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Temperature compensation in the mammalian cell cycle
Copyright @I 1982 by Academic F’ress, Inc. All rights of reproduction in any form merved 0014-4827/82/@80307-07$02.00/O
Experimental Cell Research 140 (1982) 307-313
TEMPERATURE COMPENSATION IN THE MAMMALIAN CELL CYCLE ROBERT R. KLEVECZ
and GARY A. KING
City of Hope Research Institute, Division of Biology, Duarte, CA 91010, USA
SUMMARY Random and synchronous V79 cells were shifted from 37.W to temperatures between 29” and 41°C. Intermitotic time determinations of random cultures showed an increase in generation time and a broadening in the distribution of generation times in cells whose cycle spanned the temperature shift, but only a slight increase in generation time after one generation at temperatures between 34”-4o”C. At 33.W and below there was a stepwise increase in generation time. When cells grown at non-standard temperatures were allowed to habituate for 48 h at the altered temperature prior to analysis, the increase in median intermitotic time was slightly less in comparison to analyses done after only one generation following the temperature step. The Qle for cell division of cells growing at temperatures from 38 to 40°C was between 1.15 and 1.26, suggesting that the mammalian cell cycle is temperature compensated over a limited (6-7°C) temperature span. Mammalian cells in culture appear to have the same capacity for temperature compensation in their cell cycle as do unicellular eukaryotes. The fact that cycle time at lower temperatures increases in a discrete manner is taken as evidence for a quanta1 clock.
Temperature compensation is the property of an ensemble of reactions that allows them to maintain a net reaction rate that is only slightly changed with changing temperature. It is an empirically important generalization in studies of circadian rhythmicity, and is often used to establish that a particular rhythm is under control of the biological clock [l-3]. The capacity to maintain an accurate time sense through the range of temperatures likely to be encountered is of obvious importance in poikilotherms [2]. In homeotherms the need for a temperature-compensated clock is not obvious, but it nevertheless appears that the capacity exists, as Rawson [4] showed some years ago in several rodent species whose homeothermic mechanisms had been abrogated by pentobarbital injections. In unicellular eukaryotes with generation times close to or greater than 24 h, cell
division appears to be temperature compensated and gated by the circadian rhythm. In consequence, the fraction of the population that divides in each circadian burst may be reduced with decreasing temperature, even though the period of the circadian cell division rhythm is little changed with changing temperature. In Euglena, Edmunds [4-6] has found that the rhythmic expression of cell division can be maintained at 22.9 h at temperatures from 13” to 19”C, while Gonyaulax has a Qlo of 0.9 for cell division occurring over a range of temperatures from 18.5” to 25°C. The situation in the cell cycle of cultured animal cells is not so clear. Sisken [7] found little change in G2+prophase with changing temperature, but noted a marked increase in [3H]thymidine. incorporation rate, not necessarily S-phase duration, with increasing temperature. In contrast, Wimber [S] in exExp Cell Res 140 (1982)
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amining the cycle of higher plants found S phase to be relatively insensitive to temperature changes. To explain enzyme oscillations and periodic gene expression in animal cells we have suggested that the cell cycle was under control of a timekeeping oscillator, whose period, Gq, is an integral submultiple of the cell generation time, and constant, or nearly so, in all cells [9-l 11.In extending an earlier study of temperature effects on synchronous cultures [9], we have investigated intermitotic times (IMT) of random cultures and the population median of division waves in synchronous V79 cells at temperatures between 29“ and 41°C. If the cell cycle itself were perfectly temperature compensated, then growth at any temperature in the viable range might be expected to yield unaltered generation times. Alternatively, if the putative time-keeping bscillator [9-l l] is temperature compensated and the cycle is not, then the occurrence of cell divisions should be gated at multiples of the oscillator period. Finally, if neither the cycle nor the oscillator are compensated, then the change in generation time with temperature should follow the simple kinetics predicted by the van’t Hoff equation. We have found that the cell cycle in cultured V79 cells increases with a Q10 of between 1.16 and 1.25 over the range of temperatures from 33.5 to 4OS”C, that the cell cycle increases more rapidly with decreasing temperature only at temperatures approaching the limits of reproductive viability, and that this increase in generation time tends to occur in a quantal fashion with a period of approx. 4.5 h. MATERIALS
AND METHODS
Cell lines Clonal cell lines derived from the V79 line of Chinese hamster lung fibroblasts were propagated in McCoy’s Exp Cell Res 140 (1982)
5a medium supplemented with 10% fetal bovine serum, as described previously [9, 121.This cell line displays a modal 7.75-8.25 h intermitotic generation time, median 8.25-8.75 h intermitotic time, and modal 9.25 h generation time, following mitotic selection synchrony. As characterized in this Laboratory by flow microfluorometric (FMF), autoradiographic, and [3H]thymidine incorporation rate analyses, the cell cycle could be subdivided as follows: Gl, 2.25 h; S, 4.5 h; G2+M, 1.5 h.
Synchrony
and cell culture
V79 cells were synchronized by mitotic selection from roller bottles b; manual selection, as described in detail elsewhere r9. 101. Three roller bottles were inoculated with 7:5x 16 cells/roller bottle in 100 ml McCoy’s 5a medium. Mitotic selections were performed 18 h after subculture by first removing and filtering the inoculating medium. This conditioned, cell-&ee medium was used for all subsequent operations. Prior to selection, two changes of medium were rinsed through the bottles. A rinse to remove unattached cells was followed by rotation of the bottles at 250 rpm in 30 ml medium for 2 min. This first election was discarded and after an additional t h of rotation at slow speed (0.5 rpm) a second selection was performed. These mitotic cells in 30 ml medium were inoculated into 2-4 25 cm* flasks at a concentration of 5 x l(yLcells/cm*, 50-100 cells/microscopic field. Using this method the mitotic index was 9%99%. and firstgeneration time coefficients of variation (COV) were 5-15% for V79. No change in pH was observed as a consequence of changing temperatures.
Time-lapse video tape analysis Video tapes were made using RCA TC lOOS/Ol and Hitachi HV-16S low liiht cameras and Hitachi SV512 and Panasonic NV-8030 time-lapse video tape recorders at 50-lwfold time compression [g-lo]. Cells were examined using a Nikon MS inverted microscope with a heat filter and Wrattan 25 visible light filter. Illumination was continuous at 0.2 ft cdl. In earlier experiments less sensitive cameras (>I.3 ft cdl) were used, with the result that the response to temperature shifts yielded a complex IMT result. In the earliest experiments cultures were fixed and stained at the termination of the recording to determine whether growth inhibition from the continuous microscope illumination had occurred. At high light intensities and in the absence of either the heat filter or the Wrattan filter a zone of partial growth inhibition coherent with the illuminated portion of the flask could be observed. It should be mentioned that the absence of obvious growth inhibition does not guarantee unperturbed growth. Temperature was maintained to +0.2”C using YSI model 73A temperature sensor/recorders interfaced to a Napco incubator, or Freas 818 incubators interfaced with an Intel Intercept microprocessor. Tapes were analysed for the occurrence of anaphase figures, which were the most characteristic and easily identified mitotic stage. The duration of anaphase was brief and variation in the duration of anaphase was also less than that of metaphase. In low temperature
Temperature
compensation
in thi? mammalian
cell cycle
309
experiments, mitotic stages were prolonged so that analysis became difftcult. At elevated temperatures (M@C) the third and fourth divisions were often multipolar, usually tripolar. At extreme high (>4l”C) and low temperatures (<30“(Z) only a small fraction of the cells divided and consequently these results were not included in the analysis,
RESULTS Temperature cell cycle
compensation
5
in the
The effect on cell generation time of a shift in temperature was analyzed by examining individual control and treated cultures for each temperature shift. This was necessary because of the small variations in control cell generation times which occurred over the 4-year span of these experiments. Timelapse recordings were made of randomly growing cells beginning l&24 h prior to the shift. The IMT of all cells dividing during this period were determined and pedigree charts constructed for each cell. In this way the IMT of cells growing at the normal incubator temperature, 37.X, their progeny, whose cycles spanned the temperature shift, and the progeny of these cells now growing at a new constant temperature, could be compared. In addition, these cultures were repassaged and the final generation time determinations made using cells that had been continuously grown at the test temperature for 48 h. In an earlier study mitotic cells were selected and these synchronous cultures were immediately shifted to temperatures from 33” to 41°C [9]. In that case, a quantized increase in the median generation time of the population (T,) was observed at temperatures below 35°C. At that time it was suggested that the increase in generation time was due to the gating of cells into the chromosome replication cycle and mitosis by a time-keeping oscillator with a period less than the cell cycle. Two problems with
10 IMT
15
(h)
I. Generation time increase after temperature downshift. Randomly growing V79 cells were scored to determine IMT prior to and following a temperature step from 37.5” to 33°C. Cell IMT were presented in three arouns: (1) the generation times of the nroaenitor cells-dividing ‘at 3f5”C, prior to the temperature steo (closed blocks, median IMT 7.3 h): (2) the aeneration time of cells whose cycles spanned the temperature step (open blocks); and (3) the first generation progeny dividing after one complete cell cycle at 33°C (cross-hatched blocks, median IMT 11.28 h).
Fig.
that study weakened the interpretation. Only synchronous populations were examined so that in the strictest sense the generation times of individual cells were not determined and, since the cells were examined immediately following selection, the discrete jump in generation time could have been due to the perturbation and consequent phase shift that accompanies a temperature step [9-l I]. Here, that quanta1 increase is observed again in synchronous cultures and in addition the step-wise increase in generation time is found to occur in the IMT analysis of individual cells growing at the new steady state temperature for several generations (fig. 1). There was only a slight increase in cycle time or IMT in cells growing at temperatures between 34” and 40°C in three of the four test conditions: (1) synchronous cultures shifted to the new temperature at the time of mitotic selection (fig. 2a); (2) cultures undergoing the first and second division after the temperature shift, whose entire cell cycle was spent at the new steadv state temperature (fig. 2b); Exp Cell Res I40 (1982)
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Klevecz and King
10
5
IMT or GENERATION TIME (h’) Fig. 2. Temperature compensation in the V79 cell
cycle. IMT of random exnonentisl V79 cells and aene&ion times of synchrkous cultures were dGermined using time-lapse video microscopy as described in Methods. a, Median generation time of mitotically selected cells shifted to the new temperature at mitosis; 6, median IMT of cells in the first and second generations after shift to the new steady state temperature; c, median IMT of cells after 48 h of adaptation to the new steady state temperature.
plete and the Q10 closest to 1.0 in cells grown at the new steady state for 48 h prior to analysis (fig. 2~). However, over the range where cell cycle time is compensated, 33.5”-4OS”C, these cells are not signilicantly better compensated than those grown at the new steady state for just two generations (fig. 2b). This behavior and the fact that there was no significant increment in the fraction of the population that divided at the new temperature suggests that this is an adaptive process and not selection. As shown in the histogram of fig. 3a and the summation of fig. 3b, where the results of three independent temperature shift experiments are plotted together, there is little increase in IMT as temperature is reduced from 40” to 34°C. The Q10for a cell cycle time of 8.5 h at temperatures between 40” and 34°C is between 1.16 and 1.25 in all three experimental series.
and (3) cultures which had been adapted to A quanta1 change in generation time: the new temperatures for 48 h prior to anal- the clock in animal cells ysis (fig. 2~). In cells whose cycle spanned At temperatures of 33.5”C and less, generathe temperature shift, there was a greater tion time increases, though not in a conincrease in generation time compared with tinuous manner. A step from 37.5 to 33 steady state growth, the COV of generation or 32°C gives a -4+ h increase in generatime was likewise greater, and there was no tion time, whereas a step down to 31 or evidence for a quanta1 increase in IMT (fig. 30°C gives an 8.5 h increase in generation 3d). This behavior is consistent with the time (fig. 3a). Rather than attempt to vary notion that cells are differentially phase re- temperature in smaller increments between sponsive to temperature steps depending 34” and 30°C and then to argue for a disupon the time in the cycle at which they ex- continuous fit to generation time changes, perience the step [9-111. The synchronized we chose instead to pool all generation cultures also experienced a temperature times taken from cells at steady state step in the first cycle, but prior results [9, growth at temperatures between 41” and lo] have indicated that mitosis is a point 30°C from a single experiment series (fig. of minimum sensitivity to temperature 3~). These data were then analysed for pepulses. Nevertheless it does appear that the riodicity in comparison with IMT of cells in temperature compensation to extreme tem- this population, whose cycle spanned the peratures is poorest in synchronous cul- temperature shift and which did not show tures (fig. 2a). One might expect that tem- any apparent periodicity (fig. 3d, f). This perature compensation would be more com- was done using Fourier spectral analysis. Exp Cell Res 140 (1982)
Temperature compensation in the mammalian cell cycle
a
3 11
b
32 -
5
10
15
Ilvif
20
25
fhf
d
5
IO
15
20
IMT (hf
25
IMT (hi
3216
PERIOD IN HOURS Fig. 3. A quanta1 change in generation time. (a) IMT
histogram for cells growing for one or two generations at the new steady state temperatures. Histograms of experiments performed at 36” and 38°C were left out and alternate histograms cross-hatched for clarity. Taken from (b) ser. 3, (6) IMT of three series of experiments performed between 1977and 1981were normalized to the median control (37.5“-38°C) IMT and plotted together. Numbers indicate modal IMT of members of each of the three series. -, Control IMT;
IO 76
5
4
3
2
PERIOD IN HOURS
--- , quantai increments assuming Gq=T,/Z. (c) Sum Of all steady state IMT histograms for experiments in (a, 6) ser. 3 grown at temperatures between 30’ and 41°C. Plots are normalized to the median control IMT of the population of 7.51 h. (d) Sum of a11histograms in (a, b) ser. 3 for cells whose cycle spanned the temperature shiR for temperatures between 3fP and 41°C. Plots are normalized to the median control IMT of the population. (e, ff Fourier power spectrum analysis of data presented in fc) and (d).
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Klevecz and King
In fig. 3c a histogram of the data from fig. 3a (also shown as experiment 3 of fig. 3b) are plotted together with the Fourier spectral analysis (fig. 3e). A major component appears at 4.6 h. The peak at 32 h is an artifact of reflection.
DISCUSSION In unicellular eukaryotes expressing their circadian growth mode it is commonly observed that reduction in temperature from the optimal yields a reduced fraction of cells undergoing synchronous division at 24 h intervals, but only a small change in the 7 of the culture [6, 131. In other words, division is quantized in circadian intervals, even though only a fraction of the cells divide in any 24 h period. In characterizing the circadian clock in the unicellular eukaryote Euglena, Edmunds found that the cell division cycle was temperature compensated over a 7°C temperature range from 13 to 19°C [6]. However, Bruce & Pittendrigh [2] also using Euglena showed that phototaxic rhythms were temperature compensated over a much wider range of temperatures from 16” to 33°C suggesting that certain isolated physiological and biochemical functions may be better temperature compensated than is the cell cycle, which represents the end product of a vast reaction scheme. There has been a continuing controversy over whether the cell cycle is a ‘clocked’ property of the cell [5, 12, 141. Though it is possible to construct a network of coupled reactions, whose net reaction rate shows relative temperature independence [ 151, such behavior, excluding photochemical reactions, is the exception to the rule of temperature dependence. Indeed the biological rhythm literature is the extant example of the temperature compensation Exp Cell Res 140 (1982)
phenomenon. Even so it is questionable whether the notion of compensation can be applied to the organism as a whole. For example in D. pseudoobscura the pupal eclosion rhythm is considered to be temperature compensated, because flies will emerge at the same subjective time of day, independent of the temperature at which they are raised, even though the total development time increases with decreasing temperatures [ 11. In the case of unicellular organisms growing in their circadian mode with a generation time equal to or greater than 24 h, and in the regrettably few instances where the population doubling time has been measured [6, 131, doubling time and presumably cell cycle time increases with decreasing temperature, and only the rhythm which gates cell division shows temperature compensation. We suggest that an analogous situation occurs in mammalian cells growing in an ultradian mode (with a generation time significantly less than 24 h) and that the time-keeping oscillator, which is here thought to be an integral submultiple of 24 h and of the cell cycle, similarly gates cell division in -44.5 h intervals. In addition we find that over a limited range of temperatures the cell cycle is itself temperature compensated. The need for temperature compensation in poikiiothermic organisms is readily apparent and can be rationalized on evolutionary grounds. It is regarded by Pittendrigh [l] as the central element of a biological clock and of such importance as to be likely to arise by a number of mechanisms. The existence of temperature compensation in a mammalian cell where present need is not obvious suggests its early evolutionary origin. If the clock evolved at a sufficiently early time, then certain vestiges of this primitive clock may be ‘frozen’ in the chemistry of modern organisms.
Temperature
compensation
Based on the work presented here we are led to conclude that the animal cell cycle has some capacity for temperature compensation and is as ‘clocked’ as that of unicellular eukaryotes. The span of temperatures over which compensation holds, while narrow in comparison to many rhythmic functions of poikilothermic metazoans, is much the same as that seen in unicellular eukaryotes, where temperature compensation is considered to be an established fact. More significantly we can argue that the underlying oscillator which times cell division and gives rise to quantizement of generation times is more perfectly temperature compensated. By analogy with circadian systems the underlying oscillator may compensate over a broader span of temperatures, but cell division, while gated by the clock, can also be blocked at lower temperatures by the failure of downstream, non-clock events to be completed in time to make the gate. This work was supported in part by grants GM26015 and AGO0434 from the NIH and by the Morley Benjamm Research Fund.
Prmted
I” Sweden
in the mammalian
cell cycle
3 13
REFERENCES 1. I;i6(:ydrigh, C S, Proc natl acad sci US 40 (1954) 2. Bruce, V G & Pittendrigh, C S, Proc natl acad sci US 42 (1956) 676. 3. Rawson, K S, Cold Spring Harbor symp quant biol 25 (1960) 105.
4. Sweeney, B Y & Hastings, J W, J protozool 5 (1958) 217. 5. Edmunds, L N & Adams, K J, Science 211 (1981) 1002. 6. Edmunds, L N, Chuann, L, Jarrett, R M & Terry, 0 W, J interdiscipl cycle res 2 (1972) 121. 7. Sisken, J E, Exp cell res 39 (1965) 103. 8. Wimber, D E, Am j bot 53 (1966) 21. 9. Klevecz, R R, Proc natl acad sci US 73 (1976) 4012. 10. Klevecz, R R, King, G A & Shymko, R M, J supramolec struct 14 (1980) 329. 11. Shymko, R M & Klevecz, R R, Biomathematics and cell kinetics (ed M Rotenberg) p. 329. Elsevier/North-Holland Biomedical Press, Amsterdam (1980). 12. Klevecz, R R, Kros, J & King, G A, Cytogenet cell genet 26 (1980) 236. 13. Chisholm, SW, Primary productivity in the sea (ed P Falkowski) p. 281. Plenum, New York (1980). 14. Winfree, A T, The geometry of biological time. Springer-Verlag, New York (1980). 15. Pavlidis, T, Biological oscillators: their mathematical analysis. Academic Press, New York (1973).
Received October 20, 1981 Revised version ._. received .^ .^-- March 8, 1982 Accepted March 10, lYM2
Em Cell Rrs 140 (19821
Experimental Cell Research 140 (1982) 307-313
TEMPERATURE COMPENSATION IN THE MAMMALIAN CELL CYCLE ROBERT R. KLEVECZ
and GARY A. KING
City of Hope Research Institute, Division of Biology, Duarte, CA 91010, USA
SUMMARY Random and synchronous V79 cells were shifted from 37.W to temperatures between 29” and 41°C. Intermitotic time determinations of random cultures showed an increase in generation time and a broadening in the distribution of generation times in cells whose cycle spanned the temperature shift, but only a slight increase in generation time after one generation at temperatures between 34”-4o”C. At 33.W and below there was a stepwise increase in generation time. When cells grown at non-standard temperatures were allowed to habituate for 48 h at the altered temperature prior to analysis, the increase in median intermitotic time was slightly less in comparison to analyses done after only one generation following the temperature step. The Qle for cell division of cells growing at temperatures from 38 to 40°C was between 1.15 and 1.26, suggesting that the mammalian cell cycle is temperature compensated over a limited (6-7°C) temperature span. Mammalian cells in culture appear to have the same capacity for temperature compensation in their cell cycle as do unicellular eukaryotes. The fact that cycle time at lower temperatures increases in a discrete manner is taken as evidence for a quanta1 clock.
Temperature compensation is the property of an ensemble of reactions that allows them to maintain a net reaction rate that is only slightly changed with changing temperature. It is an empirically important generalization in studies of circadian rhythmicity, and is often used to establish that a particular rhythm is under control of the biological clock [l-3]. The capacity to maintain an accurate time sense through the range of temperatures likely to be encountered is of obvious importance in poikilotherms [2]. In homeotherms the need for a temperature-compensated clock is not obvious, but it nevertheless appears that the capacity exists, as Rawson [4] showed some years ago in several rodent species whose homeothermic mechanisms had been abrogated by pentobarbital injections. In unicellular eukaryotes with generation times close to or greater than 24 h, cell
division appears to be temperature compensated and gated by the circadian rhythm. In consequence, the fraction of the population that divides in each circadian burst may be reduced with decreasing temperature, even though the period of the circadian cell division rhythm is little changed with changing temperature. In Euglena, Edmunds [4-6] has found that the rhythmic expression of cell division can be maintained at 22.9 h at temperatures from 13” to 19”C, while Gonyaulax has a Qlo of 0.9 for cell division occurring over a range of temperatures from 18.5” to 25°C. The situation in the cell cycle of cultured animal cells is not so clear. Sisken [7] found little change in G2+prophase with changing temperature, but noted a marked increase in [3H]thymidine. incorporation rate, not necessarily S-phase duration, with increasing temperature. In contrast, Wimber [S] in exExp Cell Res 140 (1982)
308
Klevecz and King
amining the cycle of higher plants found S phase to be relatively insensitive to temperature changes. To explain enzyme oscillations and periodic gene expression in animal cells we have suggested that the cell cycle was under control of a timekeeping oscillator, whose period, Gq, is an integral submultiple of the cell generation time, and constant, or nearly so, in all cells [9-l 11.In extending an earlier study of temperature effects on synchronous cultures [9], we have investigated intermitotic times (IMT) of random cultures and the population median of division waves in synchronous V79 cells at temperatures between 29“ and 41°C. If the cell cycle itself were perfectly temperature compensated, then growth at any temperature in the viable range might be expected to yield unaltered generation times. Alternatively, if the putative time-keeping bscillator [9-l l] is temperature compensated and the cycle is not, then the occurrence of cell divisions should be gated at multiples of the oscillator period. Finally, if neither the cycle nor the oscillator are compensated, then the change in generation time with temperature should follow the simple kinetics predicted by the van’t Hoff equation. We have found that the cell cycle in cultured V79 cells increases with a Q10 of between 1.16 and 1.25 over the range of temperatures from 33.5 to 4OS”C, that the cell cycle increases more rapidly with decreasing temperature only at temperatures approaching the limits of reproductive viability, and that this increase in generation time tends to occur in a quantal fashion with a period of approx. 4.5 h. MATERIALS
AND METHODS
Cell lines Clonal cell lines derived from the V79 line of Chinese hamster lung fibroblasts were propagated in McCoy’s Exp Cell Res 140 (1982)
5a medium supplemented with 10% fetal bovine serum, as described previously [9, 121.This cell line displays a modal 7.75-8.25 h intermitotic generation time, median 8.25-8.75 h intermitotic time, and modal 9.25 h generation time, following mitotic selection synchrony. As characterized in this Laboratory by flow microfluorometric (FMF), autoradiographic, and [3H]thymidine incorporation rate analyses, the cell cycle could be subdivided as follows: Gl, 2.25 h; S, 4.5 h; G2+M, 1.5 h.
Synchrony
and cell culture
V79 cells were synchronized by mitotic selection from roller bottles b; manual selection, as described in detail elsewhere r9. 101. Three roller bottles were inoculated with 7:5x 16 cells/roller bottle in 100 ml McCoy’s 5a medium. Mitotic selections were performed 18 h after subculture by first removing and filtering the inoculating medium. This conditioned, cell-&ee medium was used for all subsequent operations. Prior to selection, two changes of medium were rinsed through the bottles. A rinse to remove unattached cells was followed by rotation of the bottles at 250 rpm in 30 ml medium for 2 min. This first election was discarded and after an additional t h of rotation at slow speed (0.5 rpm) a second selection was performed. These mitotic cells in 30 ml medium were inoculated into 2-4 25 cm* flasks at a concentration of 5 x l(yLcells/cm*, 50-100 cells/microscopic field. Using this method the mitotic index was 9%99%. and firstgeneration time coefficients of variation (COV) were 5-15% for V79. No change in pH was observed as a consequence of changing temperatures.
Time-lapse video tape analysis Video tapes were made using RCA TC lOOS/Ol and Hitachi HV-16S low liiht cameras and Hitachi SV512 and Panasonic NV-8030 time-lapse video tape recorders at 50-lwfold time compression [g-lo]. Cells were examined using a Nikon MS inverted microscope with a heat filter and Wrattan 25 visible light filter. Illumination was continuous at 0.2 ft cdl. In earlier experiments less sensitive cameras (>I.3 ft cdl) were used, with the result that the response to temperature shifts yielded a complex IMT result. In the earliest experiments cultures were fixed and stained at the termination of the recording to determine whether growth inhibition from the continuous microscope illumination had occurred. At high light intensities and in the absence of either the heat filter or the Wrattan filter a zone of partial growth inhibition coherent with the illuminated portion of the flask could be observed. It should be mentioned that the absence of obvious growth inhibition does not guarantee unperturbed growth. Temperature was maintained to +0.2”C using YSI model 73A temperature sensor/recorders interfaced to a Napco incubator, or Freas 818 incubators interfaced with an Intel Intercept microprocessor. Tapes were analysed for the occurrence of anaphase figures, which were the most characteristic and easily identified mitotic stage. The duration of anaphase was brief and variation in the duration of anaphase was also less than that of metaphase. In low temperature
Temperature
compensation
in thi? mammalian
cell cycle
309
experiments, mitotic stages were prolonged so that analysis became difftcult. At elevated temperatures (M@C) the third and fourth divisions were often multipolar, usually tripolar. At extreme high (>4l”C) and low temperatures (<30“(Z) only a small fraction of the cells divided and consequently these results were not included in the analysis,
RESULTS Temperature cell cycle
compensation
5
in the
The effect on cell generation time of a shift in temperature was analyzed by examining individual control and treated cultures for each temperature shift. This was necessary because of the small variations in control cell generation times which occurred over the 4-year span of these experiments. Timelapse recordings were made of randomly growing cells beginning l&24 h prior to the shift. The IMT of all cells dividing during this period were determined and pedigree charts constructed for each cell. In this way the IMT of cells growing at the normal incubator temperature, 37.X, their progeny, whose cycles spanned the temperature shift, and the progeny of these cells now growing at a new constant temperature, could be compared. In addition, these cultures were repassaged and the final generation time determinations made using cells that had been continuously grown at the test temperature for 48 h. In an earlier study mitotic cells were selected and these synchronous cultures were immediately shifted to temperatures from 33” to 41°C [9]. In that case, a quantized increase in the median generation time of the population (T,) was observed at temperatures below 35°C. At that time it was suggested that the increase in generation time was due to the gating of cells into the chromosome replication cycle and mitosis by a time-keeping oscillator with a period less than the cell cycle. Two problems with
10 IMT
15
(h)
I. Generation time increase after temperature downshift. Randomly growing V79 cells were scored to determine IMT prior to and following a temperature step from 37.5” to 33°C. Cell IMT were presented in three arouns: (1) the generation times of the nroaenitor cells-dividing ‘at 3f5”C, prior to the temperature steo (closed blocks, median IMT 7.3 h): (2) the aeneration time of cells whose cycles spanned the temperature step (open blocks); and (3) the first generation progeny dividing after one complete cell cycle at 33°C (cross-hatched blocks, median IMT 11.28 h).
Fig.
that study weakened the interpretation. Only synchronous populations were examined so that in the strictest sense the generation times of individual cells were not determined and, since the cells were examined immediately following selection, the discrete jump in generation time could have been due to the perturbation and consequent phase shift that accompanies a temperature step [9-l I]. Here, that quanta1 increase is observed again in synchronous cultures and in addition the step-wise increase in generation time is found to occur in the IMT analysis of individual cells growing at the new steady state temperature for several generations (fig. 1). There was only a slight increase in cycle time or IMT in cells growing at temperatures between 34” and 40°C in three of the four test conditions: (1) synchronous cultures shifted to the new temperature at the time of mitotic selection (fig. 2a); (2) cultures undergoing the first and second division after the temperature shift, whose entire cell cycle was spent at the new steadv state temperature (fig. 2b); Exp Cell Res I40 (1982)
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Klevecz and King
10
5
IMT or GENERATION TIME (h’) Fig. 2. Temperature compensation in the V79 cell
cycle. IMT of random exnonentisl V79 cells and aene&ion times of synchrkous cultures were dGermined using time-lapse video microscopy as described in Methods. a, Median generation time of mitotically selected cells shifted to the new temperature at mitosis; 6, median IMT of cells in the first and second generations after shift to the new steady state temperature; c, median IMT of cells after 48 h of adaptation to the new steady state temperature.
plete and the Q10 closest to 1.0 in cells grown at the new steady state for 48 h prior to analysis (fig. 2~). However, over the range where cell cycle time is compensated, 33.5”-4OS”C, these cells are not signilicantly better compensated than those grown at the new steady state for just two generations (fig. 2b). This behavior and the fact that there was no significant increment in the fraction of the population that divided at the new temperature suggests that this is an adaptive process and not selection. As shown in the histogram of fig. 3a and the summation of fig. 3b, where the results of three independent temperature shift experiments are plotted together, there is little increase in IMT as temperature is reduced from 40” to 34°C. The Q10for a cell cycle time of 8.5 h at temperatures between 40” and 34°C is between 1.16 and 1.25 in all three experimental series.
and (3) cultures which had been adapted to A quanta1 change in generation time: the new temperatures for 48 h prior to anal- the clock in animal cells ysis (fig. 2~). In cells whose cycle spanned At temperatures of 33.5”C and less, generathe temperature shift, there was a greater tion time increases, though not in a conincrease in generation time compared with tinuous manner. A step from 37.5 to 33 steady state growth, the COV of generation or 32°C gives a -4+ h increase in generatime was likewise greater, and there was no tion time, whereas a step down to 31 or evidence for a quanta1 increase in IMT (fig. 30°C gives an 8.5 h increase in generation 3d). This behavior is consistent with the time (fig. 3a). Rather than attempt to vary notion that cells are differentially phase re- temperature in smaller increments between sponsive to temperature steps depending 34” and 30°C and then to argue for a disupon the time in the cycle at which they ex- continuous fit to generation time changes, perience the step [9-111. The synchronized we chose instead to pool all generation cultures also experienced a temperature times taken from cells at steady state step in the first cycle, but prior results [9, growth at temperatures between 41” and lo] have indicated that mitosis is a point 30°C from a single experiment series (fig. of minimum sensitivity to temperature 3~). These data were then analysed for pepulses. Nevertheless it does appear that the riodicity in comparison with IMT of cells in temperature compensation to extreme tem- this population, whose cycle spanned the peratures is poorest in synchronous cul- temperature shift and which did not show tures (fig. 2a). One might expect that tem- any apparent periodicity (fig. 3d, f). This perature compensation would be more com- was done using Fourier spectral analysis. Exp Cell Res 140 (1982)
Temperature compensation in the mammalian cell cycle
a
3 11
b
32 -
5
10
15
Ilvif
20
25
fhf
d
5
IO
15
20
IMT (hf
25
IMT (hi
3216
PERIOD IN HOURS Fig. 3. A quanta1 change in generation time. (a) IMT
histogram for cells growing for one or two generations at the new steady state temperatures. Histograms of experiments performed at 36” and 38°C were left out and alternate histograms cross-hatched for clarity. Taken from (b) ser. 3, (6) IMT of three series of experiments performed between 1977and 1981were normalized to the median control (37.5“-38°C) IMT and plotted together. Numbers indicate modal IMT of members of each of the three series. -, Control IMT;
IO 76
5
4
3
2
PERIOD IN HOURS
--- , quantai increments assuming Gq=T,/Z. (c) Sum Of all steady state IMT histograms for experiments in (a, 6) ser. 3 grown at temperatures between 30’ and 41°C. Plots are normalized to the median control IMT of the population of 7.51 h. (d) Sum of a11histograms in (a, b) ser. 3 for cells whose cycle spanned the temperature shiR for temperatures between 3fP and 41°C. Plots are normalized to the median control IMT of the population. (e, ff Fourier power spectrum analysis of data presented in fc) and (d).
312
Klevecz and King
In fig. 3c a histogram of the data from fig. 3a (also shown as experiment 3 of fig. 3b) are plotted together with the Fourier spectral analysis (fig. 3e). A major component appears at 4.6 h. The peak at 32 h is an artifact of reflection.
DISCUSSION In unicellular eukaryotes expressing their circadian growth mode it is commonly observed that reduction in temperature from the optimal yields a reduced fraction of cells undergoing synchronous division at 24 h intervals, but only a small change in the 7 of the culture [6, 131. In other words, division is quantized in circadian intervals, even though only a fraction of the cells divide in any 24 h period. In characterizing the circadian clock in the unicellular eukaryote Euglena, Edmunds found that the cell division cycle was temperature compensated over a 7°C temperature range from 13 to 19°C [6]. However, Bruce & Pittendrigh [2] also using Euglena showed that phototaxic rhythms were temperature compensated over a much wider range of temperatures from 16” to 33°C suggesting that certain isolated physiological and biochemical functions may be better temperature compensated than is the cell cycle, which represents the end product of a vast reaction scheme. There has been a continuing controversy over whether the cell cycle is a ‘clocked’ property of the cell [5, 12, 141. Though it is possible to construct a network of coupled reactions, whose net reaction rate shows relative temperature independence [ 151, such behavior, excluding photochemical reactions, is the exception to the rule of temperature dependence. Indeed the biological rhythm literature is the extant example of the temperature compensation Exp Cell Res 140 (1982)
phenomenon. Even so it is questionable whether the notion of compensation can be applied to the organism as a whole. For example in D. pseudoobscura the pupal eclosion rhythm is considered to be temperature compensated, because flies will emerge at the same subjective time of day, independent of the temperature at which they are raised, even though the total development time increases with decreasing temperatures [ 11. In the case of unicellular organisms growing in their circadian mode with a generation time equal to or greater than 24 h, and in the regrettably few instances where the population doubling time has been measured [6, 131, doubling time and presumably cell cycle time increases with decreasing temperature, and only the rhythm which gates cell division shows temperature compensation. We suggest that an analogous situation occurs in mammalian cells growing in an ultradian mode (with a generation time significantly less than 24 h) and that the time-keeping oscillator, which is here thought to be an integral submultiple of 24 h and of the cell cycle, similarly gates cell division in -44.5 h intervals. In addition we find that over a limited range of temperatures the cell cycle is itself temperature compensated. The need for temperature compensation in poikiiothermic organisms is readily apparent and can be rationalized on evolutionary grounds. It is regarded by Pittendrigh [l] as the central element of a biological clock and of such importance as to be likely to arise by a number of mechanisms. The existence of temperature compensation in a mammalian cell where present need is not obvious suggests its early evolutionary origin. If the clock evolved at a sufficiently early time, then certain vestiges of this primitive clock may be ‘frozen’ in the chemistry of modern organisms.
Temperature
compensation
Based on the work presented here we are led to conclude that the animal cell cycle has some capacity for temperature compensation and is as ‘clocked’ as that of unicellular eukaryotes. The span of temperatures over which compensation holds, while narrow in comparison to many rhythmic functions of poikilothermic metazoans, is much the same as that seen in unicellular eukaryotes, where temperature compensation is considered to be an established fact. More significantly we can argue that the underlying oscillator which times cell division and gives rise to quantizement of generation times is more perfectly temperature compensated. By analogy with circadian systems the underlying oscillator may compensate over a broader span of temperatures, but cell division, while gated by the clock, can also be blocked at lower temperatures by the failure of downstream, non-clock events to be completed in time to make the gate. This work was supported in part by grants GM26015 and AGO0434 from the NIH and by the Morley Benjamm Research Fund.
Prmted
I” Sweden
in the mammalian
cell cycle
3 13
REFERENCES 1. I;i6(:ydrigh, C S, Proc natl acad sci US 40 (1954) 2. Bruce, V G & Pittendrigh, C S, Proc natl acad sci US 42 (1956) 676. 3. Rawson, K S, Cold Spring Harbor symp quant biol 25 (1960) 105.
4. Sweeney, B Y & Hastings, J W, J protozool 5 (1958) 217. 5. Edmunds, L N & Adams, K J, Science 211 (1981) 1002. 6. Edmunds, L N, Chuann, L, Jarrett, R M & Terry, 0 W, J interdiscipl cycle res 2 (1972) 121. 7. Sisken, J E, Exp cell res 39 (1965) 103. 8. Wimber, D E, Am j bot 53 (1966) 21. 9. Klevecz, R R, Proc natl acad sci US 73 (1976) 4012. 10. Klevecz, R R, King, G A & Shymko, R M, J supramolec struct 14 (1980) 329. 11. Shymko, R M & Klevecz, R R, Biomathematics and cell kinetics (ed M Rotenberg) p. 329. Elsevier/North-Holland Biomedical Press, Amsterdam (1980). 12. Klevecz, R R, Kros, J & King, G A, Cytogenet cell genet 26 (1980) 236. 13. Chisholm, SW, Primary productivity in the sea (ed P Falkowski) p. 281. Plenum, New York (1980). 14. Winfree, A T, The geometry of biological time. Springer-Verlag, New York (1980). 15. Pavlidis, T, Biological oscillators: their mathematical analysis. Academic Press, New York (1973).
Received October 20, 1981 Revised version ._. received .^ .^-- March 8, 1982 Accepted March 10, lYM2
Em Cell Rrs 140 (19821