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Energy metabolism and ATP turnover time during the cell cycle of Ehrlich ascites tumour cells
-opyright @I 1982 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/82/090023-07$02.00/O
Experimental
ENERGY
METABOLISM
THE CELL
CYCLE
Cell Research 141 (1982) 23-29
AND ATP TURNOVER
OF EHRLICH
SVEN SKOG, BERNHARD
ASCITES
TRIBUKAIT
TIME
TUMOUR
DURING CELLS
and GUDRUN SUNDIUS
Department of Medical Radiobiology, Karolinska Instituter, S-10401 Stockholm, Sweden
SUMMARY The energy production in different parts of the cell cycle due to aerobic and aerobic glycolytic metabolism and ATP turnover time was estimated by measuring the oxygen consumption, lactate-pyruvate and ATP content of Ehrlich ascites tumour cells growing in vivo. Cell fractions of high purity fromthe various parts of the cell cycle were obtained by means of elutriator centrifuging. The total energy production for one cell cycle was estimated to be 19x lo-r2 mol ATP, 60% of which was due to the aerobic metabolism. Whereas the total ATP production is unchanged during Gl a fairly exponential increase is found during the S and G2+M phases. The total cellular ATP content increases from 12fmol ATP at early Gl to 28 fmol ATP at G2+M; this increase, however, is discontinuous and is most pronounced during Gl and during late S phase S phase/GZ+M. The ATP turnover time, as defined as the ratio between ATP content and ATP production, was found to increase significantly from 75 set in early Gl to 120 set in late Gl but was constantly 100 set during the early, middle and late S phase as well as G2+M. These variations indicate maximum energy-requiring processes during early Gl period of the cell cycle and are discussed in relation to K+Na+ flux and macromolecule synthesis.
Various sections of the energy metabolism In the experiments presented here, eluof mammalian cells, such as aerobic meta- triator centrifugation of in vivo growing bolism, aerobic glycolytic metabolism, and Ehrlich ascites tumour cells has been used ATP content in various parts of the cell to obtain sufftcient cell material from difcycle have been studied in a number of syn- ferent parts of the cell cycle. Elutriator cenchronized cell systems [l-l 11. trifugation has been shown to avoid interTo our knowledge there is, however, ference with cell growth [12], giving cell only one study on mouse L cells growing in fractions with a high degree of purity, exvitro in which both aerobic and aerobic cept in the case of mitotic cells. The oxygen glycolytic metabolism and ATP content in consumption, lactate and pyruvate contents the various parts of the cell cycle have been and the ATP concentration have been determined, enabling an estimation of the studied. From these data, aerobic and ATP turnover time [ 111. In this study con- aerobic glycolytic energy production and siderable changes in oxygen uptake were ATP turnover time have been estimated. found during the cell cycle, but less pronounced changes in the production of lactic MATERIAL AND METHODS acid and only slight changes in the ATP content. The estimated turnover time for The cell material ATP increased from 16 to 19 set during Gl, Hyperdiploid Ehrlich ascites tumour cells were to grow in vivo for 4 days after i.p. transdecreased during S, but increased again at allowed plantation of 3x 10’ IO-day-old cells into 3-month old female NMRI mice. G2+M. Exp Cell Res 141 (1982)
24
Skog, Tribukait and Sundius
J., ,,..IO 0
zoburs
I 0,
,
IO
.
20~s
various parts”bf cell
Fig. 2. The homogeneity of the various fractions is
indicated in the inset at the bottom. Cell fractions from various parts of the cell cycle were obtained by elutriator centrifuging. The ATP content was determined immediately after separation using luciferinluciferase assay. Then the cells were incubated in Ringer-phosphate-glucose buffer at 37°C and the oxygen consumption was measured by the Warburg manometric technique for 70 min. Dotted lines drawn by eye. Results from eleven and three separations respectively.
J=++s 50
150
Channel no. corresponding
to DNA amount
Fig. I. After preparation of cell nuclei (see Material
and Methods) the nuclei were stained with ethidium bromide and analysed by means of a rapid-flow cytophotometer. (n) Resulting DNA histogram of unseparated cells. The first maximum to the left corresponds to normal cells. The second maximum is formed by Gl cells of the tumour cell population. The maximum to the right is formed by the corresponding G2 and mitotic cells. In between these two maxima the S phase cells can be seen. (b-e) Examples of fractions of cells after elutriation centrifuging; (b) mainly normal cells with a small proportion of Gl tumour cells; (c) Gl cells of the tumour cell population; (d) mainly cells from the middle part of the S phase; (e) mainly G2+M cells.
Cell preparation and separation Fresh cell material was washed twice in ice-cold Ringer-phosphate-glucose buffer (0.154 M NaCI, 0.154 M KCI, 0.064 M CaC1, 0.15 M MgS0,x7Hp0, 0.1 M phosphate buffer, 0.01 M glucose pH 7.4) or Tris-EDTA buffer (0.1 M Tris, O.od7M NaCl, 0.005 M EDTA, pH 7.5) and centrifugated at 250 g for 5 min. Cell separation was carried out using a Beckman JE-G elutriator rotor with a Beckman J21B centrifuge. The rotor speed was held at 2780flO rpm. About 5x108 cells in buffer solution were added to this system, the whole of which was kept at 4”C, with an initial pump speed of 12 ml/min. Generally six cell E.xp Cell Res 141 (1982)
fractions of 100ml were obtained by stepwise increase in the pump speed up to 57 ml per min. The measurements of ATP were carried out on twenty fractions obtained in a similar manner. The cells were concentrated by centrifuging at 500 g for 5 min. The time interval between start of the separation and start of the analyses was about 40 min.
Determination of the composition of the cell fractions The composition of the cell fractions was determined using quantitative DNA analysis by means of flowcytofluorometric measurements as described earlier [13]. Generally, the cells were fixed in ethanol. After washing in Tris EDTA buffer together with RNase, suspensions of individual cell nuclei were obtained after further pepsin treatment and stained with ethidium bromide (EB). The DNA contents of individual cell nuclei were analysed using a flow-cytofluorometer (ICP 11; Phywe, West Germany). The DNA contents of the cells were sorted with the aid of a 256 channel multi-channel analyser. The proportion of cells in the different cell cycle phases (Gl, S and G2+M) was determined from the area of the histograms assuming a Gaussian function of the Gl and the G2+M maxima and attributing the remaining part of the DNA histogram to the cells of the S phase. By [3H]thymidine autoradiography of the separated cell material after pulse-labelling of the cells, in vivo, a good correspondence was found between the proportion of cells in S phase, estimated by autoradiography and from the DNA histograms respectively.
Energy metabolism and cell cycle a 02 CONSUMPTION
d
ATP CONTENT
l
O 1.4 0
.
25
’
n
00
m m
l
mm
n
b
LACTATE PR’XIUCTION
e
0 0
ATP PROWCTION
0 0 0
.
0 0
. .
OOOO C
2.i
PVRUVATE PRODUCTION d d A
f
0
.
0. .
ATP TURNOVER TIME
. L
I .
Duration
of various
. .
0
A,b,AAA
3. The homogeneity of the various fractions is indicated in the inset at the bottom. The figure summarizes the measured and estimated data. (a) Oxygen consumption; (6) lactate production; (c) pyruvate formation; (d) ATP content; (e) total ATP production; (f) ATP turnover time.
Fig.
parts of cell cycle
The DNA distribution of unseparated cells of the first cell fraction of non-tumour cells and of ascites tumour cell fractions, mainly of Gl, of the middle of S phase and G2+M are shown in fig. 1. The percentage of mitotic cells has been found to be up to 10%in the G2 fractions. The first fraction of non-tumour cells also contains the dead cells. The proportion of dead cells in the remaining fractions was below 2 %.
Cell counting and viability studies The number of cells was determined microscopically in a Biirker counting chamber; at the same time the proportion of dead cells was determined with a dye exclusion test using 0.02 ml 10% Lissamine Green B solution for 1 ml of a cell suspension.
Determination of oxygen consumption The oxygen consumption was determined by means of the Warburg technique [14]. 1&60x106 cells were incubated in 3.0 ml of Ringer-phosphate-glucose buffer at 37°C. After an initial adaptation time of 10 min the oxygen consumption was determined every 5th min, for 1 h. Unseparated controls were run in parallel with the separated cell fractions.
Determination of pyruvate and lactate After measurement of the oxygen consumption the cells were inactivated by 0.2 M PCA and centrifuged
at 10000 rpm for 5 min. To 2.0 ml of the supernatant (pH 6.9, 0.165 ml 5 M K2C03 was added. One ml of this cell extract was mixed with 1.0 ml Tris buffer (pH 7.0), 0.4 ml dist. H,O and 0.1 ml DPNH (5.0 mg/ ml). Following the addition of 0.02 ml of LDH (5.0 ms/ml) the content of pyruvate was determined spectrophotometrically (A, 340 nm). For analysis of the lactate, 0.1 ml of the cell-extract was added to 2.2 ml glycine, 0.1 ml dest. H,O and 0.1 ml DPN (10.0 mglml). To the latter 0.02 ml LDH (5.0 mg/ml) was added and the content of lactate determined by measurement of the extinction for 45 min at A, 340 nm [IS].
Determination of ATP The ATP quantity was measured from 1-6~ 104cells usmg the luciferin-luciferase method. 50-100 ~1 NRS (Lumac, Basel) was added to corresponding volumes of cell suspensions in order to liberate the ATP from the cells. The number of photons released subsequent to the luciferin-luciferase reaction with ATP was measured by a Lumac cell-tester, Model 1030, calibrated using known amounts of ATP [ 161.
RESULTS The results are presented (1) as a function of time after cell division (figs 2, 3); (2) in relation to the position of the cells in the cell Exp Cell Res 141 (1982)
26
Skog, Tribukait
and Sundius
Table 1. ATP content, uptake of oxygen and production
of lactate and pyruvate
during
cell cycle The values are expressed on a per cell basis and the oxygen uptake and lactate and pyruvate production are given per hour ATP (fmol) Unseparated cells Gl cell Early Middle Late S phase cell Early Middle Late G2+M cells
Lactate (fmol)
Pyruvate (fmol)
Lactate/ pyruvate
21+2
77+3
315f13
56&l
5.62
l2?l 15*1 17fl
59+2 57t2 49f I
211f7 216f8 20726
42+2 45+3 4352
5.02 4.80 4.81
IWI 20+2 26t 1 28f5
60t2 73+3 94&5 101*4
230&7 290f 13 33Orll2 404f 16
45+2 57f2 69+2 85f3
5.11 5.09 4.78 4.75
cycle (tables 1, 2). For the calculation of cell cycle times the following data and assumptions have been used. The cell doubling time for Cday-old tumour cells determined from the total number of cells at different times after inoculation has been found to be 23 h. The duration of the S phase, as determined by double-labelling autoradiography ([14C]thymidine 1 h followed by [3H]thymidine 40 min), was found to be 10.5 h. Assuming that the proportions of cells in the various parts of the cell cycle are proportional to the time and finding mean values for Gl , S and G2+M cells of 38.1k2.3, 47.6k1.5 and 14.3?0.5% respectively (M + SE n, 18), the mean duration for Gl is 8.4 h and for G2+M 3.2 h. The values were not corrected for exponential growth. The calculated cell cycle time of 22 h is in good agreement with the doubling time of 23 h, indicating a growth fraction of nearly 1.0. The estimated times for the various parts of the cell cycle have been adopted for the abscissa in figs 2-3, which take into consideration the fact that the various fractions contain cells from more than one stage. Each separated fraction is placed on Exp Cell RPS 141 (1982)
the time scale according to the mean DNA content found. Gl and G2+M fractions have been subdivided according to the fraction number into early, middle and late Gl and G2+M cells. Oxygen consumption, lactate and pyruvate contents, ATP content
The results of these experiments are summarized in table 1 and fig. 3. Whereas oxygen consumption, lactate and pyruvate contents were unchanged during Gl and started to increase at early S phase, the ATP content increased during Gl, but remained on a plateau during part of the S phase. All these values increased to double the initial values during late G2. Representative results, showing these differences and the reproducibility of the experiments are shown in fig. 2. The ratio lactatelpyruvate (table 1) has a constant mean value of about 5. ATP production
and turnover time
ATP production due to the aerobic glycolytic metabolism has been estimated by determination of the formation of lactic
Energy metabolism
acid. One mole of lactic acid produced corresponds to 1 mole of ATP. For the estimation of ATP generated by oxidative phosphorylation two assumptions have been made. The whole content of CO, was assumed to be produced by glucose oxidation via the TCA cycle and the oxidative phosphorylation was assumed to be complete coupling, that means that 3 moles of ATP are generated by t mole of O2 [22-251. The total ATP production in unseparated cells is 13 fmol/cell/min. Of the ATP produced, 60% is due to the aerobic metabolism. The same proportion is observed throughout the cell cycle (table 2). The ATP turnover time, given in seconds, is expressed here as the ratio between the ATP content and the ATP production (table 2, fig. 3). The mean time in unseparated cells is 95 set, whereas from early to late Gl the turnover time increases significantly from 75 to 120 set, at the borderline between Gl and S the turnover time decreases slightly and remains throughout the remaining cell cycle at a constant level of 100 sec.
and cell cycle
27
Table 2. Production of ATP by oxygen and lactate expressed per minute and the ATP turnover time in seconds during the cell cycle The values are given on a per cell basis 02 (fmol) Unseparated cells Gl cell Early Middle Late S phase cell Early Middle Late G2+M cells
Lactate (fmol)
o,+
lactate (fmol)
ATP turnover time
8
5
13
95
6 5
4 4
10 9
;: 121
6 7 9 10
4 5 6 7
10 12 15 17
100 loo 100 100
Since the oxygen uptake per 5 min interval was found to be the same throughout the incubation time of 70 min, it would appear that the metabolic function of the cells is not disturbed by in vitro conditions. An unchanged ATP content after a 70 min incubation period further supports this conclusion, since the ATP content is a sensitive DISCUSSION indicator of cell function. Thus, it can be The present investigation includes meas- assumed that our in vitro measurements of urements of ATP, aerobic and aerobic energy metabolism during the cell cycle reglycolytic metabolism. It is thus possible to flect these values occurring under in vivo estimate the total energy metabolism of the conditions. In unseparated Ehrlich ascites tumour cells in different parts of the cell cycle. The elutriation centrifugation technique cells, oxygen uptake and lactate production used in our experiments permitted us to ob- values in agreement with our results have tain sufficient numbers of cells from the been reported earlier [17, 181.However, for various parts of the cell cycle. In compari- ATP only about half the values obtained in son with other techniques, such as mitotic the present study were found after incubaselection and chemical synchronization, in tion for 45 min at a temperature of 30°C which either the number of cells is limited [191. or the cell functions are disturbed, the Nearly 50% of the energy production in elutriation technique is superior. Further- our studies was found to be due to aerobic more elutriation centrifuging separation glycolytic metabolic pathways. This result is consistent with those of other investigapermits work with cells grown in vivo. Exp Cell Res 141 f/982)
28
Skog, Tribukait
and Sundius
tions [17, 181and has been explained by a deficiency of the plasma membrane NatK+-ATPase function [20] in spite of the shift from nearly anoxic environmental conditions in vivo to complete oxic conditions in vitro. A continuously lowered oxygen consumption for 24-48 h for cells grown in conditions of lowered oxygen tension after being placed in normal oxygen tension has also been described [21] indicating no entire change of the metabolic function at the transfer from in vivo to in vitro conditions. In our studies the normal doubling of the analysis values with progression through the cell cycle has generally been found. The slight deviations from this doubling of values can be explained by the mixture of different cell types in the cell fractions. This is particularly the case in the last fraction which contains G2, late S and mitotic cells. The latter are known to exhibit a decrease in energy metabolism [5-lo]. As a rule all parameters studied start to increase in a fairly exponential way in late Gl-early S phase. The ATP content is, however, an exception as it starts to increase in early Gl. The energy consumption connected with the Na+-K+ pump, based on data from Ehrlich ascites tumour cells [26], shows that about half of the total ATP produced during one generation cycle (19x lo-” mol ATP) is consumed by Na+-K+ transport, whereas according to our calculations one-third is consumed by protein synthesis and about 1% by RNA and DNA synthesis. It is of interest to note that in synchronized mouse leukemia lymphoblasts, growing in vitro, the Na+-K+ flux varied during the division cycle, with the highest values of flux in Gl [27]. However, even other transport processes, such as the amino acid influx into the cells, require energy and must be taken into consideration when the total energy need is Exp CellRcs 141 (1982)
discussed. On a per cell basis, as shown in fig. 3, the ATP turnover time decreases from a maximum value of 120 set at late Gl to 100 set during the remaining portion of the cell cycle. Decreasing turnover times have also been found in L cells during S phase, followed by a further rapid increase during G2+M [II]. As the authors of that report [l l] point out, the degree of synchronization during the later part of the cell cycle decreased. A lack of glycose in the later stages of these experimentals might be another factor of importance, and these estimations might therefore be less dependable. The estimated total energy requirement for one complete cell cycle for Ehrlich ascites tumour cells was found to be 19x lo-l2 mol ATP per cell. In estimating this value from the collected data of Bichis et al. [17], Wu & Racker [18] and Scholnick et al. [19] in Ehrlich ascites tumour cells and assuming a cell cycle time of 24 h, 20x lo-‘* mols of ATP/per cell are found, i.e. a similar figure. Comparing these values with corresponding estimations for mouse L cells [21, 281and mouse Ls cells [29], a considerably higher energy requirement, between 38 and 45x lo-‘* mol ATP is found. This may be related to the greater size of the L cells [21] but more experience with other cell types is required to ascertain whether low requirements are associated, e.g., with malignant behaviour. In summary, the present investigation shows that the energy metabolism proceeds discontinuously during the cell cycle and does not show the direct relationship to the DNA synthesis proposed by other investigators [2]. Whereas the energy production during Gl is unchanged, the ATP content increases markedly and consequently the ATP turnover time changes most rapidly between early and late Gl.
Energy metabolism and cell cycle This work was supported by grants from the Cancer Societv in Stockholm.
REFERENCES 1. Gerschenson, L E, Strasser, F F & Rounds, D E, Life sci 4 (1965) 927. 2. Robbini, E & Morill, G A, J cell bio143 (1969) 692. 3. Blomquist, C H, Gregg, C T & Tobey, R A, Exp cell res 66 (1971) 75. 4. Chapman, J D, Webb, R G & Borsa, J, J cell biol 49 (1971) 229. 5. Holtzer, H & Zeuthen, E, Pubbl staz zoo1 Napoli 29 (1957) 285. 6. Scholander. P F. Claff. C L. Sveinsson. S L & Scholander; S 1, Biol bull 102(1952) 185. Erickson, R 0, Nature 159 (1947) 275. li Stern, H & Kirk, P, J gen physiol 31 (1948) 243. 9: Swann, M M, Cancer res 17 (1957) 727. 10. Scholander, P F, Leivestad, H & Sundnes, G, Exp cell res 15 (1958) 505. 11. Ishiguro, S, Yamaguchi, H, Oka, Y & Miyamoto, H, Cell struct and funct 3 (1978) 331. 12. Meistrich, M L, Meyn, R E & Barlogie, B, Exp cell res 105 (1977) 169. 13. Tribukait, B, Moberger, G & Zetterbere. A, First int symp pulse-cy&photometry (ed -C A M Haanen, H F P Hillen & J M C Wessels) p. 50. European Press Medikon, Ghent (1975). 14. Umbreit, W W. Burris, R H & Stauffer. J F. Manometric techniques. Burgess Publishing ‘Corn., Minneapolis (1964).
Printed
in Sweden
29
15. Hohorst, H J, Kreutz, F H & Bilcher, Th, Biochem Z 332 (1959) 18. 16. Thore, A, Sci tools 26 (1979) 30. 17. Bickis, I J, Quastel, J H & Vas, S I, Cancer res 19 (1959) 602. 18. Wu, R & Racker, E, J biol them 234 (1959) 1029. 19. Scholnick, P, Lang, D & Racker, E, J biol them 248 (1973) 5175. 20. Racker, E, J cell physiol 89 (1976) 697. 21. Paul, J, Cells and tissues in culture (ed E N Willner) vol. 7. v. 239. Academic Press. New York (1965). 22. Currie, W D & Gregg, C T, Biochem biophys res commun 21 (1965) 9. 23. Dallner, G & Ernster, L, Exp cell res 27 (1962) 373 -.-.
24. Minakami, S & Yoshikawa, H, Biochim biophys acta 74 (1963) 793. 25. Yonezu, T, Tokushima J exp med 37 (1974). 26. Aull, F & Hempling, H G, Am j physiol204 (1963) 789. 27. Jung, C & Rothstein, A, J gen physiol 50 (1967) 917. 28. Miyamoto, H, Yonezu, T, Tsuda, S, Yamaguchi, H, Ishiguro, S & Oka, Y, Tokushima j exp med 21 (1974) 61. 29. Kilburn, D G, Lilly, M D &Webb, F C, J cell sci 4 (1969) 645. Received December 10, 1981 Revised version received March 1, 1982 Accepted March 9, 1982
Exp Cell Res 141 (1982)
Experimental
ENERGY
METABOLISM
THE CELL
CYCLE
Cell Research 141 (1982) 23-29
AND ATP TURNOVER
OF EHRLICH
SVEN SKOG, BERNHARD
ASCITES
TRIBUKAIT
TIME
TUMOUR
DURING CELLS
and GUDRUN SUNDIUS
Department of Medical Radiobiology, Karolinska Instituter, S-10401 Stockholm, Sweden
SUMMARY The energy production in different parts of the cell cycle due to aerobic and aerobic glycolytic metabolism and ATP turnover time was estimated by measuring the oxygen consumption, lactate-pyruvate and ATP content of Ehrlich ascites tumour cells growing in vivo. Cell fractions of high purity fromthe various parts of the cell cycle were obtained by means of elutriator centrifuging. The total energy production for one cell cycle was estimated to be 19x lo-r2 mol ATP, 60% of which was due to the aerobic metabolism. Whereas the total ATP production is unchanged during Gl a fairly exponential increase is found during the S and G2+M phases. The total cellular ATP content increases from 12fmol ATP at early Gl to 28 fmol ATP at G2+M; this increase, however, is discontinuous and is most pronounced during Gl and during late S phase S phase/GZ+M. The ATP turnover time, as defined as the ratio between ATP content and ATP production, was found to increase significantly from 75 set in early Gl to 120 set in late Gl but was constantly 100 set during the early, middle and late S phase as well as G2+M. These variations indicate maximum energy-requiring processes during early Gl period of the cell cycle and are discussed in relation to K+Na+ flux and macromolecule synthesis.
Various sections of the energy metabolism In the experiments presented here, eluof mammalian cells, such as aerobic meta- triator centrifugation of in vivo growing bolism, aerobic glycolytic metabolism, and Ehrlich ascites tumour cells has been used ATP content in various parts of the cell to obtain sufftcient cell material from difcycle have been studied in a number of syn- ferent parts of the cell cycle. Elutriator cenchronized cell systems [l-l 11. trifugation has been shown to avoid interTo our knowledge there is, however, ference with cell growth [12], giving cell only one study on mouse L cells growing in fractions with a high degree of purity, exvitro in which both aerobic and aerobic cept in the case of mitotic cells. The oxygen glycolytic metabolism and ATP content in consumption, lactate and pyruvate contents the various parts of the cell cycle have been and the ATP concentration have been determined, enabling an estimation of the studied. From these data, aerobic and ATP turnover time [ 111. In this study con- aerobic glycolytic energy production and siderable changes in oxygen uptake were ATP turnover time have been estimated. found during the cell cycle, but less pronounced changes in the production of lactic MATERIAL AND METHODS acid and only slight changes in the ATP content. The estimated turnover time for The cell material ATP increased from 16 to 19 set during Gl, Hyperdiploid Ehrlich ascites tumour cells were to grow in vivo for 4 days after i.p. transdecreased during S, but increased again at allowed plantation of 3x 10’ IO-day-old cells into 3-month old female NMRI mice. G2+M. Exp Cell Res 141 (1982)
24
Skog, Tribukait and Sundius
J., ,,..IO 0
zoburs
I 0,
,
IO
.
20~s
various parts”bf cell
Fig. 2. The homogeneity of the various fractions is
indicated in the inset at the bottom. Cell fractions from various parts of the cell cycle were obtained by elutriator centrifuging. The ATP content was determined immediately after separation using luciferinluciferase assay. Then the cells were incubated in Ringer-phosphate-glucose buffer at 37°C and the oxygen consumption was measured by the Warburg manometric technique for 70 min. Dotted lines drawn by eye. Results from eleven and three separations respectively.
J=++s 50
150
Channel no. corresponding
to DNA amount
Fig. I. After preparation of cell nuclei (see Material
and Methods) the nuclei were stained with ethidium bromide and analysed by means of a rapid-flow cytophotometer. (n) Resulting DNA histogram of unseparated cells. The first maximum to the left corresponds to normal cells. The second maximum is formed by Gl cells of the tumour cell population. The maximum to the right is formed by the corresponding G2 and mitotic cells. In between these two maxima the S phase cells can be seen. (b-e) Examples of fractions of cells after elutriation centrifuging; (b) mainly normal cells with a small proportion of Gl tumour cells; (c) Gl cells of the tumour cell population; (d) mainly cells from the middle part of the S phase; (e) mainly G2+M cells.
Cell preparation and separation Fresh cell material was washed twice in ice-cold Ringer-phosphate-glucose buffer (0.154 M NaCI, 0.154 M KCI, 0.064 M CaC1, 0.15 M MgS0,x7Hp0, 0.1 M phosphate buffer, 0.01 M glucose pH 7.4) or Tris-EDTA buffer (0.1 M Tris, O.od7M NaCl, 0.005 M EDTA, pH 7.5) and centrifugated at 250 g for 5 min. Cell separation was carried out using a Beckman JE-G elutriator rotor with a Beckman J21B centrifuge. The rotor speed was held at 2780flO rpm. About 5x108 cells in buffer solution were added to this system, the whole of which was kept at 4”C, with an initial pump speed of 12 ml/min. Generally six cell E.xp Cell Res 141 (1982)
fractions of 100ml were obtained by stepwise increase in the pump speed up to 57 ml per min. The measurements of ATP were carried out on twenty fractions obtained in a similar manner. The cells were concentrated by centrifuging at 500 g for 5 min. The time interval between start of the separation and start of the analyses was about 40 min.
Determination of the composition of the cell fractions The composition of the cell fractions was determined using quantitative DNA analysis by means of flowcytofluorometric measurements as described earlier [13]. Generally, the cells were fixed in ethanol. After washing in Tris EDTA buffer together with RNase, suspensions of individual cell nuclei were obtained after further pepsin treatment and stained with ethidium bromide (EB). The DNA contents of individual cell nuclei were analysed using a flow-cytofluorometer (ICP 11; Phywe, West Germany). The DNA contents of the cells were sorted with the aid of a 256 channel multi-channel analyser. The proportion of cells in the different cell cycle phases (Gl, S and G2+M) was determined from the area of the histograms assuming a Gaussian function of the Gl and the G2+M maxima and attributing the remaining part of the DNA histogram to the cells of the S phase. By [3H]thymidine autoradiography of the separated cell material after pulse-labelling of the cells, in vivo, a good correspondence was found between the proportion of cells in S phase, estimated by autoradiography and from the DNA histograms respectively.
Energy metabolism and cell cycle a 02 CONSUMPTION
d
ATP CONTENT
l
O 1.4 0
.
25
’
n
00
m m
l
mm
n
b
LACTATE PR’XIUCTION
e
0 0
ATP PROWCTION
0 0 0
.
0 0
. .
OOOO C
2.i
PVRUVATE PRODUCTION d d A
f
0
.
0. .
ATP TURNOVER TIME
. L
I .
Duration
of various
. .
0
A,b,AAA
3. The homogeneity of the various fractions is indicated in the inset at the bottom. The figure summarizes the measured and estimated data. (a) Oxygen consumption; (6) lactate production; (c) pyruvate formation; (d) ATP content; (e) total ATP production; (f) ATP turnover time.
Fig.
parts of cell cycle
The DNA distribution of unseparated cells of the first cell fraction of non-tumour cells and of ascites tumour cell fractions, mainly of Gl, of the middle of S phase and G2+M are shown in fig. 1. The percentage of mitotic cells has been found to be up to 10%in the G2 fractions. The first fraction of non-tumour cells also contains the dead cells. The proportion of dead cells in the remaining fractions was below 2 %.
Cell counting and viability studies The number of cells was determined microscopically in a Biirker counting chamber; at the same time the proportion of dead cells was determined with a dye exclusion test using 0.02 ml 10% Lissamine Green B solution for 1 ml of a cell suspension.
Determination of oxygen consumption The oxygen consumption was determined by means of the Warburg technique [14]. 1&60x106 cells were incubated in 3.0 ml of Ringer-phosphate-glucose buffer at 37°C. After an initial adaptation time of 10 min the oxygen consumption was determined every 5th min, for 1 h. Unseparated controls were run in parallel with the separated cell fractions.
Determination of pyruvate and lactate After measurement of the oxygen consumption the cells were inactivated by 0.2 M PCA and centrifuged
at 10000 rpm for 5 min. To 2.0 ml of the supernatant (pH 6.9, 0.165 ml 5 M K2C03 was added. One ml of this cell extract was mixed with 1.0 ml Tris buffer (pH 7.0), 0.4 ml dist. H,O and 0.1 ml DPNH (5.0 mg/ ml). Following the addition of 0.02 ml of LDH (5.0 ms/ml) the content of pyruvate was determined spectrophotometrically (A, 340 nm). For analysis of the lactate, 0.1 ml of the cell-extract was added to 2.2 ml glycine, 0.1 ml dest. H,O and 0.1 ml DPN (10.0 mglml). To the latter 0.02 ml LDH (5.0 mg/ml) was added and the content of lactate determined by measurement of the extinction for 45 min at A, 340 nm [IS].
Determination of ATP The ATP quantity was measured from 1-6~ 104cells usmg the luciferin-luciferase method. 50-100 ~1 NRS (Lumac, Basel) was added to corresponding volumes of cell suspensions in order to liberate the ATP from the cells. The number of photons released subsequent to the luciferin-luciferase reaction with ATP was measured by a Lumac cell-tester, Model 1030, calibrated using known amounts of ATP [ 161.
RESULTS The results are presented (1) as a function of time after cell division (figs 2, 3); (2) in relation to the position of the cells in the cell Exp Cell Res 141 (1982)
26
Skog, Tribukait
and Sundius
Table 1. ATP content, uptake of oxygen and production
of lactate and pyruvate
during
cell cycle The values are expressed on a per cell basis and the oxygen uptake and lactate and pyruvate production are given per hour ATP (fmol) Unseparated cells Gl cell Early Middle Late S phase cell Early Middle Late G2+M cells
Lactate (fmol)
Pyruvate (fmol)
Lactate/ pyruvate
21+2
77+3
315f13
56&l
5.62
l2?l 15*1 17fl
59+2 57t2 49f I
211f7 216f8 20726
42+2 45+3 4352
5.02 4.80 4.81
IWI 20+2 26t 1 28f5
60t2 73+3 94&5 101*4
230&7 290f 13 33Orll2 404f 16
45+2 57f2 69+2 85f3
5.11 5.09 4.78 4.75
cycle (tables 1, 2). For the calculation of cell cycle times the following data and assumptions have been used. The cell doubling time for Cday-old tumour cells determined from the total number of cells at different times after inoculation has been found to be 23 h. The duration of the S phase, as determined by double-labelling autoradiography ([14C]thymidine 1 h followed by [3H]thymidine 40 min), was found to be 10.5 h. Assuming that the proportions of cells in the various parts of the cell cycle are proportional to the time and finding mean values for Gl , S and G2+M cells of 38.1k2.3, 47.6k1.5 and 14.3?0.5% respectively (M + SE n, 18), the mean duration for Gl is 8.4 h and for G2+M 3.2 h. The values were not corrected for exponential growth. The calculated cell cycle time of 22 h is in good agreement with the doubling time of 23 h, indicating a growth fraction of nearly 1.0. The estimated times for the various parts of the cell cycle have been adopted for the abscissa in figs 2-3, which take into consideration the fact that the various fractions contain cells from more than one stage. Each separated fraction is placed on Exp Cell RPS 141 (1982)
the time scale according to the mean DNA content found. Gl and G2+M fractions have been subdivided according to the fraction number into early, middle and late Gl and G2+M cells. Oxygen consumption, lactate and pyruvate contents, ATP content
The results of these experiments are summarized in table 1 and fig. 3. Whereas oxygen consumption, lactate and pyruvate contents were unchanged during Gl and started to increase at early S phase, the ATP content increased during Gl, but remained on a plateau during part of the S phase. All these values increased to double the initial values during late G2. Representative results, showing these differences and the reproducibility of the experiments are shown in fig. 2. The ratio lactatelpyruvate (table 1) has a constant mean value of about 5. ATP production
and turnover time
ATP production due to the aerobic glycolytic metabolism has been estimated by determination of the formation of lactic
Energy metabolism
acid. One mole of lactic acid produced corresponds to 1 mole of ATP. For the estimation of ATP generated by oxidative phosphorylation two assumptions have been made. The whole content of CO, was assumed to be produced by glucose oxidation via the TCA cycle and the oxidative phosphorylation was assumed to be complete coupling, that means that 3 moles of ATP are generated by t mole of O2 [22-251. The total ATP production in unseparated cells is 13 fmol/cell/min. Of the ATP produced, 60% is due to the aerobic metabolism. The same proportion is observed throughout the cell cycle (table 2). The ATP turnover time, given in seconds, is expressed here as the ratio between the ATP content and the ATP production (table 2, fig. 3). The mean time in unseparated cells is 95 set, whereas from early to late Gl the turnover time increases significantly from 75 to 120 set, at the borderline between Gl and S the turnover time decreases slightly and remains throughout the remaining cell cycle at a constant level of 100 sec.
and cell cycle
27
Table 2. Production of ATP by oxygen and lactate expressed per minute and the ATP turnover time in seconds during the cell cycle The values are given on a per cell basis 02 (fmol) Unseparated cells Gl cell Early Middle Late S phase cell Early Middle Late G2+M cells
Lactate (fmol)
o,+
lactate (fmol)
ATP turnover time
8
5
13
95
6 5
4 4
10 9
;: 121
6 7 9 10
4 5 6 7
10 12 15 17
100 loo 100 100
Since the oxygen uptake per 5 min interval was found to be the same throughout the incubation time of 70 min, it would appear that the metabolic function of the cells is not disturbed by in vitro conditions. An unchanged ATP content after a 70 min incubation period further supports this conclusion, since the ATP content is a sensitive DISCUSSION indicator of cell function. Thus, it can be The present investigation includes meas- assumed that our in vitro measurements of urements of ATP, aerobic and aerobic energy metabolism during the cell cycle reglycolytic metabolism. It is thus possible to flect these values occurring under in vivo estimate the total energy metabolism of the conditions. In unseparated Ehrlich ascites tumour cells in different parts of the cell cycle. The elutriation centrifugation technique cells, oxygen uptake and lactate production used in our experiments permitted us to ob- values in agreement with our results have tain sufficient numbers of cells from the been reported earlier [17, 181.However, for various parts of the cell cycle. In compari- ATP only about half the values obtained in son with other techniques, such as mitotic the present study were found after incubaselection and chemical synchronization, in tion for 45 min at a temperature of 30°C which either the number of cells is limited [191. or the cell functions are disturbed, the Nearly 50% of the energy production in elutriation technique is superior. Further- our studies was found to be due to aerobic more elutriation centrifuging separation glycolytic metabolic pathways. This result is consistent with those of other investigapermits work with cells grown in vivo. Exp Cell Res 141 f/982)
28
Skog, Tribukait
and Sundius
tions [17, 181and has been explained by a deficiency of the plasma membrane NatK+-ATPase function [20] in spite of the shift from nearly anoxic environmental conditions in vivo to complete oxic conditions in vitro. A continuously lowered oxygen consumption for 24-48 h for cells grown in conditions of lowered oxygen tension after being placed in normal oxygen tension has also been described [21] indicating no entire change of the metabolic function at the transfer from in vivo to in vitro conditions. In our studies the normal doubling of the analysis values with progression through the cell cycle has generally been found. The slight deviations from this doubling of values can be explained by the mixture of different cell types in the cell fractions. This is particularly the case in the last fraction which contains G2, late S and mitotic cells. The latter are known to exhibit a decrease in energy metabolism [5-lo]. As a rule all parameters studied start to increase in a fairly exponential way in late Gl-early S phase. The ATP content is, however, an exception as it starts to increase in early Gl. The energy consumption connected with the Na+-K+ pump, based on data from Ehrlich ascites tumour cells [26], shows that about half of the total ATP produced during one generation cycle (19x lo-” mol ATP) is consumed by Na+-K+ transport, whereas according to our calculations one-third is consumed by protein synthesis and about 1% by RNA and DNA synthesis. It is of interest to note that in synchronized mouse leukemia lymphoblasts, growing in vitro, the Na+-K+ flux varied during the division cycle, with the highest values of flux in Gl [27]. However, even other transport processes, such as the amino acid influx into the cells, require energy and must be taken into consideration when the total energy need is Exp CellRcs 141 (1982)
discussed. On a per cell basis, as shown in fig. 3, the ATP turnover time decreases from a maximum value of 120 set at late Gl to 100 set during the remaining portion of the cell cycle. Decreasing turnover times have also been found in L cells during S phase, followed by a further rapid increase during G2+M [II]. As the authors of that report [l l] point out, the degree of synchronization during the later part of the cell cycle decreased. A lack of glycose in the later stages of these experimentals might be another factor of importance, and these estimations might therefore be less dependable. The estimated total energy requirement for one complete cell cycle for Ehrlich ascites tumour cells was found to be 19x lo-l2 mol ATP per cell. In estimating this value from the collected data of Bichis et al. [17], Wu & Racker [18] and Scholnick et al. [19] in Ehrlich ascites tumour cells and assuming a cell cycle time of 24 h, 20x lo-‘* mols of ATP/per cell are found, i.e. a similar figure. Comparing these values with corresponding estimations for mouse L cells [21, 281and mouse Ls cells [29], a considerably higher energy requirement, between 38 and 45x lo-‘* mol ATP is found. This may be related to the greater size of the L cells [21] but more experience with other cell types is required to ascertain whether low requirements are associated, e.g., with malignant behaviour. In summary, the present investigation shows that the energy metabolism proceeds discontinuously during the cell cycle and does not show the direct relationship to the DNA synthesis proposed by other investigators [2]. Whereas the energy production during Gl is unchanged, the ATP content increases markedly and consequently the ATP turnover time changes most rapidly between early and late Gl.
Energy metabolism and cell cycle This work was supported by grants from the Cancer Societv in Stockholm.
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24. Minakami, S & Yoshikawa, H, Biochim biophys acta 74 (1963) 793. 25. Yonezu, T, Tokushima J exp med 37 (1974). 26. Aull, F & Hempling, H G, Am j physiol204 (1963) 789. 27. Jung, C & Rothstein, A, J gen physiol 50 (1967) 917. 28. Miyamoto, H, Yonezu, T, Tsuda, S, Yamaguchi, H, Ishiguro, S & Oka, Y, Tokushima j exp med 21 (1974) 61. 29. Kilburn, D G, Lilly, M D &Webb, F C, J cell sci 4 (1969) 645. Received December 10, 1981 Revised version received March 1, 1982 Accepted March 9, 1982
Exp Cell Res 141 (1982)