Cell, Vol. 18. 1065-l
072,
December
1979.
Copyright
0 1979
by MIT
Centriole Deciliation Associated with the Early Response of 3T3 Cells to Growth Factors But Not to sv40 R. W. Tucker*, C. D. Schert and C. D. Stile+ *Laboratory of Tumor Biology and Division of Medical Oncology TDivision of Hematology-Oncology and Department of Pediatrics Children’s Hospital Medical Center *Laboratory of Tumor Biology, Group W and Department of Microbiology and Molecular Genetics Sidney Farber Cancer Institute and Harvard Medical School Boston, Massachusetts 02115
BALB/c-3T3 cells which are growth-arrested by high cell density or low serum have ciliated, unduplicated centrioles. Stimulation of these quiescent cells by serum is associated with a rapid (within l2 hr) deciliation of the centriole, followed by reciiiation within 6-10 hr. This transient deciliation of the centrioie is induced by the platelet-derived growth factor (PDGF) component of serum. The ceils treated with PDGF became competent to replicate their DNA; most PDGF treated ceils, however, did not progress from Go toward S phase unless they were incubated with the platelet-poor plasma component of serum. Addition of CaC12 or Fibroblast Growth Factor to the media mimicked PDGF by producing both centriole deciliation and competence to replicate DNA. in fact, over a range of concentrations of each of these factors, only doses which produced centrioie deciiiation were capable of producing competence for DNA synthesis. Plasma alone or factors such as Multiplication Stimulating Activity produced neither centriole deciiiabon nor competence; these agents were, however, required for the optimum progression of competent cells into DNA synthesis. in contrast, infection with SV40 induced host cell DNA synthesis without an initial transient deciliation of the centrioie. Thus while growth factors may have to induce centriole deciliation for 3T3 cells to synthesize DNA, abortive transformation by SV40 overrides this requirement.
As proliferating cells replicate their DNA and proceed through mitosis, closely correlated events must also occur with respect to centriole duplication. in studies on mammalian “centriole cycle” in 3T3 cells, Tucker, Pardee and Fujiwara (1979) demonstrated that centrioles become ciliated during G,, then deciliate and l To whom reprint Oncology Center, 21205.
requests should be addressed at: Johns Hopkins 600 North Wolfe Street, Baltimore, Maryland
duplicate concurrently with DNA synthesis. Quiescent 3T3 cells in low serum are growth-arrested in Go/G1 and contain a centriole pair forming a primary cilium; upon serum stimulation, these cells undergo an early (l-2 hr) transient deciliation, followed by another deciliation which is associated with centriole duplication and DNA synthesis. Serum contains polypeptide growth factors which control different events in the cell cycle (Temin, Pierson and Dulak, 1972; Jiminez de Asua et al., 1977; Pledger et al., 1977). The platelet-derived growth factor (PDGF) (Kohler and Lipton, 1974; Ross et al., 1974) component of serum controls an early event in the mitogenic response. Quiescent cultures of BALB/ c-3T3 cells exposed transiently to PDGF become “competent” to replicate their DNA and divide; however, PDGF-treated BALB/c-3T3 cells do not “progress” efficiently from Go/G1 into the S phase unless they are incubated in medium containing an optimal concentration (that is, 5%) of platelet-poor plasma. In medium containing a suboptimal concentration of platelet-poor plasma (50.25%) most PDGF-treated cells remain growth-arrested; however, such cells remain competent to replicate their DNA for many hours, and will do so, following a minimum 12 hr lag time, when the plasma content of the culture medium is raised to an optimal level of 5% (Pledger et al., 1977). Thus a cellular state of “competence” occurs as an early event in the mitogenic response and requires only transient exposure to PDGF. “Progression” is a later series of events leading to DNA synthesis (Pledger et al., 1978) and cell division (Vogel et al., 1978) which requires prolonged exposure to a second set of growth factors contained in plasma. At present there is no morphologic or biochemical marker of these two functionally distinct steps in mitogenesis. Using complementation assays, Stiles et al. (1979) have demonstrated that competence and progression are mediated by two distinct classes of growth factors. Fibroblast Growth Factor (FGF) and microprecipitates of calcium phosphate [Ca3(PO&] principally regulate competence, while progression activity is predominantly controlled by growth factors contained in plasma. An important regulatory component of plasma is the somatomedin family-a group of insulin-like polypeptides whose concentration in blood is largely controlled by pituitary growth hormone. Somatomedin8 in plasma function synergistically with competence factors such as PDGF, FGF, and Ca3(PO& to induce the optimal mitogenic response. Virally transformed and normal ceils appear to differ in their growth requirement for the competence and progression factors found in serum (Scher et al., 1978). SV40 infection of quiescent 3T3 cells allows them to traverse Go/G1 and synthesize DNA in the absence of both competence and progression factors (Stiles et al., 1979). Whether SV40 produces the
Cell 1066
same or a different sequence of events as that induced by exogenous serum growth factors is unknown. In this study, we demonstrate that different events in the centriole cycle are associated with the competence and the progression steps of the cell cycle. Furthermore, centriole events in 3T3 ceils stimulated with growth factors differ from those in 3T3 cells infected with SV40. Specifically, we find that the transient deciliation of the centriole in 3T3 cells is induced by competence factors, and may be a necessary prerequisite for DNA synthesis; this transient deciliation is bypassed during SV40-induced DNA synthesis. Results Plasma and Platelet-Derived Growth Factor (PDGF) In a previous study, Tucker et al. (1979) demonstrated that the addition of fresh, serum-containing medium to quiescent 3T3 cells induced an initial transient deciliation of the centriole, followed by a later deciliation associated with DNA synthesis. In this study we have used the serum components, PDGF and plasma, to examine the control of these centriole events. As Figure 1 (top) demonstrates, PDGF together with an optimal (5%) concentration of plasma induced a transient initial deciliation followed by a later deciliation which was temporally correlated with DNA synthesis. Cells made competent by a 4 hr pulse of PDGF and incubated in medium containing a suboptimal (0.25%) concentration of plasma also showed an initial transient deciliation (Figure 1, middle); however, very little secondary deciliation or DNA synthesis was observed. Cells which were exposed continuously to an optimal concentration of plasma (5%) without PDGF (Figure 1, bottom) demonstrated neither the initial transient deciliation nor the later secondary deciliation, and did not undergo DNA synthesis. This series of experiments suggests that the initial transient deciliation is induced by a factor derived from platelets. The later secondary deciliation is associated with cells entering the S phase. This second deciliation (like DNA synthesis) requires plasma components but cannot be induced by plasma alone. PDGF and plasma control sequential events in the cell cycle, so that delaying the addition of plasma to PDGF-treated cells also delayed DNA synthesis by a corresponding time (Pledger et al., 1977, 1978). Figure 2 demonstrates that under these conditions the second centriole deciliation was also correspondingly delayed. Thus the second deciliation is closely correlated with DNA synthesis. The experiments depicted in Figures 1 and 2 were conducted with partially purified preparations of PDGF. As Table 1 shows, pure PDGF (Antoniades, Scher and Stiles, 1979) also produced an initial deciliation associated with the induction of competence. Deciliation occurred in 95% of the cells measured at 2 and 4 hr after pure PDGF addition, and competence was simultaneously induced in at least 80% of the
n
PLhsy1
Tii bsl
Figure 1. PDGF and Plasma Control Cycle of BALB/c-3T3 Cells
Separate
Events
in the Centriole
(Top) Quiescent 3T3 cells received fresh medium containing both an optimal concentration of plasma (5%) and partially purified PDGF (350 pgg/ml) at time zero. (Middle) Quiescent 3T3 cells received fresh medium containing partially purified PDGF (350 pg/ml) and a suboptimal concentration of plasma (0.25%). (Bottom) Quiescent 3T3 cells received fresh medium containing 5% plasma at time zero. The percentage of cells with ciliated centrioles in two different experiments, (0) or (O), and the percentage of cells with labeled nuclei in the continuous presence of 1 &i/ml 3H-thymidine (III) is shown as a function of time thr). Shaded area connects the data for the different experiments measuring centriole ciliation.
cells. Thus the response of centriole ciliation to serum can be resolved into two parts controlled by different components of serum; an initial transient deciliation was produced by pure PDGF, and a final deciliation associated with DNA synthesis was controlled by plasma. This second deciliation of the centriole produced by plasma occurs only in PDGF-treated cells which have already undergone an earlier deciliation (Figure 1). Fibroblast Growth Factor (FGF) and Ca3(PO& PDGF is one of a class of mitogenic agents which induces 3T3 ceils to become competent for DNA replication; other agents (or treatments) which induce
Centriole 1067
Deciliation
and
Early
Response
to Growth
Factors
Table I. Relationship Competence
Factor
(rig/ml)
between
Centriole
Deciliation
% Cells with Daciliated Centrioles”
and
% Labeled Nuclei (Competence)b
Pure PDGF 0.7 1.4 2.8 4.5 9.0 18.0
12 25 34 66 90
13 10 18 27 43 75
100 1000
7 10
17 7
1 10 100
25 40 24
2 11 16
MSA
’ Maximum percentage of cells which have deciliated their centrioles during a 4 hr period following the addition of growth factors. b Percentage of cells in medium supplemented with 5% plasma which enter DNA synthesis 24-36 hr after a 4 hr exposure to a growth factor.
Tune (hr) Figure 2. Delaying the Addition of 5% Plasma to Competent BALB/ c-3T3 Cells Delays S Phase and the Second Centriole Deciliation by a Corresponding Time (Top) Quiescent cells were treated for 4 hr with 350 pg/ml of partially purified PDGF and then incubated in medium containing suboptimal (0.25%) plasma and 3H-thymidine (1 &i/ml) so that little DNA synthesis occurred. (Middle) Same as above except that cells were fluid-changed to DME + 5% plasma at 8 hr. (Bottom) Same as above except cells were fluid-changed to DME + 5% plasma at 20 hr. The percentage of cells with ciliated centrioles (0) and with autoradiographically labeled nuclei (0) was followed as a function of time.
competence include FGF, precipitates of Ca3(PO& and “wounding” of a confluent monolayer culture (Stiles et al., 1979); in light of this data, we examined the relationship between centriole deciliation and the mitogenic response to FGF, Ca3(PO& and “wounding.” We found that wounding of the cell monolayer (data not shown) or the addition of FGF or of CaC& [which precipitates in DME as (Ca)3(P04)2] to a quiescent monolayer of 3T3 cells for 4 hr produced competence and deciliation of the centriole. Figure 3 depicts the similarity of the time course of the transient deciliation obtained with FGF and Ca3(POJ2 with that produced by RDGF, as described in Figure 1. Thus three different agents which control the same event in the mitogenic response of quiescent 3T3 cells (competence) all produced a transient deciliation of the centriole. Competence and Centriole Deciliation To further study the association between the initial transient centriole deciliation and the state of competence, we studied both parameters over a wide
range of doses for five different factors. As Figure 4 summarizes, the doses of factors [PDGF, FGF, Ca3(P0&] which produced competence also produced centriole deciliation. On the other hand, MSA and EGF at physiological concentrations produced neither competence nor substantial centriole deciliation (Table 1). It is important to note that PDGF produced substantial deciliation at concentrations lower than that required to produce competent cells (Table 1 and Figure 4) whereas Ca3(PO& induced deciliation and competence at similar concentrations (Figure 4). Thus deciliation of the centriole may be necessary for the induction of competence, but is not always accompanied by competence. Continuous Exposure to Ca3(PO& or PDGF As shown in Figures 1 and 2, 4 hr pulses of PDGF, FGF or Ca3(PO& produced an initial deciliation of the centriole which appeared to be a transient phenomenon, with reciliation occurring within 8-12 hr. To be certain that the transient nature of the deciliation was an integral part of the response to the growth factors and not simply a result of the transient application of the stimulus, we studied centriole ciliation during the continuous exposure to PDGF or Ca3(PO& in amounts capable of producing competence. As shown in Figure 5 (top), quiescent cells treated with CaCI, at concentrations greater than 2.6 mM became rapidly deciliated. The initial deciliation was only transient despite continuous exposure to the Ca3(PO& precipitate which formed in the culture medium. Higher concentrations of CaCL increased the magnitude of cell population which became deciliated
Cell 1066
01 lo
100
o\o
POGF
40
CaCI,
w* 0
-O-
20 '10
Figure 4. Centriole Become Competent Deciliation
Is Produced
by FGF and C&(PO&
(Top) 50 rig/ml of FGF in DME plus suboptimal (0.25%) plasma were added at 4 hr. Cells were then placed in DME plus 0.25% plasma and 1 &i/ml 3H-thymidine. (Bottom) 5 mM CaC& was added to DME. producing a Ca3(PO& precipitate. After 4 hr. cells were fluid-changed to TIME plus 0.25% plasma and 3H-thymidine (1 &i/ml). The percentage of cells with ciliated centrioles (0) and the percentage of autoradiographically labeled nuclei 0 were monitored versus time. In parallel cultures of both FGF- and Ca,(PO&-treated cells, plasma was added to an optimum final concentration (5%); under these conditions 75% of the FGF-treated cells and 83% of the Ca3(PO& treated cells entered the S phase within 36 hr.
without changing the time course of the transient deciliation. There was no apparent loss of Ca3(PO& precipitate during the course of the experiment. In the suboptimal concentration of plasma (0.25%) contained in the medium, very little DNA synthesis (15% labeled nuclei) or secondary deciliation (15% deciliated cells) occurred. Similarly, the initial deciliation produced by PDGF was Only trNISient, despite COntinUOUS exposure to PDGF (Figure 5, bottom). The magnitude of the deciliation increased with PDGF dosage, but the duration was roughly constant. The fact that the time course of the deciliation was not affected by PDGF dosage argues against the possibility that the deciliation was transient because the PDGF was consumed during the course of the experiment; moreover, the time course of the deciliation was similar to that produced by a “pulse” of PDGF (Figure 1). As noted previously (Pledger et al., 19771, continuous exposure to higher concentrations of these impure PDGF preparations (68 pg/ml) produces some DNA synthesis (30% labeled nuclei) and secondary deciliation (30%) even in suboptimal (0.25%) plasma concentrations; the percentage of cells induced to enter S phase and undergo secondary deciliation is, however, always greater in
/
0
/
,-;”
A
3. Transient
r
IQ
20
Figure
;
534’
40
60
80
100
Deciliation of Centrioles
Deciliation Is Necessary for BALB/c-3T3 to Replicate Their DNA
Cells to
Quiescent cells were exposed for 4 hr to C&(P04)2 or growth factors at various concentrations. Deciliation of centrioles was measured in DME plus 0.25% plasma, and competence was measured in DME plus 5% plasma. The points on the graph depict: (0) Ca3(PO& at concentrations ranging from 2.0 mM to 9.0 mM; (A) FGF at concentrations ranging from 3.0-l 00 rig/ml; (0) partially purified PDGF at concentrations ranging from 14-272 f.rg/ml. The separate lines indicate separate experiments.
the presence of optimal (5%) plasma (Pledger et al., 1977). Thus the transient nature of centriole deciliation associated with competence is characteristic of the cellular response to the agents and is not dependent on the duration of exposure. Sequential Transient Exposures to Ca3(PO& or PDGF We next asked whether a cell which has undergone one transient deciliation can again transiently deciliate. Quiescent 3T3 cells were stimulated with two consecutive pulses of either Ca3(PO& or PDGF in 0.25% plasma. As Figure 6 demonstrates, both pulses of either agent produced a transient deciliation of the centriole. Thus the transient exposure to competence factors did not produce a refractory state for further transient deciliation of the centriole. sv40 Since 3T3 cells transformed with SV40 no longer require PDGF to grow (Scher et al., 1978) and since infection of quiescent 3T3 cells with SV40 induces host cell DNA synthesis without plasma or PDGF (Stiles et al., 1979), it was of interest to follow centriole ciliation in quiescent 3T3 cells infected with SV40. As Figure 7 demonstrates, infection (abortive transformation) with SV40 did not produce an initial deciliation of the centriole. However, the second and final deci-
Centriole 1069
Deciliation
t ( COCIP
::i 4
e
and
Early
i 12
16
Response
20
to Growth
24
28
Factors
32
; 34
“- WC , 40
Time Chrs)
Figure 6. Sequential Addition quential Transient Deciliations Figure 5. Continuous Exposure Transient Initial Deciliation
to Competence
Factors
Produces
a
(Top) CaClz at indicated mM concentration [2.2 (0). 2.6 (O), 4.3 (A). 9.3 (B)] was added to cells in DME + suboptimal (0.25%) plasma at time zero. Cells were not fluid-changed. 4.3 mM CaC12 induced a Ca3(PO& precipitate and only a transient deciliation of the centriole; addition of 9.3 mM CaCI, induced more deciliation, but cells did not survive long enough to observe later effects. (Bottom) Partially purified PDGF &g/ml) [14 (0). 7 (O), 27 (A), 66 (D] was added to the cells in DME + suboptimal (0.25%) plasma at time zero. Cells were not fluid-changed. The percentage of cells with ciliated centrioles was monitored versus time.
liation of the centriole still occurred with DNA synthesis. The lack of an initial centriole deciliation correlates with the lack of dependence on PDGF by SV40-transformed mouse fibroblast cell lines (Scher et al., 1978; Stiles et al., 1979). The production of both the second centriole deciliation and DNA synthesis in these infected cells strengthens the association between these two events. Discussion At mitosis, the duplicated chromosome must be partitioned equally to the two daughter cells. To accomplish this precise distribution of the genetic material in mammalian cells, the centriole and DNA synthesis cycles must be coordinated. One aspect of the centriole cycle which may be involved in this coordination is the formation of a primary cilium by the centriole. Tucker et al. (1979) showed that ciliation of the centriole undergoes marked changes in quiescent 3T3 cells stimulated to replicate by serum addition. In this
of Competence
Factors
Produces
Se-
(Top) Quiescent BALB/c-3T3 cells were exposed to DME containing 9.3 mM CaC12 and 5% plasma for 4 hr. once at time zero and again at 16 hr. In the intervening times, the cells were incubated in DME containing a suboptimal (0.25%) concentration of plasma. The percentage of cells with ciliated centrioles (0) is shown as a function of time. (Bottom) 1.9 mg/cc (33 U/mg) of partially purified PDGF was added to DME + 0.25% plasma in 4 hr pulses starting at time zero and time 16 hr. Cells were fluid-changed to DME + 0.25% plasma to other times.
, sv 40
5
Figure 7. SV40 Induces Transient Deciliation
TIME (HRS)
Replicative
DNA Synthesis
without
an Initial
SV40 in DME + 0.25% plasma was added to quiescent BALE/c-3T3 cells at time zero at a multiplicity of infection of 100. In two separate experiments, the percentage of cells with labeled nuclei (0. A) and with deciliated centrioles (0. A) is shown versus time.
report we show that competence, an early mitogenic event produced by the PDGF component of serum, is closely associated with a transient deciliation of the centriole. In fact, this transient deciliation of the centriole may be a morphologic marker of competence. The deciliation of the centriole also marks a later
Cell 1070
period of coordination between the centriole and DNA synthesis cycles. As shown in Figure 8, the two periods (competence and progression) are controlled by separate serum factors. PDGF controls competence in the DNA synthesis cycle and an early transient deciliation of the centriole; plasma controls progression from Go (quiescence) into DNA synthesis and the final deciliation and duplication of the centriole. Centriole duplication may be a key event in the control of initiation of DNA synthesis. There is already evidence that centriole duplication occurs before the initiation of DNA synthesis in mammalian cells (Fiobbins, Jenttsch and Micali, 1968; Rattner and Phillips, 1973; Snyder and Liskay, 1978). In yeast there is also accumulating evidence that the initiation of DNA synthesis is directly dependent upon prior centriole or spindle plaque duplication (Byers and Goetsch, 1974; Hereford and Hartwell, 1974; S. Dutcher and L. Hartwell, personal communication). Similar studies have not yet been performed in mammalian cells, but in this report we have provided more evidence for the coordination of centriole events with mammalian DNA synthesis cycle during Go or G,. Centriole deciliation does not itself cause competence, but must reflect events in the cell which lead to competence and DNA synthesis. Such events may include an increased intracellular calcium concentration at the centriole which could depolymerize the ciliary microtubules and cause deciliation or cilium resorption (Tucker et al., 1979). At the same time, calcium could bind to calmodulin, a calcium-dependent regulatory protein, which has been localized near the centriole in at least one portion of the cell cycle (Marcum et al., 1978). Calmodulin-Ca++ complex might then induce changes in cell shape via actinmyosin interaction (Yerna et al., 1979), lower cyclic AMP by activation of phosphodiesterase (Tea and Wang, 1973) and even shorten the duration of increased cytoplasmic calcium by increasing calcium transport out through the plasma membrane or back into membrane vesicles (Larsen and Vincenzi, 1979). Such a transient change in intracellular calcium is I-
GoI
&S -F
I-
compatible with the transient nature of centriole deciliation (Figure 5) and the sequential deciliation produced by sequential additions of growth factors (Figure 6). Competence might then be secondary to a change in cyclic AMP or phosphorylated proteins associated with the binding of calcium to calmodulin. Centriole deciliation would therefore be necessary, but not always sufficient, for competence, as we have reported in this paper. Using the transient deciliation of the centriole as a marker for early mitogenic events, we have found differences between the cell cycle in uninfected and SV40-infected 3T3 cells. The events leading to DNA synthesis in 3T3 cells stimulated with growth factors appear to require a transient centriole deciliation, whereas those in SV40-infected cells do not. Thus viral infection does not produce all of the usual cellular prereplicative changes, and may indeed induce an alternate pathway to DNA synthesis. If deciliation of the centriole does reflect a transient increase in intracellular calcium concentration, then these results may explain in part why neoplastic and virally transformed cells have decreased growth requirements for exogenous calcium (Boynton and Whitfield, 1976; Boynton et al., 1977). Thus one of the changes during neoplastic transformation may relate to changes in location and effect of intracellular calcium (Whitfield et al., 1979). Understanding more about how calcium and other factors influence the relationships between centriole and DNA synthesis cycles may help us to better define some of the growth regulatory differences between non-neoplastic and virally transformed or neoplastic cells. Experimental
Procedures
Cell Culture BALB/c-3T3 cells (clone A31) were maintained as sparse cultures in Dulbecco’s modified Eagle’s medium (DME) (Flow Laboratories) su& plemented with 10% calf serum (Flow Laboratories). To produce density-arrested cell cultures, l-2 x 10’ cells were grown in 1 cc of DME + 10% calf serum on circular glass coverslips (12 mm diameter: Rochester Scientific Co.) for 5-7 days. Cells were then fluid-changed to DME medium containing various growth factors and ‘H-thymidine. Duplicate coverslips were processed in parallel for antitubulin staining and for autoradiography.
Centrioie Events and DNA Repof BALB/c-3T3 Cells to Serum
Autoradiography The percentage of nuclei labeled in the continuous presence of 1 pCi/ml ‘H-thymidine (1 .O mCi/0.038 mg; New England Nuclear) was measured as a function of time afler serum or growth factor stimuiation of quiescent cultures. as previously described (Tucker et al.. 1979). Briefly, cells were fixed in 100% methanol for 20 min. airdried and overlaid with photographic emulsion (1:2 dilution of Kodak NB-T). The photographic emulsion was developed (Kodak D-l 9) and fixed (Kodak rapid fixer) after 3 days, and the cells were counterstained with 10% Giemsa in PBS for 20 min. The percentage of nuclei with more than 50 grain counts above the background was recorded at each time point.
Transient centrioie deciliation occurs early during POGF-dependent sequence of events (“competence”). and centriole duplication occurs near the start of DNA synthesis. Final deciiiation in some ceils may occur after initiation of DNA synthesis, during S or later.
Growth Factors Most of these experiments were performed with partially purified (100 fold) preparations of PDGF prepared by heat treatment (100°C) as previously described (Pledger et al., 1977). During the course of
PDGF Dependent
Plasma Dependent
Ksfent Centnole Decihotion Figure 8. The Relationship between lication in the Mitogenic Response Factors
Plosmo Independent
Fmol Centnole Deciliothon ond Dupllcotion
Centriole 1071
Deciliation
and
Early
Response
to Growth
Factors
these studies, however, human PDGF was purified to homogeneity; electrophoretically homogeneous PDGF prepared by the method of Antoniades et al. (1979) was used to substantiate our main conclusions. The preparation of defibrinogenated platelet-poor plasma from human blood has been described by Pledger et al. (1977). Only preparations that maintained cell viability without stimulating growth of BALB/c-3T3 cells were used. Fibroblast Growth Factor (FGF). Multiplication Stimulating Activity(MSA)and Epidermal Growth Factor (EGF) were all obtained from Collaborative Research (Waltham. Massachusetts) and stored at 0-4’C. SW0 The preparation of SV40 has been previously described (Scher et al., 1979). Briefly, a clonal isolate of the small plaque variant (Takemoto, Kirchstein and Habel. 1966) of SV40 was grown on confluent Vero monkey cells in medium containing 5% agamma calf serum (North American Biological). Approximately 2 weeks after infection, when a cytopathic effect was noted, the cells and medium were frozen and thawed 3 times and the extracts were clarified by low speed centrifugation. SV40 was precipitated by the addition of polyethylene glycol (molecular weight 6000-7000; Fisher) (Friedman and Haas. 1970) and further purified by a centrifugation through sucrose (15%) onto a layer of 60% sucrose. The virus was titered by a plaque assay on CV-1 cells. Aliquots of virus were cryopreserved as l-2 x 10’ pfu/ ml and thawed just before use. Centriole Ciliation The percentage of cells with ciliated centrioles was determined by indirect immunofluorescence with antitubulin antibody as described by Tucker et al. (1979). Cells were stimulated with the different growth factors in DME + 0.25% plasma for 4 hr, then fluid-changed to DME + 0.25% plasma only. At various time points, 50-100 cells were counted in random fields on each of one or two coverslips: reproducability of counts was within 10%. Experiments were also conducted with the continuous presence of growth factor in DME + 0.25% plasma. Antitubulin antibody was prepared in rabbits immunized with vinblastine-induced tubulin crystals from sea urchin eggs and has been previously characterized (Fujiwara and Pollard, 1976: Sato, Ohnuki and Fujiwara, 1976). Cells were processed for indirect immunofluorescence as described by Tucker et al. (1979). Briefly, cells were fixed in 1: 10 dilutions of formaldehyde (Baker) in phosphate-buffered saline (PBS) for 30 min at room temperature, permeabilized with cold acetone for 7 min. air-dried and incubated with rabbit anti-tubulin antiserum (I:60 dilution in PBS) for 30 min at 37’C. The cells were viewed in a Zeiss epifluorescence microscope (Photomicroscope Ill) equipped with a rhodamine excitation barrier filter set (Zeiss), 50 W mercury arc lamp and 63X (N.A. 1.4. Planapo) objective lens. The fluorescent images were photographed using 35 mm Tri-X film (Kodak). Competence Each factor to be tested was added to quiescent BALE/c-3T3 cell cultures in DME + 0.25% plasma for 4 hr. then removed. The cell cultures were washed once with DME and then fluid-changed to DME + 5% plasma. Competence was determined as the percentage of the treated cells which could then enter DNA synthesis as measured by autoradiography at 36-46 hr after the addition of 5% plasma. Acknowledgments This work would not have and support provided by technical assistance of Albertini (Department of of microscope facilities. American Cancer Society three grants from the NIH. Inc.. Boston and CDS. Society.
been possible without the generous advice A. B. Pardee and the skilled and dedicated J. Hafner. We thank K. Fujiwara and D. Anatomy, Harvard Medical School) for use This work was aided by a grant from the (Massachusetts Division) to R.W.T.. and by R.W.T. is a fellow of the Medical Foundation is a Scholar of the American Leukemia
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received
August
23, 1979
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Boynton. A. L. and Whitfield. J. F. (1976). Different calcium requirements for proliferation of conditionally and unconditionally tumorigenie mouse cells. Proc. Nat. Acad. Sci. USA 73, 1651-I 654. Boynton, A. L., Whitfield, J. F.. Issacs, R. J. and Tremblay, R. (1977). The control of human WI -38 cell proliferation by extracellular calcium and its elimination by SV40 virus-induced proliferative transformation. J. Cell Physiol. 92, 241-248. Friedman, T. and Haas, M. (1970). Rapid concentration and purification of polyoma virus and SV40 with polyethylene glycol. Virology 42, 248-250. Fujiwara. K. and Pollard, T. D. (1976). Fluorescent antibody zation of myosin in the cytoplasma, cleavage furrow and spindle of human cells. J. Cell Biol. 77. 846-859. Hereford, L. M. and Hartwell, in the initiation of S. cerevisiae 461.
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Jimenez deAsua. L., O’Farrell. M. K., Clingan. D. and Rudland, P. S. (1977). Temporal sequence of hormonal interactions during the prereplicative phase of quiescent cultured 3T3 fibroblasts. Proc. Nat. Acad. Sci. USA 74, 3645-3649. Kohler, N. and Lipton, A. (1974). Platelets as a source growth-promoting activity. Exp. Cell Res. 87, 297-301. Larsen, plasma 306.
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Marcum. J. M., Dedman. J. R.. Brinkley, B. R. and Means, A. R. (1978). Control of microtubule assembly-dissassembly by calciumdependent regulator protein. Proc. Nat. Acad. Sci. USA 75, 37713775. Pledger, W. J.. Stiles, C. D., Antoniades, H. N. and Scher, C. D. (1977). Induction of DNA synthesis in Balb/c-3T3 cells by serum components: reevaluation of the commitment process. Proc. Nat. Acad. Sci. USA 74, 4481-4484. Pledger, W. J.. Stiles, C. D.. Antoniades. H. N. and Scher. C. D. (1976). An ordered sequence of events is required before Balb/c3T3 cells become committed to DNA synthesis. Proc. Nat. Acad. Sci. USA 75, 2639-2843. Rattner. J. B. and Phillips, S. G. (li73). Independence formation and DNA synthesis. J. Cell Biol. 57, 359-372.
of centriole
Robbins. E., Jentzsch, G. and Micali. A. (1968). The centriole in synchronized HeLa cells. J. Cell Biol. 36, 329-339.
cycle
Ross, R.. Glomset, J., Kariya, B. and Harker, L. (1974). A plateletdependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc. Nat. Acad. Sci. USA 77, 12071210. Sate, H.. Ohnuki. Y. and Fujiwara. K. (1976). lmmunofluorescent antitubutin staining of spindle microtubules and critique for the technique. In Cell Motility, R. Goldman, T. Pollard and J. Rosenbaum. eds. (New York: Cold Spring Harbor Laboratory), pp. 419-433. Scher, C. D., Pledger, W. J.. Martin, P.. Antoniades. H. N. and Stiles, C. D. (1978). Transforming viruses directly reduce the cellular growth requirement for a platelet derived growth factor. J. Cell Physiol. 97, 371-360.
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Snyder, J. A. and Liskay. R. M. (1978). Timing of centriole duplication in Chinese hamster cells that have no G, period. J. Cell Biol. 79, 13a. Stiles, C. D., Capone. G. T., Scher. C. D.. Antoniades. f-f. N., Van Wyk, J. J. and Pledger, W. J. (1979). Dual control of cell growth by somatomedins and platelet-derived growth factor. Proc. Nat. Acad. Sci. USA 76. 1279-l 283. Takemoto. K. K., Kirschstein. Ft. L. and Habel. K. (1966). Mutants of Simian virus 40 differing in plaque size, oncogenicity and heat sensitivity. J. Bacterial. 92, 990-994. Temin, f-l. N., Pierson, R. W. and Dulak. N. C. (1972). In Nutrition and Metabolism of Cells in Culture, 5, G. H. Ruthblat and V. J. Cristofalo, eds. (New York: Academic Press). pp. 50-61. Teo. T. S. and Wang, J. H. (1973). Mechanism of activation of a cyclic adenosine 3’:5’-monophosphate phosphodiesterase from bovine heart by calcium ions. J. Biol. Chem. 248, 5950-5955. Tucker, R. W.. Pardee. A. B. and Fujiwara, K. (1979). Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells. Cell 17, 527-535. Vogel, A., Raines. E., Kariya. B.. Rivest, M. J. and Ross, R. (1978). Coordinate control of 3T3 cell proliferation by platelet-derived growth factor and plasma components. Proc. Nat. Acad. Sci. USA 75, 281 O2814. Whitfield. J. F., Boynton, A. L.. MacManus. J. P.. Sikorska. M. and Tsang, B. K. (1979). The regulation of cell proliferation by calcium and cyclic AMP. Mol. Cell. Biochem.. in press. Yerna. M.. Dabrowska, R., Hartshorne. D. J. and Goldman, R. D. (1979). Calcium-sensitive regulation of actin-myosin interactions in baby hamster kidney (BHK-21) cells. Proc. Nat. Acad. Sci. USA 76, 184-188.
072,
December
1979.
Copyright
0 1979
by MIT
Centriole Deciliation Associated with the Early Response of 3T3 Cells to Growth Factors But Not to sv40 R. W. Tucker*, C. D. Schert and C. D. Stile+ *Laboratory of Tumor Biology and Division of Medical Oncology TDivision of Hematology-Oncology and Department of Pediatrics Children’s Hospital Medical Center *Laboratory of Tumor Biology, Group W and Department of Microbiology and Molecular Genetics Sidney Farber Cancer Institute and Harvard Medical School Boston, Massachusetts 02115
BALB/c-3T3 cells which are growth-arrested by high cell density or low serum have ciliated, unduplicated centrioles. Stimulation of these quiescent cells by serum is associated with a rapid (within l2 hr) deciliation of the centriole, followed by reciiiation within 6-10 hr. This transient deciliation of the centrioie is induced by the platelet-derived growth factor (PDGF) component of serum. The ceils treated with PDGF became competent to replicate their DNA; most PDGF treated ceils, however, did not progress from Go toward S phase unless they were incubated with the platelet-poor plasma component of serum. Addition of CaC12 or Fibroblast Growth Factor to the media mimicked PDGF by producing both centriole deciliation and competence to replicate DNA. in fact, over a range of concentrations of each of these factors, only doses which produced centrioie deciiiation were capable of producing competence for DNA synthesis. Plasma alone or factors such as Multiplication Stimulating Activity produced neither centriole deciiiabon nor competence; these agents were, however, required for the optimum progression of competent cells into DNA synthesis. in contrast, infection with SV40 induced host cell DNA synthesis without an initial transient deciliation of the centrioie. Thus while growth factors may have to induce centriole deciliation for 3T3 cells to synthesize DNA, abortive transformation by SV40 overrides this requirement.
As proliferating cells replicate their DNA and proceed through mitosis, closely correlated events must also occur with respect to centriole duplication. in studies on mammalian “centriole cycle” in 3T3 cells, Tucker, Pardee and Fujiwara (1979) demonstrated that centrioles become ciliated during G,, then deciliate and l To whom reprint Oncology Center, 21205.
requests should be addressed at: Johns Hopkins 600 North Wolfe Street, Baltimore, Maryland
duplicate concurrently with DNA synthesis. Quiescent 3T3 cells in low serum are growth-arrested in Go/G1 and contain a centriole pair forming a primary cilium; upon serum stimulation, these cells undergo an early (l-2 hr) transient deciliation, followed by another deciliation which is associated with centriole duplication and DNA synthesis. Serum contains polypeptide growth factors which control different events in the cell cycle (Temin, Pierson and Dulak, 1972; Jiminez de Asua et al., 1977; Pledger et al., 1977). The platelet-derived growth factor (PDGF) (Kohler and Lipton, 1974; Ross et al., 1974) component of serum controls an early event in the mitogenic response. Quiescent cultures of BALB/ c-3T3 cells exposed transiently to PDGF become “competent” to replicate their DNA and divide; however, PDGF-treated BALB/c-3T3 cells do not “progress” efficiently from Go/G1 into the S phase unless they are incubated in medium containing an optimal concentration (that is, 5%) of platelet-poor plasma. In medium containing a suboptimal concentration of platelet-poor plasma (50.25%) most PDGF-treated cells remain growth-arrested; however, such cells remain competent to replicate their DNA for many hours, and will do so, following a minimum 12 hr lag time, when the plasma content of the culture medium is raised to an optimal level of 5% (Pledger et al., 1977). Thus a cellular state of “competence” occurs as an early event in the mitogenic response and requires only transient exposure to PDGF. “Progression” is a later series of events leading to DNA synthesis (Pledger et al., 1978) and cell division (Vogel et al., 1978) which requires prolonged exposure to a second set of growth factors contained in plasma. At present there is no morphologic or biochemical marker of these two functionally distinct steps in mitogenesis. Using complementation assays, Stiles et al. (1979) have demonstrated that competence and progression are mediated by two distinct classes of growth factors. Fibroblast Growth Factor (FGF) and microprecipitates of calcium phosphate [Ca3(PO&] principally regulate competence, while progression activity is predominantly controlled by growth factors contained in plasma. An important regulatory component of plasma is the somatomedin family-a group of insulin-like polypeptides whose concentration in blood is largely controlled by pituitary growth hormone. Somatomedin8 in plasma function synergistically with competence factors such as PDGF, FGF, and Ca3(PO& to induce the optimal mitogenic response. Virally transformed and normal ceils appear to differ in their growth requirement for the competence and progression factors found in serum (Scher et al., 1978). SV40 infection of quiescent 3T3 cells allows them to traverse Go/G1 and synthesize DNA in the absence of both competence and progression factors (Stiles et al., 1979). Whether SV40 produces the
Cell 1066
same or a different sequence of events as that induced by exogenous serum growth factors is unknown. In this study, we demonstrate that different events in the centriole cycle are associated with the competence and the progression steps of the cell cycle. Furthermore, centriole events in 3T3 ceils stimulated with growth factors differ from those in 3T3 cells infected with SV40. Specifically, we find that the transient deciliation of the centriole in 3T3 cells is induced by competence factors, and may be a necessary prerequisite for DNA synthesis; this transient deciliation is bypassed during SV40-induced DNA synthesis. Results Plasma and Platelet-Derived Growth Factor (PDGF) In a previous study, Tucker et al. (1979) demonstrated that the addition of fresh, serum-containing medium to quiescent 3T3 cells induced an initial transient deciliation of the centriole, followed by a later deciliation associated with DNA synthesis. In this study we have used the serum components, PDGF and plasma, to examine the control of these centriole events. As Figure 1 (top) demonstrates, PDGF together with an optimal (5%) concentration of plasma induced a transient initial deciliation followed by a later deciliation which was temporally correlated with DNA synthesis. Cells made competent by a 4 hr pulse of PDGF and incubated in medium containing a suboptimal (0.25%) concentration of plasma also showed an initial transient deciliation (Figure 1, middle); however, very little secondary deciliation or DNA synthesis was observed. Cells which were exposed continuously to an optimal concentration of plasma (5%) without PDGF (Figure 1, bottom) demonstrated neither the initial transient deciliation nor the later secondary deciliation, and did not undergo DNA synthesis. This series of experiments suggests that the initial transient deciliation is induced by a factor derived from platelets. The later secondary deciliation is associated with cells entering the S phase. This second deciliation (like DNA synthesis) requires plasma components but cannot be induced by plasma alone. PDGF and plasma control sequential events in the cell cycle, so that delaying the addition of plasma to PDGF-treated cells also delayed DNA synthesis by a corresponding time (Pledger et al., 1977, 1978). Figure 2 demonstrates that under these conditions the second centriole deciliation was also correspondingly delayed. Thus the second deciliation is closely correlated with DNA synthesis. The experiments depicted in Figures 1 and 2 were conducted with partially purified preparations of PDGF. As Table 1 shows, pure PDGF (Antoniades, Scher and Stiles, 1979) also produced an initial deciliation associated with the induction of competence. Deciliation occurred in 95% of the cells measured at 2 and 4 hr after pure PDGF addition, and competence was simultaneously induced in at least 80% of the
n
PLhsy1
Tii bsl
Figure 1. PDGF and Plasma Control Cycle of BALB/c-3T3 Cells
Separate
Events
in the Centriole
(Top) Quiescent 3T3 cells received fresh medium containing both an optimal concentration of plasma (5%) and partially purified PDGF (350 pgg/ml) at time zero. (Middle) Quiescent 3T3 cells received fresh medium containing partially purified PDGF (350 pg/ml) and a suboptimal concentration of plasma (0.25%). (Bottom) Quiescent 3T3 cells received fresh medium containing 5% plasma at time zero. The percentage of cells with ciliated centrioles in two different experiments, (0) or (O), and the percentage of cells with labeled nuclei in the continuous presence of 1 &i/ml 3H-thymidine (III) is shown as a function of time thr). Shaded area connects the data for the different experiments measuring centriole ciliation.
cells. Thus the response of centriole ciliation to serum can be resolved into two parts controlled by different components of serum; an initial transient deciliation was produced by pure PDGF, and a final deciliation associated with DNA synthesis was controlled by plasma. This second deciliation of the centriole produced by plasma occurs only in PDGF-treated cells which have already undergone an earlier deciliation (Figure 1). Fibroblast Growth Factor (FGF) and Ca3(PO& PDGF is one of a class of mitogenic agents which induces 3T3 ceils to become competent for DNA replication; other agents (or treatments) which induce
Centriole 1067
Deciliation
and
Early
Response
to Growth
Factors
Table I. Relationship Competence
Factor
(rig/ml)
between
Centriole
Deciliation
% Cells with Daciliated Centrioles”
and
% Labeled Nuclei (Competence)b
Pure PDGF 0.7 1.4 2.8 4.5 9.0 18.0
12 25 34 66 90
13 10 18 27 43 75
100 1000
7 10
17 7
1 10 100
25 40 24
2 11 16
MSA
’ Maximum percentage of cells which have deciliated their centrioles during a 4 hr period following the addition of growth factors. b Percentage of cells in medium supplemented with 5% plasma which enter DNA synthesis 24-36 hr after a 4 hr exposure to a growth factor.
Tune (hr) Figure 2. Delaying the Addition of 5% Plasma to Competent BALB/ c-3T3 Cells Delays S Phase and the Second Centriole Deciliation by a Corresponding Time (Top) Quiescent cells were treated for 4 hr with 350 pg/ml of partially purified PDGF and then incubated in medium containing suboptimal (0.25%) plasma and 3H-thymidine (1 &i/ml) so that little DNA synthesis occurred. (Middle) Same as above except that cells were fluid-changed to DME + 5% plasma at 8 hr. (Bottom) Same as above except cells were fluid-changed to DME + 5% plasma at 20 hr. The percentage of cells with ciliated centrioles (0) and with autoradiographically labeled nuclei (0) was followed as a function of time.
competence include FGF, precipitates of Ca3(PO& and “wounding” of a confluent monolayer culture (Stiles et al., 1979); in light of this data, we examined the relationship between centriole deciliation and the mitogenic response to FGF, Ca3(PO& and “wounding.” We found that wounding of the cell monolayer (data not shown) or the addition of FGF or of CaC& [which precipitates in DME as (Ca)3(P04)2] to a quiescent monolayer of 3T3 cells for 4 hr produced competence and deciliation of the centriole. Figure 3 depicts the similarity of the time course of the transient deciliation obtained with FGF and Ca3(POJ2 with that produced by RDGF, as described in Figure 1. Thus three different agents which control the same event in the mitogenic response of quiescent 3T3 cells (competence) all produced a transient deciliation of the centriole. Competence and Centriole Deciliation To further study the association between the initial transient centriole deciliation and the state of competence, we studied both parameters over a wide
range of doses for five different factors. As Figure 4 summarizes, the doses of factors [PDGF, FGF, Ca3(P0&] which produced competence also produced centriole deciliation. On the other hand, MSA and EGF at physiological concentrations produced neither competence nor substantial centriole deciliation (Table 1). It is important to note that PDGF produced substantial deciliation at concentrations lower than that required to produce competent cells (Table 1 and Figure 4) whereas Ca3(PO& induced deciliation and competence at similar concentrations (Figure 4). Thus deciliation of the centriole may be necessary for the induction of competence, but is not always accompanied by competence. Continuous Exposure to Ca3(PO& or PDGF As shown in Figures 1 and 2, 4 hr pulses of PDGF, FGF or Ca3(PO& produced an initial deciliation of the centriole which appeared to be a transient phenomenon, with reciliation occurring within 8-12 hr. To be certain that the transient nature of the deciliation was an integral part of the response to the growth factors and not simply a result of the transient application of the stimulus, we studied centriole ciliation during the continuous exposure to PDGF or Ca3(PO& in amounts capable of producing competence. As shown in Figure 5 (top), quiescent cells treated with CaCI, at concentrations greater than 2.6 mM became rapidly deciliated. The initial deciliation was only transient despite continuous exposure to the Ca3(PO& precipitate which formed in the culture medium. Higher concentrations of CaCL increased the magnitude of cell population which became deciliated
Cell 1066
01 lo
100
o\o
POGF
40
CaCI,
w* 0
-O-
20 '10
Figure 4. Centriole Become Competent Deciliation
Is Produced
by FGF and C&(PO&
(Top) 50 rig/ml of FGF in DME plus suboptimal (0.25%) plasma were added at 4 hr. Cells were then placed in DME plus 0.25% plasma and 1 &i/ml 3H-thymidine. (Bottom) 5 mM CaC& was added to DME. producing a Ca3(PO& precipitate. After 4 hr. cells were fluid-changed to TIME plus 0.25% plasma and 3H-thymidine (1 &i/ml). The percentage of cells with ciliated centrioles (0) and the percentage of autoradiographically labeled nuclei 0 were monitored versus time. In parallel cultures of both FGF- and Ca,(PO&-treated cells, plasma was added to an optimum final concentration (5%); under these conditions 75% of the FGF-treated cells and 83% of the Ca3(PO& treated cells entered the S phase within 36 hr.
without changing the time course of the transient deciliation. There was no apparent loss of Ca3(PO& precipitate during the course of the experiment. In the suboptimal concentration of plasma (0.25%) contained in the medium, very little DNA synthesis (15% labeled nuclei) or secondary deciliation (15% deciliated cells) occurred. Similarly, the initial deciliation produced by PDGF was Only trNISient, despite COntinUOUS exposure to PDGF (Figure 5, bottom). The magnitude of the deciliation increased with PDGF dosage, but the duration was roughly constant. The fact that the time course of the deciliation was not affected by PDGF dosage argues against the possibility that the deciliation was transient because the PDGF was consumed during the course of the experiment; moreover, the time course of the deciliation was similar to that produced by a “pulse” of PDGF (Figure 1). As noted previously (Pledger et al., 19771, continuous exposure to higher concentrations of these impure PDGF preparations (68 pg/ml) produces some DNA synthesis (30% labeled nuclei) and secondary deciliation (30%) even in suboptimal (0.25%) plasma concentrations; the percentage of cells induced to enter S phase and undergo secondary deciliation is, however, always greater in
/
0
/
,-;”
A
3. Transient
r
IQ
20
Figure
;
534’
40
60
80
100
Deciliation of Centrioles
Deciliation Is Necessary for BALB/c-3T3 to Replicate Their DNA
Cells to
Quiescent cells were exposed for 4 hr to C&(P04)2 or growth factors at various concentrations. Deciliation of centrioles was measured in DME plus 0.25% plasma, and competence was measured in DME plus 5% plasma. The points on the graph depict: (0) Ca3(PO& at concentrations ranging from 2.0 mM to 9.0 mM; (A) FGF at concentrations ranging from 3.0-l 00 rig/ml; (0) partially purified PDGF at concentrations ranging from 14-272 f.rg/ml. The separate lines indicate separate experiments.
the presence of optimal (5%) plasma (Pledger et al., 1977). Thus the transient nature of centriole deciliation associated with competence is characteristic of the cellular response to the agents and is not dependent on the duration of exposure. Sequential Transient Exposures to Ca3(PO& or PDGF We next asked whether a cell which has undergone one transient deciliation can again transiently deciliate. Quiescent 3T3 cells were stimulated with two consecutive pulses of either Ca3(PO& or PDGF in 0.25% plasma. As Figure 6 demonstrates, both pulses of either agent produced a transient deciliation of the centriole. Thus the transient exposure to competence factors did not produce a refractory state for further transient deciliation of the centriole. sv40 Since 3T3 cells transformed with SV40 no longer require PDGF to grow (Scher et al., 1978) and since infection of quiescent 3T3 cells with SV40 induces host cell DNA synthesis without plasma or PDGF (Stiles et al., 1979), it was of interest to follow centriole ciliation in quiescent 3T3 cells infected with SV40. As Figure 7 demonstrates, infection (abortive transformation) with SV40 did not produce an initial deciliation of the centriole. However, the second and final deci-
Centriole 1069
Deciliation
t ( COCIP
::i 4
e
and
Early
i 12
16
Response
20
to Growth
24
28
Factors
32
; 34
“- WC , 40
Time Chrs)
Figure 6. Sequential Addition quential Transient Deciliations Figure 5. Continuous Exposure Transient Initial Deciliation
to Competence
Factors
Produces
a
(Top) CaClz at indicated mM concentration [2.2 (0). 2.6 (O), 4.3 (A). 9.3 (B)] was added to cells in DME + suboptimal (0.25%) plasma at time zero. Cells were not fluid-changed. 4.3 mM CaC12 induced a Ca3(PO& precipitate and only a transient deciliation of the centriole; addition of 9.3 mM CaCI, induced more deciliation, but cells did not survive long enough to observe later effects. (Bottom) Partially purified PDGF &g/ml) [14 (0). 7 (O), 27 (A), 66 (D] was added to the cells in DME + suboptimal (0.25%) plasma at time zero. Cells were not fluid-changed. The percentage of cells with ciliated centrioles was monitored versus time.
liation of the centriole still occurred with DNA synthesis. The lack of an initial centriole deciliation correlates with the lack of dependence on PDGF by SV40-transformed mouse fibroblast cell lines (Scher et al., 1978; Stiles et al., 1979). The production of both the second centriole deciliation and DNA synthesis in these infected cells strengthens the association between these two events. Discussion At mitosis, the duplicated chromosome must be partitioned equally to the two daughter cells. To accomplish this precise distribution of the genetic material in mammalian cells, the centriole and DNA synthesis cycles must be coordinated. One aspect of the centriole cycle which may be involved in this coordination is the formation of a primary cilium by the centriole. Tucker et al. (1979) showed that ciliation of the centriole undergoes marked changes in quiescent 3T3 cells stimulated to replicate by serum addition. In this
of Competence
Factors
Produces
Se-
(Top) Quiescent BALB/c-3T3 cells were exposed to DME containing 9.3 mM CaC12 and 5% plasma for 4 hr. once at time zero and again at 16 hr. In the intervening times, the cells were incubated in DME containing a suboptimal (0.25%) concentration of plasma. The percentage of cells with ciliated centrioles (0) is shown as a function of time. (Bottom) 1.9 mg/cc (33 U/mg) of partially purified PDGF was added to DME + 0.25% plasma in 4 hr pulses starting at time zero and time 16 hr. Cells were fluid-changed to DME + 0.25% plasma to other times.
, sv 40
5
Figure 7. SV40 Induces Transient Deciliation
TIME (HRS)
Replicative
DNA Synthesis
without
an Initial
SV40 in DME + 0.25% plasma was added to quiescent BALE/c-3T3 cells at time zero at a multiplicity of infection of 100. In two separate experiments, the percentage of cells with labeled nuclei (0. A) and with deciliated centrioles (0. A) is shown versus time.
report we show that competence, an early mitogenic event produced by the PDGF component of serum, is closely associated with a transient deciliation of the centriole. In fact, this transient deciliation of the centriole may be a morphologic marker of competence. The deciliation of the centriole also marks a later
Cell 1070
period of coordination between the centriole and DNA synthesis cycles. As shown in Figure 8, the two periods (competence and progression) are controlled by separate serum factors. PDGF controls competence in the DNA synthesis cycle and an early transient deciliation of the centriole; plasma controls progression from Go (quiescence) into DNA synthesis and the final deciliation and duplication of the centriole. Centriole duplication may be a key event in the control of initiation of DNA synthesis. There is already evidence that centriole duplication occurs before the initiation of DNA synthesis in mammalian cells (Fiobbins, Jenttsch and Micali, 1968; Rattner and Phillips, 1973; Snyder and Liskay, 1978). In yeast there is also accumulating evidence that the initiation of DNA synthesis is directly dependent upon prior centriole or spindle plaque duplication (Byers and Goetsch, 1974; Hereford and Hartwell, 1974; S. Dutcher and L. Hartwell, personal communication). Similar studies have not yet been performed in mammalian cells, but in this report we have provided more evidence for the coordination of centriole events with mammalian DNA synthesis cycle during Go or G,. Centriole deciliation does not itself cause competence, but must reflect events in the cell which lead to competence and DNA synthesis. Such events may include an increased intracellular calcium concentration at the centriole which could depolymerize the ciliary microtubules and cause deciliation or cilium resorption (Tucker et al., 1979). At the same time, calcium could bind to calmodulin, a calcium-dependent regulatory protein, which has been localized near the centriole in at least one portion of the cell cycle (Marcum et al., 1978). Calmodulin-Ca++ complex might then induce changes in cell shape via actinmyosin interaction (Yerna et al., 1979), lower cyclic AMP by activation of phosphodiesterase (Tea and Wang, 1973) and even shorten the duration of increased cytoplasmic calcium by increasing calcium transport out through the plasma membrane or back into membrane vesicles (Larsen and Vincenzi, 1979). Such a transient change in intracellular calcium is I-
GoI
&S -F
I-
compatible with the transient nature of centriole deciliation (Figure 5) and the sequential deciliation produced by sequential additions of growth factors (Figure 6). Competence might then be secondary to a change in cyclic AMP or phosphorylated proteins associated with the binding of calcium to calmodulin. Centriole deciliation would therefore be necessary, but not always sufficient, for competence, as we have reported in this paper. Using the transient deciliation of the centriole as a marker for early mitogenic events, we have found differences between the cell cycle in uninfected and SV40-infected 3T3 cells. The events leading to DNA synthesis in 3T3 cells stimulated with growth factors appear to require a transient centriole deciliation, whereas those in SV40-infected cells do not. Thus viral infection does not produce all of the usual cellular prereplicative changes, and may indeed induce an alternate pathway to DNA synthesis. If deciliation of the centriole does reflect a transient increase in intracellular calcium concentration, then these results may explain in part why neoplastic and virally transformed cells have decreased growth requirements for exogenous calcium (Boynton and Whitfield, 1976; Boynton et al., 1977). Thus one of the changes during neoplastic transformation may relate to changes in location and effect of intracellular calcium (Whitfield et al., 1979). Understanding more about how calcium and other factors influence the relationships between centriole and DNA synthesis cycles may help us to better define some of the growth regulatory differences between non-neoplastic and virally transformed or neoplastic cells. Experimental
Procedures
Cell Culture BALB/c-3T3 cells (clone A31) were maintained as sparse cultures in Dulbecco’s modified Eagle’s medium (DME) (Flow Laboratories) su& plemented with 10% calf serum (Flow Laboratories). To produce density-arrested cell cultures, l-2 x 10’ cells were grown in 1 cc of DME + 10% calf serum on circular glass coverslips (12 mm diameter: Rochester Scientific Co.) for 5-7 days. Cells were then fluid-changed to DME medium containing various growth factors and ‘H-thymidine. Duplicate coverslips were processed in parallel for antitubulin staining and for autoradiography.
Centrioie Events and DNA Repof BALB/c-3T3 Cells to Serum
Autoradiography The percentage of nuclei labeled in the continuous presence of 1 pCi/ml ‘H-thymidine (1 .O mCi/0.038 mg; New England Nuclear) was measured as a function of time afler serum or growth factor stimuiation of quiescent cultures. as previously described (Tucker et al.. 1979). Briefly, cells were fixed in 100% methanol for 20 min. airdried and overlaid with photographic emulsion (1:2 dilution of Kodak NB-T). The photographic emulsion was developed (Kodak D-l 9) and fixed (Kodak rapid fixer) after 3 days, and the cells were counterstained with 10% Giemsa in PBS for 20 min. The percentage of nuclei with more than 50 grain counts above the background was recorded at each time point.
Transient centrioie deciliation occurs early during POGF-dependent sequence of events (“competence”). and centriole duplication occurs near the start of DNA synthesis. Final deciiiation in some ceils may occur after initiation of DNA synthesis, during S or later.
Growth Factors Most of these experiments were performed with partially purified (100 fold) preparations of PDGF prepared by heat treatment (100°C) as previously described (Pledger et al., 1977). During the course of
PDGF Dependent
Plasma Dependent
Ksfent Centnole Decihotion Figure 8. The Relationship between lication in the Mitogenic Response Factors
Plosmo Independent
Fmol Centnole Deciliothon ond Dupllcotion
Centriole 1071
Deciliation
and
Early
Response
to Growth
Factors
these studies, however, human PDGF was purified to homogeneity; electrophoretically homogeneous PDGF prepared by the method of Antoniades et al. (1979) was used to substantiate our main conclusions. The preparation of defibrinogenated platelet-poor plasma from human blood has been described by Pledger et al. (1977). Only preparations that maintained cell viability without stimulating growth of BALB/c-3T3 cells were used. Fibroblast Growth Factor (FGF). Multiplication Stimulating Activity(MSA)and Epidermal Growth Factor (EGF) were all obtained from Collaborative Research (Waltham. Massachusetts) and stored at 0-4’C. SW0 The preparation of SV40 has been previously described (Scher et al., 1979). Briefly, a clonal isolate of the small plaque variant (Takemoto, Kirchstein and Habel. 1966) of SV40 was grown on confluent Vero monkey cells in medium containing 5% agamma calf serum (North American Biological). Approximately 2 weeks after infection, when a cytopathic effect was noted, the cells and medium were frozen and thawed 3 times and the extracts were clarified by low speed centrifugation. SV40 was precipitated by the addition of polyethylene glycol (molecular weight 6000-7000; Fisher) (Friedman and Haas. 1970) and further purified by a centrifugation through sucrose (15%) onto a layer of 60% sucrose. The virus was titered by a plaque assay on CV-1 cells. Aliquots of virus were cryopreserved as l-2 x 10’ pfu/ ml and thawed just before use. Centriole Ciliation The percentage of cells with ciliated centrioles was determined by indirect immunofluorescence with antitubulin antibody as described by Tucker et al. (1979). Cells were stimulated with the different growth factors in DME + 0.25% plasma for 4 hr, then fluid-changed to DME + 0.25% plasma only. At various time points, 50-100 cells were counted in random fields on each of one or two coverslips: reproducability of counts was within 10%. Experiments were also conducted with the continuous presence of growth factor in DME + 0.25% plasma. Antitubulin antibody was prepared in rabbits immunized with vinblastine-induced tubulin crystals from sea urchin eggs and has been previously characterized (Fujiwara and Pollard, 1976: Sato, Ohnuki and Fujiwara, 1976). Cells were processed for indirect immunofluorescence as described by Tucker et al. (1979). Briefly, cells were fixed in 1: 10 dilutions of formaldehyde (Baker) in phosphate-buffered saline (PBS) for 30 min at room temperature, permeabilized with cold acetone for 7 min. air-dried and incubated with rabbit anti-tubulin antiserum (I:60 dilution in PBS) for 30 min at 37’C. The cells were viewed in a Zeiss epifluorescence microscope (Photomicroscope Ill) equipped with a rhodamine excitation barrier filter set (Zeiss), 50 W mercury arc lamp and 63X (N.A. 1.4. Planapo) objective lens. The fluorescent images were photographed using 35 mm Tri-X film (Kodak). Competence Each factor to be tested was added to quiescent BALE/c-3T3 cell cultures in DME + 0.25% plasma for 4 hr. then removed. The cell cultures were washed once with DME and then fluid-changed to DME + 5% plasma. Competence was determined as the percentage of the treated cells which could then enter DNA synthesis as measured by autoradiography at 36-46 hr after the addition of 5% plasma. Acknowledgments This work would not have and support provided by technical assistance of Albertini (Department of of microscope facilities. American Cancer Society three grants from the NIH. Inc.. Boston and CDS. Society.
been possible without the generous advice A. B. Pardee and the skilled and dedicated J. Hafner. We thank K. Fujiwara and D. Anatomy, Harvard Medical School) for use This work was aided by a grant from the (Massachusetts Division) to R.W.T.. and by R.W.T. is a fellow of the Medical Foundation is a Scholar of the American Leukemia
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received
August
23, 1979
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