International Journal of Radiation Biology
ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20
Heat Resistance and Thermotolerance in a Radiation-resistant Cell Line T.M. Koval & D.L. Suppes To cite this article: T.M. Koval & D.L. Suppes (1992) Heat Resistance and Thermotolerance in a Radiation-resistant Cell Line, International Journal of Radiation Biology, 61:3, 425-431, DOI: 10.1080/09553009214551121 To link to this article: http://dx.doi.org/10.1080/09553009214551121
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Date: 12 May 2016, At: 12:03
INT . J . RADIAT . BIOL .,
1992,
VOL .
61,
NO.
3, 425-431
Heat resistance and thermotolerance in a radiation-resistant cell line T. M . KOVAL*t and D. L. SUPPESt
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(Received 10 May 1991 ; revision received 2 August 1991 ; accepted 7 August 1991)
Abstract . Exponentially growing TN-368 lepidopteran insect cells have a normal growth temperature of 28°C . These cells were heated in water baths at various temperatures between 33 and 44 ° C under conditions of constant or fractionated heating. Determinations of cell survival using colony formation as well as measurements of DNA and protein synthesis were performed to assess relative heat resistance and development of thermotolerance . The results demonstrate a marked heat resistance over previously reported findings from the same laboratory for dipteran Drosophila cells in culture . The degree of heat resistance is remarkable, especially when compared to the heat resistance of mammalian cells, i .e . TN-368 cell survival at 41 . 5 and 44 °C was somewhat similar to mammalian cell survival, even though these temperatures are 13 . 5 and 16° C above the normal growth temperature for TN-368 cells and 4 . 5 and 7 °C above the growth temperature of mammalian cells . Furthermore, the lepidopteran cells maintain the ability to develop a notable amount of thermotolerance in addition to this heat resistance . Thermotolerance development alone is capable of enhancing survival by an additional 10000-fold . Thermotolerance could also be detected at the level of protein synthesis as a more rapid recovery following heat treatment . In contrast, DNA synthesis inhibition was prolonged even further in cells receiving a prior heat treatment to induce thermotolerance . In summary, it appears that, in addition to their pronounced radiation resistance, the TN-368 cells are also quite resistant to heat. It remains to be seen whether a single mechanism could be responsible for resistance to these agents which act very differently.
plus 365 nm ultraviolet light, among other agents (Koval, unpublished data) . Evidence suggests that exceptional repair or recovery mechanisms in these cells may be responsible for their resistance to many of these agents (Koval et al. 1978, Koval 1986a,b, 1988) . The only previous utilization of the colony formation technique as a quantitative heat survival assay in cultured insect cells was performed with Drosophila melanogaster (Tomasovic and Koval 1985) . The line of Drosophila cells used in that study was much more sensitive to both ionizing and UV radiation than the TN-368 cells, although still more resistant than most mammalian cells (Koval 1983a,c, 1987) . Resistance of the Drosophila cells to heating was not particularly notable and thermotolerance was only slightly evident at 33 ° C and not apparent at higher temperatures (Tomasovic and Koval 1985) . Thus, the aim of this study was to investigate survival and thermotolerance in the more radioresistant TN-368 cells for comparison with the Drosophila system.
2. Materials and methods 2.1 . Cell lines
1. Introduction Cultured TN-368 lepidopteran insect cells are of the order of 100 times more resistant to cell killing by ionizing radiation than cultured mammalian cells (Koval, 1983a,b) . These cells also exhibit a pronounced resistance to 254 nm ultraviolet light, to which they are in the range of 10-25 times more resistant than mammalian cells (Koval 1986a) . In addition, the TN-368 cells are resistant to killing by methyl methanesulphonate, N-methyl-R-nitro-Nnitrosoguanidine, 1,3-propane sultone, mitomycin C, 4-nitroquinoline-l-oxide, and 8-methoxypsoralen *Author for correspondence. tDivision of Radiation Oncology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA .
The TN-368 cells are a continuous line of primarily fibroblast-like cells which were derived from minced adult ovaries of the cabbage looper, Trichoplusia ni (Hink 1970) . The cells, which were originally obtained from Dr W. F. Hink, Ohio State University, have been in culture for more than 15 years and have been subcultured more than 1500 times at a split ratio of approximately 1 :10. The cells generally grow in a loosely attached monolayer and require only moderate agitation to detach them from the bottom surface of growth vessels . Cultures are normally maintained in polystyrene tissue culture flasks (Corning) and kept in a humidified incubator at 28°C. The cell line has a doubling time of approximately 19h and is normally maintained by subculturing three times a week at a 1 :10 ratio in
0020-7616/92 $3 .00 ® 1992 Taylor & Francis Ltd
T. M. Koval and D . L . Suppes
42 6
TNM-FH medium (Koval 1989) supplemented with 8% fetal bovine serum (Hyclone) .
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2.2 .
Cell
survival experiments
Exponentially growing TN-368 cells in 25 cm 2 polystyrene tissue culture flasks (Corning) were heated for various periods of time by immersing flasks in precision-controlled (±0 .05 °C) water baths (Tecam, TU-15 ; Techne Inc ., Princeton, NJ, USA) equipped with supplementary water pumps. Standardized thermometers were used for temperature measurement . In fractionated experiments an initial 15, 30, or 60 min heat exposure was followed by a 2 h incubation at 28 ° C before additional graded heat exposures. Following fractionated or continuous heating, the cells were removed from flasks by gentle pipetting and were counted using a haemacytometer and a Model ZM Coulter Counter . Cells were then plated into 60 mm diameter polystyrene tissue culture dishes (Lux) containing conditioned TNM-P medium (Koval 1989) . A 0 . 15 ml volume of cell suspension was added to 3 .8 ml of conditioned medium . The dishes were incubated at 28°C in a humidified incubator and left undisturbed for 18-19 days to allow for colony formation . The dishes were then removed from the incubator, stained with neutral red, and colonies counted to determine cell surviving fractions (Koval 1989) . Absolute plating efficiencies were generally in the range of 40-50% .
2 .3. DNA synthesis Exponentially growing cultures were labelled with .(1961-2072MBq/mmol, [methyl- 14C]thymidine Moravek) at 2 .59 kBq/ml for aproximately 42 h . The medium was then removed, the cells were rinsed with 2 ml of fresh medium, and 5 ml of fresh medium was added. The cells were heated at 37, 41 .5 or 44°C for various periods of time . Cells were pulse-labelled for 20 min immediately prior to sampling with [methyl-3H]thymidine (1665-1813MBq/mmol, Amersham) at 296 kBq/ml by adding 0 . 1 ml of labelled medium to each tube containing 1 .4 ml of heated cell suspension . At each sampling, the cells were pelleted at 15 000 rpm for 10 s in a Beckman microfuge, tritium-labelled medium was removed, and the cell pellet was stored at -80°C until further processing . The cells were resuspended in 0 .75 ml SSC (0-15m sodium chloride, 0-015m sodium citrate) containing 100 pg/ml thymidine and 400 µg/ml BSA . The cells were precipitated with icecold 10% trichloroacetic acid (TCA) containing 1
sodium pyrophosphate . The pellet was dissolved in 0. 1 N NaOH containing excess thymidine (100 yg/ml) and incubated at 37°C for 15 min before reprecipitating with 10% TCA for 15 min . The suspension was then passed through a Whatman GF/C filter wetted with 10% TCA and washed sequentially with 10 ml volumes of ice-cold 10% TCA and twice with 95% ethanol . The filters were allowed to dry overnight and counted in Ultima Gold scintillation cocktail (Packard) using a Beckman LS 5000TA liquid scintillation system to provide disintegrations per minute (dpm) . The dpm were then analysed as 3H/ 14C ratios which were measures of specific activity of DNA, i .e. the rate of DNA synthesis. Percentage of control was calculated as the 3H/ 14C ratio for heated cells divided by the 3H/14C ratio for untreated cells .
2 .4.
Protein synthesis
Exponentially growing cells were heated at 41 .5°C for various times . The cells were pulselabelled for 30 or 60 min immediately prior to sampling with [3H]leucine (4440 GBq/mmol, RPI) by adding 0 . 1 ml of labelled medium (296 kBq/ml) to 1 . 4 ml of medium in each tube of suspended cells . At each sampling, the cells were pelleted, tritiumlabelled medium was removed, and the cell pellet was stored at -80°C until further processing as described above for DNA synthesis . Cell counts were obtained at each sampling and were performed in triplicate or quadruplicate using a haemacytometer . Percentage of control was calculated as counts per minute (cpm) per cell for heated cells divided by the cpm per cell for untreated cells . 3.
Results
Survival curves for exponentially growing TN-368 cells subjected to continuous heating at 33, 37, 41 . 5 and 44°C are shown in Figure 1 . Marked differences in thermal sensitivity at these temperatures, as assayed by colony formation, were observed . Survival was reduced to approximately 50% of control following 48 h of heating at 33 ° C, 29 h at 37°C, 3 h at 41 .5°C, and 15 min at 44°C, and to approximately 10% after 45 h at 37°C, 7 h at 41 .5°C, and 35 min at 44°C. Slopes of the curves varied greatly . Survival remained at or near control levels for 20 h of continuous heating at 33°C, and very gradually began to decrease between 20 and 50 h of heating . Survival after continuous 37°C heating was similar to that at 33°C until approximately 16 h, when it began to
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Heat resistance in radioresistant TN-368 cells
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Time (hr) Figure 1 . Exponentially growing TN-368 cells were heated for various times at 33, 37, 41 . 5, and 44 ° C and immediately plated for colony formation . Points represent the mean and relative SE from a minimum of five plate counts . These data were compiled from 18 separate experiments .
decrease at a slightly faster rate. At 41 . 5 ° C a shoulder of approximately 10h preceded exponential killing . A small shoulder was also observed for 44°C heating, though by 40 min at this temperature survival decreased sharply and cell killing was exponential . Approximate D0 , D4 , and n values for the survival curves are provided in Table 1 .
Table 1 .
The effect of heating on DNA synthesis is illustrated in Figure 2 . Depression of DNA synthesis was rapid at each temperature examined, with rates dropping to approximately 50% of control levels after 30 min at 37 ° C and to less than 10% of control levels after 30 min at 41 . 5 and 44°C . DNA synthesis recovered quickly at 37°C, rebounding to 100% by 1 h after heat exposures up to 1 h . After 6 h, DNA synthesis had recovered to control levels following an exposure of 2 h at 37 ° C. Recovery was slower at 41 . 5 and 44 ° C, with recovery rates only at 50% of control levels by 6h, after exposures of 20 and 10 min, respectively . DNA synthesis did recover to levels approaching 100% by 24 h after heating for exposures up to 60 min at 41 . 5°C and 30 min at 44°C, but no recovery was observed after being exposed to 44°C for 60 min (data not shown) . Total protein synthesis decreased marginally after heat exposures of 30, 60, and 120 min at 37°C (Figure 2), but rebounded promptly to control levels or above by 3 h . The depression in protein synthesis was more significant at 41 . 5 ° C, decreasing to about 40%, 30%, and 12% of control levels by 30 min after heating times of 30, 60, or 120 min, respectively . Recovery was gradual, returning to near 90%, 75%, and 45% by 6h . At 44°C, heating times of 15-60 min reduced protein synthesis to about 10-2% of controls by 1 h after heating, Six hours after 15 and 30 min at 44°C, protein synthesis had recovered to approximately 47% and 15% of con-
Survival curve parameters for continuous and fractionated heating
Do
Dq
n
No. of points'
1.1 9.3 45 .7 11 . 5
6 11 9 8
x Value range b
R°
Figure 1 33°C 37 °C 41 .5°C 44°C
64 . 0 h 9. 7 h 1 .3 h 0. 1 h
7.4 21 . 8 4.8 0.3
h h h h
31 . 5-48 . 25 24 .0-49 .25 8.0-16 . 00 1 . 0- 2 . 00
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0. 796 0.908 0 . 961 0.936
Figure 3 44 ° C 15 min 44 ° C//44° C 30 min 44°C //44° C
8 . 5 min 16 .9 min 10 .4 min
21 . 7 min 57 .2 min 40. 1 min
12 . 8 28. 7 45. 8
6 3 4
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0. 945 0 .964 0 . 971
8 . 5 min 50 . 2 min 17.8 min 15 .0 min 9 . 3 min 8 . 5 min
21 .7 7 .8 34.6 26.6 32.8 24.4
12 .8 1 .2 6. 9 5.9 32 . 9 17 . 6
6 4 4 3 6 4
60-120 70-130 50-110 70-110 60-120 60-105
0. 945 0 . 958 0 .942 0.999 0.966 0. 980
Figure 4 44°C 30 min 60 min 30 min 60 min 30 min
41 . 5° C //44° C 37°C//44°C 37°C//44°C 33°C //44°C 33° C //44° C
min min min min min min
:Numberof points used in delineating exponential portion of curve . 'Number of time values corresponding to points used for' . 'Correlation coefficient of line arrived at using points indicated in ' and b. //indicates a break in treatment where the cells were incubated for 2 hours at 28 ° C .
min min min min min min
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T. M . Koval and D . L . Suppes 150 125
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Figure 2 .
.! . 0
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e
DNA and protein synthesis in TN-368 cells following heat exposures of 37, 41 .5, and 44° C for various periods of time . Points represent the mean and SE of at least two experiments with three replicates per experiment .
trol, but no recovery was evident for cells heated for 60 min . Cells heated for 15 or 30 min at 44°C and then incubated at 28 ° C for 2 h before a second series of graded heat doses at 44° C showed enhanced thermal resistance compared to cells for which the heat dose was not fractionated (Figure 3) . Survival was reduced to 10% of control after 35 min of continuous 44° C heating and after fractionated exposures of 30 and 25 or 15 and 75 min, for total heating times of 55 and 90 min, respectively . Do values of the curves for fractionated doses were greater than the curve for continuous heating, the greatest Do belonging to the curve for which the cells received an initial heat dose r
of 15 min (Table 1) . By 120 min of total heating time, the curves were separated by several orders of magnitude . Induction of thermotolerance was also evident in cells that received an initial heat dose at 33, 37, or 41 . 5 ° C, were incubated at 28 ° C for 2 h, then heated for increasing times at 44°C (Figure 4), as compared to cells heated continuously at 44°C without any prior treatment . Do values for the fractionated heating curves are listed in Table 1 . No significant increase in survival ability at 44 ° C was observed for cells previously exposed to 33 ° C for 30 min, but thermal resistance was enhanced among cells previously exposed to 33 ° C for 60 min . Pretreatment of 0
10
∎
Pretreatment :
00 min/37 .0°C min/33 .0°C 0 min/33 .0 C
V 30 min/41 .5 C
0 60
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Total time at 44 ° C (min) Figure 3 . Exponentially growing TN-368 cells were heated continuously at 44 ° C or heated for 15 or 30 min at 44 ° C, incubated for 2 h at 28 ° C, and then heated again at 44° C. Points represent the mean and SE of at least two experiments with a minimum of five replicates per experiment .
Time at 44 ° C (min) Figure 4. Exponentially growing TN-368 cells were heated for either 30 or 60 min at 33, 37, and 41 .5°C, incubated for 2 h at 28 ° C, and then heated continuously at 44 °C . Points represent the mean and SE of at least two experiments with a minimum of five replicates per experiment .
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Heat resistance in radioresistant TN-368 cells
30 or 60 min at 37 ° C induced even greater thermotolerance in these cells, the 60 min dose giving rise to the more significant resistance . Of the treatments investigated, cells receiving an initial treatment of 15 min at 41 . 5°C showed the greatest thermal resistance ; survival was enhanced by 4 orders of magnitude for pretreated cells compared to cells receiving no prior treatment when heated at 44°C for 120 min . DNA and protein synthesis were also examined after conditions which induced thermotolerance (Figure 5) . Both DNA and protein synthesis were rapidly reduced, reaching their nadir by 30 min after the second heating . Protein synthesis increased steadily at varying rates depending on the primary heat dose used to induce thermotolerance . DNA synthesis, however, remained depressed for at least 6 h, rising only to approximately 5-15% of control in any of the thermotolerance-inducing regimens used .
4. Discussion The lepidopteran TN-368 cells are much more resistant to heat treatment than the previously examined Drosophila WR69-DM-1 cells, a dipteran line (Tomasovic and Koval 1985) . Both cell lines were routinely cultured at 28 ° C. However, the heating time at 33 ° C which resulted in 50% cell survival was approximately 30 min for the Drosophila cells but approximately 48 h for the TN-368 cells, nearly 100 times longer. Similar relationships existed at higher temperatures as well . At 37°C, 50% cell survival was reached after only a slightly longer exposure than at 33°C, still about I h, for the Drosophila cells, while it took about 29 h to reach the same survival in the TN-368 cells. At 42° C, Drosophila cell survival fell to 50% in about 3 min, whereas at 41 . 5°C TN-368 cell survival did not reach 50% until approximately 3 h . Even at 44°C, TN-368 cell survival fell to 50% only after about 15 min. The general shape of the heat
survival curves also presents quite a contrast between the TN-368 and Drosophila cells . The TN368 cells display a typical survival profile at each temperature studied with an initial shoulder area followed by exponential killing as heating time increases (Figure 1) . The Drosophila cells, on the other hand, were characterized by a complex heat (33, 37 and 42 °C) survival pattern with exponential killing followed by a broad inflection point or plateau in the 10 -2 to 10 -3 survival region (Tomasovic and Koval 1985) . At 42 ° C this inflection point was followed by further exponential killing and another plateau near the 10 - s survival level. Although Do values were not calculated for the Drosophila cells, due to their complex survival curves, the slope of the exponential portion of survival curves was much steeper for the Drosophila cells than the TN-368 cells at all temperatures . Thermotolerance was demonstrated in the TN368 cells at 33, 37, 41 . 5, and 44 ° C. This is in contrast to the slight thermotolerance observed in Drosophila cells at 33 ° C, but not at 37 or 42°C (Tomasovic and Koval 1985) . Thermotolerance was assessed by fractionated heating experiments for both cell types; however, both the initial heating and the later follow-up heating were performed at the same temperature for the Drosophila cells while the initial heating was usually followed by a later 'increasedtemperature' heating for the TN-368 cells . The increased-temperature heating was necessary in the case of the TN-368 cells because relatively long heating times were required to result in a decrease in survival at 33, 37 and 41 . 5 ° C . Thus, thermal resistance was more practically measured by survival at 44°C following the initial heating to induce thermotolerance at 33, 37, 41 . 5 or 44° C. It would have been otherwise impossible to detect thermotolerance at 33 and 37°C and difficult at 41 . 5 ° C. It is worth noting that the thermotolerance induction conditions in the Drosophila experiments (30 min at 33, 37 and 42°C followed by 1, 3 and 6 h incubations at 28°C before 110
30 30 15 10 15
min min min min min
0 0 0 0 0
37°C/ 2 h 0 28°C/15 min 0 44°C 41,5°C/2h 0 28°C/15 min 0 44°C 44°C/2 h 0 28°C/ 15 min 0 44°C 44°C 44°C
,oo 90
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90 y 70 t g + o C U 60 n O 50 C 91 40 '~ 30
p
d t a zo n lo 0 6 1 2 3 4 5 Time after Second Heat Exposure (hr)
7
Figure 5 . DNA and protein synthesis in thermotolerant TN-368 cells . Cells were treated with a priming heat dose, returned to incubation at 28° C, and then evaluated after 15 min at 44 ° C . Points represent the mean and SE of at least two experiments with three replicates per experiment .
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T. M . Koval and D. L . Suppes
heating again at 33, 37 or 42°C, respectively) were more damaging to the cells than the induction conditions in the TN-368 studies (30 or 60 min at 33 and 37°C, 30 min at 41 .5°C, and 15 or 30 min at 44°C followed by 2 h at 28 °C before heating again at 44°C for various times) . The corresponding survival levels were about 0 .5-0 .01 for the Drosophila cells and 1 .0-0 . 2 for the TN-368 cells . Therefore, higher tolerance levels are not correlated to more heat killing by the priming heat dose as might be expected . While it remains to be seen whether these induction conditions would make a difference in development of thermotolerance, it is clear that several distinct differences exist regarding the survival response to heat in these two cultured insect cell systems . The TN-368 insect cells used in this study demonstrate sensitivity to heating at 41 .5 and 44°C that is somewhat similar to mammalian cell heat sensitivity . For instance, survival is reduced to 50% of control after 3 h of heating at 41 .5°C for TN-368 cells, or after about 1-4 h of heating at the same temperature for mammalian cells (Dewey et al . 1977, Nielsen et al. 1982) . The D o values for the TN-368 cells are also within the range observed for mammalian cells (Gerner et al . 1976, Nielsen and Overgaard, 1982, Nielsen et al. 1982) . Additionally, the TN-368 cells develop a significant amount of thermotolerance in fractionated heating experiments at 44°C in a manner quite comparable to several types of mammalian cells (Gerner et al. 1976, Henle and Dethlefsen 1978, Nielsen and Overgaard 1982, Gerweck and DeLaney 1984) . However, development of thermotolerance was not evident during continuous heating at 41 .5°C as in mammalian cells (Henle and Dethlefsen 1978) . These similarities in thermal sensitivity become remarkable when it is considered that the normal growth temperature of the TN-368 cells is 9°C lower than that of mammalian cells, so that heating to 41 .5 and 44°C results in temperature elevations of 13 .5 and 16°C for the TN-368 cells compared to 4 .5 and 7°C for mammalian cells . Therefore, on this basis, it appears that the TN-368 cells are more resistant to damage by heat than mammalian cells . It is recognized, however, that, depending on the mechanisms involved in thermal damage, the absolute temperature of the heat stress could be more important in considering cell survival than the magnitude of the temperature elevation . The patterns of heat-induced inhibition of DNA and protein synthesis depicted in Figure 2 for the TN-368 cells are generally similar to those which have been reported for various mammalian and human cells (Henle and Leeper 1979, Warters and Stone 1983, Kern et al . 1988, Oesterreich et al. 1990) .
Inhibition of both types of synthesis increased with time and temperature . Recovery was somewhat faster for protein synthesis than DNA synthesis, again in line with the previous studies (Henle and Leeper 1979, Warters and Stone 1983) . When a priming heat exposure was used to induce thermotolerance prior to a second heat treatment, DNA synthesis was reduced to a level as low as or below the level that would have been produced by the second treatment only (Figure 5) . However, after a 41 .5°C priming dose, protein synthesis was less inhibited and its recovery was more rapid in tolerant cells than in cells receiving only a single 44°C treatment (Figure 5) . For 37 and 44°C priming regimens the tolerant cells displayed similar protein synthesis kinetics to the non-tolerant cells . The degree of inhibition in the tolerant cells was therefore dependent on the time and temperature of the priming heat exposure . This also is relatively consistent with other reports (Lee et al. 1990, Oesterreich et al. 1990), although higher tolerance levels did not correlate with the amount of heat killing by the priming dose . Investigations with other eukaryotic cell systems have also demonstrated that alterations in protein synthesis are not necessary for the expression of thermotolerance (Tomasovic et al. 1983, Watson et al . 1984) . Therefore, despite the ostensibly marked heat resistance observed in the TN-368 cells, fundamental alterations in macromolecular, DNA and protein, synthesis appear to be comparable to mammalian and human cells . The teleological significance of a rapid and prolonged depression in DNA synthesis (to allow time for cellular repair and recovery) and the perhaps more intricate balance in protein synthesis inhibition and recovery have been discussed at length elsewhere (Wong and Dewey 1982, Warters 1988, Lepock et al . 1990, Lee et al . 1990) . The TN-368 cells may provide an appropriate model system to study mechanisms of heat damage and thermotolerance . The cells display considerable resistance to heat killing and rapidly develop thermotolerance across a wide temperature range . The considerable resistance at temperatures so far above their normal growth temperature, as well as several of the other points mentioned above, make this system appealing for further study . An additional recent development which should enhance the usefulness of the TN-368 cell system in studying mechanisms of heat killing is our recent isolation of two UV- and two y-ray-sensitive strains of the TN-368 line (Koval 1991) . One of each of these types of radiation-sensitive mutant is also sensitive to heat, while the other is not . It is hoped that these cell strains can be exploited in future heat investigations .
Heat resistance in radioresistant TN-368 cells Acknowledgements
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tion damage in a lepidopteran insect cell line . Radiation
Research, 115, 413-420 .
The authors thank J . M. Lacey for technical assistance and A. L . Loechler for assistance in manuscript preparation . This work was supported by USPHS Grant R01 CA34158, awarded by the National Cancer Institute, DHHS, Bethesda, MD .
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Journal of Surgical Oncology, 37, 60-64 . KOVAL, T. M ., 1983a, Intrinsic resistance to the lethal effects of x-irradiation in insect and arachnid cells . Proceedings of the National Academy of Sciences, USA, 80, 4752-4755 . KOVAL, T . M ., 1983b, Radiosensitivity of cultured insect cells : I . Lepidoptera. Radiation Research, 96, 118-126. KOVAL, T . M ., 1983c, Radiosensitivity of cultured insect cells : II . Diptera . Radiation Research, 96, 127-134 . KOVAL, T. M ., 1986a, Enhanced survival by photoreactivation and liquid-holding following UV damage of TN-368 insect cells . Mutation Research, 166, 149-156 . KOVAL, T . M ., 1986b, Inducible repair of ionizing radiation damage in higher eukaryotic cells . Mutation Research, 173, 291-293 . KOVAL, T . M ., 1987, Photoreactivation of UV damage in cultured Drosophila cells . Experientia, 43, 446 . KOVAL, T . M ., 1988, Enhanced recovery from ionizing radia-
KOVAL, T. M ., 1989, Methods for determining the survival of cultured insect cells . Journal of Tissue Culture Methods, 12, 43-46 . KOVAL, T. M ., 1991, Gamma-ray- and UV-sensitive strains of a radioresistant cell line : isolation and cross-sensitivity to other agents. Radiation Research, 127, 58-63 . KOVAL, T. M ., MYSER, W . C ., HART, R . W. and HINK, W. F ., 1978, Comparison of survival and unscheduled DNA synthesis between an insect and a mammalian cell line following x-ray treatments . Mutation Research, 49, 431-435 . LEE, Y . J., PERLAKY, L., DEWEY, W . C ., ARMOUR, E . P. and CORRY, P . M ., 1990, Differences in thermotolerance induced by heat or sodium arsenite : cell killing and inhibition of protein synthesis . Radiation Research, 121, 295-303 . LEPOCK, J. R ., FREY, H . E ., HEYNEN, M . P., NisHIO, J., WATERS, B ., RITCHIE, K . P. and KRUUV, J., 1990, Increased thermostability of thermotolerant CHL V79 cells as determined by differential scanning calorimetry.
Journal of Cellular Physiology, 142, 628-634. NIELSEN, O . S . and OVERGAARD, J ., 1982, Influence of time and temperature on the kinetics of thermotolerance in L 1 A2 cells in vitro . Cancer Research, 42, 4190-4196 . NIELSEN, O .' S ., HENLE, K . J . and OVERGAARD, J ., 1982, Arrhenius analysis of survival curves from thermotolerant and step-down heated L1A2 cells in vitro . Radiation Research, 91, 468-482 . OESTERREICH, S ., BENNDORF, R . and BIELKA, H ., 1990, The expression of the growth-related 25kDa protein (p25) of Ehrlich ascites tumor cells is increased by hyperthermic treatment (heat shock) . Biomedica et Biochimica Acta, 49, 219-226 . ToaAsovlc, S . P. and KOVAL, T . M ., 1985, Relationship between cell survival and heat-stress protein synthesis in a Drosophila line . International Journal of Radiation Biology, 48, 635-650. ToMASOVIC, S . P., STECK, P. A . and HEITZMAN, D ., 1983, Heatstress proteins and thermal resistance in rat mammary tumor cells . Radiation Research, 95, 399-413 . WARTERS, R . L ., 1988, Hyperthermia blocks DNA processing at the nuclear matrix. Radiation Research, 115, 258-272 . WARTERS, R . L. and STONE, O . L ., 1983, Macromolecule synthesis in HeLa cells after thermal shock . Radiation Research, 96, 646-652 . WATSON, K ., DUNLOP, G. and CAVICCHIOLI, R. R ., 1984, Mitochondrial and cytoplasmic protein syntheses are not required for heat shock acquisition of ethanol and thermotolerance in yeast . FEBS Letters, 172, 299-302 . WONG, R . S . L . and DEWEY, W. C ., 1982, Molecular studies on the hyperthermic inhibition of DNA synthesis in Chinese hamster ovary cells . Radiation Research, 92, 370-395 .
ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20
Heat Resistance and Thermotolerance in a Radiation-resistant Cell Line T.M. Koval & D.L. Suppes To cite this article: T.M. Koval & D.L. Suppes (1992) Heat Resistance and Thermotolerance in a Radiation-resistant Cell Line, International Journal of Radiation Biology, 61:3, 425-431, DOI: 10.1080/09553009214551121 To link to this article: http://dx.doi.org/10.1080/09553009214551121
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Date: 12 May 2016, At: 12:03
INT . J . RADIAT . BIOL .,
1992,
VOL .
61,
NO.
3, 425-431
Heat resistance and thermotolerance in a radiation-resistant cell line T. M . KOVAL*t and D. L. SUPPESt
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(Received 10 May 1991 ; revision received 2 August 1991 ; accepted 7 August 1991)
Abstract . Exponentially growing TN-368 lepidopteran insect cells have a normal growth temperature of 28°C . These cells were heated in water baths at various temperatures between 33 and 44 ° C under conditions of constant or fractionated heating. Determinations of cell survival using colony formation as well as measurements of DNA and protein synthesis were performed to assess relative heat resistance and development of thermotolerance . The results demonstrate a marked heat resistance over previously reported findings from the same laboratory for dipteran Drosophila cells in culture . The degree of heat resistance is remarkable, especially when compared to the heat resistance of mammalian cells, i .e . TN-368 cell survival at 41 . 5 and 44 °C was somewhat similar to mammalian cell survival, even though these temperatures are 13 . 5 and 16° C above the normal growth temperature for TN-368 cells and 4 . 5 and 7 °C above the growth temperature of mammalian cells . Furthermore, the lepidopteran cells maintain the ability to develop a notable amount of thermotolerance in addition to this heat resistance . Thermotolerance development alone is capable of enhancing survival by an additional 10000-fold . Thermotolerance could also be detected at the level of protein synthesis as a more rapid recovery following heat treatment . In contrast, DNA synthesis inhibition was prolonged even further in cells receiving a prior heat treatment to induce thermotolerance . In summary, it appears that, in addition to their pronounced radiation resistance, the TN-368 cells are also quite resistant to heat. It remains to be seen whether a single mechanism could be responsible for resistance to these agents which act very differently.
plus 365 nm ultraviolet light, among other agents (Koval, unpublished data) . Evidence suggests that exceptional repair or recovery mechanisms in these cells may be responsible for their resistance to many of these agents (Koval et al. 1978, Koval 1986a,b, 1988) . The only previous utilization of the colony formation technique as a quantitative heat survival assay in cultured insect cells was performed with Drosophila melanogaster (Tomasovic and Koval 1985) . The line of Drosophila cells used in that study was much more sensitive to both ionizing and UV radiation than the TN-368 cells, although still more resistant than most mammalian cells (Koval 1983a,c, 1987) . Resistance of the Drosophila cells to heating was not particularly notable and thermotolerance was only slightly evident at 33 ° C and not apparent at higher temperatures (Tomasovic and Koval 1985) . Thus, the aim of this study was to investigate survival and thermotolerance in the more radioresistant TN-368 cells for comparison with the Drosophila system.
2. Materials and methods 2.1 . Cell lines
1. Introduction Cultured TN-368 lepidopteran insect cells are of the order of 100 times more resistant to cell killing by ionizing radiation than cultured mammalian cells (Koval, 1983a,b) . These cells also exhibit a pronounced resistance to 254 nm ultraviolet light, to which they are in the range of 10-25 times more resistant than mammalian cells (Koval 1986a) . In addition, the TN-368 cells are resistant to killing by methyl methanesulphonate, N-methyl-R-nitro-Nnitrosoguanidine, 1,3-propane sultone, mitomycin C, 4-nitroquinoline-l-oxide, and 8-methoxypsoralen *Author for correspondence. tDivision of Radiation Oncology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA .
The TN-368 cells are a continuous line of primarily fibroblast-like cells which were derived from minced adult ovaries of the cabbage looper, Trichoplusia ni (Hink 1970) . The cells, which were originally obtained from Dr W. F. Hink, Ohio State University, have been in culture for more than 15 years and have been subcultured more than 1500 times at a split ratio of approximately 1 :10. The cells generally grow in a loosely attached monolayer and require only moderate agitation to detach them from the bottom surface of growth vessels . Cultures are normally maintained in polystyrene tissue culture flasks (Corning) and kept in a humidified incubator at 28°C. The cell line has a doubling time of approximately 19h and is normally maintained by subculturing three times a week at a 1 :10 ratio in
0020-7616/92 $3 .00 ® 1992 Taylor & Francis Ltd
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TNM-FH medium (Koval 1989) supplemented with 8% fetal bovine serum (Hyclone) .
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2.2 .
Cell
survival experiments
Exponentially growing TN-368 cells in 25 cm 2 polystyrene tissue culture flasks (Corning) were heated for various periods of time by immersing flasks in precision-controlled (±0 .05 °C) water baths (Tecam, TU-15 ; Techne Inc ., Princeton, NJ, USA) equipped with supplementary water pumps. Standardized thermometers were used for temperature measurement . In fractionated experiments an initial 15, 30, or 60 min heat exposure was followed by a 2 h incubation at 28 ° C before additional graded heat exposures. Following fractionated or continuous heating, the cells were removed from flasks by gentle pipetting and were counted using a haemacytometer and a Model ZM Coulter Counter . Cells were then plated into 60 mm diameter polystyrene tissue culture dishes (Lux) containing conditioned TNM-P medium (Koval 1989) . A 0 . 15 ml volume of cell suspension was added to 3 .8 ml of conditioned medium . The dishes were incubated at 28°C in a humidified incubator and left undisturbed for 18-19 days to allow for colony formation . The dishes were then removed from the incubator, stained with neutral red, and colonies counted to determine cell surviving fractions (Koval 1989) . Absolute plating efficiencies were generally in the range of 40-50% .
2 .3. DNA synthesis Exponentially growing cultures were labelled with .(1961-2072MBq/mmol, [methyl- 14C]thymidine Moravek) at 2 .59 kBq/ml for aproximately 42 h . The medium was then removed, the cells were rinsed with 2 ml of fresh medium, and 5 ml of fresh medium was added. The cells were heated at 37, 41 .5 or 44°C for various periods of time . Cells were pulse-labelled for 20 min immediately prior to sampling with [methyl-3H]thymidine (1665-1813MBq/mmol, Amersham) at 296 kBq/ml by adding 0 . 1 ml of labelled medium to each tube containing 1 .4 ml of heated cell suspension . At each sampling, the cells were pelleted at 15 000 rpm for 10 s in a Beckman microfuge, tritium-labelled medium was removed, and the cell pellet was stored at -80°C until further processing . The cells were resuspended in 0 .75 ml SSC (0-15m sodium chloride, 0-015m sodium citrate) containing 100 pg/ml thymidine and 400 µg/ml BSA . The cells were precipitated with icecold 10% trichloroacetic acid (TCA) containing 1
sodium pyrophosphate . The pellet was dissolved in 0. 1 N NaOH containing excess thymidine (100 yg/ml) and incubated at 37°C for 15 min before reprecipitating with 10% TCA for 15 min . The suspension was then passed through a Whatman GF/C filter wetted with 10% TCA and washed sequentially with 10 ml volumes of ice-cold 10% TCA and twice with 95% ethanol . The filters were allowed to dry overnight and counted in Ultima Gold scintillation cocktail (Packard) using a Beckman LS 5000TA liquid scintillation system to provide disintegrations per minute (dpm) . The dpm were then analysed as 3H/ 14C ratios which were measures of specific activity of DNA, i .e. the rate of DNA synthesis. Percentage of control was calculated as the 3H/ 14C ratio for heated cells divided by the 3H/14C ratio for untreated cells .
2 .4.
Protein synthesis
Exponentially growing cells were heated at 41 .5°C for various times . The cells were pulselabelled for 30 or 60 min immediately prior to sampling with [3H]leucine (4440 GBq/mmol, RPI) by adding 0 . 1 ml of labelled medium (296 kBq/ml) to 1 . 4 ml of medium in each tube of suspended cells . At each sampling, the cells were pelleted, tritiumlabelled medium was removed, and the cell pellet was stored at -80°C until further processing as described above for DNA synthesis . Cell counts were obtained at each sampling and were performed in triplicate or quadruplicate using a haemacytometer . Percentage of control was calculated as counts per minute (cpm) per cell for heated cells divided by the cpm per cell for untreated cells . 3.
Results
Survival curves for exponentially growing TN-368 cells subjected to continuous heating at 33, 37, 41 . 5 and 44°C are shown in Figure 1 . Marked differences in thermal sensitivity at these temperatures, as assayed by colony formation, were observed . Survival was reduced to approximately 50% of control following 48 h of heating at 33 ° C, 29 h at 37°C, 3 h at 41 .5°C, and 15 min at 44°C, and to approximately 10% after 45 h at 37°C, 7 h at 41 .5°C, and 35 min at 44°C. Slopes of the curves varied greatly . Survival remained at or near control levels for 20 h of continuous heating at 33°C, and very gradually began to decrease between 20 and 50 h of heating . Survival after continuous 37°C heating was similar to that at 33°C until approximately 16 h, when it began to
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Heat resistance in radioresistant TN-368 cells
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Time (hr) Figure 1 . Exponentially growing TN-368 cells were heated for various times at 33, 37, 41 . 5, and 44 ° C and immediately plated for colony formation . Points represent the mean and relative SE from a minimum of five plate counts . These data were compiled from 18 separate experiments .
decrease at a slightly faster rate. At 41 . 5 ° C a shoulder of approximately 10h preceded exponential killing . A small shoulder was also observed for 44°C heating, though by 40 min at this temperature survival decreased sharply and cell killing was exponential . Approximate D0 , D4 , and n values for the survival curves are provided in Table 1 .
Table 1 .
The effect of heating on DNA synthesis is illustrated in Figure 2 . Depression of DNA synthesis was rapid at each temperature examined, with rates dropping to approximately 50% of control levels after 30 min at 37 ° C and to less than 10% of control levels after 30 min at 41 . 5 and 44°C . DNA synthesis recovered quickly at 37°C, rebounding to 100% by 1 h after heat exposures up to 1 h . After 6 h, DNA synthesis had recovered to control levels following an exposure of 2 h at 37 ° C. Recovery was slower at 41 . 5 and 44 ° C, with recovery rates only at 50% of control levels by 6h, after exposures of 20 and 10 min, respectively . DNA synthesis did recover to levels approaching 100% by 24 h after heating for exposures up to 60 min at 41 . 5°C and 30 min at 44°C, but no recovery was observed after being exposed to 44°C for 60 min (data not shown) . Total protein synthesis decreased marginally after heat exposures of 30, 60, and 120 min at 37°C (Figure 2), but rebounded promptly to control levels or above by 3 h . The depression in protein synthesis was more significant at 41 . 5 ° C, decreasing to about 40%, 30%, and 12% of control levels by 30 min after heating times of 30, 60, or 120 min, respectively . Recovery was gradual, returning to near 90%, 75%, and 45% by 6h . At 44°C, heating times of 15-60 min reduced protein synthesis to about 10-2% of controls by 1 h after heating, Six hours after 15 and 30 min at 44°C, protein synthesis had recovered to approximately 47% and 15% of con-
Survival curve parameters for continuous and fractionated heating
Do
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Figure 1 33°C 37 °C 41 .5°C 44°C
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Figure 3 44 ° C 15 min 44 ° C//44° C 30 min 44°C //44° C
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Figure 4 44°C 30 min 60 min 30 min 60 min 30 min
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min min min min min min
:Numberof points used in delineating exponential portion of curve . 'Number of time values corresponding to points used for' . 'Correlation coefficient of line arrived at using points indicated in ' and b. //indicates a break in treatment where the cells were incubated for 2 hours at 28 ° C .
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DNA and protein synthesis in TN-368 cells following heat exposures of 37, 41 .5, and 44° C for various periods of time . Points represent the mean and SE of at least two experiments with three replicates per experiment .
trol, but no recovery was evident for cells heated for 60 min . Cells heated for 15 or 30 min at 44°C and then incubated at 28 ° C for 2 h before a second series of graded heat doses at 44° C showed enhanced thermal resistance compared to cells for which the heat dose was not fractionated (Figure 3) . Survival was reduced to 10% of control after 35 min of continuous 44° C heating and after fractionated exposures of 30 and 25 or 15 and 75 min, for total heating times of 55 and 90 min, respectively . Do values of the curves for fractionated doses were greater than the curve for continuous heating, the greatest Do belonging to the curve for which the cells received an initial heat dose r
of 15 min (Table 1) . By 120 min of total heating time, the curves were separated by several orders of magnitude . Induction of thermotolerance was also evident in cells that received an initial heat dose at 33, 37, or 41 . 5 ° C, were incubated at 28 ° C for 2 h, then heated for increasing times at 44°C (Figure 4), as compared to cells heated continuously at 44°C without any prior treatment . Do values for the fractionated heating curves are listed in Table 1 . No significant increase in survival ability at 44 ° C was observed for cells previously exposed to 33 ° C for 30 min, but thermal resistance was enhanced among cells previously exposed to 33 ° C for 60 min . Pretreatment of 0
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Total time at 44 ° C (min) Figure 3 . Exponentially growing TN-368 cells were heated continuously at 44 ° C or heated for 15 or 30 min at 44 ° C, incubated for 2 h at 28 ° C, and then heated again at 44° C. Points represent the mean and SE of at least two experiments with a minimum of five replicates per experiment .
Time at 44 ° C (min) Figure 4. Exponentially growing TN-368 cells were heated for either 30 or 60 min at 33, 37, and 41 .5°C, incubated for 2 h at 28 ° C, and then heated continuously at 44 °C . Points represent the mean and SE of at least two experiments with a minimum of five replicates per experiment .
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Heat resistance in radioresistant TN-368 cells
30 or 60 min at 37 ° C induced even greater thermotolerance in these cells, the 60 min dose giving rise to the more significant resistance . Of the treatments investigated, cells receiving an initial treatment of 15 min at 41 . 5°C showed the greatest thermal resistance ; survival was enhanced by 4 orders of magnitude for pretreated cells compared to cells receiving no prior treatment when heated at 44°C for 120 min . DNA and protein synthesis were also examined after conditions which induced thermotolerance (Figure 5) . Both DNA and protein synthesis were rapidly reduced, reaching their nadir by 30 min after the second heating . Protein synthesis increased steadily at varying rates depending on the primary heat dose used to induce thermotolerance . DNA synthesis, however, remained depressed for at least 6 h, rising only to approximately 5-15% of control in any of the thermotolerance-inducing regimens used .
4. Discussion The lepidopteran TN-368 cells are much more resistant to heat treatment than the previously examined Drosophila WR69-DM-1 cells, a dipteran line (Tomasovic and Koval 1985) . Both cell lines were routinely cultured at 28 ° C. However, the heating time at 33 ° C which resulted in 50% cell survival was approximately 30 min for the Drosophila cells but approximately 48 h for the TN-368 cells, nearly 100 times longer. Similar relationships existed at higher temperatures as well . At 37°C, 50% cell survival was reached after only a slightly longer exposure than at 33°C, still about I h, for the Drosophila cells, while it took about 29 h to reach the same survival in the TN-368 cells. At 42° C, Drosophila cell survival fell to 50% in about 3 min, whereas at 41 . 5°C TN-368 cell survival did not reach 50% until approximately 3 h . Even at 44°C, TN-368 cell survival fell to 50% only after about 15 min. The general shape of the heat
survival curves also presents quite a contrast between the TN-368 and Drosophila cells . The TN368 cells display a typical survival profile at each temperature studied with an initial shoulder area followed by exponential killing as heating time increases (Figure 1) . The Drosophila cells, on the other hand, were characterized by a complex heat (33, 37 and 42 °C) survival pattern with exponential killing followed by a broad inflection point or plateau in the 10 -2 to 10 -3 survival region (Tomasovic and Koval 1985) . At 42 ° C this inflection point was followed by further exponential killing and another plateau near the 10 - s survival level. Although Do values were not calculated for the Drosophila cells, due to their complex survival curves, the slope of the exponential portion of survival curves was much steeper for the Drosophila cells than the TN-368 cells at all temperatures . Thermotolerance was demonstrated in the TN368 cells at 33, 37, 41 . 5, and 44 ° C. This is in contrast to the slight thermotolerance observed in Drosophila cells at 33 ° C, but not at 37 or 42°C (Tomasovic and Koval 1985) . Thermotolerance was assessed by fractionated heating experiments for both cell types; however, both the initial heating and the later follow-up heating were performed at the same temperature for the Drosophila cells while the initial heating was usually followed by a later 'increasedtemperature' heating for the TN-368 cells . The increased-temperature heating was necessary in the case of the TN-368 cells because relatively long heating times were required to result in a decrease in survival at 33, 37 and 41 . 5 ° C . Thus, thermal resistance was more practically measured by survival at 44°C following the initial heating to induce thermotolerance at 33, 37, 41 . 5 or 44° C. It would have been otherwise impossible to detect thermotolerance at 33 and 37°C and difficult at 41 . 5 ° C. It is worth noting that the thermotolerance induction conditions in the Drosophila experiments (30 min at 33, 37 and 42°C followed by 1, 3 and 6 h incubations at 28°C before 110
30 30 15 10 15
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Figure 5 . DNA and protein synthesis in thermotolerant TN-368 cells . Cells were treated with a priming heat dose, returned to incubation at 28° C, and then evaluated after 15 min at 44 ° C . Points represent the mean and SE of at least two experiments with three replicates per experiment .
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T. M . Koval and D. L . Suppes
heating again at 33, 37 or 42°C, respectively) were more damaging to the cells than the induction conditions in the TN-368 studies (30 or 60 min at 33 and 37°C, 30 min at 41 .5°C, and 15 or 30 min at 44°C followed by 2 h at 28 °C before heating again at 44°C for various times) . The corresponding survival levels were about 0 .5-0 .01 for the Drosophila cells and 1 .0-0 . 2 for the TN-368 cells . Therefore, higher tolerance levels are not correlated to more heat killing by the priming heat dose as might be expected . While it remains to be seen whether these induction conditions would make a difference in development of thermotolerance, it is clear that several distinct differences exist regarding the survival response to heat in these two cultured insect cell systems . The TN-368 insect cells used in this study demonstrate sensitivity to heating at 41 .5 and 44°C that is somewhat similar to mammalian cell heat sensitivity . For instance, survival is reduced to 50% of control after 3 h of heating at 41 .5°C for TN-368 cells, or after about 1-4 h of heating at the same temperature for mammalian cells (Dewey et al . 1977, Nielsen et al. 1982) . The D o values for the TN-368 cells are also within the range observed for mammalian cells (Gerner et al . 1976, Nielsen and Overgaard, 1982, Nielsen et al. 1982) . Additionally, the TN-368 cells develop a significant amount of thermotolerance in fractionated heating experiments at 44°C in a manner quite comparable to several types of mammalian cells (Gerner et al. 1976, Henle and Dethlefsen 1978, Nielsen and Overgaard 1982, Gerweck and DeLaney 1984) . However, development of thermotolerance was not evident during continuous heating at 41 .5°C as in mammalian cells (Henle and Dethlefsen 1978) . These similarities in thermal sensitivity become remarkable when it is considered that the normal growth temperature of the TN-368 cells is 9°C lower than that of mammalian cells, so that heating to 41 .5 and 44°C results in temperature elevations of 13 .5 and 16°C for the TN-368 cells compared to 4 .5 and 7°C for mammalian cells . Therefore, on this basis, it appears that the TN-368 cells are more resistant to damage by heat than mammalian cells . It is recognized, however, that, depending on the mechanisms involved in thermal damage, the absolute temperature of the heat stress could be more important in considering cell survival than the magnitude of the temperature elevation . The patterns of heat-induced inhibition of DNA and protein synthesis depicted in Figure 2 for the TN-368 cells are generally similar to those which have been reported for various mammalian and human cells (Henle and Leeper 1979, Warters and Stone 1983, Kern et al . 1988, Oesterreich et al. 1990) .
Inhibition of both types of synthesis increased with time and temperature . Recovery was somewhat faster for protein synthesis than DNA synthesis, again in line with the previous studies (Henle and Leeper 1979, Warters and Stone 1983) . When a priming heat exposure was used to induce thermotolerance prior to a second heat treatment, DNA synthesis was reduced to a level as low as or below the level that would have been produced by the second treatment only (Figure 5) . However, after a 41 .5°C priming dose, protein synthesis was less inhibited and its recovery was more rapid in tolerant cells than in cells receiving only a single 44°C treatment (Figure 5) . For 37 and 44°C priming regimens the tolerant cells displayed similar protein synthesis kinetics to the non-tolerant cells . The degree of inhibition in the tolerant cells was therefore dependent on the time and temperature of the priming heat exposure . This also is relatively consistent with other reports (Lee et al. 1990, Oesterreich et al. 1990), although higher tolerance levels did not correlate with the amount of heat killing by the priming dose . Investigations with other eukaryotic cell systems have also demonstrated that alterations in protein synthesis are not necessary for the expression of thermotolerance (Tomasovic et al. 1983, Watson et al . 1984) . Therefore, despite the ostensibly marked heat resistance observed in the TN-368 cells, fundamental alterations in macromolecular, DNA and protein, synthesis appear to be comparable to mammalian and human cells . The teleological significance of a rapid and prolonged depression in DNA synthesis (to allow time for cellular repair and recovery) and the perhaps more intricate balance in protein synthesis inhibition and recovery have been discussed at length elsewhere (Wong and Dewey 1982, Warters 1988, Lepock et al . 1990, Lee et al . 1990) . The TN-368 cells may provide an appropriate model system to study mechanisms of heat damage and thermotolerance . The cells display considerable resistance to heat killing and rapidly develop thermotolerance across a wide temperature range . The considerable resistance at temperatures so far above their normal growth temperature, as well as several of the other points mentioned above, make this system appealing for further study . An additional recent development which should enhance the usefulness of the TN-368 cell system in studying mechanisms of heat killing is our recent isolation of two UV- and two y-ray-sensitive strains of the TN-368 line (Koval 1991) . One of each of these types of radiation-sensitive mutant is also sensitive to heat, while the other is not . It is hoped that these cell strains can be exploited in future heat investigations .
Heat resistance in radioresistant TN-368 cells Acknowledgements
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tion damage in a lepidopteran insect cell line . Radiation
Research, 115, 413-420 .
The authors thank J . M. Lacey for technical assistance and A. L . Loechler for assistance in manuscript preparation . This work was supported by USPHS Grant R01 CA34158, awarded by the National Cancer Institute, DHHS, Bethesda, MD .
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