
zh'
ID
I YU CADAVERIHE ( n M )
I
an
FIGURE1 - Survival of Chinese hamster cells exposed to graded cadaverine concentrations at 42"C. The exposure times are indicated for each curve. 0-0, exposure in DMEM containing 10% horse serum; A, 1 h exposure at 37" C. Received : May 26, 1978 and in revised form August 7, 1978.
603
POLYAMINE POTENTIATION OF HEAT SENSITIVITY
Exposure to polyamines (Sigma Chemical Co, St. Louis, Mo.) was done by adding the compound (as the hydrochloride form) in a stock solution directly to plates containing growth medium and cells which had been plated about 16 h previously. Addition of polyamines caused no significant change in PH of the medium. Following treatment, the medium containing the drugs was removed by suction, the cells were rinsed, fresh medium was added and the cells were incubated for colony formation. Heat exposure was done in a CO, incubator (Tuttenauer, Jerusalem) operated at 42h0.2"C. The temperature was continuously monitored in the medium using a thermistor with a digital read-out (DigiTec, model 5810 thermometer, United System Corp., Dayton, Ohio). RESULTS
ii'l0
IJ
1.1
1.6
I
U
TIME A 1 IZ'C IUOURS 1
Figure 1 shows the survival curves of Chinese hamster cells exposed to a fixed heat (42"C) dose in the presence of varying concentrations of cadaverine (diaminopentane). Neither heat alone nor exposure to cadaverine at 37" C were effective in inducing cell killing. Simultaneous exposure to both treatments drastically reduced the proliferative capacity of the cells. The survival curve obtained following exposure for 1 h is characterized by a shoulder which is followed by exponential cell
FIGURE 2 - Survival of Chinese hamster cells as a function of exposure time at 42" C in the presence of cadaverine. The concentration of cadaverine is indicated for each curve.
measure of cell survival. Cells plated in appropriate numbers in 9-cm Petri dishes were incubated overnight before the experiment was started. This procedure ensured that the distribution of cells throughout the growth cycle was similar to that resulting from asynchronous, log-phase growth. As a result, cell multiplicities at the start of the experiment were 2-3. Following treatment, cells were incubated for 8-10 days and the colonies were then stained with methylene blue and counted. Results are expressed as the fraction of cells surviving a given treatment relative to the number of colony-forming cells in the starting suspension. The percentage of colony formers in the starting suspension (the plating efficiency) was usually over 80%. Survival curves were corrected for the average cellular multiplicity N at the start of an experiment (Elkind and Whitmore, 1967) which was determined from the increase in the number of colonies obtained when the cells after overnight growth were respread by trypsinization. Duplicate plates were employed for each datum point. Since standard errors of the mean were usually smaller than the symbols, they are not shown. The lines of the survival curves were fitted by eye. Each experiment was repeated at least twice, and variation was not greater than 5 %.
#.#OIL I
I
I
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II
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I
a1 40 C U T I S C I N E InM )
I
51
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FIGURE 3 - Survival of Chinese hamster cells exposed to graded putrescine concentrations at 42" C and 37" C for 1 or 2 h, as indicated.
604
BEN-HUR ET AL.
some toxicity at high concentration even at 37" C. The effective concentrations required to obtain cell killing vary widely: while putrescine is about half as effective as cadaverine, spermidine is 5 times more effective and spermine is 50 times more effective than cadaverine, based on the slopes of the respective survival curves, as measured by Do (Do is the concentration required to reduce survival by a factor of I/e on the exponential part of the survival curve). For 1 h exposure at 42" C Do is 7.2, 4.6, 0.82 and 0.093 mM for putrescine, cadaverine, spermidine and spermine, respectively. The effect of separating in time the combined treatment is shown in Figure 6 for cadaverine and spermine. ApparentIy, synergism is maximal for simultaneous treatment, and this is most pronounced in the case of cadaverine. Synergism decays rapidly as the time between treatments is increased, and is almost completely lost by 3 h. Whether heat precedes or follows exposure to polyamines is of minor importance only, except perhaps in the case of spermine.
2
0 U
2
2L L
3 0.
DISCUSSION
0.0
SPfRHIDIHE fmM 1
FIGURE 4 - Survival of Chinese hamster cells exposed to graded spermidine concentrations at 42" C and 37" C for 1 or 2 h, as indicated.
killing. At longer exposure times the shoulder is eliminated and the final slopes of the curves become steeper. Since bovine serum is known to contain amino oxidases (e.g. see Quash et al., 1976, for diamine oxidase) which can convert cadaverine into products toxic to the cells (aldehydes and H202), exposure was also performed in medium containing horse serum (Fig. 1). Horse serum does not contain polyamine oxidases and this was confirmed by incubating 14C-labelled polyamines in DMEM containing horse serum. Radiochromatography after 2 h incubation did not reveal the appearance of new radioactive peaks (data not shown). Since the effect was the same, regardless of the serum used, oxidation products of cadaverine apparently do not contribute to the synergism observed. Figure 2 shows the survival curves of cells exposed for varying times at 42°C in the presence of a constant cadaverine concentration. The curves are different from those of Figure 1 in two aspects: they always display a very pronounced shoulder, and no part of the curves is entirely exponential. Figures 3 to 5 show survival as a function of polyamine concentration at 37" C and 42°C. They show the survival as affected by putrescine, spermidine and spermine, respectively. These survival curves are basically the same as those shown for cadaverine (Fig. 1). They differ from cadaverine in the following ways. All show
The molecular basis for heat-inactivation of mammalian cells is still obscure in spite of the intensive effort invested in this area in recent years. The results presented in this work show that thermal sensitivity of Chinese hamster cells is greatly en-
1.1
0.
2
0
"
. I
L
Y
5
L
3 Dl
0.01
FIGURE 5 - Survival of Chinese hamster cells exposed to graded spermine concentrations at 42°C and 37°C for 1 or 2 h, as indicated. 0-0, exposure in DMEM containing 10% horse serum.
POLY AMlNE POTENTIATION OF HEAT SENSITIVITY
a
CADAVERINL
0 SP€RYINE
1lHf
AT 3 1 - C
FIGURE6 - Survival of Chinese hamster cells exposed to 35 m~ cadaverine ( 0 4 ) and 0.5 mM spermine (0-0) for 1 h at different times before or after 1 h exposure at 42°C. The shaded area indicates simultaneous exposure. hanced by addition of polyamines t o the growth medium. This synergism between heat and polyamines is maximal when exposure to both agents is simultaneous. Exposure to polyamines and heat separately is much less effective in cell killing (Fig. 6). In this respect polyamines differ markedly from radiation and drugs whose synergism with heat is affected only t o a small extent by variation of the treatment-heat sequence during, before or after treatment (Ben-Hur and Elkind, 1974; Ben-Hur et al., 1978; Dewey et af., 1977). The effectiveness of polyamines in potentiating heat-induced killing is in the order spermine >
605
spermidine > cadaverine > putrescine. This is the same order of effectiveness of polyamines in inhibiting the proliferation of mammalian cells (Katsuta et al., 1975) and in interaction with DNA (Bachrach, 1973). Heat affects chromosomes (Dewey ef aE., 1977) and its effect on this level was postulated to potentiate the effect of radiation (Ben-Hur, 1976). Therefore, it is suggested that chromosomes are also the target for the synergistic interaction between heat and polyamines. It could be argued that the effect observed is mediated via metabolites of polyamines. However, since exposure in the presence of horse serum gave the same results as with fetal calf serum (see Figs. 1 and 5), oxidation products produced by pol yamine oxidases are ruled out. The problem of metabolism is more fully discussed in the accompanying paper (Ben-Hur and Riklis, 1978). If the level of polyamines in the cell is one of the factors which determine its heat-sensitivity, then a couple of hitherto unexplained observations may now become clear. First, heat-sensitivity, which is maximal in S phase, closely correlates with the intracellular level of polyamines (spermine and spermidine) in Chinese hamster cells (Gerner and Russel, 1977). Second, cancer cells appear to be more heat-sensitive than normal cells (Giovanella et al., 1976; Kase and Hahn, 1975) and they also contain higher levels of polyamines (Russel, 1973). The spermine concentrations effective in enhancing heat-sensitivity (Fig. 5 ) are well within the range of the intracellular level, which is up to 1 mM in proliferating cells (Gerner and Russel, 1977; Tabor and Tabor, 1976). Therefore, it is likely that this is the polyamine which is effective in determining heat-sensitivity of mammalian cells. Clinical modification of the intracellular level of polyamines could greatly enhance the therapeutic value of heat application in cancer therapy. Experiments in tumor-bearing animals to achieve this purpose are presently in progress (G. Hazan and E. Ben-Hur, to be published).
ACKNOWLEDGEMENTS
We thank Dr. M. M. Elkind for supplying the cells and Miss Ziva Swisa for able technical assistance.
FACILITATION DE LA THERMODESTRUCTION PAR LES POLYAMINES. I. SURVIE DES CELLULES DE HAMSTER CHINOIS L’exposition de cellules de hamster chinois B des polyamines B temperature Clevke (42” C) entraine une destruction synergique des cellules. L’effet potentialisateur des polyamines sur la thermodestruction dkcroit dans l’ordre suivant : spermine > spermidine >cadaverine >putrescine. L’importance du synergisme augmente avec la duree de I’exposition. Les courbes de survie, calculkes en fonction de la concentration de polyamines, prksentent un palier au moment d’une exposition d’une heure B 42“ C; on observe ensuite une destruction exponentielle des cellules. Des expositions plus longues Bliminent le palier et donnent des pentes plus abruptes. L’effet est maximal lorsque l’exposition aux polyamines et B la chaleur est sirnultanbe. Si les deux traitements sont separes par un certain laps de temps, le synergisme disparatt rapidement. L’ordre d’application n’a qu’une importance mineure B cet kgard. Les rCultats donnent B penser que le niveau intracellulaire de la spermine est peut-stre un facteur important pour determiner la thermosensibilitk des cellules de hamster chinois.
606
BEN-HUR ET AL. REFERENCES
BACHRACH, U., Function of naturally occurring polyamines, Academic Press, New York (1973). BEN-HUR,E., Mechanisms of the synergistic interaction between hyperthermia and radiation in cultured mammalian cells. J . Radiat. Res., 17, 92-98 (1976). BEN-HUR,E., BRONK,B. V., and ELKIND,M. M., Thermally enhanced radiosensitivity of cultured Chinese hamster cells. Nature New Biol., 238, 209-211 (1972). BEN-HUR,E., and ELKIND,M. M., Thermal sensitization of Chinese hamster cells to methyl methanesulfonate: relation of DNA damage and repair to survival response. Cancer Biochem. Biophys., 1, 23-32 (1974). BEN-HUR,E., and ELKIND,M. M., D N A damage and repair in hyperthermic mammalian cells: relation to enhanced cell killing. In: 0. F. Nygaard, H. I. Adler and W. K. Sinclair (ed.), Radiation research-biomedical, chemical and physical perspectives, pp. 703-717, Academic Press, New York (1975). BEN-HUR,E., ELKIND, M. M., and BRONK,B. V.,Thermally enhanced radio-response of cultured Chinese hamster cells: inhibition of repair of sublethal damage and enhancement of lethal damage. Radiat. Res., 58, 38-51 (1974). BEN-HUR,E., ELKIND, M. M., and RIKLIS,E., The combined effects of hyperthermia and radiation in cultured mammalian cells. In: C. Streffer et al. (eds.), Cancer therapy by hyperthermia and radiation, Urban and Schwarzenberg, Munich (1978). BEN-HUR,E., KOL, R., and RIKLIS,E., Modification of radiation response by hyperthermia and its relation to DNA damage and repair. In: Radiobiological research and radiotherapy, Vol. 1, pp. 299-31 1, International Atomic Energy Agency, Vienna (1977). BEN-HUR,E., and RIKLIS, E., Enhancement of thermal killing by polyamines. 11. Uptake and metabolism in hypeithermic Chinese hamster cells. Int. J. Cancer, 22, 607-610 (1978). DEWEY,W. C., HOPWOOD,L. E., SAPARETO, S. A,, and L. E., Cellular responses t o combinations of GERWECK, hyperthermia and radiation. Radiology, 123, 463-474 (1977). ELKIND, M. M.,and WHITMORE, G. F., In: The radiobiology of cultured mammalian cells, pp. 74-85, Gordon and Breach, New York (1967). GERNER, E. W., and RUSSEL,D. H., The ielationship between polyamine accumulation and DNA replication in synchronized Chinese hamster ovary cells after heat shock. Cancer Res., 37, 482-489 (1977).
GERWECK,L. E., GILLETTE, E. L., and DEWEY,W. C., Effect of heat and radiation o n synchronous Chinese hamster cells: killing and repair. Radiat. Res., 64, 611-623 (1975). GERWECK, L., and ROTTINGER, E., Enhancement of mammalian cell sensitivity to hyperthermia by pH alteiation. Radiat. Res., 67, 508-51 1 (1976). GIOVANELLA, B. C., STEHLIN,J. S., and MORGAN,A. C., Selective lethal effect of supranormal temperatures on human neoplastic cells. Cancer Res., 36, 3944-3950 (1976). HAHN,G. M., Metabolic aspects of the role of hyperthermia in mammalian cells inactivation and their possible relevance to cancer treatment. Cancer Res., 34, 3117-3123 (1974). HAHN,G. M., BRAUN,J., and HAR-KEDAR, I., Thermochemotherapy: synergism between hyperthermia (42' C43" C) and adriamycin (or bleomycin) in mammalian cell inacthation. Proc. nat. Acad. Sci. (Wash.), 72, 937-941 (1975). KASE,K., and HAHN,C. M., Differential heat response of normal and transformed human cells in tissue culture. Nature (Lond.), 255, 228-230 (1975). KATSUTA,H., TAKAOKA, T., NOSE, K., and N A C A I , Y., Effect of polyamines on the proliferation of mammalian cells in tissue culture. Sap. J. exp. Med., 45, 345-354 (1975). LEITH, J. T., MILLER,R. C., GERNER,E. W., ana BOONE, M. L. M., Hyperthermic potentiation : biological aspects and application to radiation therapy. Cancer, 39, 766-779 (1977). PALZER,R. J., and HEIDELBERGER, C., Studies o n the quantitative biology of hyperthermic killing of HeLa cells. Cancer Res., 33,415-421 (1973). QUASH,G., CALOGERO, H., FOSSAR,N., FERDINAND, N., and TAYLOR, C., Modification of diamine oxidase activity in vitro by metabolites of asparagine and differences in asparagine decarboxylation in normal and virus-transformed baby hamster kidney cells. Biochem. J., 157, 599608 (1976). RUSSEL,D. H., Polyamines in growth-normal and neoplastic. In: D. H. Russel (ed.), Polyamines in normal and neoolasticgrowth, pp. 1-13, Raven Press, New York (1973). TABOR,C. W., and TABOR, H., 1,4-Diaminobutane (puttescine), spermidine, and spermine. Ann. Rev. Biochenr., 45, 285-306 (1976). WESTRA,A., and DEWEY,W. C., Variation in sensitivity to heat shock during the cell-cycle of Chinese hamster cells in vitro. Int. J . Radiat. Biol., 19, 467-477 (1971).
ENHANCEMENT OF THERMAL KILLING BY POLYAMTNES. I. SURVIVAL OF CHINESE HAMSTER CELLS E. BEN-HUR,A. PRAGER and E. RIKLIS Israel Atomic Energy Commission, Nuclear Research Center- Negev, P . 0. Box 9001, Beer-Sheva, Israel
Exposure of Chinese hamster cells t o polyamines at an elevated temperature (42'C) results i n synergistic cell killing. The effectiveness of polyamines i n potentiating thermal killing decreases in the following order: sperrnine:-sperrnidine> cadaverine>putrescine. The magnitude of t h e synergism increases w i t h exposure time. The survival curves, when plotted as a function o f polyamine concentration, display a shoulder during I h exposure at 42' C, followed by exponential cell killing. Longer exposure times eliminate t h e shoulder and result in steeper slopes of the survival curves. The effect is maximal when exposure t o polyamines and heat i s simultaneous. Separation i n t i m e between the t w o treatments causes a rapid disappearance of the synergism. The order o f application is of only minor importance in this regard. The results suggest that the intracellular level of spermine may be a major factor in determining heat sensitivity o f Chinese hamster cells.
Mammalian cells are sensitive to temperatures above 41" C (hyperthermia). Continued exposure to hyperthermia leads to cell death, measured by loss of proliferative ability. The quantitative biology of this response was studied by various laboratories for different cell lines. Heat sensitivity varies with the position of the cell in the cell cycle, S-phase cells being most heat-sensitive (Palzer and Heidelberger, 1973; Westra and Dewey, 1971). Environmental factors such as lowered PH (Gerweck and Rottinger, 1976) and nutritional deficiency (Hahn, 1974) increase heat sensitivity. In mammalian cells, heat interacts synergistically with radiation (Ben-Hur et al., 1972, 1974; Gerweck et a!., 1975) and drugs (Ben-Hur and Elkind, 1974; Hahn et al., 1975). The mechanisms of this synergism are not clear. In many cases (e.g. radiation) DNA is the target molecule inside the cell (Ben-Hur and Elkind, 1975; Ben-Hur et al., 1977). One of the effects of heat is the inhibition of the repair capacity of the cells (Ben-Hur et al., 1974). This could be due to effects on proteins associated with DNA (Ben-Hur, 1976) since proteins are considered the major target molecule for heat-inactivation (Dewey et al., 1977). Hyperthermia combined with radiation or drugs is now considered potentially useful in cancer therapy. The cellular aspects which underline the usefulness of the combined modality were reviewed (Ben-Hur et al., 1978; Leith et al., 1977). In the course of our studies we have found that polyamines cause a dramatic potentiation of heat-induced cell killing. Since this may help to explain some features of heat-inactivation, the phenomena were further explored for all the naturally-occurring
polyamines. The results suggest that spermine may play a role in determining heat-sensitivity of mammalian cells.
MATERIAL AND METHODS
Chinese hamster V79 fibroblasts were grown attached t o glass or plastic in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal calf serum. The cells doubled in number in about 9 h in 5 % CO, humidified atmosphere at 37" C. Colony-forming ability in vitro was used as a
\
I
zh'
ID
I YU CADAVERIHE ( n M )
I
an
FIGURE1 - Survival of Chinese hamster cells exposed to graded cadaverine concentrations at 42"C. The exposure times are indicated for each curve. 0-0, exposure in DMEM containing 10% horse serum; A, 1 h exposure at 37" C. Received : May 26, 1978 and in revised form August 7, 1978.
603
POLYAMINE POTENTIATION OF HEAT SENSITIVITY
Exposure to polyamines (Sigma Chemical Co, St. Louis, Mo.) was done by adding the compound (as the hydrochloride form) in a stock solution directly to plates containing growth medium and cells which had been plated about 16 h previously. Addition of polyamines caused no significant change in PH of the medium. Following treatment, the medium containing the drugs was removed by suction, the cells were rinsed, fresh medium was added and the cells were incubated for colony formation. Heat exposure was done in a CO, incubator (Tuttenauer, Jerusalem) operated at 42h0.2"C. The temperature was continuously monitored in the medium using a thermistor with a digital read-out (DigiTec, model 5810 thermometer, United System Corp., Dayton, Ohio). RESULTS
ii'l0
IJ
1.1
1.6
I
U
TIME A 1 IZ'C IUOURS 1
Figure 1 shows the survival curves of Chinese hamster cells exposed to a fixed heat (42"C) dose in the presence of varying concentrations of cadaverine (diaminopentane). Neither heat alone nor exposure to cadaverine at 37" C were effective in inducing cell killing. Simultaneous exposure to both treatments drastically reduced the proliferative capacity of the cells. The survival curve obtained following exposure for 1 h is characterized by a shoulder which is followed by exponential cell
FIGURE 2 - Survival of Chinese hamster cells as a function of exposure time at 42" C in the presence of cadaverine. The concentration of cadaverine is indicated for each curve.
measure of cell survival. Cells plated in appropriate numbers in 9-cm Petri dishes were incubated overnight before the experiment was started. This procedure ensured that the distribution of cells throughout the growth cycle was similar to that resulting from asynchronous, log-phase growth. As a result, cell multiplicities at the start of the experiment were 2-3. Following treatment, cells were incubated for 8-10 days and the colonies were then stained with methylene blue and counted. Results are expressed as the fraction of cells surviving a given treatment relative to the number of colony-forming cells in the starting suspension. The percentage of colony formers in the starting suspension (the plating efficiency) was usually over 80%. Survival curves were corrected for the average cellular multiplicity N at the start of an experiment (Elkind and Whitmore, 1967) which was determined from the increase in the number of colonies obtained when the cells after overnight growth were respread by trypsinization. Duplicate plates were employed for each datum point. Since standard errors of the mean were usually smaller than the symbols, they are not shown. The lines of the survival curves were fitted by eye. Each experiment was repeated at least twice, and variation was not greater than 5 %.
#.#OIL I
I
I
I#
II
I
I
a1 40 C U T I S C I N E InM )
I
51
I
I#
FIGURE 3 - Survival of Chinese hamster cells exposed to graded putrescine concentrations at 42" C and 37" C for 1 or 2 h, as indicated.
604
BEN-HUR ET AL.
some toxicity at high concentration even at 37" C. The effective concentrations required to obtain cell killing vary widely: while putrescine is about half as effective as cadaverine, spermidine is 5 times more effective and spermine is 50 times more effective than cadaverine, based on the slopes of the respective survival curves, as measured by Do (Do is the concentration required to reduce survival by a factor of I/e on the exponential part of the survival curve). For 1 h exposure at 42" C Do is 7.2, 4.6, 0.82 and 0.093 mM for putrescine, cadaverine, spermidine and spermine, respectively. The effect of separating in time the combined treatment is shown in Figure 6 for cadaverine and spermine. ApparentIy, synergism is maximal for simultaneous treatment, and this is most pronounced in the case of cadaverine. Synergism decays rapidly as the time between treatments is increased, and is almost completely lost by 3 h. Whether heat precedes or follows exposure to polyamines is of minor importance only, except perhaps in the case of spermine.
2
0 U
2
2L L
3 0.
DISCUSSION
0.0
SPfRHIDIHE fmM 1
FIGURE 4 - Survival of Chinese hamster cells exposed to graded spermidine concentrations at 42" C and 37" C for 1 or 2 h, as indicated.
killing. At longer exposure times the shoulder is eliminated and the final slopes of the curves become steeper. Since bovine serum is known to contain amino oxidases (e.g. see Quash et al., 1976, for diamine oxidase) which can convert cadaverine into products toxic to the cells (aldehydes and H202), exposure was also performed in medium containing horse serum (Fig. 1). Horse serum does not contain polyamine oxidases and this was confirmed by incubating 14C-labelled polyamines in DMEM containing horse serum. Radiochromatography after 2 h incubation did not reveal the appearance of new radioactive peaks (data not shown). Since the effect was the same, regardless of the serum used, oxidation products of cadaverine apparently do not contribute to the synergism observed. Figure 2 shows the survival curves of cells exposed for varying times at 42°C in the presence of a constant cadaverine concentration. The curves are different from those of Figure 1 in two aspects: they always display a very pronounced shoulder, and no part of the curves is entirely exponential. Figures 3 to 5 show survival as a function of polyamine concentration at 37" C and 42°C. They show the survival as affected by putrescine, spermidine and spermine, respectively. These survival curves are basically the same as those shown for cadaverine (Fig. 1). They differ from cadaverine in the following ways. All show
The molecular basis for heat-inactivation of mammalian cells is still obscure in spite of the intensive effort invested in this area in recent years. The results presented in this work show that thermal sensitivity of Chinese hamster cells is greatly en-
1.1
0.
2
0
"
. I
L
Y
5
L
3 Dl
0.01
FIGURE 5 - Survival of Chinese hamster cells exposed to graded spermine concentrations at 42°C and 37°C for 1 or 2 h, as indicated. 0-0, exposure in DMEM containing 10% horse serum.
POLY AMlNE POTENTIATION OF HEAT SENSITIVITY
a
CADAVERINL
0 SP€RYINE
1lHf
AT 3 1 - C
FIGURE6 - Survival of Chinese hamster cells exposed to 35 m~ cadaverine ( 0 4 ) and 0.5 mM spermine (0-0) for 1 h at different times before or after 1 h exposure at 42°C. The shaded area indicates simultaneous exposure. hanced by addition of polyamines t o the growth medium. This synergism between heat and polyamines is maximal when exposure to both agents is simultaneous. Exposure to polyamines and heat separately is much less effective in cell killing (Fig. 6). In this respect polyamines differ markedly from radiation and drugs whose synergism with heat is affected only t o a small extent by variation of the treatment-heat sequence during, before or after treatment (Ben-Hur and Elkind, 1974; Ben-Hur et al., 1978; Dewey et af., 1977). The effectiveness of polyamines in potentiating heat-induced killing is in the order spermine >
605
spermidine > cadaverine > putrescine. This is the same order of effectiveness of polyamines in inhibiting the proliferation of mammalian cells (Katsuta et al., 1975) and in interaction with DNA (Bachrach, 1973). Heat affects chromosomes (Dewey ef aE., 1977) and its effect on this level was postulated to potentiate the effect of radiation (Ben-Hur, 1976). Therefore, it is suggested that chromosomes are also the target for the synergistic interaction between heat and polyamines. It could be argued that the effect observed is mediated via metabolites of polyamines. However, since exposure in the presence of horse serum gave the same results as with fetal calf serum (see Figs. 1 and 5), oxidation products produced by pol yamine oxidases are ruled out. The problem of metabolism is more fully discussed in the accompanying paper (Ben-Hur and Riklis, 1978). If the level of polyamines in the cell is one of the factors which determine its heat-sensitivity, then a couple of hitherto unexplained observations may now become clear. First, heat-sensitivity, which is maximal in S phase, closely correlates with the intracellular level of polyamines (spermine and spermidine) in Chinese hamster cells (Gerner and Russel, 1977). Second, cancer cells appear to be more heat-sensitive than normal cells (Giovanella et al., 1976; Kase and Hahn, 1975) and they also contain higher levels of polyamines (Russel, 1973). The spermine concentrations effective in enhancing heat-sensitivity (Fig. 5 ) are well within the range of the intracellular level, which is up to 1 mM in proliferating cells (Gerner and Russel, 1977; Tabor and Tabor, 1976). Therefore, it is likely that this is the polyamine which is effective in determining heat-sensitivity of mammalian cells. Clinical modification of the intracellular level of polyamines could greatly enhance the therapeutic value of heat application in cancer therapy. Experiments in tumor-bearing animals to achieve this purpose are presently in progress (G. Hazan and E. Ben-Hur, to be published).
ACKNOWLEDGEMENTS
We thank Dr. M. M. Elkind for supplying the cells and Miss Ziva Swisa for able technical assistance.
FACILITATION DE LA THERMODESTRUCTION PAR LES POLYAMINES. I. SURVIE DES CELLULES DE HAMSTER CHINOIS L’exposition de cellules de hamster chinois B des polyamines B temperature Clevke (42” C) entraine une destruction synergique des cellules. L’effet potentialisateur des polyamines sur la thermodestruction dkcroit dans l’ordre suivant : spermine > spermidine >cadaverine >putrescine. L’importance du synergisme augmente avec la duree de I’exposition. Les courbes de survie, calculkes en fonction de la concentration de polyamines, prksentent un palier au moment d’une exposition d’une heure B 42“ C; on observe ensuite une destruction exponentielle des cellules. Des expositions plus longues Bliminent le palier et donnent des pentes plus abruptes. L’effet est maximal lorsque l’exposition aux polyamines et B la chaleur est sirnultanbe. Si les deux traitements sont separes par un certain laps de temps, le synergisme disparatt rapidement. L’ordre d’application n’a qu’une importance mineure B cet kgard. Les rCultats donnent B penser que le niveau intracellulaire de la spermine est peut-stre un facteur important pour determiner la thermosensibilitk des cellules de hamster chinois.
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