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Coordination between Cell Growth and Cell Cycle
Transit in Animal Cells
A. ZETTERBERG AND O. LARSSON
Department of Tumor Pathology, Karolinska Institutet, Karolinska Hospital, S-104 01 Stockholm, Sweden
Most studies of the control of animal cell proliferation have been performed in various model systems in
vitro, in which cell proliferation can be modulated in a
controlled fashion. Although each in vitro system has
its own particular features and limitations, and although it is unclear to what extent in vitro data can be
extrapolated to the in vivo situation, some general
features of proliferation control of animal cells have
emerged from the in vitro studies.
Normal cells usually cease to proliferate in a cellcycle-specific way. They arrest in G 1 or enter a state of
quiescence (Go) from G 1 after depletion of serum or
growth factors (Temin 1971; Pardee 1974; Baserga
1976) or nutrients (Prescott 1976) or after cell crowding
(Nielhausen and Green 1965; Zetterberg and Auer
1970). This is also consistent with the general opinion
that arrested cells in vivo, e.g., terminally differentiated cells, contain a GI amount of DNA. It has,
however, been reported that cells can occasionally be
arrested in the G 2 phase under physiological conditions
(Gelfant 1981; Melchers and Lernhardt 1985; GomezLechon and Castell 1987) and under certain experimental conditions (Yoshida and Beppu 1988).
In contrast to normal cells, cells transformed to
tumorigenicity or cells of tumor origin often respond
differently to suboptimal culture conditions, e.g.,
growth factor starvation. Instead of being arrested in
G~ or entering Go, they continue slowly through the
cell cycle until they eventually die as a consequence of
the environmental restraints (Zetterberg and Sk61d
1969; Paul 1973; Pardee and James 1975; Vogel and
Pollack 1975; Medrano and Pardee 1980). Consequently, the ability of normal cells, as opposed to tumor cells,
to arrest in Go, as a response to changes in environmental conditions, reflects a fundamental growth regulatory
mechanism that operates stringently in untransformed
cells but is defective in transformed cells. Research
focused on the processes that lie behind G 1 arrest is
therefore of particular interest in tumor biology research. Studies of the molecular basis of these growth
control events in G 1 would be facilitated if such studies
could focus on a defined and very limited stage within
G 1 that is of particular importance for the specific G O
arrest. To search for such a stage and to map its precise
location within G1 are therefore important.
In this paper, we discuss certain aspects of commitment to DNA replication and mitosis and exit from the
cell cycle, as well as the coordination between cell
growth (in size) and transit through the cell cycle.
METHODS
Cell culture. Mouse Swiss-3T3 cells, SV40-transformed derivatives (SV-3T3), and low-passage human
diploid fibroblasts (HDF) (all purchased from Flow
Laboratories) were maintained in monolayer cultures
and prepared for experiments as described elsewhere
(Zetterberg and Larsson 1985; Larsson et al. 1989a).
Human mammary epithelial cells (HMEC) were prepared from reduction plasty tissues, essentially as described by Stampfer et al. (1980). The mammary epithelium was characterized by its morphological appearance and by immunological assays. The culture medium
was composed of MCDB 170 (Flow Laboratories) supplemented with epidermal growth factor (EGF) (25
ng/ml), insulin (5 /zg/ml), hydrocortisone (0.5 /~g/
ml), ethanolamine (10 -4 M), phosphoethanolamine
(10 4 M), transferrin (5 /zg/ml), and bovine pituitary
extract (BPE) (70/xg/ml), as described by Hammond
et al. (1984). Only low-passage cell cultures with a high
growth capacity were used.
Time-lapse cinematography. Cell ages (time elapsed
from last mitosis) and intermitotic times of individual
cycling cells were determined by time-lapse video recording. Culture flasks (25 cm 2) containing growing
cells were placed in an upright microscope with an
attached video camera system for time-lapse
cinematographic analysis. The temperature was carefully maintained at 37~ by an air-stream stage incubator. A detailed description of the technique is
presented elsewhere (Larsson and Zetterberg 1986a).
Autoradiography. DNA synthesis in cells growing in
flasks containing glass coverslips was measured after
incorporation of [3H]thymidine (1 p~Ci/ml, 5 Ci/
mmole; Amersham). After fixation of the cells in 95%
ethanol (v/v), the coverslips were subjected to autoradiography, essentially as described previously (Zetterberg and Larsson 1985). Percentages of labeled nuclei were determined microscopically.
Protein synthesis. Cells cultured in 50-mm dishes
were pulse-labeled for 30 minutes with [3H]leucine (10
/~Ci/ml, 50 Ci/mmole; Amersham). Thereafter, the
cells were harvested by scraping and washed with icecold trichloracetic acid (7.5%). Acid-precipitable material was thereafter dissolved in 0.1 M NaOH, and
aliquots were taken for spectrophotometric determination of cellular protein amount and for scintillation
counting.
ColdSpringHarborSymposiaon QuantitativeBiology,VolumeLVI.~ 1991 Cold SpringHarbor LaboratoryPress 0-87969-061-5/91 $3.00
137
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138
ZETTERBERG AND LARSSON
Determination of HMG CoA reductase activity. Cells
in 50-mm dishes were, after experimental procedures,
rinsed and scraped for determination of HMG CoA
reductase activity, essentially as described by Cavenee
et al. (1981).
Quantitative microspectrophotometry. The ceils grown
on glass slides in dishes were briefly rinsed in 0.9%
NaC1 and fixed in a 10% neutral formalin solution. The protein content of individual cells was determined as follows: At least 20 mitotic (post-telophase) cells were analyzed by a rapid scanning and integrating microspectrophotometer equipped with a fieldlimiting device that allows separate measurements of
nucleus and cytoplasm (Caspersson and Lomakka
1970; Caspersson 1979; Caspersson and Kudynowski
1980) after staining with the combined Feulgen/
Naphtol Yellow-S method (Gaub et al. 1975). The total
extinction at 435 nm was selected for Naphtol Yellow-S
and used as a measure of the amount of cellular protein. Feulgen-positive material (DNA) was measured
at 546 nm.
RESULTS AND DISCUSSION
Time-lapse Cinematographic Analysis of
the Cell Cycle
To perform accurate kinetic studies on cell cycle
control, we have made use of time-lapse cinematography (Zetterberg and Larsson 1985). In contrast to
alternative methods such as thymidine labeling and
flow cytometry, which only describe the behavior of the
average cell in the population, time-lapse cinematography enables detailed measurements of individual cells
in an unperturbed, asynchronously growing cell population. In particular, this method makes it possible to
map the celt cycle with regard to response to brief
environmental manipulations on the cell cycle progression (Zetterberg and Larsson 1985; Larsson et
al. 1985a,b, 1987, 1989a; Larsson and Zetterberg
1986a,b). Our aim has been to study the consequences
of transient limited treatments (e.g., growth factor depletion, inhibition of protein synthesis, and inhibition
of mevalonic acid synthesis) on cell cycle progression,
measured as delay in intermitotic time. As is evident
from these studies, time-lapse cinematography is a
powerful method in the analysis of cell cycle kinetics.
The method allows the following aspects of the cell
cycle to be studied: (1) Response in relation to precise
cell cycle position in unperturbed, asynchronously growing cell populations. This permits exact timing of point
of commitment to go through the cell cycle (or restriction point) and its relation to initiation of DNA replication. (2) Response as a consequence of treatment for a
brief time period ( < 1 hr). This reflects readiness of
response. (3) Duration of response with respect to duration of treatment. This allows a distinction to be made
between temporary arrest in the cell cycle or set back in
the cell cycle (exit to Go). (4) Response of each in-
dividual cell. This reveals intercellular variability in
responsiveness.
Transition from Gl-Pm to Gl-ps, Commitment to
the Chromosome Cycle and Restriction Point
Time-lapse analysis of proliferating Swiss-3T3 cells
clearly reveals that cell cycle progression is rapidly
interrupted in postmitotic, early G 1 cells by a short
period of growth factor starvation (Fig. 1). This response is detected as a delayed mitosis (increased intermitotic time). Only cells younger than 3 hours (time
after mitosis) responded, whereas cells older than 4
hours were not arrested by growth factor starvation,
but advanced through the remaining part of the cell
cycle with the same speed as untreated control cells.
The subpopulation of postmitotic G 1 cells arrested by
growth factor starvation was denoted Gl-pm, and the
remaining G~ cells, which are able to initiate DNA
replication in the absence of growth factors, were denoted Gl-ps cells (pre-S phase) (Zetterberg and Larsson 1985). The transition from growth factor dependence in Gl-pm cells to growth factor independence in
Gl-ps cells is equivalent to commitment (Temin 1971)
to the chromosome cycle (DNA replication and
mitosis) (Mitchison 1971) or the restriction point (Pardee 1974) and probably corresponds to "START" (Hartwell et al. 1974; Nurse 1981) in yeast. The timeqapse
analysis reveals that virtually all G~-pm cells in the
population undergo this transition within the narrow
time period of I hour (between the third and the fourth
hour after mitosis), i.e., a small intercellular variability
with respect to commitment (or restriction point) as
opposed to a large intercellular variability seen with
respect to initiation of DNA replication (see below).
Time-lapse cinematographic analysis in combination
with very brief exposure to growth-factor-free medium
further reveals that Gl-pm cells respond quickly. Some
of these cells are in fact arrested by such a short growth
factor starvation period as 15 minutes (Fig. 1B). A
1-hour starvation period is required to arrest all G 1-pm
cells (Fig. 1C). A situation similar to that seen in
Figure 1B is observed after a partial growth factor
starvation performed in 0.5% serum (data not shown).
Of principal interest is the finding that the cells are
arrested in all parts of G~-pm and not only at the
restriction point. The synthetic program operating in
G~-pm and leading to commitment of the chromosome
cycle is thus equally sensitive to inhibition by growth
factor starvation or metabolic inhibitors (see below)
throughout the entire Gl-pm period from mitosis to the
restriction point.
To investigate whether the existence of a growthfactor-dependent G~-pm subphase and a growth-factorindependent G~-ps subphase is a general property of
postembryonic animal cells, we have carried out timelapse cinematographic experiments on two different
types of normal human cells, namely, human diploid
fibroblasts (HDF) and human mammary epithelial cells.
(HMEC). Growing populations of Swiss-3T3 cells,
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C E L L G R O W T H AND CELL CYCLE T R A N S I T
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1. Effects of transient growth factor starvation, with respect to cell age, on intermitotic times. Exponentially growing 3T3
cells cultured in medium containing serum were exposed to fresh medium containing serum for 4 hr (A) or to serum-free medium
for 15 min (B) or for 1 hr (C) or for 8 hr (D), whereupon they were re-exposed to medium supplemented with serum for an
additional 48 hr. The cell age at the time of serum depletion and the intermitotic time for each cell were determined by time-lapse
cinematography. Dotted lines and dashed lines represent the average intermitotic times of responding and nonresponding ceils,
respectively.
Figure
H D F , and H M E C were exposed to medium lacking
growth factors, and the effect on cell cycle progression
was studied (Fig. 2). Cells younger than approximately
3 - 4 hours (measured as time elapsed after last mitosis)
at onset of growth factor depletion were not capable of
undergoing a new mitosis in any of these three cell
types. This implies that these two human diploid cell
types exhibit G~-pm properties similar to those of 3T3
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cells, i.e., their cell cycle includes a 3-4-hour postmitotic phase before commitment (restriction point).
A n o t h e r finding of principal importance detected by
time-lapse analysis is the fact that the mitotic delay seen
in the G l - p m cells exceeds the actual starvation or
treatment time by approximately 8 hours. This 8-hour
set back suggests exit from the cell cycle to G O. This will
be discussed in greater depth below.
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age
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Figure 2. Effects of growth factor starvation on intermitotic times for different cell types. Exponentially growing cultures of 3T3
cells (left), HDF (middle), and HMEC (right) were exposed to a 48-hr depletion of serum (for 3T3 cells and HDF) or EGF and
insulin (for HMEC). Cell age and intermitotic times were analyzed by time-lapse cinematography.
Downloaded from symposium.cshlp.org on March 4, 2016 - Published by Cold Spring Harbor Laboratory Press
140
ZETTERBERG AND LARSSON
Ga-ps Variability: Relationship between
Restriction Point and Initiation of
DNA Replication
Time-lapse cinematographic analysis permits exact
timing of the transition between G~-pm and G~-ps
(commitment or restriction point) and transition between G~-ps and S (initiation of DNA replication) in
individual cells in the population (Fig. 3A,B). Thus,
new information about the temporary relationship between these two transitional events in the cell cycle was
obtained. Both in 3T3 cells (Fig. 3A) and in HDF (Fig.
3B), the restriction point (G~-pm/G~-ps transition) is
located between the third and the fourth hour after
mitosis. DNA replication, on the other hand, is
initiated from the third to the thirteenth hour after
mitosis in most cells in the two cell populations (Fig.
3A,B). Thus, Gl-pm is remarkably constant in length,
whereas the length of G~-ps can vary considerably. In
fact, the G~-ps variability accounts for almost all variability of the whole cell cycle. Thus, it seems as if the
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cells, which make the "yes or no" decision in G~-pm
about whether to continue through the cell cycle or not,
have the capacity to decide, in Gl-ps, "when" they will
enter the S phase. The differences in the kinetics between these two transitions (Gl-pm/Gl-ps vs. Gl-ps/
S) suggest the involvement of different mechanisms in
their control. In addition to the probable involvement
of labile proteins in the "G~-pm program" leading up
to commitment (see below, Response to Metabolic
Inhibitors), its constant length in time after mitosis
suggests that other processes initiated at or immediately after mitosis may also be involved. Such processes
might concern reorganization of the cytoskeleton or
decondensation of the chromatin. Conversely, it is conceivable that more variable events may underlie the
control of the G~-ps/S transition. Such a variable event
might comprise overall accumulation of cellular protein. As support for this hypothesis, preliminary results
in our laboratory have shown that cells fail to grow as
long as they are maintained in the Gl-pm subphase.
However, as soon as they have completed the Gl-pm/
Gl-ps transition, they start to increase in size (data not
shown). Therefore, it is tempting to speculate that the
cells adjust their cell size in G~-ps before initiating
DNA synthesis. A small Gl-ps cell would thus need a
relatively long period to accumulate sufficient protein
content in order to traverse into S phase, whereas a
large cell would require a short Gl-ps period for this
purpose. This would be in line with previous data on L
cells (Killander and Zetterberg 1965a,b).
100
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8
12
16
20
24
Cell a g e (h)
Figure 3. Cell age distribution of different cell cycle phases in
3T3 cells (derived from Zetterberg and Larsson 1985) (A) and
HDF (derived from Larsson et al. 1989b) (B). (O) Cell age
distribution for the transition from the serum-sensitive phase
(Gl-pm) to the serum-insensitive phase (G~-ps). (O) Distribution for the transition from Gl-ps to S. (A) Cell age
distribution for entrance into mitotic phase (M).
Figure 4 demonstrates that treatment with an inhibitor of protein synthesis (cycloheximide) and of 3hydroxy-3-methylglutaryl coenzyme A (HMG CoA)
reductase (25-hydroxycholesterol) can induce a mitotic
delay of Gl-pm cells similar to that obtained by growth
factor deprivation. A cycloheximide dose causing a
50% inhibition of protein synthesis was sufficient to
induce this kind of mitotic delay. In fact, an inhibition
of protein synthesis as low as approximately 20% is
sufficient to induce a mitotic delay in a limited portion
of Gl-pm cells (Zetterberg and Larsson 1985). These
data are consistent with data obtained by other investigators (Highfield and Dewey 1972; Rossow et al.
1979; Pardee et al. 1981) and suggest the existence of
labile proteins in the control of exit from the cell cycle.
This is discussed more extensively below. A dose of
25-hydroxycholesterol inducing a 90% inhibition of
HMG CoA reductase activity was needed to cause a
mitotic delay in all G~-pm cells. A similar cell cycle
arrest was obtained by treatment with mevinolin, an
alternative HMG CoA reductase inhibitor of human
diploid fibroblasts (Larsson et al. 1989a,b). The mechanisms mediating the 25-hydroxycholesterol-induced
mitotic delay are unclear. However, it is well established that mevalonate constitutes the key metabolite in
the biosynthesis of cholesterol and isoprenoid derivatives. This biosynthesis is catalyzed by HMG CoA
Downloaded from symposium.cshlp.org on March 4, 2016 - Published by Cold Spring Harbor Laboratory Press
CELL GROWTH AND CELL CYCLE TRANSIT
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Figure 4. Effects of transient exposures to different treatments on intermitotic times. 3T3 cells exponentially growing in the
presence of serum were, as indicated, shifted to either serum-free medium (no growth factors) or serum-containing medium
together with cycloheximide (100 ng/ml) or 25-hydroxycholesterol (1.5/xg/ml); 4 hr later, the cells were reshifted to serumcontaining medium without supplements. The cell ages and intermitotic times were determined by time-lapse cinematography.
reductase. Mevalonate or some downstream metabolite
is essential for initiation of D N A synthesis (Brown and
Goldstein 1980; Siperstein 1984). It has more recently
been shown that isoprene residues, synthesized from
mevalonate, are covalently linked to certain important
cellular proteins (Goldstein and Brown 1990). Of particular interest among such prenylated proteins is the
growth regulatory proto-oncogene product p21 ra~, and
data arc presented indicating that prenylation of r a s is
necessary for its activation (Schafer et al. 1989).
Whether any biochemical or molecular interconnections exist between the mechanisms of the three different types of cell cycle inhibitory agents (growth factor
depletion, cycloheximide, and 25-hydroxycholesterol)
or whether they act independently remains to be analyzed. However, we can confirm that these three principally different treatments elicit a kinetically identical
exit from the cell cycle.
Exit from the Cell Cycle
It is well known that the time from G Oto mitosis is
longer than intermitotic time in exponentially growing
cells (Baserga 1976). Time-lapse analysis performed in
our laboratory of quiescent 3T3 cells stimulated with
serum shows that the average time from G o to mitosis is
about 23 hours (data not shown). This is approximately
8 hours longer than the average intermitotic time
(about 15 hr) in exponentially proliferating 3T3 cells
(Fig. 3). As is evident from Figures 1, 4, 5 (top), and 7
(3T3 cells), the recorded mitotic delay is approximately
8 hours longer than the time of exposure to growth-
A . First Mitosis (M1)
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Figure 5. Relationship between cell age at the onset of serum
starvation and intermitotic time. Exponentially growing
cells reaching a density of 6000 cells/cm 2 were exposed to
serum-free medium for 4 hr, after which they were again
exposed to medium supplemented with serum. The cell ages at
the onset of serum starvation and intermitotic time during the
first generation (A) and second generation (B) for individual
cells were determined by time-lapse cinematography.
Downloaded from symposium.cshlp.org on March 4, 2016 - Published by Cold Spring Harbor Laboratory Press
142
ZETTERBERG AND LARSSON
factor-free medium or to metabolic inhibitors. Since a
mitotic delay of 8 hours in addition to the actual exposure time occurs after both brief exposures (15 min to 1
hr) and longer exposures (8-24 hr), these data suggest
that the cells rapidly (within less than 1 hr) exit to G O
even after a brief treatment. They remain in G Oduring
the period of treatment, and after re-addition of growth
factors or removal of metabolic inhibitors, the cells
return to the cell cycle, which takes about 8 hours.
Although Gl-pm cells respond immediately with a
mitotic delay (data above and Fig. 5A), time-lapse
analysis of the second cell cycle reveals that committed
cells beyond the restriction point (Gl-ps , S, and G 2
cells) also respond to a temporary exposure to growthfactor-free medium by a mitotic delay observed in the
second cell cycle (Fig. 5B). The indication that in fact
all cells in the population respond to growth factor
starvation, irrespective of cell cycle position, is consistent with the finding that the rate of protein synthesis is
suppressed rapidly after growth factor starvation in all
cell cycle stages (Table 1). If ability to remain in the
cell cycle depends on a high rate of protein synthesis to
maintain a critical concentration of labile proteins of
importance for the proliferative state (e.g., c-myc and
H M G CoA-reductase), one would expect these proteins to be depleted rapidly in all cells in which protein
synthesis is suppressed. A model taking all of these
observations into consideration is presented in Figure
6. Cells treated (growth-factor-starved or inhibited by
metabolic inhibition) while in Gl-pm exit immediately
from the cell cycle. Cells treated after Gl-pm, i.e., in
G~-ps, S, or G2, also leave the cell cycle. However, the
Table 1. Relationship between Cell Age and De Novo Protein Synthesis during Exposure to Serum-free Medium
Cell age group
0-4 hr (Gl-pm)
Length of serum-free Mean number of
treatment (hr)
grains/cell
0
2.0
4.0
80
62
52
50
4-8 hr (Gl-ps to early S)
0
1.0
2.0
4.0
125
107
65
65
8-12 hr (middle S)
0
1.0
2.0
4.0
170
120
87
77
12-16 hr (late S to G2)
0
1.0
2.0
4.0
215
190
120
1.0
100
Cells which were exponentially growing on glass coverslips and had
been classified by time-lapse cinematography with respect to cell age
were exposed to serum-free medium for indicated intervals. At termination of each experimental period, the cultures were pulse-labeled
(30 min) with [3H]leucine (20/~Ci/ml). The slides were processed for
autoradiography, and protein synthesis was assayed by counting the
number of silver grains covering each age-determined cell. Data from
four pooled cell-age groups (0-4, 4-8, 8-12, 12-16 hr) are presented.
15h
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Figure 6. Model describing kinetics of exit from and re-entry
to the cell cycle after brief exposure to growth-factor-free
medium or metabolic inhibitors. M0 represents mitosis before
treatment, and M 1 and M 2 represent first and second mitosis
after treatment. Arrows indicate beginning and end of treatment. Intermitotic times and times from G O to mitosis are
derived from data in Fig. 5. For further details, see text.
chromosome cycle ( D N A replication and mitosis) is
irreversibly initiated and runs on independently of the
influence of growth factors on the cell, and the exit
from the cycle is not observed until the cell enters the
second cell cycle. The second mitosis is delayed. The
time taken to proceed from Go to mitosis in cells
treated before commitment in Gl-pm is equal (23 hr) to
the time from G Oto the second mitosis (23 hr) in cells
treated after commitment in Gl-ps, S, or G 2. In these
latter cells, time to first mitosis must be ignored since
the chromosome cycle is already irreversibly initiated in
these ceils at the time of treatment and runs independently of the growth factor situation in the cellular
environment. A Gl-ps, S, and G 2 cell that has been
exposed to growth-factor-free medium or metabolic
inhibitors can thus be considered as a G O cell with
respect to proliferation and growth control but still in
the cycle with respect to the chromosome cycle.
E x i t to G O v e r s u s G 1 A r r e s t
Unlike normal 3T3 cells, the SV40-transformed derivative (SV-3T3) does not respond by a mitotic delay
upon treatment with serum-free medium or cycloheximide (Larsson and Zetterberg 1986a). In contrast, the
transformed cells are arrested in a Gl-pm-like phase
when treated with the HMG CoA reductase inhibitor
25-hydroxycholesterol (Larsson and Zetterberg 1986a).
Figure 7, top, shows the effect of an 8-hour exposure to
25-hydroxycholesterol on the intermitotic times of 3T3
and SV-3T3 cells. As can be seen, in both cell lines, all
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CELL G R O W T H AND CELL CYCLE T R A N S I T
3T3
143
SV3T3
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14
......
"6'''L
9 9. . . . .
9 9
16h
9
9
!
9!
...,,u
J
;
~ 8h
e __o.b___e..o._9_~
.........
10
0
,
e
e
4
8
12
Cell
0
age
I
I
4
8
I
12
(h)
POST - MITOTIC CELLS (cell age <4h)
SV-3T3
20
-se, ,,~
~'," 25-OH
-'"
16 '
/
/.
~' 12
A
.__o
4
~
0
0
-$e
9
A
1
I
4
r
I
I
8
12
16
Length of treatment (h)
I
20
Figure 7. (Top) Effects of exposure to 25-hydroxycholesterol on intermitotic time of 3T3 and SV-3T3 cells. Exponentially
growing 3T3 and SV-3T3 cells were shifted to medium supplemented with 25-hydroxycholesterol (1 ~g/ml) for 8 hr, whereupon
they were shifted back to 25-hydroxycholesterol-free medium for an additional 48 hr. Cell ages and intermitotic times were
determined by time-lapse cinematography. (Bottom) Relationship between treatment time with serum-free medium or 25hydroxycholesterol and intermitotic delay for 3T3 and SV-3T3 cells. The mean intermitotic delay of Gl-pm cells (i.e., ceils
younger than 3 hr) following treatment with serum-free medium or 25-hydroxycholestero! was determined from several
experiments (compare with Top).
cells younger than 3 - 4 hours at the moment of onset of
25-hydroxycholesterol treatment responded by a mitotic delay. Thus, SV-3T3 cells also possess a " G l - p m program," which must be completed before commitment to D N A synthesis and mitosis. Of particular
interest is the finding that the duration of the mitotic
delay (8 hr) of the responding SV-3T3 cells ( G l - p m
cells) is equal to the duration of the actual time of the
25-hydroxycholesterol treatment (i.e., 8 hr) and not 16
hours (i.e., 8 hr longer) as in 3T3 cells (Fig. 7, top).
This is more evident from Figure 7, bottom, in which
time-lapse data from several experiments clearly show
that duration of mitotic delay is identical to duration of
treatment. Thus, in contrast to untransformed 3T3
cells, the transformed SV-3T3 cells are not set back in
the cell cycle by the treatment, i.e., they do not exit
Downloaded from symposium.cshlp.org on March 4, 2016 - Published by Cold Spring Harbor Laboratory Press
144
ZETTERBERG AND LARSSON
from the cell cycle to G O but are instead arrested in
Gl-pm as long as they are exposed to the inhibitor. The
loss of the ability to exit from the cycle and become
Go-arrested most likely reflects some fundamental defect in the cell cycle or growth regulatory mechanisms
of tumor-transformed ceils.
120
~
100
or
..~
so
,o
Growth in Cell Size and Protein Synthesis
The role of cell size in control of cell division has
been discussed for many years but it is still obscure.
Prescott (1956) showed that division in Amoeba proteus could be postponed for several days by performing
periodic amputations of the amoeba cytoplasm. The
main conclusion from these experiments was that cells
cannot divide unless they are allowed to reach a critical
size. Killander and Zetterberg (1965a,b) presented
data indicating that cellular enlargement in G 1 w a s
somehow involved in the regulation of entry into S
phase in L cells. Further evidence for a size control
over initiation of D N A synthesis was given by Donachie (1968), who demonstrated that D N A synthesis in
Escherichia coli is initiated at a fixed size independent
of the growth rate. Similarly, a cell size control over
initiation of D N A synthesis has been suggested in other
systems such as the fission yeast Schizosaccharomyces
pombe (Fantes and Nurse 1977), the budding yeast
Saccharomyces cerevisiae (Johnston et al. 1977), the
slime mold Physarum polycephalum (Sachsenmaier
1981), and the amphibian Paramecium tetraurelia
(Berger 1982; Rasmussen and Berger 1982). More recent studies performed on yeast have dealt with
molecular aspects of cell size (Reed et al. 1985; Cross
1988; Nash et al. 1988). Data from these studies suggest
that the G 1 cyclins may be involved in coordination
between cell cycle commitment or START and cell size
in S. cerevisiae. It is reasonable that there is an interrelationship between the transit through the cell cycle and
the growth in celt size, in the sense that cells approximately double in size prior to mitosis, producing "balanced growth," when the cells are growing under physiological growth conditions. However, it has been demonstrated that it is possible to separate the two cycles
(Auer et al. 1970; Zetterberg et al. 1982; Das et al.
1983; Baserga 1984; Mercer et al. 1984). It has, for
instance, been demonstrated that quiescent Swiss-3T3
cells can be stimulated to undergo D N A replication and
mitosis in the absence of cellular enlargement ("unbalanced growth") (Zetterberg et al. 1982, 1984; Zetterberg and Engstr6m 1983; R6nning and Petterson
1984). In a further attempt to study the conditions
influencing growth of mammalian cells, we have examined the effects of growth factor depletion on mitotic
size of exponentially growing cells. Whereas exposure
to growth-factor-free medium forces Gl-pm cells to
arrest immediately in G o, cells located in subsequent
phases (Gl-ps, S, and G2) undergo the chromosome
cycle on schedule (see Figs. 1 and 2). Since the rate of
protein synthesis is decreased rapidly following growth
factor depletion in all cells, irrespective of cell age (see
9~, 4o
~
2fl
0
Oh
4h
8h
Minus serum
8h+insulin 8h+IGF1
(1001ag/ml) (10ng/m|)
Figure 8. Effect of 4- or 8-hr exposure to serum-free m e d i u m
on the protein content of mitotic cells. Proliferating Swiss-3T3
cells were rinsed and exposed to m e d i u m containing 10%
serum, serum-free medium, or serum-free medium supplemented with insulin (100/xg/ml) or IGF-1 (10 ng/ml). After 4
or 8 hr, the cells were fixed and stained with Feulgen/Naphtol
Yellow S (NYS). Mitotic cells were identified microscopically,
and DNA and protein content in individual mitotic cells was
determined by microspectrophotometry.
Table 1), it is conceivable that the increase in cell size
( = protein accumulation) of Gl-ps, S, or G 2 cells is
also reduced. To verify this hypothesis, we measured
the protein content of mitotic cells after short growthfactor-free periods (4 and 8 hr). Figure 8 shows data
from microspectrophotometric determinations of cell
size at mitosis. As a matter of fact, there is a small but
clearly detectable reduction in mitotic cell size as compared to control cells following exposure to growthfactor-free medium for 4 hours. After an 8-hour treatment, the cell size at mitosis is reduced as much as 40%
(Fig. 8). From these data and those in Table 1, it can be
concluded that short exposures to growth-factor-free
medium result in a rapid decrease in de novo protein
synthesis and a rapid inhibition of cell growth (in size)
in all stages of the cell cycle. This inhibitory effect on
cell growth by growth factor starvation could, however,
Table 2. Effects of Growth Factors on Intermitotic Delay
and Protein Synthesis
Treatment
Serum
-Serum
-Serum
-Serum
Serum +
Serum +
Serum +
+ PDGF
+ insulin
CHM
CHM + PDGF
C H M + insulin
Protein synthesis
(% of control)
Intermitotic delay
(hr)
I00
55
70
92
48
47
50
0
16.0
2.7
14.9
14.8
2.0
15.0
Exponentially growing 3T3 cells, either in flasks for time-lapse
cinematographic analysis or in dishes for determination of protein
synthesis, were shifted to serum-free medium or serum-containing
medium supplemented with cycloheximide (CHM) (100 ng/ml), with
or without PDGF (25 ng/ml) or insulin (100/~g/ml), for 8 hr. The
intermitotic delay was determined by time-lapse cinematography, and
the rate of protein synthesis was assayed by pulse-labeling with
[3H]leucine. The leucine incorporation values are expressed as percentages of the serum control.
Downloaded from symposium.cshlp.org on March 4, 2016 - Published by Cold Spring Harbor Laboratory Press
CELL GROWTH AND CELL CYCLE TRANSIT
be counteracted if supraphysiological concentrations of
insulin were added to the growth-factor-free medium
(Fig. 8). This effect of insulin is compatible with its
stimulatory effect on de novo protein synthesis (Table
2). However, the finding that insulin fails to prevent Go
arrest (see below) supports the findings of unbalanced
growth discussed above, i.e., that growth in size and
the chromosome cycle ( D N A replication and mitosis)
are two separate sets of processes under different controis. Figure 8 shows that physiological doses of insulinlike growth factor 1 (IGF-1) could substitute for insulin
in promoting growth in size. Together with the observation that insulin can bind with low affinity to the IGF-1
receptor (Massagu6 and Czech 1982), our data suggest
that the stimulatory effect of insulin on cellular protein
content may be mediated via the IGF-1 receptor.
Different Growth Factor Requirements for
Cell Cycle Progression and for
Growth in Cell Size
By exposing the cells for various time periods to
medium containing individual purified growth factors,
we have previously demonstrated that platelet-derived
growth factor (PDGF) alone could substitute for the
whole serum complement in driving 3T3 cells through
the whole of G~-pm, including the restriction point and
commitment to the chromosome cycle (Zetterberg and
Larsson 1985). In contrast, epidermal growth factor
(EGF) and insulin failed to do so. However, EGF and
insulin both exhibited a temporary effect (up to 4 hr) in
preventing exit to G O. In other words, the cells were
temporarily arrested in Gl-pm or advanced very slowly
through Gl-pm. In the present study, we also investigated whether two different types of growth factors,
insulin and PDGF, could to any extent prevent the
mitotic delay induced by transient treatments (here for
a duration of 8 hr) with growth factor depletion or
cycloheximide. The effects of these treatments on protein synthesis were also analyzed. Table 2 shows the
results from these experiments performed on Swiss-3T3
cells. Similar results were obtained from experiments
on H D F (data not shown). The mitotic delay caused by
serum depletion was efficiently prevented if P D G F was
present, whereas insulin exerted no detectable counteractive effect. In contrast, insulin counteracted the
depressive effects of treatment with growth-factor-free
medium on protein synthesis, whereas P D G F only had
a partial effect in this respect. These data suggest that a
general increase in overall protein synthesis, as induced
by insulin, is not sufficient to counteract exit from the
cell cycle. Since P D G F did not increase the overall rate
of protein synthesis much, the question may be raised
as to whether PDGF instead induces the synthesis of
specific cell cycle regulatory proteins and thereby
would overcome the mitotic delay. This would be in
line with the notion that the cellular decision to proceed
through the cell cycle instead of becoming quiescent is
dependent on the accumulation of critical cell-cyclespecific or growth-promoting proteins (Rossow et al.
145
1979; Pardee et al. 1981; Croy and Pardee 1983; Cross
1988; Nash et al. 1988; Hadwiger et al. 1989). To
investigate this, the effects of insulin and P D G F on
cycloheximide-treated cells were studied. As shown in
Figure 3, the dose of cycloheximide (100 ng/ml) that
reduced protein synthesis by approximately 50% also
induced a mitotic delay. As shown in Table 2, neither
of the two growth factors could counteract the cycloheximide-induced inhibition of protein synthesis.
However, P D G F was nevertheless capable of preventing the mitotic delay, whereas insulin had no such
effects. On the basis of these results, it is reasonable to
assume that PDGF does not prevent mitotic delay primarily by restoring the overall rate of protein synthesis
to normal levels. Rather, it is likely that PDGF exerts
its effect by altering the expression of cell-cycle-specific
or growth-promoting genes encoding for proteins required for progression through Gl-pm and commitment to the chromosome cycle. This opinion is in line
with previous reports showing a preferential effect of
P D G F on expression of c-myc when added to quiescent
cells (Kelly et al. 1983; Campisi et al. 1984).
ACKNOWLEDGMENTS
This project was supported by grants from the Swedish Council of Medical Research, the Swedish Cancer
Society, and the Stockholm Cancer Society.
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Downloaded from symposium.cshlp.org on March 4, 2016 - Published by Cold Spring Harbor Laboratory Press
Coordination between Cell Growth and Cell Cycle
Transit in Animal Cells
A. Zetterberg and O. Larsson
Cold Spring Harb Symp Quant Biol 1991 56: 137-147
Access the most recent version at doi:10.1101/SQB.1991.056.01.018
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