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Parasynchronous division of HeLa cells
Experimental
624
PARASYNCHRONOUS A. A. NEWTON Department
DIVISION
Cell Research 16, 624-635 (1959)
OF HeLa CELLS
and P. WILDY
of Pathology, University of Cambridge and Department of Bacteriology, St. Thomas’s Hospital Medical School, London, England Received July 28, 1958
Iis
recent years, the question of how various constituents of dividing cells increase has attracted the attention of biologists, and a number of investigations, many of which are concerned with synthesis of nucleic acid, have been made on a variety of cells. Two methods of approach have been used. Some have effectively studied one cell at a time using special assay methods, e.g. ultraviolet absorption [4], cytochemical methods [l], or autoradiographic techniques [ll]; others using more conventional assay methods, have studied masses of synchronously dividing cells. A few studies have been made using groups of cells which naturally divide synchronously, for example Rapkine’s [20] studies with segmenting sea urchin eggs, but most investigators have employed material in which synchronous cell division has been secured artificially. Such synchronous division may be achieved by selection of a portion of a cell population in the same stage of the growth cycle; for example, the visual selection of dividing amoebae [18], and the differential filtration of bacteria [16]. It may also be induced by applying to a randomly growing culture one of several environmental stimuli, of which the most frequently used has been some form of temperature shock. Since the pioneering studies of Spear [22] and more recently of Scherbaum and Zeuthen [al], numerous examples of temperature-induced synchrony have been reported (c.f. the recent reviews of Swann [23] and Campbell [3]) but most of these concern free living unicellular organisms. Apart from the remark that HeLa cultures “. . . manifest synchronously dividing cells following a period of cooling” [lo], we are aware of no other reports of temperature-induced synchronous division in these or any other cells of mammalian origin. The present paper extends the observations previously reported [25] describing partially synchronous cell division which follows chilling of growing HeLa cultures. Particular attention has been paid to the mitotic events occurring and to the synthesis of nucleic acids in such cultures. EzperimentaZ
CeZl Research 16
Parasynchronous division of NeLa cells MATERIALS
AND
625
METHODS
Cell line.-Human cervical carcinoma cells (strain HeLa, Gey) have been grown in this laboratory for two years by culture as cell sheets on one of the six sides of Pyrex babies’ feeding bottles (volume 250 ml). Weekly subcultures were made following dispersal of the cells by pipetting in a solution of disodium ethylene-diaminetetra-acetate (EDTA) (1:5000 w/v) in phosphate buffered saline free from calcium and magnesium, the solution A of Dulbecco and Vogt [7]. Growth medium.-The growth medium used routinely contained 20 per cent (v/v) human serum, 80 per cent (v/v) Gey’s [9] balanced salt solution and 0.5 per cent (w/v) lactalbumin hydrolysate; 0.002 per cent (w/v) phenol red, 400 units of penicillin and 100 pg streptomycin per ml were also added. Growth conditions used in experiments.-HeLa cells in the logarithmic phase of growth were removed from the stock bottles in the I: 5000 EDTA solution, suspended by pipetting and added immediately to a large volume of growth medium, so that the final cell density was 5-10 x IO4 cells/ml. Whilst this suspension was kept agitated, 2.0 ml aliquots were dispensed into several hundred screw-capped bottles of 10 ml capacity, some of which contained coverslips. All the culture bottles were then left horizontal and stationary for 24 hours to allow the cells to adhere to the glass and to begin growing. These operations were all carried out in a hot room at 37°C. Recovery and counting cells.-Preliminary experiments had shown that under most experimental conditions, only a very small proportion of cells were to be found floating free in the medium. For this reason, only the cells growing on the glass of the bottles were sampled. These were detached from the glass by replacing the medium with I: 5000 EDTA solution; after 10 min. incubation at 37°C the cells were suspen using a siliconed pasteur pipette; great care was taken to ensure that all cells bad been removed from the glass. They were then counted in a haemocytometer; at Beast 24 I mm squares, containing 10-100 cells each, were counted for each sample, giving a standard error of 2 12 per cent. Nucleic acid determinations.-Determination of the nucleic acid content was carried out on suspensions of cells in EDTA solution from not less than IO bottles, by a modification of the method of Ogur and Rosen [17]. Perchloric acid was added to the cell suspension to give a final concentration of 0.2 M perchloric acid, the whole being kept at 4°C. After half an hour the acid-insoluble precipitate was sedimented by centrifugation, and was washed twice by resuspension and sedimentation in cold 0.2 M perchloric acid. The insoluble material was suspended in 0.5 M pescbloric acid, heated at 70°C for 20 min., and the precipitate was removed by centrifugation. This hot acid extraction was repeated twice. The clear supernatant fluids obtained were combined and samples taken for determination of the total nucleic acid and deoxysibonucleic acid (DNA). Total nucleic acid was estimated by measurement of the absorption of ultraviolet light of wavelength 260 rnp by a diluted aliquot in a silica cell of light path 1 cm, using a Unicam spectrometer. The DNA content of another sample was estimated by tbe calorimetric method of Burton (21 calibrated on a sample of DNA of known pbosphorus content prepared from HeLa cells. No difference in the amount of material reacting with the diphenylamine reagent was observed following removal Experimental
Cell Research 16
A. A. Newton and P. U’ildy
626
of lipid material from the cells, so this was not normally done. The ribonucleic acid (RNA) content of the cells could be deduced from the difference between the total and DNA content; values obtained by this method agreed with determinations made by the cysteine method of Dische 161. Mitotic counts.-Cultures growing on coverslips were used to estimate the number of cells in mitosis and were fixed at 37°C by tipping off the medium and flooding at once with methanol. The fixed cells were then stained with chromalum-gallocyanin [S] and the proportions of mitotic cells and multinucleate cells determined by examining not less than 4000 cells per sample, using a i/7” oil immersion objective. The whole coverslip was then rescanned, and all the mitotic figures examined to determine the proportion of cells in the different stages of mitosis, and the proportion of multipolar cells in metaphase and anaphase. RESULTS
Growth eqeriments.-Cultures of cells which had been maintained at 37°C for 24 hours following subculture were chilled in the refrigerator at 4°C for one hour, and then returned to the incubator at 37°C. Some of the cultures, which were not chilled, served as controls. Samples of at least three bottles were taken immediately before and after chilling and thereafter at regular intervals. The growth curve obtained by estimation of the cell numbers in the culture bottles is shown in Fig. 1, and suggests that little cel1 division occurred in the chilled cultures following return to 37°C over a period of 20 hours, but that cells then divided within the next two hours. In this first Temperacure 37°C
4°C
4°C
5.7 S------: 0.
5.6 -
.
2E 55E
P l Ai
8 s 3
: 5.3 -
0
8
16
ld
I 32 Hours
I I 40 48 after chilling
Fig. l.--Growth of HeLa cells at 37°C after chilling cells in individual bottles. Erperimenfal
CeZI Research 16
I 56
I 64
at 4°C. The points
I 72
indicate
I 80
the number
of
Parasynchronous division of HeLa CC&G experiment the cells were again subjected to a temperature of 4°C for one bour when the growth cycle was repeated. However, subsecment experiments have shown that this second chilling period was unnecessary as cell division remained almost synchronous for at least two division cycles after the initiaH cold shock, as shown in Fig. 2. In one of these experiments the majority of cells divided within a period of half an hour with little cell rn~lt~~licat~o~ in the intervening period.
0I
5 I
10I
15 I
I 20 Hovrr
Fig. Z.-Growth . -. cultures
I 30
2sI
1 40
I 35
afrer chilling
of HeLa cells at 37°C after chilling chilled at 4°C for 1 hour.
at 4°C.
A-A
Control
cultures,
not chilled.
In most experiments bursts of division occurred at intervals of 18 hours after chilling, but when human serum was replaced by sera of other animabs (ox or rabbit), the intervals between division periods were longer. Tbe proportion of cells dividing during each growth period was usually 60-80 per cent, although on one occasion a 95 per cent increase in cell number has been recorded. Unfavourable treatment of the cells at subculture, i.e. lowering of the temperature or delay in adding the cells to medium, considerably reduced the number of cells which divided. However, at the end of tbe division period of chilled cultures, the number of cells always equalled that in control cultures, showing that the period of chilling had not reduced the viability of the cells. Experiments were made to determine the effect of chilling upon cells in ferent stages of the growth cycle. Cultures which had already been subjected to one period at 4°C were rechilled after different intervals at 37°C. The resuhs are given in Fig. 3 and show that when cells were recbilled 14 or more hours after the initial period of cold shock, the only effect was to delay the burst of Experimenlal
Cell Reseurch 16
A. A. Newton
628
and P. Wildy
division by about one hour (the duration of the second chilling period). When, however, the cells were rechilled less than 14 hours after the initial cold shock, the period of division was delayed for a further 18 hours. Cytological examination.-In several experiments the mitotic indices of cultures have been determined in parallel with cell counts and estimation of
56
54
i” .
Temperacure
.-.
.
.
8-g-%I
/ .-o-o Temperature
Temperature
. ./ 0-o
Fig. 3.-The effect of 2 periods of chilling on the growth of HeLa cell cultures. The uppermost line in each case indicates the periods of chilling. l - l Control cultures chilled once. O-O Cultures chilled twice.
nucleic acid content; the results of one of these are given in Fig. 4. The mitotic indices of control cultures which had not been chilled are shown in Fig. 4a, where it will be seen that there was a steady increase in the percentage of cells found in mitosis as the experiment progressed. Nevertheless, the proportion of cells found in prophase remained fairly constant suggesting that the increase in mitotic index was due to a prolongation of the later stages of mitosis rather than to an actual increase in the number of cells entering mitosis. In fact, the steady increase in metaphase index.suggests that an increasing time was being spent in metaphase. It has been estimated that the time spent in mitosis by these cells is 25 to 35 minutes. Fig. 4b illustrates the results obtained with chilled cells. Three points of interest emerge. Firstly, immediately after cooling the prophase index was markedly reduced, whilst later mitotic phases remained at levels similar to the controls. This was probably due to some temporary effect on the properties Experimenfal
CeZl Research 16
Pat-asynchronous division o/ HeLa cells
6%
of the prophase nucleus because a short time later all phases of mitosis had values similar to those observed before chilling. Secondly, the proportion of cells in all stages of mitosis gradually de during the first 12 hours after chilling, suggesting that cells actually going mitosis at the time of cooling either completed the mitotic epis
Fig. 4.-Variation of the mitotic index of HeLa cell cultures grown at 37°C after chilling at 4’C for 1 hour. x-x Cell numbers. A-A Metaphase index. *-e Mitotic index. O--6 Prophase index O-O Anaphase + early telophase index.
being returned to 37”C, or that they persisted for some time and then degenerated. The first hypothesis seems more probable, as in many experiments the cell counts increased very slightly during the first 12 hours of the experiment. Furthermore, the results shown in Fig. 3 indicate that cell division was not prevented by a period of chilling given when cells were known to have been in mitosis (see below). Thirdly, from the 13th until the 20th hour there was a considerable increase in the mitotic index of these cultures; this increase, which immediately preceded the rise in cell numbers, was characterised by successive peaks in the proportion of cells in prophase, metaphase, anaphase and telophase. These results leave no doubt that the partial synchrony in cell division is associated with a partial synchrony of the events of mitosis. The mitotic index observed in the period prior to cell division has ’ several cases, been between 6 and 8 per cent, but it has never excee these values. On two occasions no fall in the mitotic index occurred in the hours soon after chilling of the cells: however, since we have never found a Esperimenfal
Cell Research I
A. A. Newton and P. Wildy
630
significant increase in cell numbers during the first 17 hours of an experiment, nor have we observed any change in the proportion of multinucleate cells in this period, we assume that on these two occasions there was some prolongation of mitosis. The mitotic indices of cultures undergoing a second divsion cycle has also been determined; after the first period of division the mitotic index remained constant at a low value for 14 hours and then again showed a marked peak which preceded the increase in cell numbers.
Fig. 5.-Nucleic points represent
acid content of HeLa cells grown at 37’C after chilling average values obtained from 10 experiments.
for 1 hour at 4°C. The
It is well known that populations of HeLa cells contain many abnormal cells [la]. For this reason, the proportions of multinucleate cells in interphase and of multipolar cells in the later stages of mitosis were determined. In both control and chilled cultures 0.5-1.3 per cent of those in interphase were multinucleate. Although this proportion varied between experiments, there was no relationship either to the mitotic events or to cell division. In all cultures examined some multipolar, metaphasic and anaphasic configurations were noted. In some cases a variation in the proportion of these abnormal cells was observed at different times during the experiment. However, the number of such cells was usually 0.02-0.04 per cent of all the cells present. Because of the small population of multipolar cells it is believed Experimenfal
Cell Research 16
Parasynchronous
division o/
631
that they contributed very little to the overall results, and their existence has been neglected. ?Jwleic acid content.-The total nucleic acid and DNA content of tbe cult has been determined at various times during the growth of the cells, in o to relate the time of synthesis of the nucleic acid to the time of cell muhiplication in the culture. Fig. 5 illustrates the mean values of results obtained from 10 experiments. From these results it seems clear that synthesis of H>F;A
I 0
I 5
Fig. B.-Nucleic acid content The results of 2 experiments
I 10
I 15 Hours
I I 20 25 after chtiiing
of H&a cell cultures are shown.
I 30
35
grown at 37°C after chilling
for I hour at 4°C.
is inhibited for some 14 hours after cooling of the cells, whilst the synthesis of total nucleic acid, presumably RNA, continues. At 14 hours, when an increasing mitotic index of the culture is observed, there is a rapid increase of the average amount of DNA in the cells, but as the cells divide the content falls abruptly to about 60 per cent of the maximum value. Rapid synthesis of DKA is renewed after telophase and synthesis probably continues throughout interphase; however, the rate of DKA synthesis seems to be greatest just before the appearance of any prophase nuclei in the cultures. It is probable that this second cycle of cell growth gives a more exact picture of the course of events in cell division than the first cycle, where cooling the cells has obviously modified the process occurring. The total nucleic acid content of both chilled and control cells fell slightly during the course of the experiments, possibly due to depletion of same component of the growth 41 - 593703
Experimental
Cell Research
16
A. A. Newton and P. Wildy
632
medium. No changes in the nucleic acid content of the cells during the period of chilling at 4°C were observed. When these results are expressed as the amount of nucleic acid present in each culture one striking fact appears. The total amount of nucleic acid decreases sharply just before cell division occurs. In a typical experiment the total nucleic acid content of a culture of 3 x 10” cells fell from 25-20 ,ug whilst the DNA content remained constant at 7.8 ,ug. This phenomenon has been observed in all the experiments performed so far; the results of two such experiments are illustrated in Fig. 6. As the total amount of DNA in the cultures is not falling at this time, it seems probable -that the apparent disappearance of nucleic acid from the cultures is due to some change in the amount or properties of the RNA. However, no direct determination of the RNA content of these dividing cells has yet been made. DISCUSSION
It is clear from our results that exposure of cultures of HeLa cells to a temperature of 4°C is followed, some time after replacement of the cells at 37”C, by a stepwise increase in cell number. In every experiment this period of cell division has been preceded by a rise in the mitotic index of the cultures, although the greatest mitotic index recorded in various experiments has varied between 3.5 and 8 per cent. Moreover the duration of the burst of division has varied from & to & of the total growth period (18 hours). It is therefore clear that whilst most of the cells tend to fall into step after a period of chilling, the character of the growth curves is not entirely predictable and in no experiment could the term “synchronous division” be strictly applied. For this reason we propose the term parasynchronous division which we believe more accurately describes the phenomenon in which the time of division is restricted to a small portion of the total growth period. In a recent review Campbell [3] suggested that differences in degree of synchrony might be apparent when expressed as changes in cell number on the one hand or in terms of mitotic index on the other. In our experiments both determinations have been made and this has indeed proved to be the case. Thus in the experiment represented in Fig. 4 a maximum mitotic index of only 3.5 per cent preceded a burst of cell division representing 78 per cent of the cells. This discrepancy may not be as great as appears in the first instance. Thus, the results given in Fig. 4 suggest that the control cultures in this experiment had a doubling time of 26 hours; throughout the period of incubation the prophase index remained stationary at a value of 0.5 per cent which indicates that the Experimenfal
Cell Research 16
Parasynchronous
division of NeLa cells
duration of prophase was about eight minutes. If prophase lasts for a similar time in cooled cultures the number of cells in prophase, observed at intervals between the 13th and 19th hours of the experiment, suggest that 32 per cent of the cells entered mitosis during this period. Data obtained from cell. counts show that, in fact, 78 per cent of the cells divided during this time. It is interesting to compare our results with other examples in which stepwise growth has been induced by temperature change. Firstly, the regularity with which this occurs and the contrasting unpredictabihty o seems to be a general feature of the phenomenon; perhaps this is not surprising in view of the complexity of the material with which we are dealing. Secondly, the method used to induce parasynchrony is perhaps worth some consideration as the time of chilling is, in this case, a very small tion of the total growth period of the cells, and only one period of cold shock is necessary. In other systems where temperature differences have been used to secure synchronous division, it has been found necessary to give either one long period or many repeated periods of exposure to a sub-optimal growth temperature. It seems that, in our example, the mechanism responsible for producing parasynchrony cannot be the same as was suggested for iretrahymena by Scherbaum and Zeuthen [al]; namely, that the sublethal temperatures employed set back those cells in a predivision phase whilst allowing other processes to continue, thus bringing all the cells into the same state at the end of several periods of incubation at this temperature. Our results indicate that chilling affects all HeLa cells except those that have prepared for division. Thirdly, in some synchronised bacterial cultures [5, 151 division of the cells follows shortly upon their replacement at the optimal growth temperature, but in MeLa cells there is a delay corresponding to the normal interphase period before division occurs. This suggests that the effect of chilling is to place all those cells not actually in mitosis in a state corresponding to that at the end of telophase. Thus at the end of the period at 4°C most cells would be state whilst a few cells would be in earlier stages in this “pseudotelophase” of mitosis: such a situation could result in a parasynchronous form of growth. Ko chemical changes have been detected in the cells during the period of chilling, nor is any extensive cytological change apparent apart from some ‘“clumping” of chromatin material in prophase nuclei. From the preliminary results so far obtained for the changes occurring in the nucleic acid content of these parasynchronously dividing cells during the growth cycle, two points of interest emerge. The first is the apparent decrease in total nucleic acid of the culture at a time just preceding cell division. Ezperimenfal
Cell Heseurch 86
A. A. Newton and P. Wildy
634
As the medium surrounding the cells was not analysed for nucleic acid it is not clear whether this disappearance is a result of loss of nucleic acid into the medium or to breakdown within the cell. In this connection the observation by Rabinovitch and Plaut [ 191 that RNA disappears from the nuclei of amoebae at the time of division may be relevant. But whatever the mechanism of this disappearance of nucleic acid, it is of interest that it occurs at the period when RNA appears associated with the chromosomes as observed by Jacobson and Webb [13]. The values obtained for the DNA content of the cells indicate that during most of the first growth cycle following chilling, DNA synthesis is inhibited. In the second growth period, which is more likely to be related to the events occurring during natural growth, the results suggest that synthesis of DNA occurs during interphase; this synthesis does not appear to continue at the same rate throughout, there being one period of rapid production very early in interphase and another at the end of interphase. It is not yet clear whether there is any synthesis of DNA between these two periods. Although several authors have shown that synthesis of DNA occurs during interphase in many different types of cell (reviewed by Vendrely [24]), the work of Klein, Klein and Klein [14] may suggest that in another type of tumour cell synthesis of DNA occurs either just before or just after cell division. No consistent relationship between increase in DNA content and time of division is apparent in other systems of synchronous cell division (summarised by Campbell [3]) and it may be that the results obtained with HeLa cells are only a result of the “unbalanced” growth produced by the initial stress causing synchrony of division. SUMMARY
It has been observed that populations of human cervical carcinoma cells (strain HeLa) may be induced to undergo parasynchronous division by chilling of the cell culture for a short period. Replicate cultures of HeLa cells were incubated at 37°C for 24 hours after subculture and then placed at 4°C for 1 hour. Samples were taken for cell counts at intervals after replacement at 37°C. Growth curves based on these estimations of cell number show that little or no cell division occurred for 17 hours after chilling; at this time as many as 95 per cent of the cells then divided within 1 hour. No further cell division was observed in the following 18 hours, after which most of the cells again underwent division. Cytological observations of the cells at various stages during those growth cycles have been made to determine the frequency of mitosis. The results of those studies indicate that following chilling of the cells there is a gradual decrease in the number of cells in mitosis until at 10 hours this figure reaches Experimental
Cell Research 16
Parasynchronous division of
635
a minimum. There is then a rapid increase in the mitotic index which ac a maximum value just before the increase in cell numbers is observed. mum values of the prophase, metaphase, anapbase indices are observed in succession. The nucleic acid content of these dividing cells has been dete thesis of DIGA is inhibited for about 14 hours after chilling, the of the cells then increases rapidly before cell division occurs. second cycle of cell division DNA synthesis occurs during early and late interphase. The total nucleic acid in each culture increases throughout the period when cell numbers are not increasing but at the time just prior fro division there is a fall in the amount estimated. This has not been explai These results are discussed and it is concluded that although these cultures do not exhibit a complete synchrony of the events of mitosis, the time of cell division is restricted to a very small portion of tb For this reason the term parasynchronous division has artThe authors are grateful to Professor H. R. Dean for the hospitality of the ment of Pathology, Cambridge, where much of this work was carried out and to Dr. ?iz. G. P. Stoker and Mrs Simon Reuss for many helpful suggestions. We should like to acknowledge the financial support of the T. C. Beebe fund (to P. W.), the Medical Research Council and a Wheldale Onslow Research Fellowship from Newnham College, Cambridge (to A. A. N.) REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 43. 14. 15. 46. 17. P8. 19. 20. 21. 22. 23. 24. 25.
J., Arch. bid. (Likge) 44, 519 (1933). BURTQS, K., Biochem. J. 62, 315 (1956). CAMPBELL, A., Bacterial. Revs. 21, 263 (1957). CASPERSSON, T., Chromosoma 1, 147 (1939). DELAYATER, E. D., Symposia Sot. Gen. Microbial. 16, 215 (1956). DISCHE, A., irz The Nucleic Acids, Vol. I, p. 255. Academic Press, New York, 1955. DGLBECCO, R. and VOGT, M., J. ezpt2. ,lfed. 99, 167 (1954). EIXARSOX, L., Acta Pathol. Xicrobiol. Scand. 28, 82 (1951). GEY, 6. O., Bull. Tissue Culture Assoc. March 16 (1949). GEY, G. O., RASG, F. B. and GEY, RI. K., Ann. ,V.I’. Acnd. Sci. 58, 956 (1954). HOWARD, A. and PELC, S. R., Ezpfl. Cell Research 2, 178 (1951). Hlsu, T. C., Texus Repts. Biol. Med. 12, 833 (1954). JACOBSON, W;. and WEBB, M., Exptl. Cell Research 3, 163 (1952). KLEIS, G., KLEIS, E. and KLEIN, B., Cancer Research 12, 484 (1952). MAALQE, 0. and LARK, K. G., Recent developments in cell Physiology, p. 159. I3utterworCh, London, 1954. MARUYAXA, Y. and YANAGITA, T., Bacteriol Proc. 37 (1955). OGCR, M. and ROSEN, G., Arch. biochem. 25, 262 (1950). PRESCOTT, II. M., Expfl. Cell Reseurch 9, 328 (1955). KABIIOVITCH, IQ. and PLAUT, vi’., Exptl. Cell Research 10, 120 (1956). RAPKIWZ, L., Ann. Physiol. I’husiochem. Bid. 7, 382 (1931). ScaERBAunr, 0. and %&THEN,~E., Exptl. Cell Research 6, 221 (1954). SPEAR, F. G., Arch. expfl. Zeilforsch. 7, 484 (1928). SWANN, RI. Ii., Cancer Research 17, 727 (1957). YENURELU, R., in The Nucleic Acids, Vol. II, p. 155. Academic Press, New York, 1955. WILDY, 1’. and NEWTON, A. A., Biochem. J. 68, 14P (1958).
BRACHET,
Experimental
Cell Research $6
624
PARASYNCHRONOUS A. A. NEWTON Department
DIVISION
Cell Research 16, 624-635 (1959)
OF HeLa CELLS
and P. WILDY
of Pathology, University of Cambridge and Department of Bacteriology, St. Thomas’s Hospital Medical School, London, England Received July 28, 1958
Iis
recent years, the question of how various constituents of dividing cells increase has attracted the attention of biologists, and a number of investigations, many of which are concerned with synthesis of nucleic acid, have been made on a variety of cells. Two methods of approach have been used. Some have effectively studied one cell at a time using special assay methods, e.g. ultraviolet absorption [4], cytochemical methods [l], or autoradiographic techniques [ll]; others using more conventional assay methods, have studied masses of synchronously dividing cells. A few studies have been made using groups of cells which naturally divide synchronously, for example Rapkine’s [20] studies with segmenting sea urchin eggs, but most investigators have employed material in which synchronous cell division has been secured artificially. Such synchronous division may be achieved by selection of a portion of a cell population in the same stage of the growth cycle; for example, the visual selection of dividing amoebae [18], and the differential filtration of bacteria [16]. It may also be induced by applying to a randomly growing culture one of several environmental stimuli, of which the most frequently used has been some form of temperature shock. Since the pioneering studies of Spear [22] and more recently of Scherbaum and Zeuthen [al], numerous examples of temperature-induced synchrony have been reported (c.f. the recent reviews of Swann [23] and Campbell [3]) but most of these concern free living unicellular organisms. Apart from the remark that HeLa cultures “. . . manifest synchronously dividing cells following a period of cooling” [lo], we are aware of no other reports of temperature-induced synchronous division in these or any other cells of mammalian origin. The present paper extends the observations previously reported [25] describing partially synchronous cell division which follows chilling of growing HeLa cultures. Particular attention has been paid to the mitotic events occurring and to the synthesis of nucleic acids in such cultures. EzperimentaZ
CeZl Research 16
Parasynchronous division of NeLa cells MATERIALS
AND
625
METHODS
Cell line.-Human cervical carcinoma cells (strain HeLa, Gey) have been grown in this laboratory for two years by culture as cell sheets on one of the six sides of Pyrex babies’ feeding bottles (volume 250 ml). Weekly subcultures were made following dispersal of the cells by pipetting in a solution of disodium ethylene-diaminetetra-acetate (EDTA) (1:5000 w/v) in phosphate buffered saline free from calcium and magnesium, the solution A of Dulbecco and Vogt [7]. Growth medium.-The growth medium used routinely contained 20 per cent (v/v) human serum, 80 per cent (v/v) Gey’s [9] balanced salt solution and 0.5 per cent (w/v) lactalbumin hydrolysate; 0.002 per cent (w/v) phenol red, 400 units of penicillin and 100 pg streptomycin per ml were also added. Growth conditions used in experiments.-HeLa cells in the logarithmic phase of growth were removed from the stock bottles in the I: 5000 EDTA solution, suspended by pipetting and added immediately to a large volume of growth medium, so that the final cell density was 5-10 x IO4 cells/ml. Whilst this suspension was kept agitated, 2.0 ml aliquots were dispensed into several hundred screw-capped bottles of 10 ml capacity, some of which contained coverslips. All the culture bottles were then left horizontal and stationary for 24 hours to allow the cells to adhere to the glass and to begin growing. These operations were all carried out in a hot room at 37°C. Recovery and counting cells.-Preliminary experiments had shown that under most experimental conditions, only a very small proportion of cells were to be found floating free in the medium. For this reason, only the cells growing on the glass of the bottles were sampled. These were detached from the glass by replacing the medium with I: 5000 EDTA solution; after 10 min. incubation at 37°C the cells were suspen using a siliconed pasteur pipette; great care was taken to ensure that all cells bad been removed from the glass. They were then counted in a haemocytometer; at Beast 24 I mm squares, containing 10-100 cells each, were counted for each sample, giving a standard error of 2 12 per cent. Nucleic acid determinations.-Determination of the nucleic acid content was carried out on suspensions of cells in EDTA solution from not less than IO bottles, by a modification of the method of Ogur and Rosen [17]. Perchloric acid was added to the cell suspension to give a final concentration of 0.2 M perchloric acid, the whole being kept at 4°C. After half an hour the acid-insoluble precipitate was sedimented by centrifugation, and was washed twice by resuspension and sedimentation in cold 0.2 M perchloric acid. The insoluble material was suspended in 0.5 M pescbloric acid, heated at 70°C for 20 min., and the precipitate was removed by centrifugation. This hot acid extraction was repeated twice. The clear supernatant fluids obtained were combined and samples taken for determination of the total nucleic acid and deoxysibonucleic acid (DNA). Total nucleic acid was estimated by measurement of the absorption of ultraviolet light of wavelength 260 rnp by a diluted aliquot in a silica cell of light path 1 cm, using a Unicam spectrometer. The DNA content of another sample was estimated by tbe calorimetric method of Burton (21 calibrated on a sample of DNA of known pbosphorus content prepared from HeLa cells. No difference in the amount of material reacting with the diphenylamine reagent was observed following removal Experimental
Cell Research 16
A. A. Newton and P. U’ildy
626
of lipid material from the cells, so this was not normally done. The ribonucleic acid (RNA) content of the cells could be deduced from the difference between the total and DNA content; values obtained by this method agreed with determinations made by the cysteine method of Dische 161. Mitotic counts.-Cultures growing on coverslips were used to estimate the number of cells in mitosis and were fixed at 37°C by tipping off the medium and flooding at once with methanol. The fixed cells were then stained with chromalum-gallocyanin [S] and the proportions of mitotic cells and multinucleate cells determined by examining not less than 4000 cells per sample, using a i/7” oil immersion objective. The whole coverslip was then rescanned, and all the mitotic figures examined to determine the proportion of cells in the different stages of mitosis, and the proportion of multipolar cells in metaphase and anaphase. RESULTS
Growth eqeriments.-Cultures of cells which had been maintained at 37°C for 24 hours following subculture were chilled in the refrigerator at 4°C for one hour, and then returned to the incubator at 37°C. Some of the cultures, which were not chilled, served as controls. Samples of at least three bottles were taken immediately before and after chilling and thereafter at regular intervals. The growth curve obtained by estimation of the cell numbers in the culture bottles is shown in Fig. 1, and suggests that little cel1 division occurred in the chilled cultures following return to 37°C over a period of 20 hours, but that cells then divided within the next two hours. In this first Temperacure 37°C
4°C
4°C
5.7 S------: 0.
5.6 -
.
2E 55E
P l Ai
8 s 3
: 5.3 -
0
8
16
ld
I 32 Hours
I I 40 48 after chilling
Fig. l.--Growth of HeLa cells at 37°C after chilling cells in individual bottles. Erperimenfal
CeZI Research 16
I 56
I 64
at 4°C. The points
I 72
indicate
I 80
the number
of
Parasynchronous division of HeLa CC&G experiment the cells were again subjected to a temperature of 4°C for one bour when the growth cycle was repeated. However, subsecment experiments have shown that this second chilling period was unnecessary as cell division remained almost synchronous for at least two division cycles after the initiaH cold shock, as shown in Fig. 2. In one of these experiments the majority of cells divided within a period of half an hour with little cell rn~lt~~licat~o~ in the intervening period.
0I
5 I
10I
15 I
I 20 Hovrr
Fig. Z.-Growth . -. cultures
I 30
2sI
1 40
I 35
afrer chilling
of HeLa cells at 37°C after chilling chilled at 4°C for 1 hour.
at 4°C.
A-A
Control
cultures,
not chilled.
In most experiments bursts of division occurred at intervals of 18 hours after chilling, but when human serum was replaced by sera of other animabs (ox or rabbit), the intervals between division periods were longer. Tbe proportion of cells dividing during each growth period was usually 60-80 per cent, although on one occasion a 95 per cent increase in cell number has been recorded. Unfavourable treatment of the cells at subculture, i.e. lowering of the temperature or delay in adding the cells to medium, considerably reduced the number of cells which divided. However, at the end of tbe division period of chilled cultures, the number of cells always equalled that in control cultures, showing that the period of chilling had not reduced the viability of the cells. Experiments were made to determine the effect of chilling upon cells in ferent stages of the growth cycle. Cultures which had already been subjected to one period at 4°C were rechilled after different intervals at 37°C. The resuhs are given in Fig. 3 and show that when cells were recbilled 14 or more hours after the initial period of cold shock, the only effect was to delay the burst of Experimenlal
Cell Reseurch 16
A. A. Newton
628
and P. Wildy
division by about one hour (the duration of the second chilling period). When, however, the cells were rechilled less than 14 hours after the initial cold shock, the period of division was delayed for a further 18 hours. Cytological examination.-In several experiments the mitotic indices of cultures have been determined in parallel with cell counts and estimation of
56
54
i” .
Temperacure
.-.
.
.
8-g-%I
/ .-o-o Temperature
Temperature
. ./ 0-o
Fig. 3.-The effect of 2 periods of chilling on the growth of HeLa cell cultures. The uppermost line in each case indicates the periods of chilling. l - l Control cultures chilled once. O-O Cultures chilled twice.
nucleic acid content; the results of one of these are given in Fig. 4. The mitotic indices of control cultures which had not been chilled are shown in Fig. 4a, where it will be seen that there was a steady increase in the percentage of cells found in mitosis as the experiment progressed. Nevertheless, the proportion of cells found in prophase remained fairly constant suggesting that the increase in mitotic index was due to a prolongation of the later stages of mitosis rather than to an actual increase in the number of cells entering mitosis. In fact, the steady increase in metaphase index.suggests that an increasing time was being spent in metaphase. It has been estimated that the time spent in mitosis by these cells is 25 to 35 minutes. Fig. 4b illustrates the results obtained with chilled cells. Three points of interest emerge. Firstly, immediately after cooling the prophase index was markedly reduced, whilst later mitotic phases remained at levels similar to the controls. This was probably due to some temporary effect on the properties Experimenfal
CeZl Research 16
Pat-asynchronous division o/ HeLa cells
6%
of the prophase nucleus because a short time later all phases of mitosis had values similar to those observed before chilling. Secondly, the proportion of cells in all stages of mitosis gradually de during the first 12 hours after chilling, suggesting that cells actually going mitosis at the time of cooling either completed the mitotic epis
Fig. 4.-Variation of the mitotic index of HeLa cell cultures grown at 37°C after chilling at 4’C for 1 hour. x-x Cell numbers. A-A Metaphase index. *-e Mitotic index. O--6 Prophase index O-O Anaphase + early telophase index.
being returned to 37”C, or that they persisted for some time and then degenerated. The first hypothesis seems more probable, as in many experiments the cell counts increased very slightly during the first 12 hours of the experiment. Furthermore, the results shown in Fig. 3 indicate that cell division was not prevented by a period of chilling given when cells were known to have been in mitosis (see below). Thirdly, from the 13th until the 20th hour there was a considerable increase in the mitotic index of these cultures; this increase, which immediately preceded the rise in cell numbers, was characterised by successive peaks in the proportion of cells in prophase, metaphase, anaphase and telophase. These results leave no doubt that the partial synchrony in cell division is associated with a partial synchrony of the events of mitosis. The mitotic index observed in the period prior to cell division has ’ several cases, been between 6 and 8 per cent, but it has never excee these values. On two occasions no fall in the mitotic index occurred in the hours soon after chilling of the cells: however, since we have never found a Esperimenfal
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A. A. Newton and P. Wildy
630
significant increase in cell numbers during the first 17 hours of an experiment, nor have we observed any change in the proportion of multinucleate cells in this period, we assume that on these two occasions there was some prolongation of mitosis. The mitotic indices of cultures undergoing a second divsion cycle has also been determined; after the first period of division the mitotic index remained constant at a low value for 14 hours and then again showed a marked peak which preceded the increase in cell numbers.
Fig. 5.-Nucleic points represent
acid content of HeLa cells grown at 37’C after chilling average values obtained from 10 experiments.
for 1 hour at 4°C. The
It is well known that populations of HeLa cells contain many abnormal cells [la]. For this reason, the proportions of multinucleate cells in interphase and of multipolar cells in the later stages of mitosis were determined. In both control and chilled cultures 0.5-1.3 per cent of those in interphase were multinucleate. Although this proportion varied between experiments, there was no relationship either to the mitotic events or to cell division. In all cultures examined some multipolar, metaphasic and anaphasic configurations were noted. In some cases a variation in the proportion of these abnormal cells was observed at different times during the experiment. However, the number of such cells was usually 0.02-0.04 per cent of all the cells present. Because of the small population of multipolar cells it is believed Experimenfal
Cell Research 16
Parasynchronous
division o/
631
that they contributed very little to the overall results, and their existence has been neglected. ?Jwleic acid content.-The total nucleic acid and DNA content of tbe cult has been determined at various times during the growth of the cells, in o to relate the time of synthesis of the nucleic acid to the time of cell muhiplication in the culture. Fig. 5 illustrates the mean values of results obtained from 10 experiments. From these results it seems clear that synthesis of H>F;A
I 0
I 5
Fig. B.-Nucleic acid content The results of 2 experiments
I 10
I 15 Hours
I I 20 25 after chtiiing
of H&a cell cultures are shown.
I 30
35
grown at 37°C after chilling
for I hour at 4°C.
is inhibited for some 14 hours after cooling of the cells, whilst the synthesis of total nucleic acid, presumably RNA, continues. At 14 hours, when an increasing mitotic index of the culture is observed, there is a rapid increase of the average amount of DNA in the cells, but as the cells divide the content falls abruptly to about 60 per cent of the maximum value. Rapid synthesis of DKA is renewed after telophase and synthesis probably continues throughout interphase; however, the rate of DKA synthesis seems to be greatest just before the appearance of any prophase nuclei in the cultures. It is probable that this second cycle of cell growth gives a more exact picture of the course of events in cell division than the first cycle, where cooling the cells has obviously modified the process occurring. The total nucleic acid content of both chilled and control cells fell slightly during the course of the experiments, possibly due to depletion of same component of the growth 41 - 593703
Experimental
Cell Research
16
A. A. Newton and P. Wildy
632
medium. No changes in the nucleic acid content of the cells during the period of chilling at 4°C were observed. When these results are expressed as the amount of nucleic acid present in each culture one striking fact appears. The total amount of nucleic acid decreases sharply just before cell division occurs. In a typical experiment the total nucleic acid content of a culture of 3 x 10” cells fell from 25-20 ,ug whilst the DNA content remained constant at 7.8 ,ug. This phenomenon has been observed in all the experiments performed so far; the results of two such experiments are illustrated in Fig. 6. As the total amount of DNA in the cultures is not falling at this time, it seems probable -that the apparent disappearance of nucleic acid from the cultures is due to some change in the amount or properties of the RNA. However, no direct determination of the RNA content of these dividing cells has yet been made. DISCUSSION
It is clear from our results that exposure of cultures of HeLa cells to a temperature of 4°C is followed, some time after replacement of the cells at 37”C, by a stepwise increase in cell number. In every experiment this period of cell division has been preceded by a rise in the mitotic index of the cultures, although the greatest mitotic index recorded in various experiments has varied between 3.5 and 8 per cent. Moreover the duration of the burst of division has varied from & to & of the total growth period (18 hours). It is therefore clear that whilst most of the cells tend to fall into step after a period of chilling, the character of the growth curves is not entirely predictable and in no experiment could the term “synchronous division” be strictly applied. For this reason we propose the term parasynchronous division which we believe more accurately describes the phenomenon in which the time of division is restricted to a small portion of the total growth period. In a recent review Campbell [3] suggested that differences in degree of synchrony might be apparent when expressed as changes in cell number on the one hand or in terms of mitotic index on the other. In our experiments both determinations have been made and this has indeed proved to be the case. Thus in the experiment represented in Fig. 4 a maximum mitotic index of only 3.5 per cent preceded a burst of cell division representing 78 per cent of the cells. This discrepancy may not be as great as appears in the first instance. Thus, the results given in Fig. 4 suggest that the control cultures in this experiment had a doubling time of 26 hours; throughout the period of incubation the prophase index remained stationary at a value of 0.5 per cent which indicates that the Experimenfal
Cell Research 16
Parasynchronous
division of NeLa cells
duration of prophase was about eight minutes. If prophase lasts for a similar time in cooled cultures the number of cells in prophase, observed at intervals between the 13th and 19th hours of the experiment, suggest that 32 per cent of the cells entered mitosis during this period. Data obtained from cell. counts show that, in fact, 78 per cent of the cells divided during this time. It is interesting to compare our results with other examples in which stepwise growth has been induced by temperature change. Firstly, the regularity with which this occurs and the contrasting unpredictabihty o seems to be a general feature of the phenomenon; perhaps this is not surprising in view of the complexity of the material with which we are dealing. Secondly, the method used to induce parasynchrony is perhaps worth some consideration as the time of chilling is, in this case, a very small tion of the total growth period of the cells, and only one period of cold shock is necessary. In other systems where temperature differences have been used to secure synchronous division, it has been found necessary to give either one long period or many repeated periods of exposure to a sub-optimal growth temperature. It seems that, in our example, the mechanism responsible for producing parasynchrony cannot be the same as was suggested for iretrahymena by Scherbaum and Zeuthen [al]; namely, that the sublethal temperatures employed set back those cells in a predivision phase whilst allowing other processes to continue, thus bringing all the cells into the same state at the end of several periods of incubation at this temperature. Our results indicate that chilling affects all HeLa cells except those that have prepared for division. Thirdly, in some synchronised bacterial cultures [5, 151 division of the cells follows shortly upon their replacement at the optimal growth temperature, but in MeLa cells there is a delay corresponding to the normal interphase period before division occurs. This suggests that the effect of chilling is to place all those cells not actually in mitosis in a state corresponding to that at the end of telophase. Thus at the end of the period at 4°C most cells would be state whilst a few cells would be in earlier stages in this “pseudotelophase” of mitosis: such a situation could result in a parasynchronous form of growth. Ko chemical changes have been detected in the cells during the period of chilling, nor is any extensive cytological change apparent apart from some ‘“clumping” of chromatin material in prophase nuclei. From the preliminary results so far obtained for the changes occurring in the nucleic acid content of these parasynchronously dividing cells during the growth cycle, two points of interest emerge. The first is the apparent decrease in total nucleic acid of the culture at a time just preceding cell division. Ezperimenfal
Cell Heseurch 86
A. A. Newton and P. Wildy
634
As the medium surrounding the cells was not analysed for nucleic acid it is not clear whether this disappearance is a result of loss of nucleic acid into the medium or to breakdown within the cell. In this connection the observation by Rabinovitch and Plaut [ 191 that RNA disappears from the nuclei of amoebae at the time of division may be relevant. But whatever the mechanism of this disappearance of nucleic acid, it is of interest that it occurs at the period when RNA appears associated with the chromosomes as observed by Jacobson and Webb [13]. The values obtained for the DNA content of the cells indicate that during most of the first growth cycle following chilling, DNA synthesis is inhibited. In the second growth period, which is more likely to be related to the events occurring during natural growth, the results suggest that synthesis of DNA occurs during interphase; this synthesis does not appear to continue at the same rate throughout, there being one period of rapid production very early in interphase and another at the end of interphase. It is not yet clear whether there is any synthesis of DNA between these two periods. Although several authors have shown that synthesis of DNA occurs during interphase in many different types of cell (reviewed by Vendrely [24]), the work of Klein, Klein and Klein [14] may suggest that in another type of tumour cell synthesis of DNA occurs either just before or just after cell division. No consistent relationship between increase in DNA content and time of division is apparent in other systems of synchronous cell division (summarised by Campbell [3]) and it may be that the results obtained with HeLa cells are only a result of the “unbalanced” growth produced by the initial stress causing synchrony of division. SUMMARY
It has been observed that populations of human cervical carcinoma cells (strain HeLa) may be induced to undergo parasynchronous division by chilling of the cell culture for a short period. Replicate cultures of HeLa cells were incubated at 37°C for 24 hours after subculture and then placed at 4°C for 1 hour. Samples were taken for cell counts at intervals after replacement at 37°C. Growth curves based on these estimations of cell number show that little or no cell division occurred for 17 hours after chilling; at this time as many as 95 per cent of the cells then divided within 1 hour. No further cell division was observed in the following 18 hours, after which most of the cells again underwent division. Cytological observations of the cells at various stages during those growth cycles have been made to determine the frequency of mitosis. The results of those studies indicate that following chilling of the cells there is a gradual decrease in the number of cells in mitosis until at 10 hours this figure reaches Experimental
Cell Research 16
Parasynchronous division of
635
a minimum. There is then a rapid increase in the mitotic index which ac a maximum value just before the increase in cell numbers is observed. mum values of the prophase, metaphase, anapbase indices are observed in succession. The nucleic acid content of these dividing cells has been dete thesis of DIGA is inhibited for about 14 hours after chilling, the of the cells then increases rapidly before cell division occurs. second cycle of cell division DNA synthesis occurs during early and late interphase. The total nucleic acid in each culture increases throughout the period when cell numbers are not increasing but at the time just prior fro division there is a fall in the amount estimated. This has not been explai These results are discussed and it is concluded that although these cultures do not exhibit a complete synchrony of the events of mitosis, the time of cell division is restricted to a very small portion of tb For this reason the term parasynchronous division has artThe authors are grateful to Professor H. R. Dean for the hospitality of the ment of Pathology, Cambridge, where much of this work was carried out and to Dr. ?iz. G. P. Stoker and Mrs Simon Reuss for many helpful suggestions. We should like to acknowledge the financial support of the T. C. Beebe fund (to P. W.), the Medical Research Council and a Wheldale Onslow Research Fellowship from Newnham College, Cambridge (to A. A. N.) REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 43. 14. 15. 46. 17. P8. 19. 20. 21. 22. 23. 24. 25.
J., Arch. bid. (Likge) 44, 519 (1933). BURTQS, K., Biochem. J. 62, 315 (1956). CAMPBELL, A., Bacterial. Revs. 21, 263 (1957). CASPERSSON, T., Chromosoma 1, 147 (1939). DELAYATER, E. D., Symposia Sot. Gen. Microbial. 16, 215 (1956). DISCHE, A., irz The Nucleic Acids, Vol. I, p. 255. Academic Press, New York, 1955. DGLBECCO, R. and VOGT, M., J. ezpt2. ,lfed. 99, 167 (1954). EIXARSOX, L., Acta Pathol. Xicrobiol. Scand. 28, 82 (1951). GEY, 6. O., Bull. Tissue Culture Assoc. March 16 (1949). GEY, G. O., RASG, F. B. and GEY, RI. K., Ann. ,V.I’. Acnd. Sci. 58, 956 (1954). HOWARD, A. and PELC, S. R., Ezpfl. Cell Research 2, 178 (1951). Hlsu, T. C., Texus Repts. Biol. Med. 12, 833 (1954). JACOBSON, W;. and WEBB, M., Exptl. Cell Research 3, 163 (1952). KLEIS, G., KLEIS, E. and KLEIN, B., Cancer Research 12, 484 (1952). MAALQE, 0. and LARK, K. G., Recent developments in cell Physiology, p. 159. I3utterworCh, London, 1954. MARUYAXA, Y. and YANAGITA, T., Bacteriol Proc. 37 (1955). OGCR, M. and ROSEN, G., Arch. biochem. 25, 262 (1950). PRESCOTT, II. M., Expfl. Cell Reseurch 9, 328 (1955). KABIIOVITCH, IQ. and PLAUT, vi’., Exptl. Cell Research 10, 120 (1956). RAPKIWZ, L., Ann. Physiol. I’husiochem. Bid. 7, 382 (1931). ScaERBAunr, 0. and %&THEN,~E., Exptl. Cell Research 6, 221 (1954). SPEAR, F. G., Arch. expfl. Zeilforsch. 7, 484 (1928). SWANN, RI. Ii., Cancer Research 17, 727 (1957). YENURELU, R., in The Nucleic Acids, Vol. II, p. 155. Academic Press, New York, 1955. WILDY, 1’. and NEWTON, A. A., Biochem. J. 68, 14P (1958).
BRACHET,
Experimental
Cell Research $6