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Cytokinetic studies on the effect of hyperthermia on Chinese hamster lung cells
Europ. J. Cancer Vol. 12, pp. 827-831. Pergamon Press 1976. Printed in Great Britain
Cytokinetic Studies on the Effect of Hyperthermia on Chinese Hamster Lung Cells H E L M U T SCHLAG and CHRISTINE LI)CKE-HUHLE Kernforschungszentrum Karlsruhe, Institut fiir Strahlenbiologie, Postfach 3640, 7500 Karlsruhe, Federal Republic of Germany Abstract--Hyperthermic killing of V 79 cells is dependent on the state of growth: while survival of plateau phase cells decreases to 0.45, only 0.03 of the exposed exponentially growing cells survive 4 hr at 42°C. Flow-microfluorometric analysis during and after heating yields the fraction of cells in the individual phases of cell cycle and allows conclusions about changes in the proliferation kinetics of the exposed cell population. Mild heat treatment (1 hr at 42°C) leads to a synchronisation effect caused by a block in S and G 2 + M, while after a stronger heat exposure (4 hr at 42°C) cell death in S-phase is predominant.
INTRODUCTION
It is hoped that the results might contribute to selecting optimum sequential application of heat-X-ray or heat-chemotherapy.
PUBLICATIONS concerning the use of elevated temperatures in therapy date back until the middle of the last century (review: 1). More recently, there is renewed interest in hyperthermia for treatment of cancer, especially in combination with radiation therapy. Such a combined treatment might possibly offer an alternative to high L E T radiations, since the respective dose-effect curves show quantitative and qualitative similarities. It is not yet clear whether the increased efficiency for cell killing by the heat-X-ray therapy is of additive or synergistic kind, but cytokinetic studies on cells after hyperthermic treatment should reveal some of the underlying mechanism. Though iI is known that the response of cells to hyperthermia depends on their stage in the cell cycle [2, 3] cell survival curves of several cell lines tested either in the logarithmic or plateau phase of growth show contradictory results [4-6]. The present study was undertaken to examine the influence of the state of growth on thermal sensitivity using V79 cells and to investigate the effect of heat on cell kinetics during and after exposure to a temperature (42°C) within acceptable limits for potential clinical treatment.
MATERIAL AND METHODS Cell culture
Chinese hamster lung cells (line V 7 9 , obtained from Dr. R. B. Painter, San Francisco) were grown as monolayer cultures on plastic tissues culture dishes in Eagle's basal medium supplemented with 15% foetal calf serum (Gibco Biocult, BCL 005a) and neomycin sulfate (0.1 g/l). Exponentially growing cultures were obtained by plating ceils at a density of 1 x 10 4 cells/cm z and letting them proliferate to about 4 x 10 4 cells/cm 2. For the studies on plateau phase cultures cells were incubated at 37°C without exchanging medium until they attained inhibition of growth at approximately 3 x 10 5 cells/cm 2 with at least 65 % of the cells being in a Gl-like phase of the cell cycle [7]. Experimental procedures
Cell monolayers were exposed to an elevated temperature of 42°C by placing the culture flask into a waterbath temperature of which was kept constant within _ 0.1 °C. Immediately before heat exposure fresh medium was applied to all cultures to avoid differences in the thermal
Accepted 14 April 1976. 827
828
Helmut Schlag and Christine Liicke-Huhle
sensitivity caused by depletion of nutritional factors [4]. After different periods of heat treatment (up to 4 h r ) cells were either immediately trypsinized using 0.05% trypsin or the culture flasks were returned to 37°C for various periods and trypsinized afterwards. Both clonogenity and the number of progeny cells were determined from the obtained single cell suspensions as described in full detail elsewhere [7]. To determine the effect of heat on cell population kinetics during and after exposure to 42°C the changes in the DNA-distribution of the cell population were analysed by flowmicrofluorometry with the "Cytofluorograf" (Model 4801; Bio/Physics Instr.) coupled to a multichannel analyzer. For that purpose single cell suspensions were washed with tris buffer, fixed in ethanol (70 %) for 30 min and treated with RNase (0.1 °/o in tris buffer) for 1 hr at 37°C. After washing once more with tris buffer cells were stained with ethidium bromide (0.01 mg/ml). Under these conditions, the amount of dye is proportional to the DNA content of the cells [8]. The distribution of fluorescence intensity of the nuclear DNA (usually of 2-5 x 104 cells) was displayed as conventional histograms (cell number versus fluorescence intensity). The percentage of cells in the individual phases of the cell cycle (G1, S, G 2 + M ) was calculated from the area under the DNA histograms assuming a rectangular distribution of the S-cells between the G 1 and G2-peak [9]. The reproducibility of independent experiments was better than
LO
cells 0.5
---o
°~o
0.2
> F-
Exponentially growing cells
0.I
-
g, 0.05
0.02
I
r t
l
T
r 2
3
4
Hours o t 4 2 ° C
Fig. 1. Survival of exponentially growing and unfed plateau phase V 79 cell, exposedfor various times to 42°C.
~
10 2
&
IO I
B
/o/
c
z
/;,""
#7
:g
+3%.
24
48
72
96
Hours after hyperthermia
RESULTS
Figure 1 demonstrates a marked difference for survival of exponentially growing cells compared to plateau phase cells after the same exposure to 42°C. While survival of plateau phase cells decreases to 45 %, only 3 °/o of the exposed exponentially growing cells survive 4 hr of heat treatment. Figure 2 compares growth curves of exponentially growing cell cultures either untreated (control) or previously exposed to 42°C for 30 and 240 min, respectively. Shorter hyperthermic treatment (30 min) leads, after a mitotic lag period of 4 hr to a synchronisation effect on cell growth, clearly demonstrated by the stepwise increase of cell number; 240 min at 42°C results in an extension of the mitotic lag to about 24 hr; 48 hr later, however, the growth rate is normalized. Figure 3 shows the results of a cytofluorometric analysis on cells during and after heating.
Fig. 2.
Growth curves of exponentially growing V 79 cells • • ©
after hyperthermic treatment. controlcells cells exposedfor 30 min to 42°C cells exposedfor 240 rain to 42°C
The percentages of cells in the individual phases (GI, S, Gz + M) are plotted against the time of treatment. During heat exposure we find a depletion of Gl-cells indicating a block in G2 + M. (see Fig. 4, second cytofluorogram) The accumulation of cells in G2 + M is only moderate because cells are arrested already in S-phase as evident from the strongly increasing level of S- phase cells. Since these cell samples also show a pronounced loss of clonogenity (see Fig. 1) we may conclude that cells die during S-phase. This conclusion presupposes, of course, that the staining is sufficiently gently not to prevent the dead cells from being
Cytokinetic Studies of Hyperthermia on Chinese Hamster Lung Cells
829
10 8 6 4
~5os,
I
40--
[
lC
]
G2 + M (
a.
20 z
ioI
2 3 4
Hours o t 4 2 e C
I0
20
30 40 50 60 70 Hours ofter hyperthermia
1
80
I
90
I00
Fig. 3. Variation of the DNA-distribution with time during and after heat exposure (4 hours at 42°C) • Cells in S-phase © cells in Gx-phase A cells in G2 + M-phase
recorded by cytofluorometry [10]. Cell cultures returned to 37°C show 10 hr after heating a pronounced increase of cells in the G2 + M phase and reach normal DNA-distribution values about 48 hr later, a time at which dead cells degrade and are no longer recorded within the normal distribution (cf. LiickeHuhle and Derringer, manuscript in preparation). Though treatment of 4 h r at 42°C gives satisfactory results for cell kinetical examinations during heat exposure, the survival rate of 3 % is too low for studying the proliferation kinetics following heat treatment. We therefore used a lower exposure time of 1 hr (at 42°C) also which led to not more than 3 5 % cell killing. A selection of cytofluorograms (analog print-outs from the multichannel analyzer) illustrating the changes on proliferation kinetics with the time after heating is presented in Fig. 4. The DNA-distributions exhibit a partial synchronisation of cell growth due to a block in S and G z + M. Figure 5 shows the results in more d e t a i l : T h e frequencies of the individual phases of the cell cycle are plotted against the time after hyperthermia. Four hours after the return to 37°C the block in S is released and cells enter the G 2 + M phase causing a steady increase in that fraction of cells for the next 10 hr reaching maximal values of 61%. This accumulation of cells is accompanied by a decrease in G1 and S populations an indication for the block in G z + M . The release of that block at 10 hr after heating generates a wave of synchronous cells which repopulates the Gx-compartment to a first maximum at 14 hr and a second peak at 30 hr after hyperthermia.
K
z
i
Channel n u m b e r ( p r o p o r t i o n a l to D N A c o n f e n t )
Fig. 4. DNA-histograms of cells after different periods of time following hyperthermic treatment (1 hour at 42°C).
The time interval between the two Gl-peaks gives us the generation time for the second division after hyperthermic treatment, amounting to 16 hr instead of 11 hr for the control cells. The wave of synchronization is not equally clear cut in the S and G2 + M compartments possibly due to dead cells contaminating results. Beyond 36 hr DNA-distributions approximate that of the control. DISCUSSION The results presented in this paper demonstrate that heat treatment causes a block in S and G 2 + M; but, while the block in G 2 + M is transient (10hr after hyperthermia cells proceed into G1) most of the cells blocked in S die in that phase. This conclusion is supported by the finding that the accumulation of cells in S-phase during 4 h r at 42°C (cf. Fig. 3) corresponds to a drastic loss in clonogenity as demonstrated in Fig. 1. After a mild heat treatment (1 hr at 42°C: 70% survivors) the release of the G2 + M block leads to a partial synchronization of cell growth (Fig. 5). Evidently, exact knowledge of its amount for a tumor cell population would greatly facilitate
830
Helmut Schlag and Christine Liicke-Huhle
7oh~ ' [
l
t
l
I
I
I
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I
. I--
30
70.~1~ IM
l
I Fd
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g
IH
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•
o ~o
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1
I
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70
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. s0
/ %
z;" (.9
2 IO
I
6 Ih42"C
I 12
I
18
I
f
24
30
36
Hours after hyperfhermia
Fig. 5. Percentages of V79 cells in G1, S and G 2 + M phases as a function of time after hyperthermic treatment (1 hr at 42°C). • cells in S-phase © cells in Gl-phase LX cells in G 2 + M-phase
the design of optimum schedules for combined treatment. This synchronizing effect of hyperthermia is certainly one of the synergistic factors which influence a following X-ray therapy. After a more rigorous treatment (4 hr at 42°C) cell killing in S-phase is the major effect (survival 0.03). The unchanged kinetics of Gl-cells during heating and after the return to 37°C confirms the relative thermal resistance of that cell fraction which also seems to be responsible for the higher survival rate of heat exposed plateau phase cultures. Figure 1 clearly illustrates that the effect ofhyperthermic treatment exhibits a strong dependency on the state of growth in which the cell cultures were exposed to heating: The survival of exponentially growing cells is much lower than that for plateau phase cells. This agrees well with the findings of Palzer and Heidelberger [2] and Westra and Dewey [3], that S-phase cells are the most sensitive cell fraction to heat shock.
Thus we may conclude that in our experiments mainly S-phase cells are killed and that the apparent differences in sensitivity between plateau phase cells and exponentially growing cells is due to the difference in the frequency of S-cells. Similar dose-effect curves have been reported by Kase and Hahn [6] for Wi 38 cells. In contrast to the just mentioned results, however, are the findings of Schulman and Hall [5] and Hahn [4] who found V 79 cells and HA1 cells, respectively, exposed to 43°C to be more sensitive in the plateau phase of growth than in an exponentially growing culture. This discrepancy could result from differences in the oxygen supply of the plateau phase cultures. Therefore, special care was taken in our experiments to provide a sufficient supply of oxygen and nutrients during hyperthermia (see material and methods), since the greater thermal sensitivity of hypoxic cells has been described [4, 5]. The results of our cytofluorometrical analysis of V 79 monolayer ceils are in good agreement not only with the findings of Kal et al. [11] who describes a similar kinetical response to hyperthermia for murine sarcoma cells ( E M T 6) but also with own experiments on V 79 spheroids (Lticke-Huhle and Dertinger, manuscript in preparation). The exponentially growing cells of the outer shell of such spheroids show the same changes in proliferation kinetics after hyperthermic treatment as found for the monolayer cultures in spite of an important difference: A much higher thermo-resistance caused by the three-dimensional contact. In conclusion, the cell kinetics following heating suggest, that hyperthermia may be a useful adjunct to radiotherapy since the cells most resistant to conventional X-ray therapy (S-phase cells) are sensitive to heat and the synchronization effect might probably be used to hit the cycling tumour ceils in their most radiosensitive phase. For the same reason combined treatment by heat and conventional X- or "l-rays might eventually compete successfully with therapy by high LET-radiations such as neutrons or ~--mesons.
authors wish to thank Prof. K. G. Zimmer for critically reading the manuscript and Mrs. Katharina Roth for skillful technical assistance. Acknowledgements--The
Cytokinetic Studies of Hyperthermia on Chinese Hamster Lung Cells
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11.
F. DmTZEL, Tumor und Temperatur. Urban & Schwarzenberg, MiinchenBerlin-Wien 1975. R . J . PALZERand CH. HEIDELBEROER,Influence of drugs and synchrony on the hyperthermic killing of HeLa ceils. Cancer Res. 3:t, 422 (1973). A. WESTRA and W. C. DEWEY, Variation in sensitivity to heat shock during the cell-cycle of Chinese hamster cells in vitro. Int. J. Rad. Biol. 19, 467 (1971). G . M . HAHN, Metabolic aspects of the role of hypertherrnia in mammalian cell inactivation and. their possible relevance to cancer treatment. Cancer Res. 34, 3117 (1974). N. SCHULMANand E. J. HALL, Hyperthermia: its effect on proliferative and plateau phase cell cultures. Radiology 113, 209 (1974). K. KASE and G. M. HAHN, Differential heat response of normal and transformed human cells in tissue culture. Nature (Lond.) 225, 228 (1975). H. DERTINGERand CH. LUCKE-HUHLE,A comparative study of post-irradiation growth kinetics of spheroids and monolayers. Int. J. Rad. Biol. 28, 255 (1975). J.-B. LE PECO and C. PAOLETTI, A fluorescent complex between ethidium bromide and nucleic acids. J. tool. Biol. 27, 87 (1967). H. BAISCH,W. GOHDE and W. A. LINDEN, Analysis of PCP-data to determine the fraction of cells in the various phases of cell cycle. Rad. and environm. Biophys. 12, 31 (1975). CH. L0CKE-HuI-ILE, Proliferation-dependent cytotoxicity of diethylenetriaminepentaacetate (DTPA) in vitro. Health Physics in press (1976). H . B . KAL, M. HATrIELD and G. M. HAHN, Cell cycle progression of routine sarcoma ceils after X-irradiation or heat shock. Radiology 117, 215 (1975).
831
Cytokinetic Studies on the Effect of Hyperthermia on Chinese Hamster Lung Cells H E L M U T SCHLAG and CHRISTINE LI)CKE-HUHLE Kernforschungszentrum Karlsruhe, Institut fiir Strahlenbiologie, Postfach 3640, 7500 Karlsruhe, Federal Republic of Germany Abstract--Hyperthermic killing of V 79 cells is dependent on the state of growth: while survival of plateau phase cells decreases to 0.45, only 0.03 of the exposed exponentially growing cells survive 4 hr at 42°C. Flow-microfluorometric analysis during and after heating yields the fraction of cells in the individual phases of cell cycle and allows conclusions about changes in the proliferation kinetics of the exposed cell population. Mild heat treatment (1 hr at 42°C) leads to a synchronisation effect caused by a block in S and G 2 + M, while after a stronger heat exposure (4 hr at 42°C) cell death in S-phase is predominant.
INTRODUCTION
It is hoped that the results might contribute to selecting optimum sequential application of heat-X-ray or heat-chemotherapy.
PUBLICATIONS concerning the use of elevated temperatures in therapy date back until the middle of the last century (review: 1). More recently, there is renewed interest in hyperthermia for treatment of cancer, especially in combination with radiation therapy. Such a combined treatment might possibly offer an alternative to high L E T radiations, since the respective dose-effect curves show quantitative and qualitative similarities. It is not yet clear whether the increased efficiency for cell killing by the heat-X-ray therapy is of additive or synergistic kind, but cytokinetic studies on cells after hyperthermic treatment should reveal some of the underlying mechanism. Though iI is known that the response of cells to hyperthermia depends on their stage in the cell cycle [2, 3] cell survival curves of several cell lines tested either in the logarithmic or plateau phase of growth show contradictory results [4-6]. The present study was undertaken to examine the influence of the state of growth on thermal sensitivity using V79 cells and to investigate the effect of heat on cell kinetics during and after exposure to a temperature (42°C) within acceptable limits for potential clinical treatment.
MATERIAL AND METHODS Cell culture
Chinese hamster lung cells (line V 7 9 , obtained from Dr. R. B. Painter, San Francisco) were grown as monolayer cultures on plastic tissues culture dishes in Eagle's basal medium supplemented with 15% foetal calf serum (Gibco Biocult, BCL 005a) and neomycin sulfate (0.1 g/l). Exponentially growing cultures were obtained by plating ceils at a density of 1 x 10 4 cells/cm z and letting them proliferate to about 4 x 10 4 cells/cm 2. For the studies on plateau phase cultures cells were incubated at 37°C without exchanging medium until they attained inhibition of growth at approximately 3 x 10 5 cells/cm 2 with at least 65 % of the cells being in a Gl-like phase of the cell cycle [7]. Experimental procedures
Cell monolayers were exposed to an elevated temperature of 42°C by placing the culture flask into a waterbath temperature of which was kept constant within _ 0.1 °C. Immediately before heat exposure fresh medium was applied to all cultures to avoid differences in the thermal
Accepted 14 April 1976. 827
828
Helmut Schlag and Christine Liicke-Huhle
sensitivity caused by depletion of nutritional factors [4]. After different periods of heat treatment (up to 4 h r ) cells were either immediately trypsinized using 0.05% trypsin or the culture flasks were returned to 37°C for various periods and trypsinized afterwards. Both clonogenity and the number of progeny cells were determined from the obtained single cell suspensions as described in full detail elsewhere [7]. To determine the effect of heat on cell population kinetics during and after exposure to 42°C the changes in the DNA-distribution of the cell population were analysed by flowmicrofluorometry with the "Cytofluorograf" (Model 4801; Bio/Physics Instr.) coupled to a multichannel analyzer. For that purpose single cell suspensions were washed with tris buffer, fixed in ethanol (70 %) for 30 min and treated with RNase (0.1 °/o in tris buffer) for 1 hr at 37°C. After washing once more with tris buffer cells were stained with ethidium bromide (0.01 mg/ml). Under these conditions, the amount of dye is proportional to the DNA content of the cells [8]. The distribution of fluorescence intensity of the nuclear DNA (usually of 2-5 x 104 cells) was displayed as conventional histograms (cell number versus fluorescence intensity). The percentage of cells in the individual phases of the cell cycle (G1, S, G 2 + M ) was calculated from the area under the DNA histograms assuming a rectangular distribution of the S-cells between the G 1 and G2-peak [9]. The reproducibility of independent experiments was better than
LO
cells 0.5
---o
°~o
0.2
> F-
Exponentially growing cells
0.I
-
g, 0.05
0.02
I
r t
l
T
r 2
3
4
Hours o t 4 2 ° C
Fig. 1. Survival of exponentially growing and unfed plateau phase V 79 cell, exposedfor various times to 42°C.
~
10 2
&
IO I
B
/o/
c
z
/;,""
#7
:g
+3%.
24
48
72
96
Hours after hyperthermia
RESULTS
Figure 1 demonstrates a marked difference for survival of exponentially growing cells compared to plateau phase cells after the same exposure to 42°C. While survival of plateau phase cells decreases to 45 %, only 3 °/o of the exposed exponentially growing cells survive 4 hr of heat treatment. Figure 2 compares growth curves of exponentially growing cell cultures either untreated (control) or previously exposed to 42°C for 30 and 240 min, respectively. Shorter hyperthermic treatment (30 min) leads, after a mitotic lag period of 4 hr to a synchronisation effect on cell growth, clearly demonstrated by the stepwise increase of cell number; 240 min at 42°C results in an extension of the mitotic lag to about 24 hr; 48 hr later, however, the growth rate is normalized. Figure 3 shows the results of a cytofluorometric analysis on cells during and after heating.
Fig. 2.
Growth curves of exponentially growing V 79 cells • • ©
after hyperthermic treatment. controlcells cells exposedfor 30 min to 42°C cells exposedfor 240 rain to 42°C
The percentages of cells in the individual phases (GI, S, Gz + M) are plotted against the time of treatment. During heat exposure we find a depletion of Gl-cells indicating a block in G2 + M. (see Fig. 4, second cytofluorogram) The accumulation of cells in G2 + M is only moderate because cells are arrested already in S-phase as evident from the strongly increasing level of S- phase cells. Since these cell samples also show a pronounced loss of clonogenity (see Fig. 1) we may conclude that cells die during S-phase. This conclusion presupposes, of course, that the staining is sufficiently gently not to prevent the dead cells from being
Cytokinetic Studies of Hyperthermia on Chinese Hamster Lung Cells
829
10 8 6 4
~5os,
I
40--
[
lC
]
G2 + M (
a.
20 z
ioI
2 3 4
Hours o t 4 2 e C
I0
20
30 40 50 60 70 Hours ofter hyperthermia
1
80
I
90
I00
Fig. 3. Variation of the DNA-distribution with time during and after heat exposure (4 hours at 42°C) • Cells in S-phase © cells in Gx-phase A cells in G2 + M-phase
recorded by cytofluorometry [10]. Cell cultures returned to 37°C show 10 hr after heating a pronounced increase of cells in the G2 + M phase and reach normal DNA-distribution values about 48 hr later, a time at which dead cells degrade and are no longer recorded within the normal distribution (cf. LiickeHuhle and Derringer, manuscript in preparation). Though treatment of 4 h r at 42°C gives satisfactory results for cell kinetical examinations during heat exposure, the survival rate of 3 % is too low for studying the proliferation kinetics following heat treatment. We therefore used a lower exposure time of 1 hr (at 42°C) also which led to not more than 3 5 % cell killing. A selection of cytofluorograms (analog print-outs from the multichannel analyzer) illustrating the changes on proliferation kinetics with the time after heating is presented in Fig. 4. The DNA-distributions exhibit a partial synchronisation of cell growth due to a block in S and G z + M. Figure 5 shows the results in more d e t a i l : T h e frequencies of the individual phases of the cell cycle are plotted against the time after hyperthermia. Four hours after the return to 37°C the block in S is released and cells enter the G 2 + M phase causing a steady increase in that fraction of cells for the next 10 hr reaching maximal values of 61%. This accumulation of cells is accompanied by a decrease in G1 and S populations an indication for the block in G z + M . The release of that block at 10 hr after heating generates a wave of synchronous cells which repopulates the Gx-compartment to a first maximum at 14 hr and a second peak at 30 hr after hyperthermia.
K
z
i
Channel n u m b e r ( p r o p o r t i o n a l to D N A c o n f e n t )
Fig. 4. DNA-histograms of cells after different periods of time following hyperthermic treatment (1 hour at 42°C).
The time interval between the two Gl-peaks gives us the generation time for the second division after hyperthermic treatment, amounting to 16 hr instead of 11 hr for the control cells. The wave of synchronization is not equally clear cut in the S and G2 + M compartments possibly due to dead cells contaminating results. Beyond 36 hr DNA-distributions approximate that of the control. DISCUSSION The results presented in this paper demonstrate that heat treatment causes a block in S and G 2 + M; but, while the block in G 2 + M is transient (10hr after hyperthermia cells proceed into G1) most of the cells blocked in S die in that phase. This conclusion is supported by the finding that the accumulation of cells in S-phase during 4 h r at 42°C (cf. Fig. 3) corresponds to a drastic loss in clonogenity as demonstrated in Fig. 1. After a mild heat treatment (1 hr at 42°C: 70% survivors) the release of the G2 + M block leads to a partial synchronization of cell growth (Fig. 5). Evidently, exact knowledge of its amount for a tumor cell population would greatly facilitate
830
Helmut Schlag and Christine Liicke-Huhle
7oh~ ' [
l
t
l
I
I
I
[
I
I
. I--
30
70.~1~ IM
l
I Fd
\
g
IH
o.Wo
•
o ~o
."
\..,,
I
I
1
I
[
i
1
l
1
I--
[0
1
70
i
AoA,,a
. s0
/ %
z;" (.9
2 IO
I
6 Ih42"C
I 12
I
18
I
f
24
30
36
Hours after hyperfhermia
Fig. 5. Percentages of V79 cells in G1, S and G 2 + M phases as a function of time after hyperthermic treatment (1 hr at 42°C). • cells in S-phase © cells in Gl-phase LX cells in G 2 + M-phase
the design of optimum schedules for combined treatment. This synchronizing effect of hyperthermia is certainly one of the synergistic factors which influence a following X-ray therapy. After a more rigorous treatment (4 hr at 42°C) cell killing in S-phase is the major effect (survival 0.03). The unchanged kinetics of Gl-cells during heating and after the return to 37°C confirms the relative thermal resistance of that cell fraction which also seems to be responsible for the higher survival rate of heat exposed plateau phase cultures. Figure 1 clearly illustrates that the effect ofhyperthermic treatment exhibits a strong dependency on the state of growth in which the cell cultures were exposed to heating: The survival of exponentially growing cells is much lower than that for plateau phase cells. This agrees well with the findings of Palzer and Heidelberger [2] and Westra and Dewey [3], that S-phase cells are the most sensitive cell fraction to heat shock.
Thus we may conclude that in our experiments mainly S-phase cells are killed and that the apparent differences in sensitivity between plateau phase cells and exponentially growing cells is due to the difference in the frequency of S-cells. Similar dose-effect curves have been reported by Kase and Hahn [6] for Wi 38 cells. In contrast to the just mentioned results, however, are the findings of Schulman and Hall [5] and Hahn [4] who found V 79 cells and HA1 cells, respectively, exposed to 43°C to be more sensitive in the plateau phase of growth than in an exponentially growing culture. This discrepancy could result from differences in the oxygen supply of the plateau phase cultures. Therefore, special care was taken in our experiments to provide a sufficient supply of oxygen and nutrients during hyperthermia (see material and methods), since the greater thermal sensitivity of hypoxic cells has been described [4, 5]. The results of our cytofluorometrical analysis of V 79 monolayer ceils are in good agreement not only with the findings of Kal et al. [11] who describes a similar kinetical response to hyperthermia for murine sarcoma cells ( E M T 6) but also with own experiments on V 79 spheroids (Lticke-Huhle and Dertinger, manuscript in preparation). The exponentially growing cells of the outer shell of such spheroids show the same changes in proliferation kinetics after hyperthermic treatment as found for the monolayer cultures in spite of an important difference: A much higher thermo-resistance caused by the three-dimensional contact. In conclusion, the cell kinetics following heating suggest, that hyperthermia may be a useful adjunct to radiotherapy since the cells most resistant to conventional X-ray therapy (S-phase cells) are sensitive to heat and the synchronization effect might probably be used to hit the cycling tumour ceils in their most radiosensitive phase. For the same reason combined treatment by heat and conventional X- or "l-rays might eventually compete successfully with therapy by high LET-radiations such as neutrons or ~--mesons.
authors wish to thank Prof. K. G. Zimmer for critically reading the manuscript and Mrs. Katharina Roth for skillful technical assistance. Acknowledgements--The
Cytokinetic Studies of Hyperthermia on Chinese Hamster Lung Cells
REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 11.
F. DmTZEL, Tumor und Temperatur. Urban & Schwarzenberg, MiinchenBerlin-Wien 1975. R . J . PALZERand CH. HEIDELBEROER,Influence of drugs and synchrony on the hyperthermic killing of HeLa ceils. Cancer Res. 3:t, 422 (1973). A. WESTRA and W. C. DEWEY, Variation in sensitivity to heat shock during the cell-cycle of Chinese hamster cells in vitro. Int. J. Rad. Biol. 19, 467 (1971). G . M . HAHN, Metabolic aspects of the role of hypertherrnia in mammalian cell inactivation and. their possible relevance to cancer treatment. Cancer Res. 34, 3117 (1974). N. SCHULMANand E. J. HALL, Hyperthermia: its effect on proliferative and plateau phase cell cultures. Radiology 113, 209 (1974). K. KASE and G. M. HAHN, Differential heat response of normal and transformed human cells in tissue culture. Nature (Lond.) 225, 228 (1975). H. DERTINGERand CH. LUCKE-HUHLE,A comparative study of post-irradiation growth kinetics of spheroids and monolayers. Int. J. Rad. Biol. 28, 255 (1975). J.-B. LE PECO and C. PAOLETTI, A fluorescent complex between ethidium bromide and nucleic acids. J. tool. Biol. 27, 87 (1967). H. BAISCH,W. GOHDE and W. A. LINDEN, Analysis of PCP-data to determine the fraction of cells in the various phases of cell cycle. Rad. and environm. Biophys. 12, 31 (1975). CH. L0CKE-HuI-ILE, Proliferation-dependent cytotoxicity of diethylenetriaminepentaacetate (DTPA) in vitro. Health Physics in press (1976). H . B . KAL, M. HATrIELD and G. M. HAHN, Cell cycle progression of routine sarcoma ceils after X-irradiation or heat shock. Radiology 117, 215 (1975).
831