- Email: [email protected]
The effect of serum starvation on DNA, RNA and protein synthesis during interphase in L-cells
Experimental Cell Research 57 (1969) 114-l 18
THE EFFECT
OF SERUM SYNTHESIS
STARVATION DURING
ON DNA,
INTERPHASE
A. ZETTERBERG
RNA AND
PROTEIN
IN L-CELLS
and 0. SKOLD
Institute for Medical Cell Research and Genetics, Karolinska Institute& and Department of Microbiology, Farmaceutiska Institutet, Stockholm, Sweden
SUMMARY The effect of withdrawal of the serum complement from tissue culture growth medium (serum starvation) on synthesis of DNA, RNA and protein and on cell multiplication was studied in mouse fibroblasts in vitro (cell lme L-929) by a combination of chemical and quantitative cytochemical methods. The first effect of serum starvation was seen on the synthesis of RNA, which was moderately depressed as early as 4-6 h after serum removal. Protein synthesis was relatively unaffected by serum starvation. The rate of DNA synthesis was unaffected for the first 10 h of serum starvation, but was depressed to about 4 of the normal value after 20 h of starvation. This effect on DNA synthesis was shown to be a combined effect on the initiation and rate of DNA synthesis. Cvtophotometric DNA measurements on individual cells revealed that the observed inhibition of mitotic activity during serum starvation was not secondary to the inhibited DNA synthesis. Instead a direct effect on the conversion of GZ-phase cells into the mitotic stage was observed to be at work, i.e. the serum complement in the growth medium seemed to be a-factor of importance for the initiation of mitosis.
It is well known that growth media used for tissue culture of mammalian cells must contain serum in order to support rapid proliferation of cells in vitro. Very little is known, however, about the mechanisms whereby serum factors are involved in the metabolic processes of the interphase and mitosis of the growing cell. The aim of the present investigation was to find out if some of the metabolic processesin the cell cycle were particularly sensitive to serum starvation. For this purpose cell proliferation and synthesis of DNA, RNA and protein in serum starved L-cell cultures were followed by a combination of biochemical and cytochemical methods. While serum starvation had a very small effect on the biosynthesis of protein, RNA synthesis was rapidly inhibited to some degree. A pronounced inhibition of DNA synthesis was observed during serum starvation but only after a Exptl Cell Res 57
certain delay. The observed inhibitory effect on cell proliferation was shown, however, not to be secondary to the inhibited DNA synthesis. MATERIALS AND METHODS Cells Monolayer cultures of mouse fibroblasts (line L-929) with a population doubling time of approx. 20 h in the exponential phase of growth were used. The cells were cultivated at 37°C in the medium of Eagle [7] as described earlier [II]. Each culture flask contained 10 ml, and each petri dish 5 ml of medium. At the time of inoculation the cell concentration was 10,000 cells/ml.
Radioisotopes W-uridine, specific activity 17.4,uC/pmole was purchased from New England Nuclear Corp. ‘H-DL-Lysine 307 &/,umole and BH-uL-tryptophan 500 yC/pmole were obtained from The Radiochemical Centre, Amersham.
Biochemical analyses After pouring off the old medium, the cell monolayer was washed with 10 ml of sterile 0.15 M NaCl and the final
Effects of serum starvation on L-cells
115
prewarmed media (with and without 10 % of dialyzed calf serum (Gibco)) containing the radioactive precursors were added. The cells from each flask were harvested by scraping, and then suspended in 2 ml of 0.15 M NaCl. A 0.3 ml sample of the suspension was withdrawn and added to 1.2 ml of 0.1 M NaOH to give a clear solution, and the absorbance at 265 nm of this was used to normalize experimental variations in total cell mass between different culture flasks. The incorporation of 8H-amino acids and of 14C-uridine into trichloroacetic acid insoluble product was used as a measure of protein and nucleic acid synthesis. RNA and DNA were separated by alkaline hydrolysis as described earlier [l 11. Determination of 8H- and 14C-activity was done in a Packard TriCarb liquid-scintillation counter and the spectrometric separation of 8H- and IV-activity was performed as described previously [ 111.
Cytochemical analyses For the cell multiplication and cytochemical analyses the cells were cultivated directly on Biirker slides in petri dishes as described previously [9]. On each slide carrying the multiplying cell populations one microscopic field was repeatedly photographed for about 60 h under low power magnification by means of time-lapse-cinematography. When the cell populations had entered the exponential phase of growth the cell cultures were briefly washed in isotonic saline and the medium was changed. Eagle’s medium with and without 10 % of dialyzed calf serum (Gibco) was added to the control and experimental cultures, respectively. The temperature of the cells was carefully kept at 37°C through the whole experiment. At the end of the experiment, the slide cultures were washed in saline and fixed in a mixture of acetone and absolute ethanol (1: 1) at room temperature for 30 mm, and then kept in the fixation mixture at 4°C until the cells were stained. The cells in the photographed areas of the slide cultures were analyzed individually for their DNA content, as represented by their total extinction at 546 nm after Feulgen staining, by use of a rapid scanning microspe&ophotome& [$ 6, IO]. To obtain the mitoiic index, 5000 hematoxylin-stained cells were counted at x 800 magnification by phase contrast microscopy on each slide culture in a separate experiment. Cells from late prophase through late telophase were counted.
RESULTS Cell multiplication
The effect of serum starvation on cell multiplication is illustrated by the growth curves presented in fig. 1. During the first 10 h of serum starvation the cell number of the treated population increased by approx. 30 % compared to 75 % in the control population. During the following 35 h of serum starvation there was no increase in cell number in the treated culture. This was due to an inhibited cell division indicated by a drop in the mitotic index from 2.5 to 0.4 after 45 h of serum starvation.
i0
2.0
i0
i0
SO
BO
7b
Abscissa: Hours; ordinate: number of cells/unit area. Fig. 1. Effect of serum starvation on cell multiplication. Number of cells per unit surface area in relative units plotted against experimental time in hours. The arrow indicates the time at which the growth medium was changed and serum starvation was introduced. 0 -- 0, serum starved cell population; l - l , control population.
Incorporation of labelled precursors into DNA, RNA and protein
The effect of serum starvation on the synthesis of nucleic acid and protein was studied by the incorporation of labelled precursors into acid precipitable material. The incorporation was followed during two different time periods. Firstly, over the initial 11 h of serum starvation, i.e. when some cell multiplication was taking place. Secondly, between the 1lth and the 20th h of starvation, i.e. during the time period when the mitotic activity was greatly reduced. The results presented in fig. 2 show the incorporation of 3H-lysine and 14C-uridine into protein, RNA and DNA, respectively, during the first 11 h of serum starvation. The lack of serum did not seem to have any effect on the incorporation of 3Hlysine into protein. Similarly, no significant decrease in the rate of 14C-uridine incorporation into DNA during the first 11 h of serum starvation was observed. The effect on the synthesis of RNA was different. Already after 5 h 30 % less 14C-uridine had been incorporated into RNA of the starved cells as compared to the control. Exptl Cell Res 57
116 A. Zetterberg & 0. Skiild
10000
2oooc
15001
15ooc
7500
1oooc
5000
5000
2500
12345678
9
10
11
Ir’
7500
1oooc I -
5000
5000 , -
2500
12
Hours; ordinate: (left) cpm (3H-lysine); (right) cpm (W-uridine). Fig. 2. Effect of serum starvation on protein, RNA and DNA synthesis in L cells. To each tissue culture flask containing 10 ml of Eagle’s medium was added Wuridine and unlabelled uridine to a final concentration of 0.10 pmoles/ml and final specific activity of 0.5 pC/pmole, and 3H-lysine to a concentration of 0.5 pmoleslml and a final specific activity of 4 &/pmole of L form. Each experimental point represents one tissue culture flask. All flasks were inoculated at the same time with a standardized inoculum and the cell populations had entered the logarithmic phase of growth before the addition of the radioactive precursors (time zero). Serum starvation was instituted in one experimental series at time zero. The values along the ordinate are expressed as cpm per all harvested cells from each flask, see the experimental section. A - A , 8H-lysine incorporation + serum; A -- A , 8H-lysine incorporation - serum; 0 - 0, W-uridine incorporation into RNA + serum; 0 --- 0, W-uridine incorporation into RNA - serum; x - x , Y-uridine incorporation into DNA + serum; x --- x , W-uridine incorporation into DNA - serum.
x
Abscissa:
Fig. 3 demonstrates the effect of serum starvation on the synthesis of nucleic acid and protein during the period of mitotic inhibition. The cells were starved for serum during 11 h before the labelled precursors were added to the medium. The rate of protein synthesis was followed by incorporation of 3H-tryptophan into acid precipitable material. Nine h after the addition of SH-tryptophan, i.e. 20 h after the removal of serum from the medium, 20% less 3H-tryptophan had been incorporated than in the control. The inhibition of RNA synthesis, which was apparent as early as 5 h after the removal of serum from the medium (fig. 2) was further increasedslightly between the 1Ith and the 20th h. Exptl Cell Res 57
-x---------x 1
2
3
L
5
6
7
*
g HOURS
Abscissa: Hours; ordinate: (left) cpm/SH-tryptophan; (right) cpm (14C-uridine). Fig. 3. Effect of serum starvation on protein, RNA and DNA synthesis in L cells. In both experimental series W-uridine was added to a final concentration of 0.10 pmoles/ml and a specific activity of 0.5 ,uC/~mole. 8Htryptophan was added to a final concentration of 0.5 ,umoles/ml and a specific activity of 24 @Z/pmole of L form. Serum starvation was instituted 11 h before the addition of radioactive precursors which were added at time zero. The values along the ordinate and procedures otherwise were as described in the legend to fig. 2. 8H-tryptophan incorporation + serum; A -- A , A-A, SH-tryptophan incorporation -serum; 0 - l , r*C-uridine incorporation into RNA + serum; 0 - - - 0, W-uridine incorporation into RNA -serum; x - x , W-uridine incorporation into DNA + serum; x - - - - x , W-uridine incorporation into DNA -serum.
During the 20 h of serum starvation the incorporation of 14C-uridine into RNA was 40 % less than in the control. The rate of 14C-uridine incorporation into DNA decreaseddramatically between the 11th and the 20th h. During this period about one-third as much DNA was synthesized in the serum starved cells as in the control cells. In summary these incorporation data seemto indicate that serum starvation introduced an early and moderate inhibitory effect on the synthesis of RNA, a late and substantial inhibitory effect on the synthesis of DNA and a late and only slight inhibitory effect on the synthesis of protein.
Effects of serum starvation on L.-cells
the Gl phase, S phase and G2 phase respectively, of the cell cycle. It is thus obvious from the results presented in fig. 4 that the population which was serum-starved for 24 h is composed of Gl, S and G2 cells in the same proportion as in the non-treated population.
20 CONTROL N=130 15
117
D==35.6 i
DISCUSSION 20 MINUS N.130 15
SERUM
D%=31.L
10
5~
J10
20
30
LO
50
) 70 DNA
Abscissa: DNA; ordinate: cell number, %. FOP. 4. Intercellular distribution of DNA. Number of ceils in per cent of total number of cells analyzed, plotted against cellular Feulgen-DNA values in relative units. Upper histogram: non serum starved, multiplying control population. Lower histogram: non-multiplying cell population, serum starved for 48 h.
Intercellular
distribution of DNA
In order to investigate the relationship between inhibition of DNA synthesis and mitotic activity, the intercellular distribution of DNA content in a cell population starved for serum during 48 h was compared to that of a control population. The results which are presented in fig. 4 show the typical two-peak distribution in both the control (fig. 4, upper part) and in the serum starved population (fig. 4, lower part). Identical results were obtained from a cell population starved for serum for 24 h. There does not appear to be any significant difference between the two cell populations with respect to the proportion of cells with postmitotic amounts of DNA (the peak with the DNA values of about 20 relative units), with premitotic amounts of DNA (the peak with DNA values about 40 relative units) and with intermediate DNA values. The cells with postmitotic, intermediate and premitotic amounts of DNA represent the cells in
From the results presented in this study it was shown that the number of cells growing on a glass surface ceasedto increase a short time after the serum was removed from the growth medium. This effect was accompanied by a marked decrease in the mitotic index. It is thus obvious that serum starvation had a pronounced inhibitory effect on cell multiplication, Serum starvation could theoretically inhibit cell multiplication by affecting a number of different steps in the cell cycle. It could prevent cell multiplication by disturbing for instance the division process as such. This should then lead to an accumulation of cells in the mitotic stage i.e. an increased mitotic index. This was, however, not observed. Instead a marked drop in the mitotic index was demonstrated after some time of serum starvation. The inhibited cell multiplication was thus not primarily due to a disturbed division process. Some mitotic cells were, however, observed in the absence of any detectable increase in cell number. A small influence of serum starvation on the mitotic process could therefore not be completely ruled out. Furthermore, the inhibited cell multiplication could be the result of an inhibited DNA synthesis as is the casewith 5-fluorodeoxyuridinetreated cells [3]. An inhibited DNA synthesis was in fact observed. Measurements of cellular DNA content revealed, however, that the serumstarved cell population contained a relatively large proportion of cells with premitotic amounts of DNA (G2 phase cells). The arrested mitotic activity can thus not be a secondary phenomenon to the inhibited DNA synthesis. It rather appeared to be caused by a failure of the serum starved G2 phase cells to initiate mitosis. In this respect the effect of serum starvation seemsto Exptl Cell Res 57
118 A. Zetterberg & 0. Skiild be similar to that of X-irradiation [4, 81 and nitrogen mustard [l. 21. If exclusively the initiation of mitosis would have been inhibited by the serum starvation the proportion of Gl and S-phasecells of the population would have decreasedand the proportion of G2 phase cells increased becauseof the failure of these latter cells to enter mitosis and divide. This was not found to be the case. The cellular DNA measurements showed that both Gl and S-phase cells were found in the serum-starved population in the same proportion as in the control. In conjunction with the data showing a reduced rate of nucleoside incorporation into DNA of the serum-starved population, the cellular DNA measurements indicated that not only the rate but also the initiation of DNA synthesis was inhibited by the removal of serum from the growth medium. The inhibited cell multiplication could furthermore be due to a nonspecific inhibition of cellular growth. This is, however, an unlikely explanation taking into consideration the relatively moderate inhibitory effect of serum starvation on RNA synthesis and in particular on protein synthesis.Irrespective of the precise mechan-
Exptl Cell Res 57
ism by which serum starvation inhibits cell multiplication it appeared primarily to affect the sequenceof processesadvancing the cell towards mitosis (initiation of DNA synthesis, DNA synthesis and initiation of mitosis), while ythesynthetic processes responsible for cellular growth (RNA and protein synthesis) appeared to be relatively unaffected.
REFERENCES 1. Brewer, H B, Comstock, J P & Aronow, L, Biochem pharmacol 8 (1962) 281. 2. Caspersson, T, Farber, S, Foley, G E & Killander, D, Exptl cell res 32 (1963) 529. 3. Caspersson, T, Farber, S, Foley, G E, Killander, D & Zetterberg, A, Exptl cell res 39 (1965) 365. 4. Caspersson, T, Klein, E & Ringertz, N R, Cancer res 18 (1958) 857. 5. Caspersson, T & Lomakka, G, N Y acad sci 97 (1962) 449. 6. Caspersson, T, Lomakka, G & Caspersson, 0, Biothem pharmacol4 (1960) 113. 7. Eagle, H, Science 130 (1959) 432. a. Killander, D, Richards, B M & Ringertz, N R, Exptl cell res 27 (1962) 321. 9. Killander, D & Zetterberg, A, Exptl cell res 38 (1965) 272. :::
Lomakka, G, Acta histochem Suppl. VI (1965) 393. SkGld, 0 & Zetterberg, A, Exptl cell res 55 (1969) 289.
Received March 21, 1969
THE EFFECT
OF SERUM SYNTHESIS
STARVATION DURING
ON DNA,
INTERPHASE
A. ZETTERBERG
RNA AND
PROTEIN
IN L-CELLS
and 0. SKOLD
Institute for Medical Cell Research and Genetics, Karolinska Institute& and Department of Microbiology, Farmaceutiska Institutet, Stockholm, Sweden
SUMMARY The effect of withdrawal of the serum complement from tissue culture growth medium (serum starvation) on synthesis of DNA, RNA and protein and on cell multiplication was studied in mouse fibroblasts in vitro (cell lme L-929) by a combination of chemical and quantitative cytochemical methods. The first effect of serum starvation was seen on the synthesis of RNA, which was moderately depressed as early as 4-6 h after serum removal. Protein synthesis was relatively unaffected by serum starvation. The rate of DNA synthesis was unaffected for the first 10 h of serum starvation, but was depressed to about 4 of the normal value after 20 h of starvation. This effect on DNA synthesis was shown to be a combined effect on the initiation and rate of DNA synthesis. Cvtophotometric DNA measurements on individual cells revealed that the observed inhibition of mitotic activity during serum starvation was not secondary to the inhibited DNA synthesis. Instead a direct effect on the conversion of GZ-phase cells into the mitotic stage was observed to be at work, i.e. the serum complement in the growth medium seemed to be a-factor of importance for the initiation of mitosis.
It is well known that growth media used for tissue culture of mammalian cells must contain serum in order to support rapid proliferation of cells in vitro. Very little is known, however, about the mechanisms whereby serum factors are involved in the metabolic processes of the interphase and mitosis of the growing cell. The aim of the present investigation was to find out if some of the metabolic processesin the cell cycle were particularly sensitive to serum starvation. For this purpose cell proliferation and synthesis of DNA, RNA and protein in serum starved L-cell cultures were followed by a combination of biochemical and cytochemical methods. While serum starvation had a very small effect on the biosynthesis of protein, RNA synthesis was rapidly inhibited to some degree. A pronounced inhibition of DNA synthesis was observed during serum starvation but only after a Exptl Cell Res 57
certain delay. The observed inhibitory effect on cell proliferation was shown, however, not to be secondary to the inhibited DNA synthesis. MATERIALS AND METHODS Cells Monolayer cultures of mouse fibroblasts (line L-929) with a population doubling time of approx. 20 h in the exponential phase of growth were used. The cells were cultivated at 37°C in the medium of Eagle [7] as described earlier [II]. Each culture flask contained 10 ml, and each petri dish 5 ml of medium. At the time of inoculation the cell concentration was 10,000 cells/ml.
Radioisotopes W-uridine, specific activity 17.4,uC/pmole was purchased from New England Nuclear Corp. ‘H-DL-Lysine 307 &/,umole and BH-uL-tryptophan 500 yC/pmole were obtained from The Radiochemical Centre, Amersham.
Biochemical analyses After pouring off the old medium, the cell monolayer was washed with 10 ml of sterile 0.15 M NaCl and the final
Effects of serum starvation on L-cells
115
prewarmed media (with and without 10 % of dialyzed calf serum (Gibco)) containing the radioactive precursors were added. The cells from each flask were harvested by scraping, and then suspended in 2 ml of 0.15 M NaCl. A 0.3 ml sample of the suspension was withdrawn and added to 1.2 ml of 0.1 M NaOH to give a clear solution, and the absorbance at 265 nm of this was used to normalize experimental variations in total cell mass between different culture flasks. The incorporation of 8H-amino acids and of 14C-uridine into trichloroacetic acid insoluble product was used as a measure of protein and nucleic acid synthesis. RNA and DNA were separated by alkaline hydrolysis as described earlier [l 11. Determination of 8H- and 14C-activity was done in a Packard TriCarb liquid-scintillation counter and the spectrometric separation of 8H- and IV-activity was performed as described previously [ 111.
Cytochemical analyses For the cell multiplication and cytochemical analyses the cells were cultivated directly on Biirker slides in petri dishes as described previously [9]. On each slide carrying the multiplying cell populations one microscopic field was repeatedly photographed for about 60 h under low power magnification by means of time-lapse-cinematography. When the cell populations had entered the exponential phase of growth the cell cultures were briefly washed in isotonic saline and the medium was changed. Eagle’s medium with and without 10 % of dialyzed calf serum (Gibco) was added to the control and experimental cultures, respectively. The temperature of the cells was carefully kept at 37°C through the whole experiment. At the end of the experiment, the slide cultures were washed in saline and fixed in a mixture of acetone and absolute ethanol (1: 1) at room temperature for 30 mm, and then kept in the fixation mixture at 4°C until the cells were stained. The cells in the photographed areas of the slide cultures were analyzed individually for their DNA content, as represented by their total extinction at 546 nm after Feulgen staining, by use of a rapid scanning microspe&ophotome& [$ 6, IO]. To obtain the mitoiic index, 5000 hematoxylin-stained cells were counted at x 800 magnification by phase contrast microscopy on each slide culture in a separate experiment. Cells from late prophase through late telophase were counted.
RESULTS Cell multiplication
The effect of serum starvation on cell multiplication is illustrated by the growth curves presented in fig. 1. During the first 10 h of serum starvation the cell number of the treated population increased by approx. 30 % compared to 75 % in the control population. During the following 35 h of serum starvation there was no increase in cell number in the treated culture. This was due to an inhibited cell division indicated by a drop in the mitotic index from 2.5 to 0.4 after 45 h of serum starvation.
i0
2.0
i0
i0
SO
BO
7b
Abscissa: Hours; ordinate: number of cells/unit area. Fig. 1. Effect of serum starvation on cell multiplication. Number of cells per unit surface area in relative units plotted against experimental time in hours. The arrow indicates the time at which the growth medium was changed and serum starvation was introduced. 0 -- 0, serum starved cell population; l - l , control population.
Incorporation of labelled precursors into DNA, RNA and protein
The effect of serum starvation on the synthesis of nucleic acid and protein was studied by the incorporation of labelled precursors into acid precipitable material. The incorporation was followed during two different time periods. Firstly, over the initial 11 h of serum starvation, i.e. when some cell multiplication was taking place. Secondly, between the 1lth and the 20th h of starvation, i.e. during the time period when the mitotic activity was greatly reduced. The results presented in fig. 2 show the incorporation of 3H-lysine and 14C-uridine into protein, RNA and DNA, respectively, during the first 11 h of serum starvation. The lack of serum did not seem to have any effect on the incorporation of 3Hlysine into protein. Similarly, no significant decrease in the rate of 14C-uridine incorporation into DNA during the first 11 h of serum starvation was observed. The effect on the synthesis of RNA was different. Already after 5 h 30 % less 14C-uridine had been incorporated into RNA of the starved cells as compared to the control. Exptl Cell Res 57
116 A. Zetterberg & 0. Skiild
10000
2oooc
15001
15ooc
7500
1oooc
5000
5000
2500
12345678
9
10
11
Ir’
7500
1oooc I -
5000
5000 , -
2500
12
Hours; ordinate: (left) cpm (3H-lysine); (right) cpm (W-uridine). Fig. 2. Effect of serum starvation on protein, RNA and DNA synthesis in L cells. To each tissue culture flask containing 10 ml of Eagle’s medium was added Wuridine and unlabelled uridine to a final concentration of 0.10 pmoles/ml and final specific activity of 0.5 pC/pmole, and 3H-lysine to a concentration of 0.5 pmoleslml and a final specific activity of 4 &/pmole of L form. Each experimental point represents one tissue culture flask. All flasks were inoculated at the same time with a standardized inoculum and the cell populations had entered the logarithmic phase of growth before the addition of the radioactive precursors (time zero). Serum starvation was instituted in one experimental series at time zero. The values along the ordinate are expressed as cpm per all harvested cells from each flask, see the experimental section. A - A , 8H-lysine incorporation + serum; A -- A , 8H-lysine incorporation - serum; 0 - 0, W-uridine incorporation into RNA + serum; 0 --- 0, W-uridine incorporation into RNA - serum; x - x , Y-uridine incorporation into DNA + serum; x --- x , W-uridine incorporation into DNA - serum.
x
Abscissa:
Fig. 3 demonstrates the effect of serum starvation on the synthesis of nucleic acid and protein during the period of mitotic inhibition. The cells were starved for serum during 11 h before the labelled precursors were added to the medium. The rate of protein synthesis was followed by incorporation of 3H-tryptophan into acid precipitable material. Nine h after the addition of SH-tryptophan, i.e. 20 h after the removal of serum from the medium, 20% less 3H-tryptophan had been incorporated than in the control. The inhibition of RNA synthesis, which was apparent as early as 5 h after the removal of serum from the medium (fig. 2) was further increasedslightly between the 1Ith and the 20th h. Exptl Cell Res 57
-x---------x 1
2
3
L
5
6
7
*
g HOURS
Abscissa: Hours; ordinate: (left) cpm/SH-tryptophan; (right) cpm (14C-uridine). Fig. 3. Effect of serum starvation on protein, RNA and DNA synthesis in L cells. In both experimental series W-uridine was added to a final concentration of 0.10 pmoles/ml and a specific activity of 0.5 ,uC/~mole. 8Htryptophan was added to a final concentration of 0.5 ,umoles/ml and a specific activity of 24 @Z/pmole of L form. Serum starvation was instituted 11 h before the addition of radioactive precursors which were added at time zero. The values along the ordinate and procedures otherwise were as described in the legend to fig. 2. 8H-tryptophan incorporation + serum; A -- A , A-A, SH-tryptophan incorporation -serum; 0 - l , r*C-uridine incorporation into RNA + serum; 0 - - - 0, W-uridine incorporation into RNA -serum; x - x , W-uridine incorporation into DNA + serum; x - - - - x , W-uridine incorporation into DNA -serum.
During the 20 h of serum starvation the incorporation of 14C-uridine into RNA was 40 % less than in the control. The rate of 14C-uridine incorporation into DNA decreaseddramatically between the 11th and the 20th h. During this period about one-third as much DNA was synthesized in the serum starved cells as in the control cells. In summary these incorporation data seemto indicate that serum starvation introduced an early and moderate inhibitory effect on the synthesis of RNA, a late and substantial inhibitory effect on the synthesis of DNA and a late and only slight inhibitory effect on the synthesis of protein.
Effects of serum starvation on L.-cells
the Gl phase, S phase and G2 phase respectively, of the cell cycle. It is thus obvious from the results presented in fig. 4 that the population which was serum-starved for 24 h is composed of Gl, S and G2 cells in the same proportion as in the non-treated population.
20 CONTROL N=130 15
117
D==35.6 i
DISCUSSION 20 MINUS N.130 15
SERUM
D%=31.L
10
5~
J10
20
30
LO
50
) 70 DNA
Abscissa: DNA; ordinate: cell number, %. FOP. 4. Intercellular distribution of DNA. Number of ceils in per cent of total number of cells analyzed, plotted against cellular Feulgen-DNA values in relative units. Upper histogram: non serum starved, multiplying control population. Lower histogram: non-multiplying cell population, serum starved for 48 h.
Intercellular
distribution of DNA
In order to investigate the relationship between inhibition of DNA synthesis and mitotic activity, the intercellular distribution of DNA content in a cell population starved for serum during 48 h was compared to that of a control population. The results which are presented in fig. 4 show the typical two-peak distribution in both the control (fig. 4, upper part) and in the serum starved population (fig. 4, lower part). Identical results were obtained from a cell population starved for serum for 24 h. There does not appear to be any significant difference between the two cell populations with respect to the proportion of cells with postmitotic amounts of DNA (the peak with the DNA values of about 20 relative units), with premitotic amounts of DNA (the peak with DNA values about 40 relative units) and with intermediate DNA values. The cells with postmitotic, intermediate and premitotic amounts of DNA represent the cells in
From the results presented in this study it was shown that the number of cells growing on a glass surface ceasedto increase a short time after the serum was removed from the growth medium. This effect was accompanied by a marked decrease in the mitotic index. It is thus obvious that serum starvation had a pronounced inhibitory effect on cell multiplication, Serum starvation could theoretically inhibit cell multiplication by affecting a number of different steps in the cell cycle. It could prevent cell multiplication by disturbing for instance the division process as such. This should then lead to an accumulation of cells in the mitotic stage i.e. an increased mitotic index. This was, however, not observed. Instead a marked drop in the mitotic index was demonstrated after some time of serum starvation. The inhibited cell multiplication was thus not primarily due to a disturbed division process. Some mitotic cells were, however, observed in the absence of any detectable increase in cell number. A small influence of serum starvation on the mitotic process could therefore not be completely ruled out. Furthermore, the inhibited cell multiplication could be the result of an inhibited DNA synthesis as is the casewith 5-fluorodeoxyuridinetreated cells [3]. An inhibited DNA synthesis was in fact observed. Measurements of cellular DNA content revealed, however, that the serumstarved cell population contained a relatively large proportion of cells with premitotic amounts of DNA (G2 phase cells). The arrested mitotic activity can thus not be a secondary phenomenon to the inhibited DNA synthesis. It rather appeared to be caused by a failure of the serum starved G2 phase cells to initiate mitosis. In this respect the effect of serum starvation seemsto Exptl Cell Res 57
118 A. Zetterberg & 0. Skiild be similar to that of X-irradiation [4, 81 and nitrogen mustard [l. 21. If exclusively the initiation of mitosis would have been inhibited by the serum starvation the proportion of Gl and S-phasecells of the population would have decreasedand the proportion of G2 phase cells increased becauseof the failure of these latter cells to enter mitosis and divide. This was not found to be the case. The cellular DNA measurements showed that both Gl and S-phase cells were found in the serum-starved population in the same proportion as in the control. In conjunction with the data showing a reduced rate of nucleoside incorporation into DNA of the serum-starved population, the cellular DNA measurements indicated that not only the rate but also the initiation of DNA synthesis was inhibited by the removal of serum from the growth medium. The inhibited cell multiplication could furthermore be due to a nonspecific inhibition of cellular growth. This is, however, an unlikely explanation taking into consideration the relatively moderate inhibitory effect of serum starvation on RNA synthesis and in particular on protein synthesis.Irrespective of the precise mechan-
Exptl Cell Res 57
ism by which serum starvation inhibits cell multiplication it appeared primarily to affect the sequenceof processesadvancing the cell towards mitosis (initiation of DNA synthesis, DNA synthesis and initiation of mitosis), while ythesynthetic processes responsible for cellular growth (RNA and protein synthesis) appeared to be relatively unaffected.
REFERENCES 1. Brewer, H B, Comstock, J P & Aronow, L, Biochem pharmacol 8 (1962) 281. 2. Caspersson, T, Farber, S, Foley, G E & Killander, D, Exptl cell res 32 (1963) 529. 3. Caspersson, T, Farber, S, Foley, G E, Killander, D & Zetterberg, A, Exptl cell res 39 (1965) 365. 4. Caspersson, T, Klein, E & Ringertz, N R, Cancer res 18 (1958) 857. 5. Caspersson, T & Lomakka, G, N Y acad sci 97 (1962) 449. 6. Caspersson, T, Lomakka, G & Caspersson, 0, Biothem pharmacol4 (1960) 113. 7. Eagle, H, Science 130 (1959) 432. a. Killander, D, Richards, B M & Ringertz, N R, Exptl cell res 27 (1962) 321. 9. Killander, D & Zetterberg, A, Exptl cell res 38 (1965) 272. :::
Lomakka, G, Acta histochem Suppl. VI (1965) 393. SkGld, 0 & Zetterberg, A, Exptl cell res 55 (1969) 289.
Received March 21, 1969