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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 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 Downloaded from symposium.cshlp.org on March 4, 2016 - Published by Cold Spring Harbor Laboratory Press 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, Downloaded from symposium.cshlp.org on March 4, 2016 - Published by Cold Spring Harbor Laboratory Press C E L L G R O W T H AND CELL CYCLE T R A N S I T A 38 B 3s 139 C " D 38 A t'= 34 .]4 v 34 34 Mitotic derby 99 : w ; ......................... 4) E ~o 3 0 " 9 30 De 9 26 26 9 26" , eeoeo 9 30" .~ 0 0u ~ ~ 22 ~" 22 0 9 18 E "._..__:._'_.'._ e- l0 ,, 9 9 i 9 i 4 8 I/ 9 9 9 9 m - --e--e" - -~ W -- - - "e-- 18 9 ..... 14 9 9 0 i 4 , , 8 9 F 12 Cell 22 18 / ;J ,, .....: ? 1O [0 9 12 I 2Z 9 10 9 9 ~ 1 7 6 1 7 i6 g ~04k 9 Mitotic doisy 2 6 ...................... i 4 0 age i 8 12 i 4 0 i 8 F 12 (h) 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 31"3 :.30 Z""" ! |:8 "'~ 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. I-K)F 8 8 .,o ~ oOoOo 9 30" 30 30- 26 26 26' 22 22 Oil 9 e-- E ~ ~ el 22 0 0 I ]8 ~ 18 i P J9 E L_ 14 ,08.. t!0 10 I0 I 0 i 8 i 12 0 i 8 4 Cell age i 12 T 0 i 4 i 8 T 12 (h) 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 ,oo A ,/ / /o .I" f 50 V Response to Metabolic Inhibitors 3T3 ,-- E = . _ ~ I ~ 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 B 0 5O 0 4 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 Nopvwthf m 30 9 9 .... 9 P~ Mitotic dolly 9 ~ .... 9 ~176 .~176176176 .... 30' 26 Mitotic delay ...o. | 25-hytkoxycholesterol cycle 30' 26' ..... . ...... . ..... .. ........ Mitotic dolly ~.~.A.,. ........................ 26 .,~ 22 22 22' 18 18 18 14 " 9 !__._: 141 14 . . . . . e _ t0t 08 o _- .~_,..~. ; :..-_ _ ~. 14" . . . . . ~ f _ -o- - - ,.a- 10 i 4 i l 12 i i i 4 8 12 Cell age 4 i i 8 12 (h) 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) ~ 1 4 99 Z v Oo 9 O0 9 O9 ........ O.._ 0 4 9 JOg v ........ E .m ._o I B. I I Second Mitosis (M2) 9 O OOo0 k. t"- 9 9 gig 9 9 O00 9 9 9 9 9 9 9 I 4 U 8 I 12 Cell age (h) 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 J Mo O-.... : ! I .. M1 15h * 7. ..... : I I I M'ol t Treatment in: M,i [ ' i ' " i .............. ~*-:'h , Go Mo / G1 pm ,3 It T It Go Mo 9 ..... Control (no treatment) M2 A : M2 : G1 p s - s / ........ i ........2 3 h t 1 ~1 ~. IT' Go M= G2 ~ 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 Downloaded from symposium.cshlp.org on March 4, 2016 - Published by Cold Spring Harbor Laboratory Press CELL G R O W T H AND CELL CYCLE T R A N S I T 3T3 143 SV3T3 25 OH, 8h 2 5 O H , 8h 38 34 "-" r O M i t o t i c delay , ....~176 30 9 9 9 9 Mitotic delay ~176176176176176176176176176176176176176176176176176 9 9 & / / "i ! 26 22 O 18 99 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. 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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 References This article cites 59 articles, 19 of which can be accessed free at: http://symposium.cshlp.org/content/56/137.refs.html Article cited in: http://symposium.cshlp.org/content/56/137#related-urls Email alerting service Receive free email alerts when new articles cite this article sign up in the box at the top right corner of the article or click here To subscribe to Cold Spring Harbor Symposia on Quantitative Biology go to: http://symposium.cshlp.org/subscriptions Copyright © 1991 Cold Spring Harbor Laboratory Press