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Survival of synchronized mammalian cells following exposure to cold
Cell Research 70 (1972) 417-422
Experimental
SURVIVAL
OF SYNCHRONIZED
Department
of Physics,
MAMMALIAN
EXPOSURE
TO COLD
R. J.,NELSON
and J. KRUUV
University
of Waterloo,
Waterloo,
CELLS
Ontario,
FOLLO
Canada
SUMMARY Synchronized Chinese hamster cells (V79) exposed to suboptimal temperatures for 3-10 days exhibit variations in survival depending upon the position in the cell cycle in which the exposure was initiated. At 5 and 25°C cells are most sensitive in Gl and early S, while at 45°C 61 is the most susceptible phase. In all cases G2 and M cells are resistant to cold damage. Factors of up to 5 can be obtained between minimum and maximum survival in the cell cycle, these factors increasing with the time the cells are left at the temperature in question.
It has been found that survival of mammalian cells following freezing in liquid nitrogen is a function of the time in the cell cycle at which the freezing takes place [l]. It is also known that a fraction of the cell population dies following or during exposures of several days to temperatures of 3-10°C [2-41. Although there are several modes of damage to cells from freezing injury [5], none of the mechanisms for death or possible repair of damage have clearly been elucidated. The possibility exists that there may be some common mechanisms between cold and freezing injuries. With the above in mind, it was decided to investigate if the survival of synchronized mammalian cells following exposure to cold (5°C) varied in a similar fashion as exposure to liquid nitrogen. Experiments were also done at 15 and 25°C to determine if the pattern would shift. MATERIALS
AND METHODS
The cells employed were those of a subline (V79S171) derived from the V79 line of near-diploid
Chinese hamster lung cells. They were grown on plastic Petri dishes in-Eagle medium with 15 % fetal calf serum in a humid atmosphere of 5 % CO, and air. Under these conditions the cells grew ii log phase with a generation time of about 10 h, which was subdivided into Gl, S, G2, and M periods of 2-2&, 6-7,lt and $ h respectively. During a synchrony experiment the generation time was about 12 h. The deviation was due to trypsinization and frequent opening of the incubator during the experiment. These times have been determined autoradiographically, by liquid scintillating counting techniques, and by-monitoring the radiation colonysurvivalthroughout the cell cycle [S]. The plating efficiency was usually about 80 % for asynchronous populations and 60 % for synchronous populations with mitotic indexes of 65-85 % in the latter. The number of nonviable (nondividing) cells in the plated population, as determined by cinemicrography, was negligible [7:. Cells synchronized at or close to division were obtained by selecting for less firmly attached, dividing cells by using a combination of precooling and mecbanical shaking with a 0.035 % trypsin solution [6, 81. Many synchrony experiments were performed without the precooling (1 h cold shock) and with prewarmed medium used as the “shake-off” liquid instead of the trypsin solution. The latter technique yielded a lower mitotic yield but a higher mitotic index [9]. These synchronized cells were then inoculated into temperature (37°C) and pH (7.4) equilibrated medium in plastic Petri dishes and allowed to attach for 2 h in the 37°C incubator. At various times thereafter, three dishes were removed from the 37°C incubator and placed in a refrigerated CO, incubator which had been pre-set to the correct temperature. The CO, flow in the incubator was adjusted so that the correct pH range (7.4-7.5) was maintained, the latter being
41X
R. J. Nelson & J. Kruuv
Table 1. Experimental Expt 1 2 3 4 5 6 7 8
conditions of synchrony
Time (days) at y”C 5 I 1: 7 3 4 6
experiments
Y”C
“Shake-off” technique
Mitotic index ( %)
Mitotic yield ( %>
Plating efficiency ( %)
5 5 5 5 15 25 25 25
T&PC= Mb M M T&PC M T&PC M
60 70 75 87
3.4
45 48 36
70 G
65 0.9 3 3 0.9
i; 48 61 38
a Trypsin and precooling. b Medium.
monitored by a pH-meter. The dishes were kept at the test temperature for X number of days (X= constant). After this time, the plates were removed from the refrigerated incubator in the same time sequence in which they were put in, and placed in the 37°C incubator for colony growth. After an incubation period of 5 to 7 days, the colonies were stained with methylene blue and counted. Cell concentrations were adjusted at the time of inoculation to suit the treatment to which the cells were to be exposed, so that 100 to 200 colonies resulted per dish (100 mm diameter). The colony surviving fraction (with respect to the controls) for exposure of cells to temperature Y for X days was plotted versus time at 37°C after synchronization (i.e. time in the cell cycle). With precooling and trypsin “shake-off” the average cellular multiplicity in the first cell cycle was typically 1.8, whereas with no precooling and a medium “shakeoff” it was usually 1.6. The survival data were not corrected for the multiplicity [lo] during the first or subsequent cell cycles. To find the position in the cell cycle, the easiest and least expensive method was to do a synchronous radiation survival experiment with the same cell population and conditions that were used in the “cold” experiments. The cells were irradiated with a constant dose of 810 rad from an 800 Ci cesium source. Since the radiation survival as a function of position in cell cycle is well known for this cell subline [ll, 121, the approximate lengths of the fractions of the cell cycle (as previously determined by pulse labeling with 3H-thymidine) are indicated at the foot of the graphs. Standard errors (S.E.) were used to express experimental accuracy and were represented as vertical bars in all figures unless smaller than the points as plotted.
RESULTS Fraction colony 5°C for various Exptl Cell Res 70
survival for cells exposed to lengths of time as a function
of position in cell cycle is shown in fig. 1. The experimental conditions are listed in table 1. The longer exposures to 5°C yield lower survivals and the variations in the cell cycle become more pronounced. Gl
and early S are the most sensitive phases whereas M is the most resistant. Figs 2 and 3 show results for similar experiments at 15 and 25°C respectively. At 15°C the minimum is in G 1 and the maximum in M whereas at 25°C they are at Gl or early S and M respectively. The experimental conditions are listed in table 1. Many of the experiments listed in table 1 were done simultaneously using the same synchronous cell population. These were experiments number 4 and 8, 2 and 6, and finally 5 and 7. Fig. 4 shows the results of the radiation survival experiment done concurrently with experiment number 3 (i.e. 9 days at 5°C). The cell cycle age-responses and the approximate lengths of the fractions of the cell cycle are illustrated. Fig. 5 shows fraction colony survival of asychronous cells after exposure for various time intervals to 5°C 15°C and 25°C. The detailed experimental technique for these experiments is described elsewhere [2]. It should be noted that the length of exposure in experiments number 2, 3, 4, 5 and 8
Survival
placed the population on the linear portion of the semi-log survival curve shown in fig. 5. Experiments 1, 6 and 7 were performed in the “shoulder” region of these curves. Only the synchronous experiments performed in the linear region of the curves in fig. 5 show substantial variations in survival throughout the cell cycle although cyclic changes can be detected in all experiments. After 10 days at 5°C factors up to 5 can be obtained between minimum and maximum survival in the cell cycle. After 6 days at 25°C a factor of 4 was obtained.
1.0 0.9 0.8 0.7 1, 0.6 j 0.54 0.4 0.3
0.2 4
0.1 M J 0
DISCUSSION Various investigations with mammalian cells suggest that as the temperature decreases from 37 to 25°C there is a marked increase
0
2
4
6
8
IO
12
14
16
18
Figs 1-2. Abscissa: time in cell cycle (hours); ordinate: fraction survival. Fig. 1. Fraction colony survival after exposure to 5°C as a function of age in the cell cycle. The length of time the cells were exposed to 5°C is noted beside each curve. Vertical bars reoresent S.E. unless smaller than the points as plotted. The approximate durations of the fractions of the cell cycle (as determined by X-ray survival data) are indicated at the foot of the graph.
41
of synchronized cells after cold-exposure
“ 2
J 4
, 6
s 8
1‘2 10
jMj 1-r
G?Ii
Fig. 2. Fraction colony survival after a I day exposure to 15°C as a function of age in the cell cycle. Vertical bars represent S.E. unless smaller than the points as plotted. The approximate durations of the fractions of the cell cycle are indicated at the foot of the graph
in the length of the cell cycle and that most of this increase in time occurs in Gl E&3-I6]” Sisken et al. 1131, with human amnion cells, found Gl and metaphase the most sensitive with 62 and prophase the least sensitive to changes in temperature (37 to 30°C). Sensitivity in their experiments referred to increased time for progression through that portion of the cell cycle. Slaapiro & Lubennikova 1141, using mouse fibroblast cells, found that the ratios of times for progression through the cycle at 25°C compared to 37°C were 40 for Gl and only 10 for G2. Watanabe [15], using mouse leukemic cells, that by lowering the temperature from 37 to 31°C the duration of the G 1 stage was increased 2.8 times. It is also interesting to note that plateau phase cells in monolayer cultures at 37°C accumulate in G 1 [t 7, 1E]. This effect of low temperatures on suggested to us that the survival of cells in GI might be affected. by temperature. The possibility that medium broke down after 5 days at 25°C (the highest temperature in our experiments) and thus led to decreased survival was eliminated in earlier experiments [2].
420 R. J. Nelson & J. Kruuv l-
O.l-
6days
0.01 1M1 / G1, J , s 0
2
4
6
\G2jMl 8
IO
Gl 12
14
IS 16
Figs 3-4. Abscissa: time in cell cycle (hours); oudin&e: fraction survival. Fig. 3. Fraction colony survival after exposure to 25°C as a function of age in the cell cycle. The period of exposure to 25°C is noted beside each curve. Vertical bars represent S.E. unless smaller than the points as plotted. The approximate durations of the fractions of the cell cycle are indicated at the foot of the graph.
it is probable that this is only of concern in the 25°C experiments. It has been noted that a decrease in temperature did not significantly influence the chronology of chromosome reproduction during the second half of S phase [22]. This is in agreement with the relative resistance in survival to sub-optimal temperature in the second half of S as shown in our experiments. A partial synchrony in G 1 in mouse fibroblast cells after 4 days at 25°C has been reported by Shapiro & Lubennikova [14]. From our data it appears that this synchrony did not result from preferential killing of S, G2 and M cells. It might be due to some progression through the cell cycle with a block in Gl since they concluded that all cells in G2 underwent division during 3 days of incubation at 25°C. Multiplicity studies with our cells revealed no division of cells at 25°C. It is also possible that the survival age-responsesof the two cell lines may differ. 1
1 9 days at ST
Many investigators believe that in the region between 25 and O”C, cell cycle movement is almost completely stopped [14, 16, 191.Certainly at 25°C there is cell movement in S and from S to G2 [12, 141.Also, anywhere from 3 to 25°C there is considerable DNA, RNA and protein synthesis-at least in the S phase [2, 20, 211. Whether the latter can be considered “movement” in the cell cycle by every criterion, i.e. shifts in ageresponse patterns for radiation [6], UV [IO] and freezing [l] survival, is not known at the present time. Thus the possibility must be considered that some cell cycle movement may take place during the exposure of the synchronized cultures to sub-optimal temperatures. From earlier labeling data [2, 211, Exptl Cell Res 70
y-Radiation survival (810rads)
0.01 0
M ' I G1 > ' 2 4
S 6
8
I10
G21Mj 12
Gl 14
s 16
18
Fig. 4. Determination of position in the cell cycle at various times after synchrony using a radiation survival experiment as a reference. The aooroximate durations of the fractions of the cell cycleA(as determined by pulse labeling with 3H-thymidine) are indicated at the foot of the graph. Vertical bars represent S.E. unless smaller than the points as plotted.
Survival of synchronized
When the age-distribution in the cell cycle is known [23], it is possible to weight this by the age-response of single cells [lo] to a particular cold “dose” from synchronous data and by summing the components, to arrive at the fraction of survival for an asynchronous population for that cold “dose”. Similar analyses have been done in radiobiology [IO]. When this is done, using our synchronous data, the theoretical values obtained agree closely with the asynchronous data in fig. 5 in all cases except experiments 1 and 2 at 5°C. The reason for the latter was the difficulty in reproducing the asynchronous curves (and presumably also the synchronous curves) from experiment to experiment at that temperature 121. This was due to the difficulty in precise temperature and especially humidity control at 5°C; therefore there was occasionally some water loss from the plates (< 10 “/o). The choice of cell selection procedure (i.e. medium “‘shake-off” versus precooling with trypsin “shake-off”) did not alter the response to cold. Results in this (Mruuv, unpublished) and other laboratories [24] have shown that neither the cell cycle length, the pattern of pulse labeling with 3M-thymide, the plating efficiency nor the X-ray age-response are altered by either precooling or the presence of trypsin. The medium “shake-off” was used exclusively in the later experiments because of higher mitotic indexes and shorter pre-experiment procedures. Examination of the lowest survival experiments in figs 1, 2 and 3 reveals that as the temperature decreases from 25 to 5°C the minimum in survival appears to shift from GI toward S. It is interesting to note that as far as survival after freezing in liquid nitrogen is concerned, G2 is the most sensitive phase in these cells when partially protected by Glycerol, although Gl or early S also exhibit sensitivity [l]. The results at
4.21
ceils afier cold-exposure
0.1 1
O.OO’ 01----7-;;
14
Fig. 5. Abscissa time (days); ordinate: fraction SIXvival. Fraction colony survival oi asynchronous cells after exposure formvarious intervals io YC, 15°C and 25°C. Vertical bars represent SE unless smaller than the points as plotted.
- 196°C hold even for unprotecte From these experiments it is difficult to make any comments about common met nisms between cold and freezing injuries. The presence of a sensitive phase; G 1; in both types of damage may suggest some common mechanisms whereas the absence of a sensitive phase during G2 in cold injury indicates vast differences between cold and freezing injuries. Nypertonicity, which is a major cause of death in freezing damage CS], probably does not play a large part in cold injury as the synchronous survival curves of cells exposed to hypertonic salt solutions alone resemble those of cells frozen to - 196°C [25]. Since the variation in response in the celi cycle after X-rays is at least partly due to free intracellular nonprotein sulfhydryl con-
422 R. J. Nelson h J. Kruuv tent [ll], it is possible that some internally produced compound may also exist for the differential protection in the cell cycle against cold damage. If so, isolation of this chemical may add to our understanding of the molecular nature of cold damage.
Sinclair, W K & Morton, R A, Biophys lo. (1965) 1. 11. Sinclair, W K. Radiat res 39 (1969) 135. Koch, C J & Kruuv, J, Radiat res’48 (1971) ::. Sisken, J E. Moraska, L & Kibby.-, S, Exntl _ * res 39 (1965) 103. I M & Lubennikova, E I, Exptl 14. Shapiro, res 49 (1968) 305.
This investigation was aided by a grant from the Ontario Department of University Affairs.
16. Rao, P N & Engelberg, J, Science 1092. 17. Chapman, J D, Todd, P & Sturrock, res 42 (1970) 590. 18. Tobey, R A & Ley, K D, J cell bio146 19. Shapiro, I M & Lubenniko, E I, Dokl
j 5 74. cell cell
15. Watanabe, I & Okada, S, J cell biol 32 (1967) 309.
REFERENCES
SSR
1. Koch, C J, Kruuv, J & Bruckschwaiger, C W, Exptl cell res 63 (1970) 476. 2. Nelson, R J, Kruuv, J, Koch, C J & Frey, H E, Exptl cell Fs 68 (1971) 247. 3. Michl, J, RezaEova, D & HoleEkova, E, Exptl cell res 44 (1968) 680. 4. HoleEkova, E, BaudySova, M & CinnerovB, 0, Exptl cell res 40 (1965) 396. 5. Mazur, P, Science 168 (1970) 939. 6. Kruuv, J & Sinclair, W K, Radiat res 36 (1968) 45.
Sinclair, W K, Biophysical aspects of radiation quality (Technical Report Series 58). p. 21-43. International Atomic Energy Agency, Vienna (1966). 8. Sinclair, W K & Morton, R A, Nature 199 (1963) 1158. 9. Nelson, R J, M Sci thesis, University of Waterloo, Waterloo, Ontario, Canada (1971).
7.
Exptl Cell Res 70
148 (1965) J, Radiat (1970) 151. akad nauk
169(1966)467.
20. Cerny, M, BaudySova, M & Holeckova, E, Exptl cell res 40 (1965) 673. 21. Kruuv, J & Sinclair, W K, Argonne National Laboratory Biological and Medical Research Division Annual Report, p. 6. US Atomic Energy Commission, Argonne, Ill. (1967). 22. Shapiro, I M & Polikarpova, S I. Chromosoma 27 fi96$409.
23.
-
.
Stanners, C P & Till, J E, Biochim biophys acta
24 37 (1960)406. Sinclair, W K, Japan j genet, suppl. 40 (1965) 141. 25. McGann, L E, Kruuv, J & Frey, H E, Cryobiology. In press.
Received June 2, 1971 Revised version received September 20, 1971
Experimental
SURVIVAL
OF SYNCHRONIZED
Department
of Physics,
MAMMALIAN
EXPOSURE
TO COLD
R. J.,NELSON
and J. KRUUV
University
of Waterloo,
Waterloo,
CELLS
Ontario,
FOLLO
Canada
SUMMARY Synchronized Chinese hamster cells (V79) exposed to suboptimal temperatures for 3-10 days exhibit variations in survival depending upon the position in the cell cycle in which the exposure was initiated. At 5 and 25°C cells are most sensitive in Gl and early S, while at 45°C 61 is the most susceptible phase. In all cases G2 and M cells are resistant to cold damage. Factors of up to 5 can be obtained between minimum and maximum survival in the cell cycle, these factors increasing with the time the cells are left at the temperature in question.
It has been found that survival of mammalian cells following freezing in liquid nitrogen is a function of the time in the cell cycle at which the freezing takes place [l]. It is also known that a fraction of the cell population dies following or during exposures of several days to temperatures of 3-10°C [2-41. Although there are several modes of damage to cells from freezing injury [5], none of the mechanisms for death or possible repair of damage have clearly been elucidated. The possibility exists that there may be some common mechanisms between cold and freezing injuries. With the above in mind, it was decided to investigate if the survival of synchronized mammalian cells following exposure to cold (5°C) varied in a similar fashion as exposure to liquid nitrogen. Experiments were also done at 15 and 25°C to determine if the pattern would shift. MATERIALS
AND METHODS
The cells employed were those of a subline (V79S171) derived from the V79 line of near-diploid
Chinese hamster lung cells. They were grown on plastic Petri dishes in-Eagle medium with 15 % fetal calf serum in a humid atmosphere of 5 % CO, and air. Under these conditions the cells grew ii log phase with a generation time of about 10 h, which was subdivided into Gl, S, G2, and M periods of 2-2&, 6-7,lt and $ h respectively. During a synchrony experiment the generation time was about 12 h. The deviation was due to trypsinization and frequent opening of the incubator during the experiment. These times have been determined autoradiographically, by liquid scintillating counting techniques, and by-monitoring the radiation colonysurvivalthroughout the cell cycle [S]. The plating efficiency was usually about 80 % for asynchronous populations and 60 % for synchronous populations with mitotic indexes of 65-85 % in the latter. The number of nonviable (nondividing) cells in the plated population, as determined by cinemicrography, was negligible [7:. Cells synchronized at or close to division were obtained by selecting for less firmly attached, dividing cells by using a combination of precooling and mecbanical shaking with a 0.035 % trypsin solution [6, 81. Many synchrony experiments were performed without the precooling (1 h cold shock) and with prewarmed medium used as the “shake-off” liquid instead of the trypsin solution. The latter technique yielded a lower mitotic yield but a higher mitotic index [9]. These synchronized cells were then inoculated into temperature (37°C) and pH (7.4) equilibrated medium in plastic Petri dishes and allowed to attach for 2 h in the 37°C incubator. At various times thereafter, three dishes were removed from the 37°C incubator and placed in a refrigerated CO, incubator which had been pre-set to the correct temperature. The CO, flow in the incubator was adjusted so that the correct pH range (7.4-7.5) was maintained, the latter being
41X
R. J. Nelson & J. Kruuv
Table 1. Experimental Expt 1 2 3 4 5 6 7 8
conditions of synchrony
Time (days) at y”C 5 I 1: 7 3 4 6
experiments
Y”C
“Shake-off” technique
Mitotic index ( %)
Mitotic yield ( %>
Plating efficiency ( %)
5 5 5 5 15 25 25 25
T&PC= Mb M M T&PC M T&PC M
60 70 75 87
3.4
45 48 36
70 G
65 0.9 3 3 0.9
i; 48 61 38
a Trypsin and precooling. b Medium.
monitored by a pH-meter. The dishes were kept at the test temperature for X number of days (X= constant). After this time, the plates were removed from the refrigerated incubator in the same time sequence in which they were put in, and placed in the 37°C incubator for colony growth. After an incubation period of 5 to 7 days, the colonies were stained with methylene blue and counted. Cell concentrations were adjusted at the time of inoculation to suit the treatment to which the cells were to be exposed, so that 100 to 200 colonies resulted per dish (100 mm diameter). The colony surviving fraction (with respect to the controls) for exposure of cells to temperature Y for X days was plotted versus time at 37°C after synchronization (i.e. time in the cell cycle). With precooling and trypsin “shake-off” the average cellular multiplicity in the first cell cycle was typically 1.8, whereas with no precooling and a medium “shakeoff” it was usually 1.6. The survival data were not corrected for the multiplicity [lo] during the first or subsequent cell cycles. To find the position in the cell cycle, the easiest and least expensive method was to do a synchronous radiation survival experiment with the same cell population and conditions that were used in the “cold” experiments. The cells were irradiated with a constant dose of 810 rad from an 800 Ci cesium source. Since the radiation survival as a function of position in cell cycle is well known for this cell subline [ll, 121, the approximate lengths of the fractions of the cell cycle (as previously determined by pulse labeling with 3H-thymidine) are indicated at the foot of the graphs. Standard errors (S.E.) were used to express experimental accuracy and were represented as vertical bars in all figures unless smaller than the points as plotted.
RESULTS Fraction colony 5°C for various Exptl Cell Res 70
survival for cells exposed to lengths of time as a function
of position in cell cycle is shown in fig. 1. The experimental conditions are listed in table 1. The longer exposures to 5°C yield lower survivals and the variations in the cell cycle become more pronounced. Gl
and early S are the most sensitive phases whereas M is the most resistant. Figs 2 and 3 show results for similar experiments at 15 and 25°C respectively. At 15°C the minimum is in G 1 and the maximum in M whereas at 25°C they are at Gl or early S and M respectively. The experimental conditions are listed in table 1. Many of the experiments listed in table 1 were done simultaneously using the same synchronous cell population. These were experiments number 4 and 8, 2 and 6, and finally 5 and 7. Fig. 4 shows the results of the radiation survival experiment done concurrently with experiment number 3 (i.e. 9 days at 5°C). The cell cycle age-responses and the approximate lengths of the fractions of the cell cycle are illustrated. Fig. 5 shows fraction colony survival of asychronous cells after exposure for various time intervals to 5°C 15°C and 25°C. The detailed experimental technique for these experiments is described elsewhere [2]. It should be noted that the length of exposure in experiments number 2, 3, 4, 5 and 8
Survival
placed the population on the linear portion of the semi-log survival curve shown in fig. 5. Experiments 1, 6 and 7 were performed in the “shoulder” region of these curves. Only the synchronous experiments performed in the linear region of the curves in fig. 5 show substantial variations in survival throughout the cell cycle although cyclic changes can be detected in all experiments. After 10 days at 5°C factors up to 5 can be obtained between minimum and maximum survival in the cell cycle. After 6 days at 25°C a factor of 4 was obtained.
1.0 0.9 0.8 0.7 1, 0.6 j 0.54 0.4 0.3
0.2 4
0.1 M J 0
DISCUSSION Various investigations with mammalian cells suggest that as the temperature decreases from 37 to 25°C there is a marked increase
0
2
4
6
8
IO
12
14
16
18
Figs 1-2. Abscissa: time in cell cycle (hours); ordinate: fraction survival. Fig. 1. Fraction colony survival after exposure to 5°C as a function of age in the cell cycle. The length of time the cells were exposed to 5°C is noted beside each curve. Vertical bars reoresent S.E. unless smaller than the points as plotted. The approximate durations of the fractions of the cell cycle (as determined by X-ray survival data) are indicated at the foot of the graph.
41
of synchronized cells after cold-exposure
“ 2
J 4
, 6
s 8
1‘2 10
jMj 1-r
G?Ii
Fig. 2. Fraction colony survival after a I day exposure to 15°C as a function of age in the cell cycle. Vertical bars represent S.E. unless smaller than the points as plotted. The approximate durations of the fractions of the cell cycle are indicated at the foot of the graph
in the length of the cell cycle and that most of this increase in time occurs in Gl E&3-I6]” Sisken et al. 1131, with human amnion cells, found Gl and metaphase the most sensitive with 62 and prophase the least sensitive to changes in temperature (37 to 30°C). Sensitivity in their experiments referred to increased time for progression through that portion of the cell cycle. Slaapiro & Lubennikova 1141, using mouse fibroblast cells, found that the ratios of times for progression through the cycle at 25°C compared to 37°C were 40 for Gl and only 10 for G2. Watanabe [15], using mouse leukemic cells, that by lowering the temperature from 37 to 31°C the duration of the G 1 stage was increased 2.8 times. It is also interesting to note that plateau phase cells in monolayer cultures at 37°C accumulate in G 1 [t 7, 1E]. This effect of low temperatures on suggested to us that the survival of cells in GI might be affected. by temperature. The possibility that medium broke down after 5 days at 25°C (the highest temperature in our experiments) and thus led to decreased survival was eliminated in earlier experiments [2].
420 R. J. Nelson & J. Kruuv l-
O.l-
6days
0.01 1M1 / G1, J , s 0
2
4
6
\G2jMl 8
IO
Gl 12
14
IS 16
Figs 3-4. Abscissa: time in cell cycle (hours); oudin&e: fraction survival. Fig. 3. Fraction colony survival after exposure to 25°C as a function of age in the cell cycle. The period of exposure to 25°C is noted beside each curve. Vertical bars represent S.E. unless smaller than the points as plotted. The approximate durations of the fractions of the cell cycle are indicated at the foot of the graph.
it is probable that this is only of concern in the 25°C experiments. It has been noted that a decrease in temperature did not significantly influence the chronology of chromosome reproduction during the second half of S phase [22]. This is in agreement with the relative resistance in survival to sub-optimal temperature in the second half of S as shown in our experiments. A partial synchrony in G 1 in mouse fibroblast cells after 4 days at 25°C has been reported by Shapiro & Lubennikova [14]. From our data it appears that this synchrony did not result from preferential killing of S, G2 and M cells. It might be due to some progression through the cell cycle with a block in Gl since they concluded that all cells in G2 underwent division during 3 days of incubation at 25°C. Multiplicity studies with our cells revealed no division of cells at 25°C. It is also possible that the survival age-responsesof the two cell lines may differ. 1
1 9 days at ST
Many investigators believe that in the region between 25 and O”C, cell cycle movement is almost completely stopped [14, 16, 191.Certainly at 25°C there is cell movement in S and from S to G2 [12, 141.Also, anywhere from 3 to 25°C there is considerable DNA, RNA and protein synthesis-at least in the S phase [2, 20, 211. Whether the latter can be considered “movement” in the cell cycle by every criterion, i.e. shifts in ageresponse patterns for radiation [6], UV [IO] and freezing [l] survival, is not known at the present time. Thus the possibility must be considered that some cell cycle movement may take place during the exposure of the synchronized cultures to sub-optimal temperatures. From earlier labeling data [2, 211, Exptl Cell Res 70
y-Radiation survival (810rads)
0.01 0
M ' I G1 > ' 2 4
S 6
8
I10
G21Mj 12
Gl 14
s 16
18
Fig. 4. Determination of position in the cell cycle at various times after synchrony using a radiation survival experiment as a reference. The aooroximate durations of the fractions of the cell cycleA(as determined by pulse labeling with 3H-thymidine) are indicated at the foot of the graph. Vertical bars represent S.E. unless smaller than the points as plotted.
Survival of synchronized
When the age-distribution in the cell cycle is known [23], it is possible to weight this by the age-response of single cells [lo] to a particular cold “dose” from synchronous data and by summing the components, to arrive at the fraction of survival for an asynchronous population for that cold “dose”. Similar analyses have been done in radiobiology [IO]. When this is done, using our synchronous data, the theoretical values obtained agree closely with the asynchronous data in fig. 5 in all cases except experiments 1 and 2 at 5°C. The reason for the latter was the difficulty in reproducing the asynchronous curves (and presumably also the synchronous curves) from experiment to experiment at that temperature 121. This was due to the difficulty in precise temperature and especially humidity control at 5°C; therefore there was occasionally some water loss from the plates (< 10 “/o). The choice of cell selection procedure (i.e. medium “‘shake-off” versus precooling with trypsin “shake-off”) did not alter the response to cold. Results in this (Mruuv, unpublished) and other laboratories [24] have shown that neither the cell cycle length, the pattern of pulse labeling with 3M-thymide, the plating efficiency nor the X-ray age-response are altered by either precooling or the presence of trypsin. The medium “shake-off” was used exclusively in the later experiments because of higher mitotic indexes and shorter pre-experiment procedures. Examination of the lowest survival experiments in figs 1, 2 and 3 reveals that as the temperature decreases from 25 to 5°C the minimum in survival appears to shift from GI toward S. It is interesting to note that as far as survival after freezing in liquid nitrogen is concerned, G2 is the most sensitive phase in these cells when partially protected by Glycerol, although Gl or early S also exhibit sensitivity [l]. The results at
4.21
ceils afier cold-exposure
0.1 1
O.OO’ 01----7-;;
14
Fig. 5. Abscissa time (days); ordinate: fraction SIXvival. Fraction colony survival oi asynchronous cells after exposure formvarious intervals io YC, 15°C and 25°C. Vertical bars represent SE unless smaller than the points as plotted.
- 196°C hold even for unprotecte From these experiments it is difficult to make any comments about common met nisms between cold and freezing injuries. The presence of a sensitive phase; G 1; in both types of damage may suggest some common mechanisms whereas the absence of a sensitive phase during G2 in cold injury indicates vast differences between cold and freezing injuries. Nypertonicity, which is a major cause of death in freezing damage CS], probably does not play a large part in cold injury as the synchronous survival curves of cells exposed to hypertonic salt solutions alone resemble those of cells frozen to - 196°C [25]. Since the variation in response in the celi cycle after X-rays is at least partly due to free intracellular nonprotein sulfhydryl con-
422 R. J. Nelson h J. Kruuv tent [ll], it is possible that some internally produced compound may also exist for the differential protection in the cell cycle against cold damage. If so, isolation of this chemical may add to our understanding of the molecular nature of cold damage.
Sinclair, W K & Morton, R A, Biophys lo. (1965) 1. 11. Sinclair, W K. Radiat res 39 (1969) 135. Koch, C J & Kruuv, J, Radiat res’48 (1971) ::. Sisken, J E. Moraska, L & Kibby.-, S, Exntl _ * res 39 (1965) 103. I M & Lubennikova, E I, Exptl 14. Shapiro, res 49 (1968) 305.
This investigation was aided by a grant from the Ontario Department of University Affairs.
16. Rao, P N & Engelberg, J, Science 1092. 17. Chapman, J D, Todd, P & Sturrock, res 42 (1970) 590. 18. Tobey, R A & Ley, K D, J cell bio146 19. Shapiro, I M & Lubenniko, E I, Dokl
j 5 74. cell cell
15. Watanabe, I & Okada, S, J cell biol 32 (1967) 309.
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Received June 2, 1971 Revised version received September 20, 1971