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Protein synthesis and protein degradation through the cell cycle of human NHIK 3025 cells in vitro
Printed in Sweden Copyright 0 1979 by Academic Press. Inc. All rights of reproduction in any form rraenned
0014-4827/79/1100h3-lO$OZ.Ml/O
Experimental Cell Research 123 (1979) 63-72
PROTEIN
SYNTHESIS
THE CELL
CYCLE
0YSTEIN Department
AND PROTEIN OF HUMAN
DEGRADATION
NHIK
3025 CELLS
THROUGH IN VITRO
W. R@NNING, ERIK 0. PETTERSEN and PER 0. SEGLEN of Tissue Culture, Norsk Hydro’s Institute for Cancer Research, The Norwegian Radium Hospital, Oslo 3, Norway
SUMMARY The rates of protein synthesis and protein degradation through the cell cycle of human NHIK 3025 cells in balanced growth were studied in cells synchronized by mitotic selection. The rates of protein synthesis and protein degradation per cell were found to increase smoothly from the first measurements after selection (mitosis) and until the beginning of the next mitosis. By the end of the cell cycle, both the rate of protein synthesis per cell and the rate of protein degradation per cell had doubled their values relative to the extrapolated values at the beginning of the cell cycle. The increase in the rates of protein synthesis and protein degradation seemed to follow the increase in total protein content closely, i.e. the rates of protein synthesis and protein degradation given as percentage of total protein were constant throughout the cell cycle of NHIK 3025 cells in balanced growth. The rate of protein accumulation, either measured directly or calculated as the difference between the rates of protein synthesis and protein degradation, allowed for a doubling of the protein content during the median time period required to complete the cell cycle.
The cell cycle of proliferating cells seems to consist of two partially independent cycles: (i) the growth cycle, which represents the increase in macromolecules (mainly protein); and (ii) the DNA/division cycle, which represents the events leading to DNA replication and cell division [ 1,2]. The basis for this model are observations that inhibitors of DNA synthesis such as hydroxyurea [3, 4, 51, excess thymidine [5, 6, 71, and others [5, 8, 91, have no immediate effect on protein metabolism. How these two cycles are coordinated during balanced growth has not been clarified. In order to study such coordination it is important to analyse both cycles thoroughly in cells in balanced growth (cf the definition of Anderson et al. [IO]). However, the majority of reports concerning cell cycle kinetics have dealt only with the 5-791812
aspects of DNA synthesis and cell division, i.e. the DNA/division cycle, and there is a lack of studies which include all the aspects of protein metabolism (i.e. protein synthesis, protein degradation and the total amount of protein) throughout the cell cycle of proliferating cells. In the present work we have measured the total protein content and the rates of protein synthesis and degradation through the cell cycle of cells of a human line (NHIK 3025), grown in vitro and synchronized by selective detachment of mitotic cells from exponentially growing populations. MATERIALS
AND METHODS
Cell culture and synchronization NHIK 3025 is a cell line, derived from hyperplastic human cervix tissue (carcinoma in situ) [ll, 121. The cells were grown as monolayers in Puck’s E2a medium Exn Cell Res 123 f 1979)
64
R@nning, Pettersen and Seglen
sunn1emented with 20 % human serum and 10% horse serum [13]. They were synchronized by shake-off of mitotic cells as described elsewhere 1141. Brieflv. NHIK 3025 cells were seeded into plasticflasks on the day preceding synchronization, at a density which gives little cell-to-cell contact. Next day the flasks were shaken on a reciprocating shaker, and the medium containing the detached cells was removed and divided among an appropriate number of tissue culture flasks. In these flasks the cells reattached and proceeded through the cell cycle with the same cycle and phase durations as exponentially growing NHIK 3025 cells [ 141. The number of reattachment-capable cells in the suspension was measured by colony formation: a fixed amount of cell suspension was divided among 5 Petri dishes containing 5 ml E2a medium. The cells were incubated for 7 days, fixed in absolute alcohol, and stained with methylene blue. The number of colonies/dish was counted and used to estimate the number of cells/flask. The plating efficiency of cells of this kind is near to 100% [14].
DNA synthesis and cell number DNA synthesis in synchronized NHIK 3025 cells was measured by pulsed incorporation of [3H]thymidine (Amersham TRA 120, 5.0 Cilmmol). After a pulse duration of 15 min with a tracer dose of r3H]thymidine (10 uCi/ml). the cells were trvosinized 1141. aonhed onto Millipore filters, and precipitated with 2 ml’cold 5% (w/v) trichloroacetic acid (TCA) for 30 min. The acid-soluble radioactivity was‘ washed out, and the radioactivity on the Millipore filters was counted in a liquid scintillation counter. The increase in cell number in the population of synchronized cells was registered by counting the number of cells within a circumscribed area on the bottom of the flask, using an inverted microscope in an incubation room at 37°C.
Protein synthesis The rate of protein synthesis in synchronized NHIK 3025 cells was measured by pulsed incorporation of [W]valine (Amersham CFB 75, 285 Cilmol). In order to keep the soecific radioactivitv at a constant level, a high concentration of valine in-the medium (1.0 mM, 1.0 Cilmol) was used (see Results). After a pulse duration of 30 min, the flasks were rapidly chilled in icecold water, the medium removed, and the cells washed five times with Hanks’ balanced salt solution at OXC (5 ml in 25 cm2 flasks and 10 ml in 75 cm* flasks). The cells were then precipitated with 10% (w/v) perchloric acid (PCA) (7 and 15 ml respectively) and left at 0-4”C for 15 min. 0.5 ml aliauots of the acid extracts were taken for measurement of intracellular [Ylvaline (acid-soluble radioactivity) by liquid scintillation countine. The orecinitates. still attached to the flasks. were washed with 2-% PCA (5 and 10 ml respectively) three times. dissolved in 0.1 M NaOH/0.4% (w/v) sodium deoxycholate (24 ml depending on’ ceil number) during a 1 h incubation at 37”C, and 0.5 ml aliquots were neutralized with HCl and taken for measI_rp Cell Res 123 (1979)
urement of protein radioactivity by liquid scintillation counting. In the cases where the intracellular [W]valine radioactivity was not measured, the cells were precipitated with ice-cold 10% PCA immediately after the medium was removed. The precipitates were washed five times in 2 % PCA and dissolved as described above.
Total protein content The relativelv low vield of svnchronized cells as well as the trapping of serum protein to cells and flasks [IS] makes it difficult to determine total cell protein by conventional spectrophotometric methods. Instead the cells were allowed to grow in medium with [Wlvaline of constant specific radioactivity (1.OmM, 0.5 Ci/mol) for at least 45 h before the shake-off of mitotic cells. During these 45 h the intracellular protein was labelled to saturation, i.e. the specific radioactivity in proteinincorporated valine was the same as the specific radioactivity of valine in the growth medium (see Results). The amount of radioactivity in protein could thus be taken as an estimate of the total cellular protein content.
Protein degradation NHIK 3025 cells were ore-labelled bv growth in E2a medium containing [14C]valine of co&ant specific radioactivity (1.0 mM, 0.5 Cilmol) for at least 45 h. The exchangeable intracellular [Y]vahne pool was emptied by repeated medium changes: i.e. one rapid wash with- Hanks’ balanced salt solution followed‘by five consecutive 10 min incubations in fresh medium containing 1 mM unlabelled valine, all at 37°C. Protein degradation was subsequently measured as the net release of [14C]valine (PCA-sohtble radioactivity) from the cells into the medium containing 1 mM unlabelled valine (to prevent re-incorporation>f isotope) during a 60 min incubation at 37°C with reciprocating shaking (60 rpm; 1 cm amplitude). The extracellular accumulation of [Ylvaline can be taken as representative of total release, since the intracellular [‘*C]valine pool is constant during the release period (unpublished results). For determination of acid-soluble radioactivity in the medium, samples of medium (3 ml) were put into centrifuge tubes containing 1.0 ml ice-cold 10% PCA and precipitated for 30 min at 0-4”C. The tubes were then centrifuged at 5 000 rpm and 0°C for 10 min, and duplicate samples of the supernatant (1.0 ml) were taken for counting of radioactivity in 10 ml scintillation liquid.
RESULTS Determination of cell cycle and phase durations Fig. 1 shows the rate of [3H]thymidine incorporation and the increase in cell number for a population of synchronized NHIK
Protein metabolism through the cell cycle
65
Table 1. Release of acid-soluble radioactivity from degraded protein into media with dif ferent concentrations of unlabelled valine NHIK 3025 cells were grown in E2a medium with [Wlvaline of constant specific radioactivity (0.5 Cilmol) for 24 h, followed by a wash-out period of 5x10 min as described in Materials and Methods. The cells were then incubated for 60 min with various concentrations of unlabelled valine, and net release of acid-soluble radioactivity into the medium was measured. Values are given as percentage of remaining radioactivity in the cells, and represent the mean t S.E. of 3 flasks Cont. of unlabelled vahne in the medium (mM) Release of [‘*C]radioactivity to the medium
1.0
2.0
5.0
10.0
1.9f0.2
1.9fO. 1
1.7kO.l
1.720.1
3025 cells. The S phase was determined as the time interval when the rate of incorporation of [3H]thymidine into DNA was more than 25% of maximum as determined by liquid scintillation counting [ 141. In the experiments described in this report, both the absolute rate of protein synthesis and the length of the cell cycles might vary slightly (see table 2). In order to make it possible to compare results obtained in different experiments, the results were normalized in the following manner: The length
I I I
of the cell cycle was defined as the time interval required to increase the relative cell number from 0.75 to 1.5 [14]. The relative cell number is 0.75 at 0.5 h after mitotic selection [14], and this time was defined as a relative cell age of 0. Kinetics of [14C]valine incorporation The amount of radioactivity that is incorporated into protein is proportional to the specific radioactivity in the precursor pool
-15
i ,'
50 J i
I I
I'
-10 s
: \9+4 \'
0
5
*I I I I I I , , , , ,,, , , , 1 10 15 20
0.5
Fig. 1. Abscissa: (fop) rel. cell age; (bottom) time after mitotic selection (hours); ordinate: (lefr) radioactivity in DNA (0) (% of maximum); (right) rel. cell no. (A). Determination of cell cycle and phase duration of synchronized NHIK 3025 cells. The median cell age is defined as the time required to increase the relative cell number from 0.75 to 1.5, and the S phase is taken to be the time interval when the incorporation of r3H]thymidine into DNA is more than 25% of maximum r141.
Fig. 2. Abscissa: (A) valine cont. (mM); (B) time (mitt); ordinate: (left) (O-O) radioactivity in protein; (right) (0- - 0) acid-soluble radioactivity in the cells
(102xcpmlflask). Intracellular accumulation and incorporation of [Wlvaline. NHIK 3025 cells were grown in E2a medium with [Wlvaline of constant specific radioactivity (1.0 Cilmol). (A) Cells grown at various concentrations of extracellular valine for 30 min; (B) cells exposed to [‘*C]valine (1.O mM) for various periods of time. Each point represents the mean of duplicate samples in the same experiment. E-xl, Cell
Res 123 119791
66
Renning,
Pettersen and Seglen
Table 2. Relation of the doubling time for protein synthesis, protein degradation and total protein to the median cell cycle time in four different experiments Expt no.
Median cell cycle time (hours)
1 2 3
16.0 18.0 19.0
4
19.0
Measured parameters
Doubling time (hours)
Protein synthesis Protein synthesis Protein degradation Total protein Protein degradation Total protein
16.5 18.0 19.8 19.8 18.7 17.6
[ 161.Therefore, measurement of the rate of protein synthesis requires that the specific radioactivity in the precursor pool is constant during the incorporation period. For an amino acid like valine, which equilibrates rapidly across the cell membrane [16], this can be achieved by maintaining a valine concentration in the medium which is sufficiently high to minimize the effects of isotope consumption and dilution of isotope by proteolytically released valine [16, 171. Fig. 2A shows that in NHIK 3025 cells the rate of incorporation of [14C]valine into protein is at a maximum for valine concentrations above 1.0 mM, indicating that under these conditions the specific radioactivity of the precursor is constant during the 30 min incorporation period used. The intracellular, acid-soluble [14C]valine concentration increases as a function of the extracellular valine concentration even beyond 1.OmM, indicating that limitation in the amino acid transport across the cell membrane is not responsible for the flattening of the incorporation curve. The valine uptake is rapid, with complete equilibration of radioactivity in 5 min (fig. 2B). This time is the same as found in liver cells [16, 181 but somewhat shorter than the 10 Exp Cell Res (23 (1979)
min required to reach equilibration in HTC cells [ 191. At 1.0 mM valine, the radioactivity in the intracellular pool is maintained at a constant level, and incorporation proceeds linearly throughout a 30 min incubation period (fig. 2B); these experimental conditions have therefore been used in subsequent incorporation studies. Saturation
labelling
of protein
By labelling cellular protein to saturation at constant specific precursor radioactivity, the incorporated radioactivity can be taken as a direct expression of the total amount of protein, independent of the rates of synthesis and degradation, and undisturbed by the presence of serum protein [15]. In addition, the release of radioactivity from saturation-labelled protein provides a measure of the absolute rate of overall protein degradation, with no selective dominance by proteins with rapid turnover [20]. As shown in fig. 3, at least 45 h of incubation with [14C]valine was necessary to achieve saturation labelling of the protein of freshly subcultured NHIK 3025 cells. Protein accumulation cell cycle
through the
The total protein content/cell increases gradually through the cell cycle (fig. 4). For mathematical convenience the data were fitted to an exponential equation Pt =POekt
(1)
where k is a constant and Pt and PO are the amounts of radioactivity in protein/cell at a relative cell age oft and at the beginning of the cell cycle (t =0) respectively. By the method of linearization and least square fit P,, and k were found to be 0.43 cpmlcell and 0.75 respectively. Inserted in eq. (1):
Protein metabolism through the cell cycle
.
03 l
02
/
/
‘/ 6’ /
o/-
67
3 2
P” /O
01
0’s
04I/,!
/’
/
00
0
10
20
30
‘0
50
60
0
70
Fig. S. Abscissa: time (hours); ordinare: (left) radioact. in protein (cpm/cell); (right) rel. cell no. 0, Rel. cell no.; 0, radioact. incorporated into cellular protein as a function of time. NHIK 3025 cells were grown in E2a medium with [i4C]valine of constant specific radioactivity (1.0 mM; 0.5 Ci/mol). Each point is normalized to 1.0 Ci/mol and gives the mean value of duplicate samples.
(2)
(the coefficient of correlation, P=0.95). From eq. (2) the protein content is found to be doubled during (ln2)/0.75=0.92 parts of the cell cycle. The median cell cycle time for the cells in this experiment was 19.0 h, and therefore the protein doubling time was 17.6 h. In another experiment the protein doubling time and median cell cycle time were 19.8 h and 19 h respectively (table 2). These results indicate that the cellular protein content doubles during the cell cycle of NHIK 3025 cells in balanced growth.
02
0.‘
0.6
0.8
10
I 2
rel. cell age; ordinate: (lefr) radioact. in protein (cpm/cell); (right) rel. cell no. A, Relative cell no.; the total amount of 0, protein/culture (arbitrary units; 0, protein/cell in NHIK 3025 cells synchronized by mitotic selection. The cells were grown in E2a medium with [“Clvaline of constant specific radioactivity (1.0 mM; 0.5 mCi/mmol) for 45 h before the selection took place, and until the actual times after selection. Each point is normalized to 1.0 Cilmol and gives the mean value of duplicate samples. The solid curve is an exponential curve fitted to the results by the method of least squares (eq. (2)).
Fig. 4. Abscissa:
cell age 0.0) when the data are fitted to exponential equations similar to eq. (1). The solid line shows the path of a theoretically exponential increase in the rate of protein synthesis/cell. The rate of protein synthesis/cell declines as the cells start to divide at a relative cell age of approx. 0.8 (cf fig. 1). The decline in protein synthesis may also partly result from a depressed rate of protein synthesis in mitosis [21].
Measurement of [‘“cl valine release Protein synthesis through the (protein degradation) cell cycle When [14C]valine release from prelabelled Fig. 5 shows the rate of protein synthesis/ cells is to be used as a measure of protein cell through the cell cycle of NHIK 3025 degradation, it is necessary to empty the cells synchronized by mitotic selection. pool of intracellular [14C]valine. This can The rate of protein synthesis/cell increases be done by repeated incubations of the cells smoothly throughout the entire cell cycle, in non-radioactive medium under condiand has approximately doubled by the end tions which permit equilibration of isotope of the cycle. In fig. 5 the rate of protein syn- between the extracellular medium and the thesis is given as percent of the extrapolated intracellular exchangeable valine pool [ 16, values 0.5 h after mitotic selection (relative 221. As shown in fig. 6, four consecutive Exp Cell Res 123 (1979)
68
Rutnning, Pettersen and Seglen
G, 0
1 0
’ 02
s
\ ’
’ 0‘
1
1 06
\G.M 1
1 cm
1 10
Fig. 5. Abscissa:
rel. cell age; ordinate: radioact. in protein (% of the extrapolated value at cell age 0). The rate of protein synthesis/cell as a function of cell age in a population NHIK 3025 cells synchronized by mitotic selection. The cells were exposed to [“Clvaline of constant specific radioactivity (1.0 mM; 1.0 Cilmol) for 30 min at different times after mitotic selection. The data represent two different experiments (0, 0).
Fig.
10 min incubations at 37°C in medium containing 1 mM unlabelled valine was sufficient to bring the background radioactivity to a minimum level. The release of [‘“Clvaline during a subsequent incubation proceeded linearly for at least 2 h (fig. 7). One mM valine in the medium was apparently sufficient to prevent reincorporation of isotope, since raising the concentration up to
10 mM did not increase the net amount of [14C]valine released to the medium (table 1). One hour of incubation in the presence of 1 mM unlabelled valine was therefore chosen as standard conditions for the measurement of [14C]valine release. These [14C]valine release experiments, performed with exponentially growing cells prelabelled for 24 h, indicate a protein degradation rate of 1.7-2.3%/h (fig. 7, table 1). However, measurement of protein degradation performed on exponentially growing NHIK 3025 cells prelabelled for
7. Abscissa: time (min); ordinate: release of [Wlvaline (% of the total radioactivity in protein). Release of [Wlvaline from pre-labelled protein as a function of incubation time. NHIK 3025 cells were labelled for 24 h in E2a medium containing 1.0 mM [W]valine (0.5 Cilmol). The cells were washed as described in Materials and Methods (and the caption to fig. 6).
3D
,
,
'
' 02
,
,
,
,
'
' 0.4
'
' 06
,
,
,
'
' OS
'
20-
no. of medium changes; ordinate: acid-soluble radioact. (103cpmlflask). Extraction of intracellular acid-soluble [i4C]radioactivity by repeated changes of medium. NHIK 3025 cells were grown in E2a medium with [‘*C]valine (1.0 mM; 0.5 mCi/mmol) for 24 h. The cells were then rapidly washed with Hanks’ solution at 3PC before medium was added. The medium was removed after 10 min at 3PC, and fresh medium added. This procedure was repeated the indicated number of times, and the acid-soluble radioactivity in the medium was measured as described in Materials and Methods, Each point gives the mean value f S.E.M. for 4 flasks.
Fig. 6. Abscissa:
ExpCell Res 123(1979)
0
.M
s
G! 0
1 10
rel. cell age; ordinate: release of [“Clvaline (%/h of the total radioactivity in protein). The rate of protein degradation (% of total protein degraded per h) as a function of cell age in a population of NHIK 3025 cells synchronized by mitotic selection. The cells were pre-labelled with [YZ]valine as described in the caption to fg 4, and treated as described in the caption to fig. 6. The data represent two different experiments (0, 0).
Fig. 8. Abscissa:
Protein metabolism
0
20
7
through the cell cycle
69
Increase in the rate ofprotein synthesis, the rate of protein degradation, and total cellular protein through the cell cycle
As illustrated in fig. 8, the rate of protein degradation expressed as percent of total protein content seems to be constant (1.5 %) throughout the cell cycle of NHIK 3025 cells. This can be expressed in the following equation:
15
10
0.5
1
I
I
I
1
I
I
I
1
0 0.2
0.L
06
0.6
1.0
rel. cell age; ordinate: rel. values. Rel. values for the rate of 0, 0, protein synthesis/ cell; A, A, rate of protein degradation/cell and l , 0, total protein in the cells during the cell cycle of NHIK 3025 cells synchronized by mitotic selection. The curve is an illustration of eq. (5).
Fig. 9. Abscissa:
45 h resulted in a degradation rate of 1.41.7 %/h. However, a prelabelling period of 24 h which is too short to achieve saturation labelling may result in a larger proportion of label being released from proteins with short half-lives compared with label released from proteins with longer halflives [23]. Protein degradation
through the cell cycle
Fig. 8 shows that the rate of protein degradation, related to the total protein content, is constant during the cell cycle of NHIK 3025 cells. The degradation rate is about 1.5 %/h in the experiments depicted, which is consistent with the observed rate of protein degradation in exponentially growing cells. For technical reasons no measurements of protein degradation could be made before 2 h after shake-off of mitotic cells (the time required for reattachment of cells, washout of intracellular [ 14C]valine and measurement of [ 14C]valine release).
where P, is cpm in protein (proportional to total protein content) and dP,/dt is the rate of protein degradation/cell at a relative cell age, t. Eq. (2) substituted into eq. (3) yields: dP, ~0 ()()6je0.75t dt *
(4)
It can be seen from eq. (4) that, in the case of exponential increase in protein content, the rate of protein degradation/cell will increase in the same manner as total protein through the cell cycle of NHIK 3025 cells, and that both the total amount of protein/ cell and the rate of protein degradation/cell have a doubling time equal to the cell cycle time when the cells are in balanced growth. In the results illustrated in fig. 5 the median cell cycle times were 16.0 and 18.0 h, respectively, and the rate of protein synthesis/cell had a doubling time of 16.5 and 18.0, respectively, if the data were fitted to exponential curves similar to eq. (1). This indicates that the rate of protein synthesis also doubles during the cell cycle, and that the increase occurs in a smooth manner. These observations are all presented in table 2. In fig. 9 all parameters reflecting protein metabolism, i.e., protein synthesis, protein Exp CdRes 123 (1979)
70
Renning, Pettersen and Seglen
degradation and total protein are given in ponents and process rates should be mainthe same diagram. The data are plotted as tained at constant values, and each comrelative values (related to the extrapolated ponent or process rate should double during value at relative cell age 0), and as a func- the time period between mitoses. This retion of relative cell age. The solid line quirement imposes certain kinetic restricrepresents the plot of the following equa- tions, particularly because many cellular tion: processes are interdependent. For example, the rate of protein synthesis/cell is a function of the cellular protein content (the protein-synthesizing machinery consisting largely of protein and RNA), and the where Y is the relative value of formally any amount of cellular protein is in turn a funcvalue a, that has the corresponding value tion of the rate of protein synthesis and a, at the beginning of the cell cycle, and that degradation. For populations in balanced growth it is increases exponentially during the cell cycle to twice the initial value at the end of likely that the protein synthesizing cathe cell cycle. t is relative cell age. The data pacity, and hence the rate of protein synare consistent with an exponential increase thesis, is directly proportional to the total in all three parameters of protein metabo- protein content. Thereby the increase in lism through the cell cycle, with a doubling both cellular protein and the rate of protein of total protein as well as of the rates of synthesis will take place in an exponential protein synthesis and degradation by the manner. Our data are compatible with this way of time of cell division. The doubling times of the protein content maintaining balanced growth, if one asin synchronized NHIK 3025 cells (176 sumes that NHIK 3025 cells synchronized 19.8 h, table 2) correspond to a protein ac- by mitotic selection proceed through the cumulation rate of 3 S-4 %/h in the case of cell cycle in the same manner as cells in exponential mass growth. The rate of pro- asynchronous, exponentially growing poputein accumulation can also be calculated as lations. Earlier observations [ 141 indicate the difference between the rate of protein that this is the case. Other reports have also been published synthesis and the rate of protein degradation. The rate of protein synthesis, as com- which are compatible with the simple model puted from the rate of [14C]valine incorpora- of proportionality between protein syntion relative to the radioactivity in satura- thesizing capacity and total protein content. tion labelled protein, was 5-6 %/h. With the A smooth increase during the cell cycle was measured degradation rate of 1.5 %/h, the observed for the cellular dry mass of mouse rate of protein accumulation would thus be L cells [24, 251, for the protein content of V79 cells [26, 271, and for the rate of protein 3.5-4.5 %Ih, i.e. as observed. and RNA synthesis in HeLa S3 cells synchronized by mitotic selection [28]. In exponentially growing L cells, Zetterberg [29] DISCUSSION furthermore observed a constant rate of In exponentially growing cell pppulations, protein synthesis/unit area of outspread the ratios between different cellular com- cells. In the yeast Saccharomyces cereExp Cell Res 123 (1979)
Protein metabolism through the cell cycle visiae the rate of protein synthesis related to the total amount of protein was found to be constant throughout the cell cycle [30]. However, other patterns of protein synthesis through the cell cycle have also been reported. In Chinese hamster lung cells [3 l] and mouse P815Y cells [32] the increase in the rate of protein synthesis was greatest in the first half of the cell cycle, and in colcemid-synchronized hepatoma cells [33] or Chinese hamster Don C cells [34] a sharp rate increase during S phase was observed. However, all of these investigations employed cells synchronized by methods involving some kind of chemical treatment, which is likely to cause unbalanced growth, in contrast to the mitotic selection method used in the present work. The maintenance of a constant specific radioactivity in the precursor pool for protein synthesis was furthermore not documented, leaving open the possibility that the measured incorporation rates might be a function of amino acid transport as well as of protein degradation. Non-exponential volume increase or mass growth has been demonstrated in several protists [35, 36, 371, but this may not be comparable to the balanced growth of metazoan cell populations. The simplest relationship between protein degradation and total protein content, compatible with balanced growth, would also be a direct proportionality and hence an exponential increase in both parameters. The degradation of cellular protein seems to be closely related to lysosomal activity [38]. Berg et al. [39] found constant specific activities of lysosomal enzymes through the HeLa cell cycle, indicating that the lysosomal capacity for e.g. protein degradation increased at the same rate as total cell protein. This observation on HeLa cells is in line with our findings on NHIK 3025 cells that protein degradation relative to total
11
protein content is constant through the cell cycle. In contrast to our findings several investigators have found a gradual decline in the amount of protein/cell during exponential growth [26, 27, 401. These measurements may, however, have included serum proteins attached to the cells and incubation vessels, the relative contribution by which is greater when the cell numbers are low. Lee & Engelhardt, avoiding the inclusion of such trapped serum protein by using saturation-labelled cells like in the present experiments, found that the average protein content/cell was constant during exponential growth, i.e. cellular protein accumulated by the same exponential function as did the cell number [ 151.Similar observations were made by Baxter & Stanners [41] who also reported that the difference between protein synthesis and protein degradation, i.e. the rate of protein accumulation paralleled the increase in population DNA content, i.e. proliferation rate. This is consistent with our findings that the protein content doubles during the cell cycle, and that the increase in protein content is expressed by the difference between protein synthesis and protein degradation. The quoted observations and our own data for cells growing in a balanced state are compatible with an exponential increase in all parameters of protein metabolism, i.e. protein synthesis, protein degradation and protein accumulation. Any other kinetic interrelationship would require continuous rate adjustments in order to maintain constant ratios (i.e. balanced growth), a mode of control which would seem inordinately complicated. This work was supported by grants from the Norwegian Cancer Society and The Norwegian Research Council for Science and the Humanities. Exp Cell Res 123 (1979)
72
Rmning, Pettersen and Seglen REFERENCES
1. Mitchison, J M, The biology of the cell cycle. Cambridge University PressyCambridge (197i). 2. Mitchison, J M, Mitosis, facts and questions (ed M Little, N Paweletz, C Petzelt, H Ponstingl, D Schroeter & H-P Zimmermann) pp. l-13. Springer Verlag, Heidelberg (1977). 3. Young, C W & Hodas, S, Science 146(1%4) 1172. 4. Fleckman, P, Bailyn, R S & Kaufman, S, J biol them 253 (1978) 3320. 5. Cohen. L S & Studzinski, G P, J cell nhvsiol 69 . I (1967) 331. 6. Kim, J H, Kim, S H & Eidinoff, M L, Biochem pharmac 14 (1%5) 1821. 7. Studzinski, G P & Lambert, W C, J cell physio173 (1969) 109. _ 8. Rueckert, R R & Mueller, G C, Cancer res 20 (1960) 1584. 9. Liebhaber, S A & Schlessinger, D, Exp cell res 112 (1978) 353. 10. Anderson, E C, Petersen, D F & Tobey, R A, Nature 215 (1967) 1083. 11. Nordbye, K & Oftebro, R, Exp cell res 58 (1969) 458. 12. Oftebro, R & Nordbye, K, Exp cell res 58 (1969) 459. 13. Puck, T T, Cieciura, S J & Fisher, H, J exp med 106 (1957) 145. 14. Pettersen, E 0, Bakke, 0, Lindmo, T & Oftebro, R. Cell tissue kinet 10 (1977) 511. 15. Lee, G T-Y & Engelhardt,‘D L, J cell physiol 92 (1977) 293. 16. Seglen, P 0 & Solheim, A E, Eur j biochem 85 (1978) 15. 17. Vidrich, A, Aihart, J, Bruno, M K & Khairallah, E A, Biochem j 162 (1977) 257. 18. Mortimore, G E, Woodside, K H & Henry, J E, J biol them 247 (1972) 2776. 19. Hod, Y & Hershko, A, J biol them 251 (1976) 4458. 20. Poole, B & Wibo, M, J biol them 248 (1973) 6221.
Exp Cell Res 123 (1979)
21. Prescott, D M & Bender, M A, Exp cell res 26 (1962) 260. 22. Bradley, M 0, Hayflick, L & Schimke, R T, J biol them 251 (1976) 3521. Hendil, K B, J cell physio192 (1977) 353. ::: Killander, D & Zetterberg, A, Exp cell res 38 (1%5) 278. 25. Zetterberg, A & Killander, D, Exp cell res 39 (1965) 22. 26. Kimball, R F, Perdue, SW, Chu, E H Y & Ortiz, R J, Exp cell res 66 (1971) 17. 27. Kimball, R F, Perdue, S W & Chu, E H Y, Exp cell res 84 (1974) 111. 28. Schartf, M D & Robbins, E, Nature 208 (1965) 464. 29. Zetterberg, A, Exp cell res 42 (1966) 500. 30. Elliott, S G & McLaughlin, C S, Proc natl acad sci US 75 (1978) 4384. 31. Robbins, E & Scharff, M D, J cell biol 34 (1%7) 684. 32. Warmsley, A M H & Pasternak, C A, Biochem j 119 (1970) 493. 33. Martin Jr, D, Tomkins, G M & Granner, D, Proc natl acad sci US 62 (1969) 248. 34. Stubblefield, E, Klevecz, R & Deaven, L, J cell physio169 (1967) 345. 35. Prescott, D M, Exp cell res 9 (1955) 328. 36. Mittermayer, C, Braun, R, Chayka, T C & Rusch, H P, Nature 210 (1966) 1133. 37. Kemnner. E S & Marr, A G. J bact lOl(l970) 561. 38. Dean, R T & Barret, A J, Essays in bibchemistry (ed P N Campbell & W N Aldrige) vol. 12, p. 1. London (1976). 39. Berg, T, Melbye, B, Johnsen, S R & Prydz, H, Exp cell res 94 (1975) 106. 40. Meisler, A I, J cell sci 12 (1973) 847. 41. Baxter, G C & Stanners, C P, J cell physiol 96 (1978) 139. Received January 30, 1979 Revised version received April 10, 1979 Accepted April 17, 1979
0014-4827/79/1100h3-lO$OZ.Ml/O
Experimental Cell Research 123 (1979) 63-72
PROTEIN
SYNTHESIS
THE CELL
CYCLE
0YSTEIN Department
AND PROTEIN OF HUMAN
DEGRADATION
NHIK
3025 CELLS
THROUGH IN VITRO
W. R@NNING, ERIK 0. PETTERSEN and PER 0. SEGLEN of Tissue Culture, Norsk Hydro’s Institute for Cancer Research, The Norwegian Radium Hospital, Oslo 3, Norway
SUMMARY The rates of protein synthesis and protein degradation through the cell cycle of human NHIK 3025 cells in balanced growth were studied in cells synchronized by mitotic selection. The rates of protein synthesis and protein degradation per cell were found to increase smoothly from the first measurements after selection (mitosis) and until the beginning of the next mitosis. By the end of the cell cycle, both the rate of protein synthesis per cell and the rate of protein degradation per cell had doubled their values relative to the extrapolated values at the beginning of the cell cycle. The increase in the rates of protein synthesis and protein degradation seemed to follow the increase in total protein content closely, i.e. the rates of protein synthesis and protein degradation given as percentage of total protein were constant throughout the cell cycle of NHIK 3025 cells in balanced growth. The rate of protein accumulation, either measured directly or calculated as the difference between the rates of protein synthesis and protein degradation, allowed for a doubling of the protein content during the median time period required to complete the cell cycle.
The cell cycle of proliferating cells seems to consist of two partially independent cycles: (i) the growth cycle, which represents the increase in macromolecules (mainly protein); and (ii) the DNA/division cycle, which represents the events leading to DNA replication and cell division [ 1,2]. The basis for this model are observations that inhibitors of DNA synthesis such as hydroxyurea [3, 4, 51, excess thymidine [5, 6, 71, and others [5, 8, 91, have no immediate effect on protein metabolism. How these two cycles are coordinated during balanced growth has not been clarified. In order to study such coordination it is important to analyse both cycles thoroughly in cells in balanced growth (cf the definition of Anderson et al. [IO]). However, the majority of reports concerning cell cycle kinetics have dealt only with the 5-791812
aspects of DNA synthesis and cell division, i.e. the DNA/division cycle, and there is a lack of studies which include all the aspects of protein metabolism (i.e. protein synthesis, protein degradation and the total amount of protein) throughout the cell cycle of proliferating cells. In the present work we have measured the total protein content and the rates of protein synthesis and degradation through the cell cycle of cells of a human line (NHIK 3025), grown in vitro and synchronized by selective detachment of mitotic cells from exponentially growing populations. MATERIALS
AND METHODS
Cell culture and synchronization NHIK 3025 is a cell line, derived from hyperplastic human cervix tissue (carcinoma in situ) [ll, 121. The cells were grown as monolayers in Puck’s E2a medium Exn Cell Res 123 f 1979)
64
R@nning, Pettersen and Seglen
sunn1emented with 20 % human serum and 10% horse serum [13]. They were synchronized by shake-off of mitotic cells as described elsewhere 1141. Brieflv. NHIK 3025 cells were seeded into plasticflasks on the day preceding synchronization, at a density which gives little cell-to-cell contact. Next day the flasks were shaken on a reciprocating shaker, and the medium containing the detached cells was removed and divided among an appropriate number of tissue culture flasks. In these flasks the cells reattached and proceeded through the cell cycle with the same cycle and phase durations as exponentially growing NHIK 3025 cells [ 141. The number of reattachment-capable cells in the suspension was measured by colony formation: a fixed amount of cell suspension was divided among 5 Petri dishes containing 5 ml E2a medium. The cells were incubated for 7 days, fixed in absolute alcohol, and stained with methylene blue. The number of colonies/dish was counted and used to estimate the number of cells/flask. The plating efficiency of cells of this kind is near to 100% [14].
DNA synthesis and cell number DNA synthesis in synchronized NHIK 3025 cells was measured by pulsed incorporation of [3H]thymidine (Amersham TRA 120, 5.0 Cilmmol). After a pulse duration of 15 min with a tracer dose of r3H]thymidine (10 uCi/ml). the cells were trvosinized 1141. aonhed onto Millipore filters, and precipitated with 2 ml’cold 5% (w/v) trichloroacetic acid (TCA) for 30 min. The acid-soluble radioactivity was‘ washed out, and the radioactivity on the Millipore filters was counted in a liquid scintillation counter. The increase in cell number in the population of synchronized cells was registered by counting the number of cells within a circumscribed area on the bottom of the flask, using an inverted microscope in an incubation room at 37°C.
Protein synthesis The rate of protein synthesis in synchronized NHIK 3025 cells was measured by pulsed incorporation of [W]valine (Amersham CFB 75, 285 Cilmol). In order to keep the soecific radioactivitv at a constant level, a high concentration of valine in-the medium (1.0 mM, 1.0 Cilmol) was used (see Results). After a pulse duration of 30 min, the flasks were rapidly chilled in icecold water, the medium removed, and the cells washed five times with Hanks’ balanced salt solution at OXC (5 ml in 25 cm2 flasks and 10 ml in 75 cm* flasks). The cells were then precipitated with 10% (w/v) perchloric acid (PCA) (7 and 15 ml respectively) and left at 0-4”C for 15 min. 0.5 ml aliauots of the acid extracts were taken for measurement of intracellular [Ylvaline (acid-soluble radioactivity) by liquid scintillation countine. The orecinitates. still attached to the flasks. were washed with 2-% PCA (5 and 10 ml respectively) three times. dissolved in 0.1 M NaOH/0.4% (w/v) sodium deoxycholate (24 ml depending on’ ceil number) during a 1 h incubation at 37”C, and 0.5 ml aliquots were neutralized with HCl and taken for measI_rp Cell Res 123 (1979)
urement of protein radioactivity by liquid scintillation counting. In the cases where the intracellular [W]valine radioactivity was not measured, the cells were precipitated with ice-cold 10% PCA immediately after the medium was removed. The precipitates were washed five times in 2 % PCA and dissolved as described above.
Total protein content The relativelv low vield of svnchronized cells as well as the trapping of serum protein to cells and flasks [IS] makes it difficult to determine total cell protein by conventional spectrophotometric methods. Instead the cells were allowed to grow in medium with [Wlvaline of constant specific radioactivity (1.OmM, 0.5 Ci/mol) for at least 45 h before the shake-off of mitotic cells. During these 45 h the intracellular protein was labelled to saturation, i.e. the specific radioactivity in proteinincorporated valine was the same as the specific radioactivity of valine in the growth medium (see Results). The amount of radioactivity in protein could thus be taken as an estimate of the total cellular protein content.
Protein degradation NHIK 3025 cells were ore-labelled bv growth in E2a medium containing [14C]valine of co&ant specific radioactivity (1.0 mM, 0.5 Cilmol) for at least 45 h. The exchangeable intracellular [Y]vahne pool was emptied by repeated medium changes: i.e. one rapid wash with- Hanks’ balanced salt solution followed‘by five consecutive 10 min incubations in fresh medium containing 1 mM unlabelled valine, all at 37°C. Protein degradation was subsequently measured as the net release of [14C]valine (PCA-sohtble radioactivity) from the cells into the medium containing 1 mM unlabelled valine (to prevent re-incorporation>f isotope) during a 60 min incubation at 37°C with reciprocating shaking (60 rpm; 1 cm amplitude). The extracellular accumulation of [Ylvaline can be taken as representative of total release, since the intracellular [‘*C]valine pool is constant during the release period (unpublished results). For determination of acid-soluble radioactivity in the medium, samples of medium (3 ml) were put into centrifuge tubes containing 1.0 ml ice-cold 10% PCA and precipitated for 30 min at 0-4”C. The tubes were then centrifuged at 5 000 rpm and 0°C for 10 min, and duplicate samples of the supernatant (1.0 ml) were taken for counting of radioactivity in 10 ml scintillation liquid.
RESULTS Determination of cell cycle and phase durations Fig. 1 shows the rate of [3H]thymidine incorporation and the increase in cell number for a population of synchronized NHIK
Protein metabolism through the cell cycle
65
Table 1. Release of acid-soluble radioactivity from degraded protein into media with dif ferent concentrations of unlabelled valine NHIK 3025 cells were grown in E2a medium with [Wlvaline of constant specific radioactivity (0.5 Cilmol) for 24 h, followed by a wash-out period of 5x10 min as described in Materials and Methods. The cells were then incubated for 60 min with various concentrations of unlabelled valine, and net release of acid-soluble radioactivity into the medium was measured. Values are given as percentage of remaining radioactivity in the cells, and represent the mean t S.E. of 3 flasks Cont. of unlabelled vahne in the medium (mM) Release of [‘*C]radioactivity to the medium
1.0
2.0
5.0
10.0
1.9f0.2
1.9fO. 1
1.7kO.l
1.720.1
3025 cells. The S phase was determined as the time interval when the rate of incorporation of [3H]thymidine into DNA was more than 25% of maximum as determined by liquid scintillation counting [ 141. In the experiments described in this report, both the absolute rate of protein synthesis and the length of the cell cycles might vary slightly (see table 2). In order to make it possible to compare results obtained in different experiments, the results were normalized in the following manner: The length
I I I
of the cell cycle was defined as the time interval required to increase the relative cell number from 0.75 to 1.5 [14]. The relative cell number is 0.75 at 0.5 h after mitotic selection [14], and this time was defined as a relative cell age of 0. Kinetics of [14C]valine incorporation The amount of radioactivity that is incorporated into protein is proportional to the specific radioactivity in the precursor pool
-15
i ,'
50 J i
I I
I'
-10 s
: \9+4 \'
0
5
*I I I I I I , , , , ,,, , , , 1 10 15 20
0.5
Fig. 1. Abscissa: (fop) rel. cell age; (bottom) time after mitotic selection (hours); ordinate: (lefr) radioactivity in DNA (0) (% of maximum); (right) rel. cell no. (A). Determination of cell cycle and phase duration of synchronized NHIK 3025 cells. The median cell age is defined as the time required to increase the relative cell number from 0.75 to 1.5, and the S phase is taken to be the time interval when the incorporation of r3H]thymidine into DNA is more than 25% of maximum r141.
Fig. 2. Abscissa: (A) valine cont. (mM); (B) time (mitt); ordinate: (left) (O-O) radioactivity in protein; (right) (0- - 0) acid-soluble radioactivity in the cells
(102xcpmlflask). Intracellular accumulation and incorporation of [Wlvaline. NHIK 3025 cells were grown in E2a medium with [Wlvaline of constant specific radioactivity (1.0 Cilmol). (A) Cells grown at various concentrations of extracellular valine for 30 min; (B) cells exposed to [‘*C]valine (1.O mM) for various periods of time. Each point represents the mean of duplicate samples in the same experiment. E-xl, Cell
Res 123 119791
66
Renning,
Pettersen and Seglen
Table 2. Relation of the doubling time for protein synthesis, protein degradation and total protein to the median cell cycle time in four different experiments Expt no.
Median cell cycle time (hours)
1 2 3
16.0 18.0 19.0
4
19.0
Measured parameters
Doubling time (hours)
Protein synthesis Protein synthesis Protein degradation Total protein Protein degradation Total protein
16.5 18.0 19.8 19.8 18.7 17.6
[ 161.Therefore, measurement of the rate of protein synthesis requires that the specific radioactivity in the precursor pool is constant during the incorporation period. For an amino acid like valine, which equilibrates rapidly across the cell membrane [16], this can be achieved by maintaining a valine concentration in the medium which is sufficiently high to minimize the effects of isotope consumption and dilution of isotope by proteolytically released valine [16, 171. Fig. 2A shows that in NHIK 3025 cells the rate of incorporation of [14C]valine into protein is at a maximum for valine concentrations above 1.0 mM, indicating that under these conditions the specific radioactivity of the precursor is constant during the 30 min incorporation period used. The intracellular, acid-soluble [14C]valine concentration increases as a function of the extracellular valine concentration even beyond 1.OmM, indicating that limitation in the amino acid transport across the cell membrane is not responsible for the flattening of the incorporation curve. The valine uptake is rapid, with complete equilibration of radioactivity in 5 min (fig. 2B). This time is the same as found in liver cells [16, 181 but somewhat shorter than the 10 Exp Cell Res (23 (1979)
min required to reach equilibration in HTC cells [ 191. At 1.0 mM valine, the radioactivity in the intracellular pool is maintained at a constant level, and incorporation proceeds linearly throughout a 30 min incubation period (fig. 2B); these experimental conditions have therefore been used in subsequent incorporation studies. Saturation
labelling
of protein
By labelling cellular protein to saturation at constant specific precursor radioactivity, the incorporated radioactivity can be taken as a direct expression of the total amount of protein, independent of the rates of synthesis and degradation, and undisturbed by the presence of serum protein [15]. In addition, the release of radioactivity from saturation-labelled protein provides a measure of the absolute rate of overall protein degradation, with no selective dominance by proteins with rapid turnover [20]. As shown in fig. 3, at least 45 h of incubation with [14C]valine was necessary to achieve saturation labelling of the protein of freshly subcultured NHIK 3025 cells. Protein accumulation cell cycle
through the
The total protein content/cell increases gradually through the cell cycle (fig. 4). For mathematical convenience the data were fitted to an exponential equation Pt =POekt
(1)
where k is a constant and Pt and PO are the amounts of radioactivity in protein/cell at a relative cell age oft and at the beginning of the cell cycle (t =0) respectively. By the method of linearization and least square fit P,, and k were found to be 0.43 cpmlcell and 0.75 respectively. Inserted in eq. (1):
Protein metabolism through the cell cycle
.
03 l
02
/
/
‘/ 6’ /
o/-
67
3 2
P” /O
01
0’s
04I/,!
/’
/
00
0
10
20
30
‘0
50
60
0
70
Fig. S. Abscissa: time (hours); ordinare: (left) radioact. in protein (cpm/cell); (right) rel. cell no. 0, Rel. cell no.; 0, radioact. incorporated into cellular protein as a function of time. NHIK 3025 cells were grown in E2a medium with [i4C]valine of constant specific radioactivity (1.0 mM; 0.5 Ci/mol). Each point is normalized to 1.0 Ci/mol and gives the mean value of duplicate samples.
(2)
(the coefficient of correlation, P=0.95). From eq. (2) the protein content is found to be doubled during (ln2)/0.75=0.92 parts of the cell cycle. The median cell cycle time for the cells in this experiment was 19.0 h, and therefore the protein doubling time was 17.6 h. In another experiment the protein doubling time and median cell cycle time were 19.8 h and 19 h respectively (table 2). These results indicate that the cellular protein content doubles during the cell cycle of NHIK 3025 cells in balanced growth.
02
0.‘
0.6
0.8
10
I 2
rel. cell age; ordinate: (lefr) radioact. in protein (cpm/cell); (right) rel. cell no. A, Relative cell no.; the total amount of 0, protein/culture (arbitrary units; 0, protein/cell in NHIK 3025 cells synchronized by mitotic selection. The cells were grown in E2a medium with [“Clvaline of constant specific radioactivity (1.0 mM; 0.5 mCi/mmol) for 45 h before the selection took place, and until the actual times after selection. Each point is normalized to 1.0 Cilmol and gives the mean value of duplicate samples. The solid curve is an exponential curve fitted to the results by the method of least squares (eq. (2)).
Fig. 4. Abscissa:
cell age 0.0) when the data are fitted to exponential equations similar to eq. (1). The solid line shows the path of a theoretically exponential increase in the rate of protein synthesis/cell. The rate of protein synthesis/cell declines as the cells start to divide at a relative cell age of approx. 0.8 (cf fig. 1). The decline in protein synthesis may also partly result from a depressed rate of protein synthesis in mitosis [21].
Measurement of [‘“cl valine release Protein synthesis through the (protein degradation) cell cycle When [14C]valine release from prelabelled Fig. 5 shows the rate of protein synthesis/ cells is to be used as a measure of protein cell through the cell cycle of NHIK 3025 degradation, it is necessary to empty the cells synchronized by mitotic selection. pool of intracellular [14C]valine. This can The rate of protein synthesis/cell increases be done by repeated incubations of the cells smoothly throughout the entire cell cycle, in non-radioactive medium under condiand has approximately doubled by the end tions which permit equilibration of isotope of the cycle. In fig. 5 the rate of protein syn- between the extracellular medium and the thesis is given as percent of the extrapolated intracellular exchangeable valine pool [ 16, values 0.5 h after mitotic selection (relative 221. As shown in fig. 6, four consecutive Exp Cell Res 123 (1979)
68
Rutnning, Pettersen and Seglen
G, 0
1 0
’ 02
s
\ ’
’ 0‘
1
1 06
\G.M 1
1 cm
1 10
Fig. 5. Abscissa:
rel. cell age; ordinate: radioact. in protein (% of the extrapolated value at cell age 0). The rate of protein synthesis/cell as a function of cell age in a population NHIK 3025 cells synchronized by mitotic selection. The cells were exposed to [“Clvaline of constant specific radioactivity (1.0 mM; 1.0 Cilmol) for 30 min at different times after mitotic selection. The data represent two different experiments (0, 0).
Fig.
10 min incubations at 37°C in medium containing 1 mM unlabelled valine was sufficient to bring the background radioactivity to a minimum level. The release of [‘“Clvaline during a subsequent incubation proceeded linearly for at least 2 h (fig. 7). One mM valine in the medium was apparently sufficient to prevent reincorporation of isotope, since raising the concentration up to
10 mM did not increase the net amount of [14C]valine released to the medium (table 1). One hour of incubation in the presence of 1 mM unlabelled valine was therefore chosen as standard conditions for the measurement of [14C]valine release. These [14C]valine release experiments, performed with exponentially growing cells prelabelled for 24 h, indicate a protein degradation rate of 1.7-2.3%/h (fig. 7, table 1). However, measurement of protein degradation performed on exponentially growing NHIK 3025 cells prelabelled for
7. Abscissa: time (min); ordinate: release of [Wlvaline (% of the total radioactivity in protein). Release of [Wlvaline from pre-labelled protein as a function of incubation time. NHIK 3025 cells were labelled for 24 h in E2a medium containing 1.0 mM [W]valine (0.5 Cilmol). The cells were washed as described in Materials and Methods (and the caption to fig. 6).
3D
,
,
'
' 02
,
,
,
,
'
' 0.4
'
' 06
,
,
,
'
' OS
'
20-
no. of medium changes; ordinate: acid-soluble radioact. (103cpmlflask). Extraction of intracellular acid-soluble [i4C]radioactivity by repeated changes of medium. NHIK 3025 cells were grown in E2a medium with [‘*C]valine (1.0 mM; 0.5 mCi/mmol) for 24 h. The cells were then rapidly washed with Hanks’ solution at 3PC before medium was added. The medium was removed after 10 min at 3PC, and fresh medium added. This procedure was repeated the indicated number of times, and the acid-soluble radioactivity in the medium was measured as described in Materials and Methods, Each point gives the mean value f S.E.M. for 4 flasks.
Fig. 6. Abscissa:
ExpCell Res 123(1979)
0
.M
s
G! 0
1 10
rel. cell age; ordinate: release of [“Clvaline (%/h of the total radioactivity in protein). The rate of protein degradation (% of total protein degraded per h) as a function of cell age in a population of NHIK 3025 cells synchronized by mitotic selection. The cells were pre-labelled with [YZ]valine as described in the caption to fg 4, and treated as described in the caption to fig. 6. The data represent two different experiments (0, 0).
Fig. 8. Abscissa:
Protein metabolism
0
20
7
through the cell cycle
69
Increase in the rate ofprotein synthesis, the rate of protein degradation, and total cellular protein through the cell cycle
As illustrated in fig. 8, the rate of protein degradation expressed as percent of total protein content seems to be constant (1.5 %) throughout the cell cycle of NHIK 3025 cells. This can be expressed in the following equation:
15
10
0.5
1
I
I
I
1
I
I
I
1
0 0.2
0.L
06
0.6
1.0
rel. cell age; ordinate: rel. values. Rel. values for the rate of 0, 0, protein synthesis/ cell; A, A, rate of protein degradation/cell and l , 0, total protein in the cells during the cell cycle of NHIK 3025 cells synchronized by mitotic selection. The curve is an illustration of eq. (5).
Fig. 9. Abscissa:
45 h resulted in a degradation rate of 1.41.7 %/h. However, a prelabelling period of 24 h which is too short to achieve saturation labelling may result in a larger proportion of label being released from proteins with short half-lives compared with label released from proteins with longer halflives [23]. Protein degradation
through the cell cycle
Fig. 8 shows that the rate of protein degradation, related to the total protein content, is constant during the cell cycle of NHIK 3025 cells. The degradation rate is about 1.5 %/h in the experiments depicted, which is consistent with the observed rate of protein degradation in exponentially growing cells. For technical reasons no measurements of protein degradation could be made before 2 h after shake-off of mitotic cells (the time required for reattachment of cells, washout of intracellular [ 14C]valine and measurement of [ 14C]valine release).
where P, is cpm in protein (proportional to total protein content) and dP,/dt is the rate of protein degradation/cell at a relative cell age, t. Eq. (2) substituted into eq. (3) yields: dP, ~0 ()()6je0.75t dt *
(4)
It can be seen from eq. (4) that, in the case of exponential increase in protein content, the rate of protein degradation/cell will increase in the same manner as total protein through the cell cycle of NHIK 3025 cells, and that both the total amount of protein/ cell and the rate of protein degradation/cell have a doubling time equal to the cell cycle time when the cells are in balanced growth. In the results illustrated in fig. 5 the median cell cycle times were 16.0 and 18.0 h, respectively, and the rate of protein synthesis/cell had a doubling time of 16.5 and 18.0, respectively, if the data were fitted to exponential curves similar to eq. (1). This indicates that the rate of protein synthesis also doubles during the cell cycle, and that the increase occurs in a smooth manner. These observations are all presented in table 2. In fig. 9 all parameters reflecting protein metabolism, i.e., protein synthesis, protein Exp CdRes 123 (1979)
70
Renning, Pettersen and Seglen
degradation and total protein are given in ponents and process rates should be mainthe same diagram. The data are plotted as tained at constant values, and each comrelative values (related to the extrapolated ponent or process rate should double during value at relative cell age 0), and as a func- the time period between mitoses. This retion of relative cell age. The solid line quirement imposes certain kinetic restricrepresents the plot of the following equa- tions, particularly because many cellular tion: processes are interdependent. For example, the rate of protein synthesis/cell is a function of the cellular protein content (the protein-synthesizing machinery consisting largely of protein and RNA), and the where Y is the relative value of formally any amount of cellular protein is in turn a funcvalue a, that has the corresponding value tion of the rate of protein synthesis and a, at the beginning of the cell cycle, and that degradation. For populations in balanced growth it is increases exponentially during the cell cycle to twice the initial value at the end of likely that the protein synthesizing cathe cell cycle. t is relative cell age. The data pacity, and hence the rate of protein synare consistent with an exponential increase thesis, is directly proportional to the total in all three parameters of protein metabo- protein content. Thereby the increase in lism through the cell cycle, with a doubling both cellular protein and the rate of protein of total protein as well as of the rates of synthesis will take place in an exponential protein synthesis and degradation by the manner. Our data are compatible with this way of time of cell division. The doubling times of the protein content maintaining balanced growth, if one asin synchronized NHIK 3025 cells (176 sumes that NHIK 3025 cells synchronized 19.8 h, table 2) correspond to a protein ac- by mitotic selection proceed through the cumulation rate of 3 S-4 %/h in the case of cell cycle in the same manner as cells in exponential mass growth. The rate of pro- asynchronous, exponentially growing poputein accumulation can also be calculated as lations. Earlier observations [ 141 indicate the difference between the rate of protein that this is the case. Other reports have also been published synthesis and the rate of protein degradation. The rate of protein synthesis, as com- which are compatible with the simple model puted from the rate of [14C]valine incorpora- of proportionality between protein syntion relative to the radioactivity in satura- thesizing capacity and total protein content. tion labelled protein, was 5-6 %/h. With the A smooth increase during the cell cycle was measured degradation rate of 1.5 %/h, the observed for the cellular dry mass of mouse rate of protein accumulation would thus be L cells [24, 251, for the protein content of V79 cells [26, 271, and for the rate of protein 3.5-4.5 %Ih, i.e. as observed. and RNA synthesis in HeLa S3 cells synchronized by mitotic selection [28]. In exponentially growing L cells, Zetterberg [29] DISCUSSION furthermore observed a constant rate of In exponentially growing cell pppulations, protein synthesis/unit area of outspread the ratios between different cellular com- cells. In the yeast Saccharomyces cereExp Cell Res 123 (1979)
Protein metabolism through the cell cycle visiae the rate of protein synthesis related to the total amount of protein was found to be constant throughout the cell cycle [30]. However, other patterns of protein synthesis through the cell cycle have also been reported. In Chinese hamster lung cells [3 l] and mouse P815Y cells [32] the increase in the rate of protein synthesis was greatest in the first half of the cell cycle, and in colcemid-synchronized hepatoma cells [33] or Chinese hamster Don C cells [34] a sharp rate increase during S phase was observed. However, all of these investigations employed cells synchronized by methods involving some kind of chemical treatment, which is likely to cause unbalanced growth, in contrast to the mitotic selection method used in the present work. The maintenance of a constant specific radioactivity in the precursor pool for protein synthesis was furthermore not documented, leaving open the possibility that the measured incorporation rates might be a function of amino acid transport as well as of protein degradation. Non-exponential volume increase or mass growth has been demonstrated in several protists [35, 36, 371, but this may not be comparable to the balanced growth of metazoan cell populations. The simplest relationship between protein degradation and total protein content, compatible with balanced growth, would also be a direct proportionality and hence an exponential increase in both parameters. The degradation of cellular protein seems to be closely related to lysosomal activity [38]. Berg et al. [39] found constant specific activities of lysosomal enzymes through the HeLa cell cycle, indicating that the lysosomal capacity for e.g. protein degradation increased at the same rate as total cell protein. This observation on HeLa cells is in line with our findings on NHIK 3025 cells that protein degradation relative to total
11
protein content is constant through the cell cycle. In contrast to our findings several investigators have found a gradual decline in the amount of protein/cell during exponential growth [26, 27, 401. These measurements may, however, have included serum proteins attached to the cells and incubation vessels, the relative contribution by which is greater when the cell numbers are low. Lee & Engelhardt, avoiding the inclusion of such trapped serum protein by using saturation-labelled cells like in the present experiments, found that the average protein content/cell was constant during exponential growth, i.e. cellular protein accumulated by the same exponential function as did the cell number [ 151.Similar observations were made by Baxter & Stanners [41] who also reported that the difference between protein synthesis and protein degradation, i.e. the rate of protein accumulation paralleled the increase in population DNA content, i.e. proliferation rate. This is consistent with our findings that the protein content doubles during the cell cycle, and that the increase in protein content is expressed by the difference between protein synthesis and protein degradation. The quoted observations and our own data for cells growing in a balanced state are compatible with an exponential increase in all parameters of protein metabolism, i.e. protein synthesis, protein degradation and protein accumulation. Any other kinetic interrelationship would require continuous rate adjustments in order to maintain constant ratios (i.e. balanced growth), a mode of control which would seem inordinately complicated. This work was supported by grants from the Norwegian Cancer Society and The Norwegian Research Council for Science and the Humanities. Exp Cell Res 123 (1979)
72
Rmning, Pettersen and Seglen REFERENCES
1. Mitchison, J M, The biology of the cell cycle. Cambridge University PressyCambridge (197i). 2. Mitchison, J M, Mitosis, facts and questions (ed M Little, N Paweletz, C Petzelt, H Ponstingl, D Schroeter & H-P Zimmermann) pp. l-13. Springer Verlag, Heidelberg (1977). 3. Young, C W & Hodas, S, Science 146(1%4) 1172. 4. Fleckman, P, Bailyn, R S & Kaufman, S, J biol them 253 (1978) 3320. 5. Cohen. L S & Studzinski, G P, J cell nhvsiol 69 . I (1967) 331. 6. Kim, J H, Kim, S H & Eidinoff, M L, Biochem pharmac 14 (1%5) 1821. 7. Studzinski, G P & Lambert, W C, J cell physio173 (1969) 109. _ 8. Rueckert, R R & Mueller, G C, Cancer res 20 (1960) 1584. 9. Liebhaber, S A & Schlessinger, D, Exp cell res 112 (1978) 353. 10. Anderson, E C, Petersen, D F & Tobey, R A, Nature 215 (1967) 1083. 11. Nordbye, K & Oftebro, R, Exp cell res 58 (1969) 458. 12. Oftebro, R & Nordbye, K, Exp cell res 58 (1969) 459. 13. Puck, T T, Cieciura, S J & Fisher, H, J exp med 106 (1957) 145. 14. Pettersen, E 0, Bakke, 0, Lindmo, T & Oftebro, R. Cell tissue kinet 10 (1977) 511. 15. Lee, G T-Y & Engelhardt,‘D L, J cell physiol 92 (1977) 293. 16. Seglen, P 0 & Solheim, A E, Eur j biochem 85 (1978) 15. 17. Vidrich, A, Aihart, J, Bruno, M K & Khairallah, E A, Biochem j 162 (1977) 257. 18. Mortimore, G E, Woodside, K H & Henry, J E, J biol them 247 (1972) 2776. 19. Hod, Y & Hershko, A, J biol them 251 (1976) 4458. 20. Poole, B & Wibo, M, J biol them 248 (1973) 6221.
Exp Cell Res 123 (1979)
21. Prescott, D M & Bender, M A, Exp cell res 26 (1962) 260. 22. Bradley, M 0, Hayflick, L & Schimke, R T, J biol them 251 (1976) 3521. Hendil, K B, J cell physio192 (1977) 353. ::: Killander, D & Zetterberg, A, Exp cell res 38 (1%5) 278. 25. Zetterberg, A & Killander, D, Exp cell res 39 (1965) 22. 26. Kimball, R F, Perdue, SW, Chu, E H Y & Ortiz, R J, Exp cell res 66 (1971) 17. 27. Kimball, R F, Perdue, S W & Chu, E H Y, Exp cell res 84 (1974) 111. 28. Schartf, M D & Robbins, E, Nature 208 (1965) 464. 29. Zetterberg, A, Exp cell res 42 (1966) 500. 30. Elliott, S G & McLaughlin, C S, Proc natl acad sci US 75 (1978) 4384. 31. Robbins, E & Scharff, M D, J cell biol 34 (1%7) 684. 32. Warmsley, A M H & Pasternak, C A, Biochem j 119 (1970) 493. 33. Martin Jr, D, Tomkins, G M & Granner, D, Proc natl acad sci US 62 (1969) 248. 34. Stubblefield, E, Klevecz, R & Deaven, L, J cell physio169 (1967) 345. 35. Prescott, D M, Exp cell res 9 (1955) 328. 36. Mittermayer, C, Braun, R, Chayka, T C & Rusch, H P, Nature 210 (1966) 1133. 37. Kemnner. E S & Marr, A G. J bact lOl(l970) 561. 38. Dean, R T & Barret, A J, Essays in bibchemistry (ed P N Campbell & W N Aldrige) vol. 12, p. 1. London (1976). 39. Berg, T, Melbye, B, Johnsen, S R & Prydz, H, Exp cell res 94 (1975) 106. 40. Meisler, A I, J cell sci 12 (1973) 847. 41. Baxter, G C & Stanners, C P, J cell physiol 96 (1978) 139. Received January 30, 1979 Revised version received April 10, 1979 Accepted April 17, 1979