Inr J. Rodmron Oncology EmI. Phys Vol. Printed in the U.S.A. All rights reserved.
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036%3016/92 $5.00 + .oO Copyright 0 1992 Pergamon Press Ltd.
0 Hyperthermia Original Contribution RECOVERY OF SUBLETHAL RADIATION DAMAGE AND ITS INHIBITION HYPERTHERMIA IN NORMAL AND TRANSFORMED MOUSE CELLS G. P. RAAPHORST,
BY
PH.D.
Medical Physics Department, Ottawa Regional Cancer Centre, 190 Melrose Ave., Ottawa, Ontario KlY 4K7 Canada Radiation resistance may be related in part to the capacity of cells to repair radiation damage which can usually be characterized by the survival curve shoulder and split dose recovery. C3H-lOT1/2 normal and transformed cells and V79 cells were evaluated for their ability to recover from sublethal radiation damage and for hyperthermia to affect this recovery. The transformed cell line of the lOT1/2 cell system displayed a much huger capacity to recover from radiation damage than did the parental strain. This was also correlated with an enlarged shoulder on the radiation survival curve. When hyperthermia was given, recovery of sublethal radiation damage could be inhibited in both cell lines. This inhibition was dependent on the sequence of hyperthermia treatment and correlated with the removal of the shoulder of the radiation survival curve. In Chinese hamster V79 cells, recovery was much smaller than in the mouse C3H-lOT1/2 cell system. In the hamster cells, recovery could also be inhibited by a hyperthermia treatment. In all three cell lines the degree of inhibition was dependent on thermal dose and indicated that very small hyperthermia treatments would not inhibit recovery of sublethal damage but may in fact cause some increase possibly due to increasing the amount of damage available for repair. Thus if hyperthermia doses are sufficiently large, it may be used to overcome sublethal damage repair and may result in therapeutic ‘gain in tumors which have a large capacity for such repair. Hyperthermia,
Radiosensitization,
Inhibition of repair of sublethal radiation damage.
INTRODUCTION
Several investigations have shown that hyperthermia can inhibit repair of potentially lethal and sublethal radiation damage (1, 11, 13, 19, 22). Such inhibition was found to depend on the severity of the hyperthermia treatment and on the sequencing of hyperthermia and radiation. In addition, several reports show that hyperthermia can induce a greater degree of radiation sensitization under low dose-rate conditions (1, 8). These data indicate that perhaps the modification of repair of sublethal damage (SLD) by hyperthermia can play an extensive role in the outcome of cell survival. In this study the effect of hyperthermia on repair of sublethal radiation damage in the mouse C3H- lOT1/2 cell system and the Chinese hamster V79 cell system was evaluated. In the mouse system we have used both a normal and a transformed strain. The transformed strain displayed melanoma-type characteristics, including a larger capacity for repair of sublethal radiation damage. In addition, the Chinese hamster cells showed smaller repair capacity than each of the C3H-lOT1/2 cell lines. Thus, effects of hyperthermia on SLD repair in cells with different repair capacities could be evaluated to determine whether SLD repair could be inhibited completely in both cells exhibiting and high and low capacity.
Many
studies using in vitro cultured mammalian cells have shown that hyperthermia can cause radiation sensitization. This sensitization is generally characterized by a decrease in both the survival curve slope and the survival curve shoulder (1,2, 14, 15,23). As the thermal treatment is increased, generally the degree of radiosensitization is also increased and in many experiments it was shown that the survival curve shoulder was almost completely eliminated. Radiobiological studies have shown that the survival curve shoulder is usually related to the ability of a cell to accumulate sublethal radiation damage and that the shoulder can be indicative of repair of sublethal radiation damage (1, 6, 13, 23). Such repair can influence the outcome of cellular survival after irradiation. It has been shown by several investigators that cellular radiation response at 2 Gy is most indicative of the expected clinical response (6). The 2 Gy dose level falls on the shoulder of the survival curve for most cell lines studied in vitro. Thus, methods of modifying the shoulder of the survival curve and possibly the capacity for repair of sublethal radiation damage may lead to improved clinical response in radiation resistant tumors.
Accepted for publication 3 September 199 1. 1035
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METHODS
AND MATERIALS
The C3H-lOT1/2 mouse embryo cells used in these experiments were developed by Reznikoff et al. (24) and were obtained from the American Type Culture Collection at passage 7. Experiments with this cell system were done with culture passages below 15. Stock cultures were grown as monolayers in 75 cm2 flasks containing 30 ml of culture growth medium. The growth medium consisted of a 1: 1 mixture of Dulbecco’s modified medium and F12 medium obtained from the Grand Island Biological Company. Growth medium was supplemented with 10% heat inactivated fetal calf serum and contained no antibiotics. The pH of the medium was maintained at 7.4 by using 20 mM Hepes and 10 mM sodium bicarbonate buffers. Under these conditions the cells were incubated at 37°C in an atmosphere of 2% CO2 and 98% air. The transformed clone labeled R25 was selected from the parental C3H- 10T l/2 cell population as follows: cells were irradiated to 4 Gy and then incubated for 6-8 weeks, during which time the cell culture reached plateau phase and maintained itself in plateau phase during the last 6 weeks of incubation. During this time transformed foci developed. Such transformed clones were removed from the monolayer using a sterile spatula. These clones were trypsinized into single cells and then inoculated into an agarose solution as follows: a thin layer of 1% agarose and medium solution was placed into a 75 cm2 flask and allowed to solidify. Then cells were suspended in a 0.34% agarose and medium solution and layered on top of the 1% solution. These cultures were incubated from 6- 16 weeks and fresh medium was added to the culture every 2 weeks. In one culture, black spheroids formed after 68 weeks of incubation. One such spheroid was selected and cultured continuously. This cell line was further identified as being able to produce melanin and having melanoma characteristics (16, 28). The Chinese hamster V79 cells were cultured in the same medium and atmospheric conditions described for the C3H- 10T l/2 cell system. For the three cell lines, survival experiments were conducted as follows: cells were grown exponentially in monolayer cultures and were trypsinized and suspended as single cells. These were inoculated into 25 cm2 flasks at numbers estimated to give 1OO- 150 surviving cells after treatment. Such flasks were then incubated overnight and treatment was commenced the following day, usually 12 hr after plating. At this time the multiplicity of the cell cultures was determined and survival data were corrected for multiplicity as described before (26). All the data presented in the results section were corrected for multiplicity and, thus, fraction survival represents single cell survival. The multiplicity of the V79 cells ranged around 1.5, while the multiplicity for the two mouse cells ranged from 1.1 to 1.4. The plating efficiency for the V79 cells ranged from 60-95% while the plating efficiencies for the C3H1OT l/2 normal and R25 cells ranged from 20-40 and 30-
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50%, respectively. The survival experiments presented in the results section were repeated three times. The results of representative experiments are shown in each figure and each data point represents 4-6 replicate flasks with the standard error of the mean shown in each figure when greater than the datum point symbol. Irradiation was done using an x-ray machine operating at 250 kV and 15 mA with a 1 mm aluminum filter. The dose rate was about 3.5 Gy/min and all cell cultures were maintained at 37°C during irradiation. Heating was done using a temperature controlled water bath (+O.O2”C). After overnight incubation, the 25 cm2 flasks containing 5 ml of medium were sealed with wax and transferred to temperature controlled baths. Heating times shown on the figures indicate the total time the flasks were immersed in the temperature controlled water baths. After treatment flasks were incubated for 7- 10 days for the Chinese hamster cells and 8-14 days for the mouse cells, depending on the severity of treatment and the length of time required to form visible colonies. After incubation the flasks were rinsed, fixed, stained, and the colonies were scored. Only colonies with 50 or more cells were counted in this assay. RESULTS
The radiation responses of the C3H normal and R25 cell line are shown in Figure 1. The survival curve for
----SPLIT
I
2.0
DOSE
I
I
I
4.0 a0 DOSE6.’ (GY)
I
I
100
12.0
Fig. 1. The radiation survival curves of C3H-lOT1/2 normal and transformed cells exposed to single or split doses of radiation. For the split-dose radiation, the first dose was 4.5 Gy and the cells were incubated at 37°C for 8 hr, after which graded doses of radiation were given.
Sublethal radiation damage 0 G. P.RAAPHORST
R25 shows a much larger shoulder than the survival curve for the parental cell line, indicating a larger capacity to accumulate sublethal radiation damage. The n and Do values for the R25 and normal cell line are 8, 2, 1.3 Gy, and 1.6 1 Gy, respectively. The figure shows the survival of the normal and R25 cell line after single and split radiation doses. For split dose treatment, cells were irradiated to 4.5 Gy and then incubated for 8 hr at 37°C before additional radiation doses were given. The data showing the larger shoulder of the survival curve for R25 reflects a larger ability to repair sublethal radiation damage is supported by a greater level of recovery. For example, the dashed curves compared to the solid lines for each of the cell lines at the 8 and 10 Gy dose survival levels reflect recovery ratios of 1.6 and 1.9 for the normal and 7.2 and 9.7 for the R25 cell line, respectively. When a 2 hr heat treatment at 42.5”C was given after the first dose of irradiation, radiosensitization occurred in both R25 and the normal cell line (Fig. 2). This sensitization was characterized by removal of the radiation survival curve shoulder and elimination of the difference between the response of R25 and the normal cell line. At doses between 4 and 6 Gy the transformed cell line was
-I
Fig. 2. The radiation survival curves of cells given single or split doses of radiation. In these studies, cells also received a 2 hr hyperthermia treatment at 42S”C. Hyperthermia was started 10 min after the first dose of irradiation and survival after hyperthermia alone was 2 1 + 4%. For the split-dose radiation studies, a first dose of 3.0 Gy was given followed by hyperthetmia, which was followed by 8 hr of incubation, after which time further radiation doses were given. The curve for radiation plus heat was adjusted to account for the toxicity due to heating alone.
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slightly more sensitized by hyperthermia than the normal cell line. The Do for the survival curve after heat and radiation could not be calculated because of the non-uniform nature of the survival curve. For the split dose experiments, cells were irradiated then heated and then incubated for 8 hr after the hyperthermia treatment, before the second radiation dose was given. These data show the complete elimination of recovery of the survival curve shoulder (dashed curves). At the higher doses, such as 5 and 6 Gy, there is a slight increase in radiation resistance resulting from a split-dose treatment. Such a change in the survival curve was also observed with CHO cells (22). However, this resistance does not display the recovery of the survival curve shoulder usually observed for recovery of sublethal damage that is characterized by a shoulder. This resistance may be due, to the fact that hyperthermia can influence radiation response after long incubation periods and that some sensitization may be lost during this time. In addition, thermal tolerance can develop and has been shown to result in a reduced degree of radiation sensitization (9, 10, 17). Figures 3 and 4 show the effect of sequencing of radiation with hyperthermia treatment at 42.5”C. The data in each of the figures show that R25 had a greater ability to recover from sublethal radiation damage than the normal parental cell line. Recovery ratios at 8 hr were 3.3 and 5.4, respectively, for the normal and the R25 cell lines. When hyperthermia (2 hr at 42.5’C) was given before irradiation (Fig. 3) recovery of sublethal damage was extensively inhibited and the recovery ratio was reduced to 1.5-1.7 after 8 hr incubation for both cell lines. When the 2 hr hyperthermia treatment was given after the first radiation dose (Fig. 4), recovery of sublethal damage was completely inhibited in both the R25 and normal cell lines. When small heat treatments were given ( 15 min at 42.5”C), recovery appeared to be greater for both treatment sequences. Recovery ratios at 8 hr were 6.2 and 4.0 for heating R25 and the normal line before irradiation and 3.8 for heating the normal line after irradiation. Since survival of combined treatment was lower (reduced by factor of 2) this increase in recovery may be related to an increase in damage, which can be repaired while repair systems remain functional. For more severe heat treatments repair systems may be damaged thus inhibiting repair. These data indicate that sequencing of hyperthermia with radiation and the size of the thermal treatments play an important role in determining the degree of inhibition of recovery of sublethal damage. Such sequence and thermal dose dependence has also been shown when hyperthermia was evaluated in the inhibition of recovery of potentially lethal radiation damage (11, 19). In order to evaluate the effect of degree of thermal treatment on recovery of sublethal damage, the V79 cells were used since these were easier to culture and more manageable for such a large-scale experiment. Figure 5 shows survival curves of V79 cells after irradiation alone or after hyperthermia combined with ra-
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fact that under these conditions survival was lower than for irradiation alone so that survival was further off the survival curve shoulder (see Fig. 5) and perhaps there was more damage that could be repaired. The recovery ratios after 4 hr of incubation were 2.6,3.3 and 6.1 for the curves of no heat, 45.5”C for 4 min and 42.5”C for 30 min, respectively. When the heat treatments were increased to 42.5”C for 2 hr or 45.5”C for 15 min, hyperthermia treatment before irradiation completely eliminated the recovery of sublethal radiation damage. The recovery ratios at 4 hr were 2.6, 1.2 and 0.9 for no heat, for 45.5”C for 15 min and 42.5”C for 120 min, respectively. These data show that thermal dose can play a role in the degree of inhibition of recovery of sublethal radiation damage.
&’
3.5Gy
DISCUSSION
0.1
o
I
I
2
4
INCUBATION
1
The experimental data presented show that hyperthermia treatment can inhibit recovery of sublethal radiation damage. Such inhibition was dependent on the dose of the hyperthermia treatment and this is supported by earlier work showing these effects on recovery of sublethal dam-
I
8
TIME’ (Hours
Fig. 3. The recovery ratios of C3H-IOT1/2
1
normal and trans-
formed cells exposed to split dose radiation (closed triangles and circles). Each dose of radiation was 3.5 Gy and incubation between doses is indicated by time on the abscissa. Cells were either given radiation alone (closed triangles and circles) or given hyperthermia terminating 10 min before irradiation. Hyperthermia (indicated by large open triangle) was for 15 min at 42.5”C (dashed curves, open triangles and circles) or 2 hr at 42.5”C (solid curves, closed and open squares). Survival after hyperthermia alone was 80 + 3% and 20 + 3% for the 15 min and 2 hr treatment, respectively.
diation as indicated on the figure. The data show that as the hyperthermia treatment at 425°C was increased from 30 min to 2 hr, there was a large reduction in the survival curve shoulder and the Do as indicated in the figure legend. When hyperthermia was given before split dose irradiation the degree of inhibition of recovery increased with increasing thermal treatments. In addition for 425°C for 30 min there was also an increase in survival curve slope, indicating other effects beyond those on the recovery of SLD. These data show that hyperthermia treatment at 425°C for 30 min would have a much smaller effect on repair of sublethal radiation damage than hyperthermia treatment for 2 hr and that other effects (change in survival curve slope) may be involved. The data in Figure 6, in fact, show the effect of thermal dose on the inhibition of recovery of sublethal damage. When very small thermal treatments were given, such as 425°C for 30 min or an equivalent heat treatment of 45.5”C for 4 min, the recovery from sublethal radiation damage, as evaluated by the split-dose technique, increased and this agrees with the results observed in the C3H- lOT1/2 cells. This increase was probably due to the
X = 3.5 Gy t
0.11 IiJCUBATION
TIME
(Hokl
’
Fig. 4. Recovery ratios of C3H-IOT1/2 normal and transformed cells given split dose irradiation (open and closed circles). Each dose was 3.5 Gy and incubation between doses is indicated on the abscissa. Cells were given irradiation alone (open and closed circles) or given a first dose of radiation that was followed 10 min later by hyperthermia (represented by the large open triangle), followed immediately by incubation until the final radiation dose was given. Hyperthermia was 15 min at 425°C (dashed curve, semi-filled circle, normal only) or 2 hr at 42.5’C (squares). Survival after hyperthermia alone is given in legend of Figure 3.
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Sublethal radiation damage 0 G. P. RAAPHORST
t
L
1-i
I 2.0
I 4.0
I
6.0
DOSE
I
8.0
I
10.0
I
12.0
I
14.0
(Gy)
Fig. 5. The irradiation survival of V79 cells exposed to radiation alone or combined with various heat treatments at 425°C. Heat treatment was given such that it was completed 10 min before irradiation. The n and Do values are as follows: X rays (X) alone, 9 and 1.24 Gy; 425°C 30 min plus X, 6 and 0.89 Gy; 425°C 75 min plus X, 3 and 0.84 Gy; 42.5”C, 120 min, 2 and 0.59 Gy (solid curves). The solid and dashed curves show results for cells heated before receiving single and split dose irradiation, respectively. Incubation between doses was 8 hr. Survival after hyperthermia alone was 52 f 3%, 19 f 4%, and 5.1 -t 2% for the 30, 75, and 120 min heat treatments, respectively.
age, can be overcome by hyperthermia treatment. Several studies have shown that the clinical response of tumors to radiotherapy can be correlated to the radiation resistance of cells measured at the low dose level (2.0 Gy) and that this resistance can be related to the ability to recover from sublethal radiation damage (6, 29, 3 1). Our results with two squamous cell carcinoma lines also support this concept and show that the most sensitive line has a smaller capacity to recover from radiation damage than the resistant line and such resistance was also correlated to the clinical findings (20). The data presented in this study further support the idea that thermosensitization occurs, at least in part, through the inhibition of cellular recovery from radiation damage. Other work in the literature also supports this concept. It was shown that thermoradiosen$itization could be achieved with low LET radiation, but not with high LET radiation (7). In addition, when hyperthermia was combined with low dose-rate irradiation, it induced a much greater enhancement of radiation sensitivity than when hyperthermia was combined with acute irradiation (1, 8). It is well-known than the low dose-rate irradiation allows enhanced recovery from sublethal radiation damage and, when under these conditions, hyperthermia is given and there is a greater potential for the inhibition of
10.0
8.0
A&=-x XI 5061
age in Chinese hamster cells and as well, other work showing a dose dependent effect on the recovery of potentially lethal damage in mouse and hamster cell lines (1, 11, 13, 19,22). In addition, the degree of inhibition of sublethal damage recovery by hyperthermia was dependent on the sequence of treatment. Hyperthermia treatment after irradiation resulted in greater inhibition than hyperthermia given before radiation. Such an effect was also found for the recovery of potentially lethal radiation damage (11, 19). The results also show that the degree of recovery of sublethal radiation damage was different for the transformed and the normal cell line. The transformed cell line had a large shoulder on the radiation survival curve and also displayed a larger capacity for recovery of sublethal damage. When these two cell lines were given hyperthermia treatment, complete inhibition of recovery of sublethal radiation damage could be achieved in both cell lines, when hyperthermia was given after radiation. These results indicate that cellular radioresistance, which can manifest itself in enhanced capacity for recovery of radiation dam-
2. rw I
&-;I; 0
1.0 2.0 4.0 INCUBATION TIME (hours)
Fig. 6. Recovery ratios of V79 cells given split dose irradiation (each dose 3.0 Gy). Cells were given radiation alone or hyperthermia at 42.5”C for 30 min or 45.5”C for 4 min, which was completed 10 min before the first dose of radiation, upper panel. In the lower panel, cells were given hypertherniia for 45.5”C for 15 min or 42.5“C for 120 min, which was completed 10 min before the first dose of irradiation. Survival abler heating alone was51+-3%,4.7+ 1.8%,53+4%,and3.1+0.8%fortreatments of 42.5, 30 min and 120 min; and 45.5, 4 min and 15 min, respectively.
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such recovery. This is confirmed through the increased thermal enhancement of radiation sensitivity under these conditions. In addition, there is a considerable amount of evidence showing that hyperthermia can inhibit the recovery of DNA strand breaks induced by radiation. The degree of such recovery is dependent on temperature and time of heating. These data have been summarized in a review (15). In addition, several studies also show that hyperthermia can inhibit the activity of DNA polymerase B and that inhibition is correlated to the thermal dose given (12, 27). Our own results with glioma cells, to be published later, show that the degree of inhibition of polymerase B by hyperthermia can be correlated with the degree of inhibition of recovery of potentially lethal damage in this cell system (5). The recovery of sublethal damage in normal C3HlOT1/2, R25, and V79 cells showed an increase when very small thermal doses, such as 15 or 30 min at 42.5”C, or 4 min at 45.5”C were given. Such an increase may be due to the fact that the interaction of hyperthermia and radiation cause a greater number of damage sites to be available for repair and that such mild thermal treatments might not be adequate for the inhibition of the repair system. In fact, in an earlier study we showed that combined hyperthermia and radiation treatment resulted in a greater amount of damage available for post-irradiation fixation ( 18). There is evidence, in fact, that very low doses
Volume 22, Number 5, I992
of hyperthermia given at low temperatures immediately after irradiation can increase the rate of repair of DNA strand breaks (3). In addition, very small treatments of hyperthermia do not significantly inhibit the activity of polymerase B (4, 12,27). These results are consistent with our finding that there is an increase in recovery under conditions of very small thermal doses. Other explanations for the increase in recovery could be that small heat treatments induce thermal tolerance without damage to repair systems. Then when the second dose is given after 8 hr incubation thermal tolerance resulted in reduced radiosensitization. In addition, as the second radiation dose is given at later times after irradiation, there may be a loss of sensitization that appears like an increase in recovery. The mouse C3H-lOT1/2, R25, and hamster V79 cells show a change in survival curve slope when low dose hyperthermia was given in a splitdose regimen, indicating that the effect is not likely due to SLD recovery alone. In summary, our data show that hyperthermia can inhibit recovery of sublethal damage in cell lines displaying large capacity for sublethal damage repair. This indicates that hyperthermia may be especially useful in the sensitization of radiation resistant tumors that display their resistance through increased ability to repair radiation damage. This has been found in cells derived from such tumors as gliomas and melanomas (2 1, 25, 30, 3 1).
REFERENCES 1. Ben Hur, E.; Elkind, M. M.; Bronk, B. V. Thermally enhanced radioresponse of cultured Chinese hamster cells. Inhibition of repair of sublethal damage and enhancement of lethal damage. Radiat. Res. 58: 38-51; 1974. 2. Dewey, W. C.; Freeman, M. L.; Raaphorst, Cl. P.; Clark, E. P.; Wong, R. S. L.; Highfield, D. P.; Spiro, I. J.; Tomasovic, S. P.; Denman, D. L.; Goss, R. A. Cell biology of hyperthermia and radiation. In: Meyn, R., Withers, H. R., eds. Radiation biology in cancer research. New York: Raven Press; 1980: 589-62 1. 3. Dikomey, E. Effect of hyperthermia at 42 and 45°C on repair of radiation induced DNA strand breaks in CHO cells. Int. J. Radiat. Biol. 41: 603-614; 1982. 4. Dikomey, E.; Becker, W.; Wielckens, K. Reduction of DNApolymerase B activity of CHO cells by single and combined heat treatments. Int. J. Radiat. Biol. 52: 775-785; 1987. 5. Feeley, M. M.; Raaphorst, G. P.; Dewey, W. C.; Chu, G. L.; Danjoux, C. E.; Gerig, L. H. Thermal radiosensitization in human glioma cells. Radiat. Res. 38th Proc.; 1990. 6. Fertil, G.; Malaise, E. P. Intrinsic radiosensitivity of human cell lines is correlated with radioresponsiveness of human tumors. Analysis of 101 published survival curves. Int. J. Radiat. Oncol. 11: 1699-1707; 1985. 7. Gemer, E. W.; Leith, J. T. Interaction of hyperthermia with radiations of different linear energy transfer. Int. J. Radiat. Biol. 31: 283-288; 1977. 8. Harisiadis, L.; Sung, D.; Kessaris, N.; Hall, E. J. Hyperthermia and low dose rate irradiation. Radiol. 129: 195198; 1978. 9. Henle, K. J.; Tomasovic, S. P.; Dethlefsen, L. A. Fractionation of combined heat and radiation in synchronous CHO cells. Radiat. Res. 80: 369-377; 1979.
10. Holahan, E. V.; Highfield, D. P.; Dewey, W. C. Induction during Gl of heat radiosensitization in Chinese hamster ovary cells following single and fractionated heat doses. Natl. Cancer Inst. Monogr. 61: 123-125; 1982. Il. Li, G. C.; Evans, R. G.; Hahn, G. M. Modification and inhibition of repair of potentially lethal X-ray damage by hyperthermia. Radiat. Res. 67: 491-501; 1976. 12. Mivechi, N. F.; Dewey, W. C. DNA polymerase LYand 8 activities during the cell cycle and their role in heat radiosensitization in Chinese hamster ovary cells. Radiat. Res. 103: 337-350; 1985. 13. Murthy, A. K.; Harris, J. R.; Belli, J. A. Hyperthermia and radiation response of plateau phase cells. Radiat. Res. 70: 241-247, 1977. 14. Overgaard, J. (ed.) Proc. Fourth Int. Symp. Hypertherm. Oncol. London: Taylor and Francis; 1984: Vol. 1 and 2. 15. Raaphorst, G. P. Thermal radiosensitization in vitro. Hyperther. Oncol. 2: 17-5 1; 1989. 16. Raaphorst, G. P.; Azzam, E. I. Radiation heat and antimelanin drug response of a transformed mouse embryo cell line with varying melanin content. Br. J. Cancer 56: 622624; 1987. 17. Raaphorst, G. P.; Azzam, E. I. Thermal radiosensitization in Chinese hamster (V79) and mouse C3H-lOTl/2 cells. The thermotolerance effect. Br. J. Cancer 48: 45-54; 1983. 18. Raaphorst, G. P.; Azzam, E. I. The effect of hypo- and hypertonic NaCl solutions on cellular damage resulting from combined treatments of heat plus X-rays. Int. J. Radiat. Biol. 40: 633-643; 1981. 19. Raaphorst, G. P.; Azzam, E. I.; Feeley, M. M. Potentially lethal radiation damage repair and its inhibition by hyper-
Sublethal radiation damage 0 G. P.RAAPHORST thermia in normal hamster cells, mouse cells and transformed mouse cells. Radiat. Res. 113: 17 l-182; 1988. 20. Raaphorst, G. P.; Feeley, M. M.; Danjoux, C. E.; Martin, L.; Maroun, J.; DeSanctis, A. J. The effect of lonidamine on radiation and thermal responses of human and rodent cell lines. Int. J. Radiat. Oncol. 20: 509-5 15; 1991. 21. Raaphorst, G. P.; Feeley, M. M.; DaSilva, V. F.; Danjoux, C. E.; Gerig, L. H. Thermal radiosensitization and repair inhibition in three human ghoma cell lines. Radiat. Res. 36th Proc. 5; 1988. 22. Raaphorst, G. P.; Freeman, M. L.; Dewey, W. C. Radiosensitivity and recovery from radiation damage in cultured CHO cells exposed to hyperthermia at 42.5 and 45.5”C. Radiat. Res. 79: 390-402; 1979. 23. Raaphorst, G. P.; Romano, S. L.; Mitchell, J. B.; Bedford, .I. S.; Dewey, W. C. Intrinsic differences in heat and/or X-ray sensitivity of seven mammalian cell lines cultured and treated under identical conditions. Cancer Res. 39: 396401; 1979. 24. Reznikoff, C. A.; Brankow, D. W.; Heidelberger, C. Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division. Cancer Res. 33: 323 l-3238; 1973.
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25. Rofstad, E. K.; Brustad, T. Radiation response in vitro of cells from five human malignant melanoma xenografts. Int. J. Radiat. Biol. 40: 677-680; 198 1. 26. Sinclair, W. K.; Morton, R. A. X-ray and ultraviolet sensitivity of synchronized Chinese hamster cells at various stages of the cell cycle. Biophys. J. 5: l-25; 1965. 27. Spiro, I. J.; Denman, D. L.; Dewey, W. C. Effect of hyperthermia on CHO, DNA polymerases (Yand p. Radiat. Res. 89: 134-149; 1982. 28. Szekely, J. G.; Raaphorst, G. P.; Lobreau, A. U.; Azzam, E. I. and Vadasz, J. A. Growth of a radiation transformed clone of C3H- 10T l/2 cells into melanin-producing colonies. J. Scan. Elect. Micros. IV: 1631-1640; 1985. 29. Weichselbaum, R. R.; Dahlberg, W.; Beckett, M.; Karrison, T.; Miller, D.; Clark, J.; Ervin, J. Radiation resistant and repair proficient human tumour cells may be associated with radiotherapy failure in head and neck cancer patients. Proc. Natl. Acad. Sci. 83: 2684-2688; 1986. 30. Weichselbaum, R. R.; Malcolm, A. W.; Little, J. B. Fraction size and the repair of potentially lethal radiation damage in a human melanoma cell line. Radiol. 142: 225-227; 1982. 31. Weichselbaum, R. R.; Schmit, A.; Little, J. B. Cellular repair factors influencing radiocurability of human malignant tumors. Br. J. Cancer 45: 10-16; 1972.
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0 Hyperthermia Original Contribution RECOVERY OF SUBLETHAL RADIATION DAMAGE AND ITS INHIBITION HYPERTHERMIA IN NORMAL AND TRANSFORMED MOUSE CELLS G. P. RAAPHORST,
BY
PH.D.
Medical Physics Department, Ottawa Regional Cancer Centre, 190 Melrose Ave., Ottawa, Ontario KlY 4K7 Canada Radiation resistance may be related in part to the capacity of cells to repair radiation damage which can usually be characterized by the survival curve shoulder and split dose recovery. C3H-lOT1/2 normal and transformed cells and V79 cells were evaluated for their ability to recover from sublethal radiation damage and for hyperthermia to affect this recovery. The transformed cell line of the lOT1/2 cell system displayed a much huger capacity to recover from radiation damage than did the parental strain. This was also correlated with an enlarged shoulder on the radiation survival curve. When hyperthermia was given, recovery of sublethal radiation damage could be inhibited in both cell lines. This inhibition was dependent on the sequence of hyperthermia treatment and correlated with the removal of the shoulder of the radiation survival curve. In Chinese hamster V79 cells, recovery was much smaller than in the mouse C3H-lOT1/2 cell system. In the hamster cells, recovery could also be inhibited by a hyperthermia treatment. In all three cell lines the degree of inhibition was dependent on thermal dose and indicated that very small hyperthermia treatments would not inhibit recovery of sublethal damage but may in fact cause some increase possibly due to increasing the amount of damage available for repair. Thus if hyperthermia doses are sufficiently large, it may be used to overcome sublethal damage repair and may result in therapeutic ‘gain in tumors which have a large capacity for such repair. Hyperthermia,
Radiosensitization,
Inhibition of repair of sublethal radiation damage.
INTRODUCTION
Several investigations have shown that hyperthermia can inhibit repair of potentially lethal and sublethal radiation damage (1, 11, 13, 19, 22). Such inhibition was found to depend on the severity of the hyperthermia treatment and on the sequencing of hyperthermia and radiation. In addition, several reports show that hyperthermia can induce a greater degree of radiation sensitization under low dose-rate conditions (1, 8). These data indicate that perhaps the modification of repair of sublethal damage (SLD) by hyperthermia can play an extensive role in the outcome of cell survival. In this study the effect of hyperthermia on repair of sublethal radiation damage in the mouse C3H- lOT1/2 cell system and the Chinese hamster V79 cell system was evaluated. In the mouse system we have used both a normal and a transformed strain. The transformed strain displayed melanoma-type characteristics, including a larger capacity for repair of sublethal radiation damage. In addition, the Chinese hamster cells showed smaller repair capacity than each of the C3H-lOT1/2 cell lines. Thus, effects of hyperthermia on SLD repair in cells with different repair capacities could be evaluated to determine whether SLD repair could be inhibited completely in both cells exhibiting and high and low capacity.
Many
studies using in vitro cultured mammalian cells have shown that hyperthermia can cause radiation sensitization. This sensitization is generally characterized by a decrease in both the survival curve slope and the survival curve shoulder (1,2, 14, 15,23). As the thermal treatment is increased, generally the degree of radiosensitization is also increased and in many experiments it was shown that the survival curve shoulder was almost completely eliminated. Radiobiological studies have shown that the survival curve shoulder is usually related to the ability of a cell to accumulate sublethal radiation damage and that the shoulder can be indicative of repair of sublethal radiation damage (1, 6, 13, 23). Such repair can influence the outcome of cellular survival after irradiation. It has been shown by several investigators that cellular radiation response at 2 Gy is most indicative of the expected clinical response (6). The 2 Gy dose level falls on the shoulder of the survival curve for most cell lines studied in vitro. Thus, methods of modifying the shoulder of the survival curve and possibly the capacity for repair of sublethal radiation damage may lead to improved clinical response in radiation resistant tumors.
Accepted for publication 3 September 199 1. 1035
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METHODS
AND MATERIALS
The C3H-lOT1/2 mouse embryo cells used in these experiments were developed by Reznikoff et al. (24) and were obtained from the American Type Culture Collection at passage 7. Experiments with this cell system were done with culture passages below 15. Stock cultures were grown as monolayers in 75 cm2 flasks containing 30 ml of culture growth medium. The growth medium consisted of a 1: 1 mixture of Dulbecco’s modified medium and F12 medium obtained from the Grand Island Biological Company. Growth medium was supplemented with 10% heat inactivated fetal calf serum and contained no antibiotics. The pH of the medium was maintained at 7.4 by using 20 mM Hepes and 10 mM sodium bicarbonate buffers. Under these conditions the cells were incubated at 37°C in an atmosphere of 2% CO2 and 98% air. The transformed clone labeled R25 was selected from the parental C3H- 10T l/2 cell population as follows: cells were irradiated to 4 Gy and then incubated for 6-8 weeks, during which time the cell culture reached plateau phase and maintained itself in plateau phase during the last 6 weeks of incubation. During this time transformed foci developed. Such transformed clones were removed from the monolayer using a sterile spatula. These clones were trypsinized into single cells and then inoculated into an agarose solution as follows: a thin layer of 1% agarose and medium solution was placed into a 75 cm2 flask and allowed to solidify. Then cells were suspended in a 0.34% agarose and medium solution and layered on top of the 1% solution. These cultures were incubated from 6- 16 weeks and fresh medium was added to the culture every 2 weeks. In one culture, black spheroids formed after 68 weeks of incubation. One such spheroid was selected and cultured continuously. This cell line was further identified as being able to produce melanin and having melanoma characteristics (16, 28). The Chinese hamster V79 cells were cultured in the same medium and atmospheric conditions described for the C3H- 10T l/2 cell system. For the three cell lines, survival experiments were conducted as follows: cells were grown exponentially in monolayer cultures and were trypsinized and suspended as single cells. These were inoculated into 25 cm2 flasks at numbers estimated to give 1OO- 150 surviving cells after treatment. Such flasks were then incubated overnight and treatment was commenced the following day, usually 12 hr after plating. At this time the multiplicity of the cell cultures was determined and survival data were corrected for multiplicity as described before (26). All the data presented in the results section were corrected for multiplicity and, thus, fraction survival represents single cell survival. The multiplicity of the V79 cells ranged around 1.5, while the multiplicity for the two mouse cells ranged from 1.1 to 1.4. The plating efficiency for the V79 cells ranged from 60-95% while the plating efficiencies for the C3H1OT l/2 normal and R25 cells ranged from 20-40 and 30-
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50%, respectively. The survival experiments presented in the results section were repeated three times. The results of representative experiments are shown in each figure and each data point represents 4-6 replicate flasks with the standard error of the mean shown in each figure when greater than the datum point symbol. Irradiation was done using an x-ray machine operating at 250 kV and 15 mA with a 1 mm aluminum filter. The dose rate was about 3.5 Gy/min and all cell cultures were maintained at 37°C during irradiation. Heating was done using a temperature controlled water bath (+O.O2”C). After overnight incubation, the 25 cm2 flasks containing 5 ml of medium were sealed with wax and transferred to temperature controlled baths. Heating times shown on the figures indicate the total time the flasks were immersed in the temperature controlled water baths. After treatment flasks were incubated for 7- 10 days for the Chinese hamster cells and 8-14 days for the mouse cells, depending on the severity of treatment and the length of time required to form visible colonies. After incubation the flasks were rinsed, fixed, stained, and the colonies were scored. Only colonies with 50 or more cells were counted in this assay. RESULTS
The radiation responses of the C3H normal and R25 cell line are shown in Figure 1. The survival curve for
----SPLIT
I
2.0
DOSE
I
I
I
4.0 a0 DOSE6.’ (GY)
I
I
100
12.0
Fig. 1. The radiation survival curves of C3H-lOT1/2 normal and transformed cells exposed to single or split doses of radiation. For the split-dose radiation, the first dose was 4.5 Gy and the cells were incubated at 37°C for 8 hr, after which graded doses of radiation were given.
Sublethal radiation damage 0 G. P.RAAPHORST
R25 shows a much larger shoulder than the survival curve for the parental cell line, indicating a larger capacity to accumulate sublethal radiation damage. The n and Do values for the R25 and normal cell line are 8, 2, 1.3 Gy, and 1.6 1 Gy, respectively. The figure shows the survival of the normal and R25 cell line after single and split radiation doses. For split dose treatment, cells were irradiated to 4.5 Gy and then incubated for 8 hr at 37°C before additional radiation doses were given. The data showing the larger shoulder of the survival curve for R25 reflects a larger ability to repair sublethal radiation damage is supported by a greater level of recovery. For example, the dashed curves compared to the solid lines for each of the cell lines at the 8 and 10 Gy dose survival levels reflect recovery ratios of 1.6 and 1.9 for the normal and 7.2 and 9.7 for the R25 cell line, respectively. When a 2 hr heat treatment at 42.5”C was given after the first dose of irradiation, radiosensitization occurred in both R25 and the normal cell line (Fig. 2). This sensitization was characterized by removal of the radiation survival curve shoulder and elimination of the difference between the response of R25 and the normal cell line. At doses between 4 and 6 Gy the transformed cell line was
-I
Fig. 2. The radiation survival curves of cells given single or split doses of radiation. In these studies, cells also received a 2 hr hyperthermia treatment at 42S”C. Hyperthermia was started 10 min after the first dose of irradiation and survival after hyperthermia alone was 2 1 + 4%. For the split-dose radiation studies, a first dose of 3.0 Gy was given followed by hyperthetmia, which was followed by 8 hr of incubation, after which time further radiation doses were given. The curve for radiation plus heat was adjusted to account for the toxicity due to heating alone.
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slightly more sensitized by hyperthermia than the normal cell line. The Do for the survival curve after heat and radiation could not be calculated because of the non-uniform nature of the survival curve. For the split dose experiments, cells were irradiated then heated and then incubated for 8 hr after the hyperthermia treatment, before the second radiation dose was given. These data show the complete elimination of recovery of the survival curve shoulder (dashed curves). At the higher doses, such as 5 and 6 Gy, there is a slight increase in radiation resistance resulting from a split-dose treatment. Such a change in the survival curve was also observed with CHO cells (22). However, this resistance does not display the recovery of the survival curve shoulder usually observed for recovery of sublethal damage that is characterized by a shoulder. This resistance may be due, to the fact that hyperthermia can influence radiation response after long incubation periods and that some sensitization may be lost during this time. In addition, thermal tolerance can develop and has been shown to result in a reduced degree of radiation sensitization (9, 10, 17). Figures 3 and 4 show the effect of sequencing of radiation with hyperthermia treatment at 42.5”C. The data in each of the figures show that R25 had a greater ability to recover from sublethal radiation damage than the normal parental cell line. Recovery ratios at 8 hr were 3.3 and 5.4, respectively, for the normal and the R25 cell lines. When hyperthermia (2 hr at 42.5’C) was given before irradiation (Fig. 3) recovery of sublethal damage was extensively inhibited and the recovery ratio was reduced to 1.5-1.7 after 8 hr incubation for both cell lines. When the 2 hr hyperthermia treatment was given after the first radiation dose (Fig. 4), recovery of sublethal damage was completely inhibited in both the R25 and normal cell lines. When small heat treatments were given ( 15 min at 42.5”C), recovery appeared to be greater for both treatment sequences. Recovery ratios at 8 hr were 6.2 and 4.0 for heating R25 and the normal line before irradiation and 3.8 for heating the normal line after irradiation. Since survival of combined treatment was lower (reduced by factor of 2) this increase in recovery may be related to an increase in damage, which can be repaired while repair systems remain functional. For more severe heat treatments repair systems may be damaged thus inhibiting repair. These data indicate that sequencing of hyperthermia with radiation and the size of the thermal treatments play an important role in determining the degree of inhibition of recovery of sublethal damage. Such sequence and thermal dose dependence has also been shown when hyperthermia was evaluated in the inhibition of recovery of potentially lethal radiation damage (11, 19). In order to evaluate the effect of degree of thermal treatment on recovery of sublethal damage, the V79 cells were used since these were easier to culture and more manageable for such a large-scale experiment. Figure 5 shows survival curves of V79 cells after irradiation alone or after hyperthermia combined with ra-
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fact that under these conditions survival was lower than for irradiation alone so that survival was further off the survival curve shoulder (see Fig. 5) and perhaps there was more damage that could be repaired. The recovery ratios after 4 hr of incubation were 2.6,3.3 and 6.1 for the curves of no heat, 45.5”C for 4 min and 42.5”C for 30 min, respectively. When the heat treatments were increased to 42.5”C for 2 hr or 45.5”C for 15 min, hyperthermia treatment before irradiation completely eliminated the recovery of sublethal radiation damage. The recovery ratios at 4 hr were 2.6, 1.2 and 0.9 for no heat, for 45.5”C for 15 min and 42.5”C for 120 min, respectively. These data show that thermal dose can play a role in the degree of inhibition of recovery of sublethal radiation damage.
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3.5Gy
DISCUSSION
0.1
o
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2
4
INCUBATION
1
The experimental data presented show that hyperthermia treatment can inhibit recovery of sublethal radiation damage. Such inhibition was dependent on the dose of the hyperthermia treatment and this is supported by earlier work showing these effects on recovery of sublethal dam-
I
8
TIME’ (Hours
Fig. 3. The recovery ratios of C3H-IOT1/2
1
normal and trans-
formed cells exposed to split dose radiation (closed triangles and circles). Each dose of radiation was 3.5 Gy and incubation between doses is indicated by time on the abscissa. Cells were either given radiation alone (closed triangles and circles) or given hyperthermia terminating 10 min before irradiation. Hyperthermia (indicated by large open triangle) was for 15 min at 42.5”C (dashed curves, open triangles and circles) or 2 hr at 42.5”C (solid curves, closed and open squares). Survival after hyperthermia alone was 80 + 3% and 20 + 3% for the 15 min and 2 hr treatment, respectively.
diation as indicated on the figure. The data show that as the hyperthermia treatment at 425°C was increased from 30 min to 2 hr, there was a large reduction in the survival curve shoulder and the Do as indicated in the figure legend. When hyperthermia was given before split dose irradiation the degree of inhibition of recovery increased with increasing thermal treatments. In addition for 425°C for 30 min there was also an increase in survival curve slope, indicating other effects beyond those on the recovery of SLD. These data show that hyperthermia treatment at 425°C for 30 min would have a much smaller effect on repair of sublethal radiation damage than hyperthermia treatment for 2 hr and that other effects (change in survival curve slope) may be involved. The data in Figure 6, in fact, show the effect of thermal dose on the inhibition of recovery of sublethal damage. When very small thermal treatments were given, such as 425°C for 30 min or an equivalent heat treatment of 45.5”C for 4 min, the recovery from sublethal radiation damage, as evaluated by the split-dose technique, increased and this agrees with the results observed in the C3H- lOT1/2 cells. This increase was probably due to the
X = 3.5 Gy t
0.11 IiJCUBATION
TIME
(Hokl
’
Fig. 4. Recovery ratios of C3H-IOT1/2 normal and transformed cells given split dose irradiation (open and closed circles). Each dose was 3.5 Gy and incubation between doses is indicated on the abscissa. Cells were given irradiation alone (open and closed circles) or given a first dose of radiation that was followed 10 min later by hyperthermia (represented by the large open triangle), followed immediately by incubation until the final radiation dose was given. Hyperthermia was 15 min at 425°C (dashed curve, semi-filled circle, normal only) or 2 hr at 42.5’C (squares). Survival after hyperthermia alone is given in legend of Figure 3.
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Sublethal radiation damage 0 G. P. RAAPHORST
t
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1-i
I 2.0
I 4.0
I
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(Gy)
Fig. 5. The irradiation survival of V79 cells exposed to radiation alone or combined with various heat treatments at 425°C. Heat treatment was given such that it was completed 10 min before irradiation. The n and Do values are as follows: X rays (X) alone, 9 and 1.24 Gy; 425°C 30 min plus X, 6 and 0.89 Gy; 425°C 75 min plus X, 3 and 0.84 Gy; 42.5”C, 120 min, 2 and 0.59 Gy (solid curves). The solid and dashed curves show results for cells heated before receiving single and split dose irradiation, respectively. Incubation between doses was 8 hr. Survival after hyperthermia alone was 52 f 3%, 19 f 4%, and 5.1 -t 2% for the 30, 75, and 120 min heat treatments, respectively.
age, can be overcome by hyperthermia treatment. Several studies have shown that the clinical response of tumors to radiotherapy can be correlated to the radiation resistance of cells measured at the low dose level (2.0 Gy) and that this resistance can be related to the ability to recover from sublethal radiation damage (6, 29, 3 1). Our results with two squamous cell carcinoma lines also support this concept and show that the most sensitive line has a smaller capacity to recover from radiation damage than the resistant line and such resistance was also correlated to the clinical findings (20). The data presented in this study further support the idea that thermosensitization occurs, at least in part, through the inhibition of cellular recovery from radiation damage. Other work in the literature also supports this concept. It was shown that thermoradiosen$itization could be achieved with low LET radiation, but not with high LET radiation (7). In addition, when hyperthermia was combined with low dose-rate irradiation, it induced a much greater enhancement of radiation sensitivity than when hyperthermia was combined with acute irradiation (1, 8). It is well-known than the low dose-rate irradiation allows enhanced recovery from sublethal radiation damage and, when under these conditions, hyperthermia is given and there is a greater potential for the inhibition of
10.0
8.0
A&=-x XI 5061
age in Chinese hamster cells and as well, other work showing a dose dependent effect on the recovery of potentially lethal damage in mouse and hamster cell lines (1, 11, 13, 19,22). In addition, the degree of inhibition of sublethal damage recovery by hyperthermia was dependent on the sequence of treatment. Hyperthermia treatment after irradiation resulted in greater inhibition than hyperthermia given before radiation. Such an effect was also found for the recovery of potentially lethal radiation damage (11, 19). The results also show that the degree of recovery of sublethal radiation damage was different for the transformed and the normal cell line. The transformed cell line had a large shoulder on the radiation survival curve and also displayed a larger capacity for recovery of sublethal damage. When these two cell lines were given hyperthermia treatment, complete inhibition of recovery of sublethal radiation damage could be achieved in both cell lines, when hyperthermia was given after radiation. These results indicate that cellular radioresistance, which can manifest itself in enhanced capacity for recovery of radiation dam-
2. rw I
&-;I; 0
1.0 2.0 4.0 INCUBATION TIME (hours)
Fig. 6. Recovery ratios of V79 cells given split dose irradiation (each dose 3.0 Gy). Cells were given radiation alone or hyperthermia at 42.5”C for 30 min or 45.5”C for 4 min, which was completed 10 min before the first dose of radiation, upper panel. In the lower panel, cells were given hypertherniia for 45.5”C for 15 min or 42.5“C for 120 min, which was completed 10 min before the first dose of irradiation. Survival abler heating alone was51+-3%,4.7+ 1.8%,53+4%,and3.1+0.8%fortreatments of 42.5, 30 min and 120 min; and 45.5, 4 min and 15 min, respectively.
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such recovery. This is confirmed through the increased thermal enhancement of radiation sensitivity under these conditions. In addition, there is a considerable amount of evidence showing that hyperthermia can inhibit the recovery of DNA strand breaks induced by radiation. The degree of such recovery is dependent on temperature and time of heating. These data have been summarized in a review (15). In addition, several studies also show that hyperthermia can inhibit the activity of DNA polymerase B and that inhibition is correlated to the thermal dose given (12, 27). Our own results with glioma cells, to be published later, show that the degree of inhibition of polymerase B by hyperthermia can be correlated with the degree of inhibition of recovery of potentially lethal damage in this cell system (5). The recovery of sublethal damage in normal C3HlOT1/2, R25, and V79 cells showed an increase when very small thermal doses, such as 15 or 30 min at 42.5”C, or 4 min at 45.5”C were given. Such an increase may be due to the fact that the interaction of hyperthermia and radiation cause a greater number of damage sites to be available for repair and that such mild thermal treatments might not be adequate for the inhibition of the repair system. In fact, in an earlier study we showed that combined hyperthermia and radiation treatment resulted in a greater amount of damage available for post-irradiation fixation ( 18). There is evidence, in fact, that very low doses
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of hyperthermia given at low temperatures immediately after irradiation can increase the rate of repair of DNA strand breaks (3). In addition, very small treatments of hyperthermia do not significantly inhibit the activity of polymerase B (4, 12,27). These results are consistent with our finding that there is an increase in recovery under conditions of very small thermal doses. Other explanations for the increase in recovery could be that small heat treatments induce thermal tolerance without damage to repair systems. Then when the second dose is given after 8 hr incubation thermal tolerance resulted in reduced radiosensitization. In addition, as the second radiation dose is given at later times after irradiation, there may be a loss of sensitization that appears like an increase in recovery. The mouse C3H-lOT1/2, R25, and hamster V79 cells show a change in survival curve slope when low dose hyperthermia was given in a splitdose regimen, indicating that the effect is not likely due to SLD recovery alone. In summary, our data show that hyperthermia can inhibit recovery of sublethal damage in cell lines displaying large capacity for sublethal damage repair. This indicates that hyperthermia may be especially useful in the sensitization of radiation resistant tumors that display their resistance through increased ability to repair radiation damage. This has been found in cells derived from such tumors as gliomas and melanomas (2 1, 25, 30, 3 1).
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10. Holahan, E. V.; Highfield, D. P.; Dewey, W. C. Induction during Gl of heat radiosensitization in Chinese hamster ovary cells following single and fractionated heat doses. Natl. Cancer Inst. Monogr. 61: 123-125; 1982. Il. Li, G. C.; Evans, R. G.; Hahn, G. M. Modification and inhibition of repair of potentially lethal X-ray damage by hyperthermia. Radiat. Res. 67: 491-501; 1976. 12. Mivechi, N. F.; Dewey, W. C. DNA polymerase LYand 8 activities during the cell cycle and their role in heat radiosensitization in Chinese hamster ovary cells. Radiat. Res. 103: 337-350; 1985. 13. Murthy, A. K.; Harris, J. R.; Belli, J. A. Hyperthermia and radiation response of plateau phase cells. Radiat. Res. 70: 241-247, 1977. 14. Overgaard, J. (ed.) Proc. Fourth Int. Symp. Hypertherm. Oncol. London: Taylor and Francis; 1984: Vol. 1 and 2. 15. Raaphorst, G. P. Thermal radiosensitization in vitro. Hyperther. Oncol. 2: 17-5 1; 1989. 16. Raaphorst, G. P.; Azzam, E. I. Radiation heat and antimelanin drug response of a transformed mouse embryo cell line with varying melanin content. Br. J. Cancer 56: 622624; 1987. 17. Raaphorst, G. P.; Azzam, E. I. Thermal radiosensitization in Chinese hamster (V79) and mouse C3H-lOTl/2 cells. The thermotolerance effect. Br. J. Cancer 48: 45-54; 1983. 18. Raaphorst, G. P.; Azzam, E. I. The effect of hypo- and hypertonic NaCl solutions on cellular damage resulting from combined treatments of heat plus X-rays. Int. J. Radiat. Biol. 40: 633-643; 1981. 19. Raaphorst, G. P.; Azzam, E. I.; Feeley, M. M. Potentially lethal radiation damage repair and its inhibition by hyper-
Sublethal radiation damage 0 G. P.RAAPHORST thermia in normal hamster cells, mouse cells and transformed mouse cells. Radiat. Res. 113: 17 l-182; 1988. 20. Raaphorst, G. P.; Feeley, M. M.; Danjoux, C. E.; Martin, L.; Maroun, J.; DeSanctis, A. J. The effect of lonidamine on radiation and thermal responses of human and rodent cell lines. Int. J. Radiat. Oncol. 20: 509-5 15; 1991. 21. Raaphorst, G. P.; Feeley, M. M.; DaSilva, V. F.; Danjoux, C. E.; Gerig, L. H. Thermal radiosensitization and repair inhibition in three human ghoma cell lines. Radiat. Res. 36th Proc. 5; 1988. 22. Raaphorst, G. P.; Freeman, M. L.; Dewey, W. C. Radiosensitivity and recovery from radiation damage in cultured CHO cells exposed to hyperthermia at 42.5 and 45.5”C. Radiat. Res. 79: 390-402; 1979. 23. Raaphorst, G. P.; Romano, S. L.; Mitchell, J. B.; Bedford, .I. S.; Dewey, W. C. Intrinsic differences in heat and/or X-ray sensitivity of seven mammalian cell lines cultured and treated under identical conditions. Cancer Res. 39: 396401; 1979. 24. Reznikoff, C. A.; Brankow, D. W.; Heidelberger, C. Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division. Cancer Res. 33: 323 l-3238; 1973.
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25. Rofstad, E. K.; Brustad, T. Radiation response in vitro of cells from five human malignant melanoma xenografts. Int. J. Radiat. Biol. 40: 677-680; 198 1. 26. Sinclair, W. K.; Morton, R. A. X-ray and ultraviolet sensitivity of synchronized Chinese hamster cells at various stages of the cell cycle. Biophys. J. 5: l-25; 1965. 27. Spiro, I. J.; Denman, D. L.; Dewey, W. C. Effect of hyperthermia on CHO, DNA polymerases (Yand p. Radiat. Res. 89: 134-149; 1982. 28. Szekely, J. G.; Raaphorst, G. P.; Lobreau, A. U.; Azzam, E. I. and Vadasz, J. A. Growth of a radiation transformed clone of C3H- 10T l/2 cells into melanin-producing colonies. J. Scan. Elect. Micros. IV: 1631-1640; 1985. 29. Weichselbaum, R. R.; Dahlberg, W.; Beckett, M.; Karrison, T.; Miller, D.; Clark, J.; Ervin, J. Radiation resistant and repair proficient human tumour cells may be associated with radiotherapy failure in head and neck cancer patients. Proc. Natl. Acad. Sci. 83: 2684-2688; 1986. 30. Weichselbaum, R. R.; Malcolm, A. W.; Little, J. B. Fraction size and the repair of potentially lethal radiation damage in a human melanoma cell line. Radiol. 142: 225-227; 1982. 31. Weichselbaum, R. R.; Schmit, A.; Little, J. B. Cellular repair factors influencing radiocurability of human malignant tumors. Br. J. Cancer 45: 10-16; 1972.
