Effect of sub-optimal temperatures on survival of mammalian cells

Experimental Cell Research 68 (1971) 247-252

EFFECT OF SUB-OPTIMAL

TEMPERATURES

MAMMALIAN R. J. NELSON, J. KRUUV, Department

ON SURVIVAL

OF

CELLS

C. J. KOCH and H. E. FREY

of Physics, University of Waterloo, Waterloo, Ontario, Canada

SUMMARY When survival at low temperatures, in terms of colony-forming ability, is measured in Chinese hamster lung cells (V79), it varies inversely with temperature in the lo-25°C range; i.e. survival at 10°C is greater than that at 25°C. These survival-time curves on semi-log plots havea‘shoulder” region followed by a linear region. Survival at these temperatures varies inversely with the macromolecular synthesis rate. Results with cells at 5°C break the above patterns.

In many experiments it is necessary to store cells for short or long periods at sub-optimal temperatures-usually either room temperature or 4°C. At such temperatures, the survival of the cells in the exposed population depends upon the degree of cold, the type of cells, the length of exposure and the position of the cell cycle in which the exposure was initiated [l-4]. In moderate cold many cells die when the exposure is sufficiently long, but even after several weeks at this temperature some cells remain alive and multiply when reincubated at 37°C. The understanding of the effects of sub-optimal temperature is also important in the use of hypothermia in medicine and surgery. It was decided to investigate how time, temperature, and rate of macromolecular synthesis were related to survival of mammalian cells exposed to sub-optimal temperatures. It was felt that the data from the following experiments was necessary to proceed with future investigations on the molecular nature of death of cells exposed to moderately low temperatures.

MATERIALS

AND METHODS

The cells employed are those of a subline (V79-S I7 I) from the V79 line of near-diploid Chinese hamster lung cells. They are grown on- plastic Petri dishes in Eagle medium with 15 “b fetal calf serum in a humid atmosphere of 5 (I,)CO, and air. Under these conditions the cells grow in log phase with a generation time about 10 h. The plating efficiency is usually about 80 ‘lo for asynchronous populations and 60 o,, for synchronous populations with mitotic indexes of 80 % in the latter. For the asynchronous survival studies, log-phase cells are trypsinized and inoculated into temperature (37’C) and pH (7.4) equilibrated medium in plastic Petri dishes. These cells are allowed to attach for 2 h at 37°C in the incubator. The average cellular multiplicity at this time is usually I .02. Cell concentrations are adjusted at the time of inoculation to suit the treatment to which the cells are to be exposed, so that 100 to 200 colonies will result per dish (100 mm diameter). After the 2 h attachment period all the plates, except the controls, are put into a refrigerated COB incubator which has been pre-set to the correct temperature. The CO? flow in the incubator is adjusted so that the correct pH range (7.4-7.5) is maintained and the latter is monitored bv a Beckman Expandomatic pH-meter. Three plates are removed everv 24 h and returned to the 37°C incubator for colony growth. After an incubation period of 5 to 7 days, the colonies are stained with methylene blue and counted. The colonv surviving fraction (with respect to the controls) is -plotted on a log scale versus time at temperature “x” on a linear scale. The resulting curve is known as a survival curve [5]. For the labeling experiments, a synchronous ceil Expil

Cell Res 68

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R. J. Nelson et al.

population in the S phase of the cycle is used. This phase is used since this is where the maximum DNA synthesis occurs. Also RNA and protein synthesis proceed at a substantial rate at this time in our cell line [6, 71. Cells synchronized at or close to division are obtained by selecting for less firmly attached, dividing cells by using a combination of precooling and shaking [8, 91. Mitotic cells, selected as above, are incubated for 6 h at 37’C to allow progression into the S phase. At this stage, all the plates are moved into a 5°C refrigerated CO, incubator. The temperature and pH of the medium is monitored on a strip chart recorder. Once the temperature in the medium reaches 5”C, the heating elements in the incubator are turned on, causing a temperature rise of about 20°C in 2 h. At various times during this rise, plates are pulse-labeled for 15 min in this incubator with various tritium-labeled molecules following which the plates are prepared for autoradiography [IO]. An event marker on the recorder notes the time during which the plates are labeled thus allowing determination of the temperature range during the labeling procedure. For DNA synthesis, the label is 3H-thymidine-methyl (3H-TdR, 6.7 Ci/mM, 0.1 ,&i/ ml of medium). For RNA synthesis, “H-5-uridine (3H-lJdR, 5.0 Ci/mM, 0.4 (G/ml) is used and for protein synthesis, 3H-4,5-uL-leucine (15.8 Ci/mlM, 2.0 /G/ml) is employed. After developing the film emulsion, the nuclear tracks (grains) above or in 100 cells per experimental point are counted. Both labeled and unlabeled cells are counted. In these investigations the average background count per cell area (- 0. I grains) is significantly less than the experimental counts and is neglected. Standard errors (SE.) have been used to express experimental accuracy and are represented as vertical bars in all figures unless smaller than the points as plotted.

RESULTS Fig. I shows a combined plot of all the asynchronous survival experiments. The cellular multiplicity in these experiments is about 1.02, so that single-cell survival is essentially the same as the “colony surviving fraction”. All the curves are reproducible from experiment to experiment with the exception of the 5°C curves which varied to either side of the curve shown. The reason for the latter is the difficulty in precise temperature and especially humidity control at 5°C; therefore there was some water loss from the plates (< 10 ?a). Extrapolation of the linear portion of the curves back to the log axis will yield a number which we will call the extrapolation number, Exptl Cdl Rrs 68

k

2

4

6

13

IO

12

14

Fig. I. Abscissa: time (days); or&u/r: fraction survival. Fraction colony survival of asynchronous cells after exposure for various time intervals to 5’C, 10°C 15”C, 20°C and 25°C. The vertical bars represent S.E. unless smaller than the points as plotted.

n. The D, will be defined as the time (or the number of degree-days) required to reduce the survival by a factor of I/e on the linear portion of the semi-log graph. Similar definitions are used in radiobiology [l I]: this does not imply that the mechanisms of damage are similar. The II,, and n values are listed in table I. The IO, 15, 20 and 25°C results follow a systematic pattern, i.e. n x T and D,, z 11T. The 5’C results break this pattern. The Table 1. Extrapolation numbers and D,, for z?auioustemperatures ,from fig. I Temperature c Cl 5 10 15 20 25

n

D” (days)

3.6 2.3 2.4 15 23

2.2 4.3 3.3 1.7 I .2

Survival of cells exposed to sub-optimal temperatures

249

Table 2. E.utrapolation numbers and D, from degree-day curves (fig. 2) Temperature (“C) 5 IO 25 20 25

0.001

! 0

40

I

80

I 120

I

160

200

240

1

280

Fig. 2. Ahscissu: degree-days; ordinate: fraction survival. Fraction colony survival of synchronous cells versus degree-days for various temperatures. Degreedays A ’ B where A is time in days at temperature Y, B, temperature difference between 0°C and YT. The vertical bars represent S.E., unless smaller than the points as plotted.

variation between experiments at 5°C is due mainly to changes in n number. In fig. 2 fraction survival is plotted against degree-days for the various temperatures. Degree-days are obtained by multiplying the temperature by the number of days at that temperature. The degree-day unit is widely used to compare the effect of varying temperatures on a process to some standard temperature. It can be seen from fig. 2 that three families of curves are formed. The 20 and 25°C curves are virtually inseparable. The 10 and 15 ‘C curves are nearly parallel and form another family. The 5°C curve is by itself. The D, and n values obtained from fig. 2 are listed in table 2. Again only the 5°C results break the pattern. The possibility existed that medium breaks down at 25°C and thus leads to decreased

II

D, (days)

2.6 2.3 3.8 I5 16

I3 42 42 34 33

survival. An investigation was done where medium exposed to 25°C for 5 days was used in the experimental group and medium stored at 4°C (as usual) was used in the control group. After 5 days for colony growth the 37°C incubator, there was no significant difference in colony survival between the two groups. 8 7

Protein

6 5 4 3 2 I 0

t+ ‘i c ,’ ’ t ‘+

-t -.--+-

5 4 3 2 I: 0 3

RNA

2 I 0

L 0

4

8

12

16

20

24

Fig. 3. Abscissa:

temperature (“C); ordinute: grains per cell per 15 min label. Autoradiographic studies of DNA, RNA and protein synthesis at low temperatures using a 15 min pulse label of 3H-TdR, 3H-UdR and 8H-leucine, respectively. Vertical bars represent SE. Horizontal bars represent the temperature range during the pulse (see text). Exptl Cell Rrs 68

250

R. J. Nelson et al.

Experiments were also done with a cell line of the same origin as the one discussed except that this line had been kept in log phase in phosphate buffered medium (Puck’s Saline G) for 4 years in this laboratory. The survival curves were similar [12] to those shown in fig. 1. The DNA, RNA and protein synthesis data are plotted in fig. 3. As the labeling was done for a constant time of 15 min across a small temperature range and the temperature rise in the incubator was not constant, the temperature bars vary in length along the abscissa. The RNA data is more variable than the DNA and protein synthesis data due partly to the fact that possibly as much as 20”; of the tritium label on uridine could eventually be incorporated into DNA [l3]. DISCUSSION The general pattern of the survival curves agree with the data of Kukaine & Nagayeva [14] who found that rabbit cells retained viability for 12 days at 4°C but oniy 5 days at 18-20°C. Survival-time curves for a single temperature (4°C) have been reported for several cell lines. If one replots the data of Michl et al. [I51 on semi-log paper one finds that the human diploid cells derived from embryonic lung have n ~~1 and DO--5 days. The Syrian hamster embryonic cells had n- 2 and DC,-2 days. The only thing one can deduce from the HeLa cell data is that IZis much greater than one. These parameters are only approximations since the lowest survival in the above experiments was 209,, and only five points were done per cell line. If one replots the data of Holerkovri et al. [3] on semi-log paper one finds that for Detriot-6 cells n --2 and D, 10 days. For HeLa cells n ~ I and D, =~5 days. The only Exptl Cell Rrs 68

thing one can deduce from the L cell data is that n is approx. 10. Again less than one log scale is covered for survival and five points were done per cell line. In all the survival studies cited, the criteria for survival was viable cell counts by staining methods and not colony formation as in our studies. Furthermore, there are no indications of standard errors or reproducibility. Despite this, it can be seen that our results at 5’C, 11~3.6 and D,-~-2.2 days, are of the same order of magnitude. It is possible that the surviving cells in our experiments become “cold-adapted” [3, 151. If so, there may be changes in metabolic pathways [16, 171 and in the number of chromosomes [IX]. It is important to realize that colony survival is a measure of proliferating integrity and that the surviving cells may be altered. For example, the plates from the 25’C experiments contain colonies of greatly varying size, an effect very similar to that caused by large doses of radiation [ 191. Also cells which have been subjected to low temperatures take more time to form countable colonies at 37’C than controls. The longer the exposure to the low temperature, the longer it takes for the formation of countable colonies. This suggests that cold “doses” might cause subsequent division delay periods at 37°C. The possibility also exists that the first few generations of growth at 37’C have longer cycle times. It is not known if exposure to sub-optimal temperature for long periods of time is related to the conflicting results reported for I h cold shocks [20-221. The asynchronous survival curves shown are very similar to radiation survival curves for mammalian cells [9, Ill. On a semi-log plot both types of curves exhibit a “shoulder” followed by a linear portion. From the 20 and 25°C results, this linearity continues

Surciual of cells exposed to sub-optimal temperatures down to at least 0.5 “/Asurvival. The presence of a shoulder suggests that sub-lethal damage is accumulated in this region or that a threshold exists. If “sub-lethal cold damage” is accumulated as suggested, then it would be of interest to know if this damage can be repaired by the cell when it is returned to 37°C. We have shown that this intracellular repair indeed takes place (paper in preparation). Because of the similarity between radiation and “cold damage” curves, the obvious question is whether the kind and sites of damage are the same. If a radiation survival experiment is done immediately on cells which have been exposed to 5°C for 5 days (thus placing them on the linear portion of the cold survival curve), one should obtain no shoulder on the radiation survival curve if the sites of damage are the same. In an experiment of this kind, the radiation survival curve obtained was virtually identical to the radiation control curve suggesting that the nature and sites of damage are different [ 121. One of the main factors affecting survival at low temperatures may be the rate of protein and DNA synthesis. As seen in fig. 3, cells synthesize very little DNA, RNA and protein at 5510°C. However, at 23°C the amounts of DNA and protein being synthesized are 4 and 33 times, respectively, the amounts being synthesized at 5°C. This change in rate of synthesis in this cell line is in agreement with observations by Kruuv & Sinclair [23]. As multiplicity studies revealed no significant division of cells at even 25”C, these metabolic products build up and may contribute to cell death. Even at 4’C. cells synthesize enough DNA for up to 30% of the cells to double the number of chromosomes [I 81. After 5 days at 25”C, giant cells are observed thus supporting this theory. Similar cells are seen after radiation damage [I I]. Since li

511814

2.5I

protein synthesis is greater at 25°C than 20°C (by a factor of two), survival at 20°C should be greater than at 25°C. This was observed. If the above theory is correct survival curves should show a minimum in the 25531°C range, since at least HeLa cells grow exponentially, although not at an optimum rate, above 31’C [2l]. By the criterion used for our experiments, survival is 100°b at 37’C. At the other end of the scale, one would expect a maximum in survival between 0-10°C since survival starts to decrease as one approaches temperatures where freezing and possibly “pre-freezing” damage starts to occur. While the cells are being exposed to the sub-optimal temperatures, there are differential effects on the cells depending upon their position in the cell cycle. First, cells exposed to long periods of cold in G 1 are the least likely to survive [4]. Secondly, at least in the 25537’C range, the duration of G 1 is increased disproportionately to G2 and S [2l, 24, 25, 261. Several investigators also claim that in the region between 0 and 25°C cell cycle movement in mammalian cells is almost completely stopped [21, 26. 271. The effects of low temperatures on plant cells, bacteria and viruses will not be discussed because of the greatly varied responses between these and mammalian cells. It should be noted, however, that in plants the balance between unsaturated and saturated fats in the mitochondrial membranes has been shown to be an important factor in determining their survival when exposed to low temperatures [28-311. Thus the results reported here with the cells of the heterothermic Chinese hamster cannot be extrapolated to human cells without further investigations, since the lipid composition in the mitochondrial membranes may differ between the two species. Exptl Cell Res 6X

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This investigation was aided by a grant from the Ontario Department of University Affairs.

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