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c-3T3 cells
Cellular Signalling Vol. 4, No, 6, pp. 675-686, 1992. Printed in Great Britain.
DISSOCIATION SUBSEQUENT
0898-6568/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd
BETWEEN EARLY LOSS OF ACTIN CELL DEATH IN SERUM-DEPRIVED BALB/C-3T3
FIBRES AND QUIESCENT
CELLS
IGOR TAMM, TOYOKO KIKUCHI, JAMES KRUEGER and JAMES S. MURPHY Cell Physiology and Virology Laboratory, The Rockefeller University, New York, NY 10021, U.S.A. (Received 25 March 1992; and accepted 22 June 1992)
A~tract--Serum withdrawal from either growing or quiescent Balb/c-3T3 murine fibroblasts causes a loss of F-actin fibres and focal adhesions within 30 min. Cells that are growing survive serum deprivation, whereas the great majority of density-arrested quiescent cells die during a period of up to 5 h from serum withdrawal. During this time an approximately constant fraction of the quiescent cell population dies per unit time. The population half-life is 60-70 min during this time. Addition of an appropriate cell growth factor or second messenger agonist at the time of serum withdrawal or within 2 h after serum withdrawal protects a similar fraction of viable cells. These findings suggest a model according to which withdrawal of serum (i.e. growth factors) initiates the death process in cells of the population with kinetics that approximate first-order kinetics. We postulate that appropriate growth factors or second messenger agonists block the initiating event that starts the cell death process. Key words: Actin fibres, vinculin, focal contacts, PDGF, bFGF, Br-cAMP, TPA.
second messenger agonists [1,2,4]. The evidence indicates that this can be achieved via a multiplicity of pathways and that different growth factors use distinct pathways which presumably converge on a mechanism critical for the maintenance of cellular integrity. Platelet-derived growth factor (PDGF) or 8bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP) can each activate one or more protein synthesis-independent pathways whereas basic fibroblast growth factor ( b F G F ) or 12-0tetradecanoyl phorbol-13-acetate (TPA) each activates an obligatorily protein synthesisdependent pathway as demonstrated in experiments with cycloheximide [2] and anisomycin (Tamm and Kikuchi, unpublished observations). Kinetic analysis has shown that the requirement for protein synthesis in the survival-mediating action of b F G F is similar during the first, second and third hours of incubation with b F G F [2]. This is consistent with the approximately first-order cell death kinetics, which indicate that during the first several hours o f serum deprivation, a constant fraction of the population enters the death
INTRODUCTION DENSXTY-rNmmTED quiescent Balb/c-3T3 murine fibroblasts are critically dependent for their survival on the presence o f growth factors (reviewed [1]). In their absence, most of the cells die within 5 h following a change to fresh serum-free Dulbecco's medium. The decrease in viable cells incubated in serum-free medium follows approximately first-order kinetics until only a residual fraction remains in steady state [2]. The death process in the individual cells is short (usually ~ 0.5 h) and is characterized by contraction and rounding of cells with vesiculation at the plasma membrane and loss of cytoplasmic material as vesicles are shed [2,3]. The death process can be prevented in the majority of cells by the addition to the serum-free medium of one or more growth factors or Abbreviations: bFGF--basic fibroblast growth factor; Br-cAMP--8-bromoadenosine3', Y-cyclicrnonophosphate; EGF---epidermal growth factor; IGF--insulin-like growth factor; PBS----phosphate-buffered saline; PDGF--platelet-derived growth factor; TPA--12-Otetradecanoyl phorbol-I3-acetate. 675
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process per unit time. I f a protective growth factor or second messenger agonist is removed after 5 h incubation of quiescent cells in serumfree medium, the protective effect is lost [1]. Thus, as with serum, continued presence of factors is necessary to maintain cellular integrity. That growth factors can function as cell survival factors has become apparent in several systems [5-9]. Murine 3T3 fibroblasts represent a widely used system for studies of signal transduction, regulation of gene expression, and the cell cycle. For serum-deprived quiescent cells to survive it is not necessary that all the processes required for activation of the cell cycle and progression to S-phase take place [1-4]. Thus, P D G F (competence factor) used alone or insulin-like growth factor (IGF) + epidermal growth factor (EGF) (progression factors) without P D G F have marked survival factor activity but are not sufficient to enable cells to enter S-phase. In the present studies we show that Balb/c-3T3 cells deprived of serum rapidly lose actin fibres and focal adhesions (adhesion plaques). This occurs over a period of 30 min. These striking changes precede cell death, but are not obligatorily linked to deaths which occur over a period of several hours with kinetics that approximate first-order kinetics. Evidence is presented that survival factors added at 0, 1 or 2 h after serum removal produce a protective effect of similar magnitude on the remaining living cells. We propose that activation of one or more appropriate signal transduction pathway(s) blocks the initiating event that starts the cell death process.
MATERIALS AND METHODS Materials
PDGF and EGF were obtained from Collaborative Research, Inc. (Bedford, MA, U.S.A.), bFGF from Biosource International (Westlake Village, CA, U.S.A.), and IGF-I from Amgen (Thousand Oaks, CA, U.S.A.). TPA, Br-cAMP, cholera toxin and sodium orthovanadate were purchased from Sigma Chemical Co. (St Louis, MO,
U.S.A.). Pertussis toxin was purchased from Calbiochem (San Diego, CA, U.S.A.). Affinity-isolated goat F(ab')z anti(mouse IgG) rhodamine B conjugate was obtained from Tago, Inc. (Burlingame, CA, U.S.A.), fluorescein phalIoidin from Molecular Probes (Eugene, OR, U.S.A.), and monoelonal anti-phosphotyrosine from Upstate Biological (Lake Placid, NY, U.S.A.). Fibronectin was obtained from the New York Blood Center and okadaic acid from Dr Stuart Decker.
Cell culture and survival assays
Baib/c-3T3 cells were obtained from Dr W. J. Pledger, Vanderbilt University, TN, U.S.A., and stock cultures passaged as previously described [4]. Cultures for experiments were seeded at a density of 3500 cells/cm 2 in 96-well or 6-well plates or in 25 cm' flasks. The growth medium was Dulbecco's modified Eagle's medium and supplemented with 10% bovine serum (Hyclone, UT, U.S.A.). After planting, cultures were incubated for 3 days at 37°C. The serum-containing medium was changed and incubation continued for four more days at which point they were used. Cultures were washed once with serum-free medium prior to the addition of survival factors. After varying periods, cell survival was assayed by neutral red uptake as previously described [1,4,10] or by Coulter count of trypsinized samples.
Time-lapse cinemicrography
Time-lapse cinemicrographic observations were made on cells grown to quiescence in 25-GaTI 2 flasks. The monolayers were washed once with serum-free medium before the addition of serum-free medium. Cultures were photographed every 30 s using a x 6.3 planar phase-contrast objective. Initial cell counts were performed on the image of the negative projected on a ground-glass plate. Rapid rounding in association with detachment of a cell was considered as the death end-point and the time was noted.
lmmunofluorescence Cells were grown and treated as described in the figure legends and then were fixed with 4 % formalin in phosphate-buffered saline (PBS) for 20 rain, treated with 0.50 Triton in PBS for ~ 5 min, and washed twice with PBS. They were stained for F-actin with FITC-phalloidin for 30 min at 4°C. For double staining, cells were incubated with the primary antibody, anti-phosphotyrosine, overnight at 4°C before the addition of the secondary antibody,
Cell death processinitiation rhodamine-conjugated anti-mouse IgG, and FITC-phalloidin for 2 h. After washing with PBS, the coverslips were mounted in PBS--glycerol, 1:1. The preparations were viewed and photographed using a x 63 objective with epifluorescenceor reflection interference optics.
RESULTS
Early morphological change following serum deprivation of quiescent Balb/c-3 T3 cells Time-lapse cinemicrographic observation of quiescent cultures (data not shown) reveals that most cells respond to serum deprivation within minutes by contraction of the cytoplasm, separation from neighbouring cells, and loss of the extended overall shape characteristic of fibroblasts. There does not appear to be any time relationship between these initial changes and the previously described death process characterized by vesiculation, cell shrinkage, and detachment [2,3].
Serum deprivation causes loss of actin fibres Withdrawal of serum from quiescent densityarrested Balb/c-3T3 cells leads to a rapid loss of actin fibres with a simultaneous marked increase in the punctate distribution of F-actin in the cytoplasm (Fig. 1). Some cells display a heavy concentration of punctate F-actin staining in the perinuclear region. F-actin in the cell cortex is increased in serum-deprived cells. These changes are complete by 20-30 min, by which time most cells have lost actin fibres. The F-actin fibres in apposed control cells sometimes appear to continue from one cell into another. This appearance is probably due to the fact that in each of the apposed cells, actin fibres are anchored in focal adhesions which may be aligned to fibronectin in the extracellular matrix. The concentration of F-actin in serum-deprived cells is heaviest at those cell corners or extensions that probably had a heavy concentration of focal adhesions before serum deprivation. Clusters of fine brightly stained spikes can be seen on the sides of some serum-deprived cells. CELLS 4:6-F
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Serum deprivation causes loss of focal adhesions (adhesion plaques) Actin fibres are anchored in adhesion plaques which form focal adhesions with the underlying substratum. Vinculin mediates binding of actin filaments to membranes. Because vinculin contains phosphotyrosine, immunocytochemical staining for phosphotyrosine provides a sensitive means for the detection of focal adhesions. Reflection interference microscopy can then be used to establish the presence of focal adhesions, close contacts, and areas of cell-substratum separation [11]. The effects of 30 min serum deprivation on actin fibres and focal adhesions in densityarrested quiescent Balb/c-3T3 cells are illustrated in Fig. 2. The 0-h control cells contain numerous well-formed actin fibres that in many instances span the cell (Fig. 2A). Along the cell periphery and also elsewhere, streaks of rhodamine anti-phosphotyrosine-stained structures are present in precisely the same orientation as actin fibres (Fig. 2C). In many instances location of the phosphotyrosine-containing structure coincides with the termination of an actin fibre. At such sites, reflection interference microscopy reveals the presence of focal adhesions (dark grey areas in Fig. 2E). After 30 min serum deprivation the actin fibres (Fig. 2B) have disappeared as have the associated phosphotyrosine-stained structures (Fig. 2D). Phosphotyrosine staining is limited to segments of the cell border and a diffusely stained mass in the centre of the cell. Reflection interference microscopy reveals a remarkable change in cell adherence to the substratum (Fig. 2F). No streak-shaped focal adhesions can be seen but there are extensive areas of close contacts. These findings emphasize the role that actin fibres and focal adhesions play in maintaining the fibroblast in an extended form in which it adheres strongly to the substratum and moves little. Loss of these structures, as through serum deprivation, then results in shape-change and an increase in motility. Growing Balb/c-3T3 cells undergo a similar set of changes when serum is withdrawn (Fig.
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3). Again, it can be seen that in 0-h control cells actin fibres (Fig. 3A) terminate in streak-shaped structures that can be stained with phosphotyrosine (Fig. 3C) and recognized as focal adhesions by reflection interference microscopy (Fig. 3E). The growing cells, too, lose actin fibres after serum withdrawal and F-actin assumes a punctate distribution with a heavy concentration in the perinuclear region or it relocates to the cell margin (Fig. 3B). In the serum-deprived cells, phosphotyrosine staining is heavy and diffuse over and also beyond the nucleus and in cell corners (Fig. 3D). Reflection interference microscopy (Fig. 3F) indicates the presence of close contacts in some of the cell corners. To summarize, serum-deprived cells, either quiescent or growing, rapidly lose focal adhesions and actin fibres and separate from each other. This process is complete within only 30 min of serum withdrawal. In contrast, the occurrence of cell death in the population of quiescent cells is distributed over a period of 5 h ([2] and present results); growing cells survive serum withdrawal.
Approaches to the mechanism of actin fibres dissolution in serum-deprived cells We determined that supplementation of serum-free medium with PDGF (5ng/ml), bFGF (20 ng/ml), IGF-I (40 ng/ml), Br-cAMP (1.45 mM), or TPA (100nM) did not prevent the disappearance of actin fibres, although, as previously reported [1,2,4], each of these factors has a marked cell survival-enhancing effect on quiescent Balb/c-3T3 cells that have been deprived of serum in the medium. Indeed, PDGF caused cell contraction over and above that caused by serum withdrawal, as did bFGF and Br-cAMP. TPA had a lesser contractionaccentuating effect and IGF-l-treated cells did not display such an effect. Actin fibres were rare in all cases. In these experiments cells were incubated in serum-free medium for 30 min and examined by phase-contrast and fluorescence microscopy after staining for F-actin. It has been shown previously that PDGF
itself causes disappearance of actin fibres in quiescent Balb/c-3T3 cells with redistribution of vinculin from focal adhesions to the cytoplasm around the nucleus where it displays a punctate distribution [12,13]. These effects of PDGF are dose dependent in the range 5-50ng/ml; at 3ng/ml it has little or no effect [12,13]. We therefore used PDGF at 3 ng/ml and found that at this relatively low concentration it partially protected actin fibres against dissolution in serum-free medium. This effect was somewhat enhanced by the simultaneous presence of EGF at 10ng/ml. As serum contains PDGF and EGF at low concentrations, it is likely that their presence is at least in part responsible for the ability of serum to maintain an actin cytoskeleton in cells. In these experiments, as previously, quiescent cells were incubated with various agents for 30 min and then examined. To investigate the possibility that the rapid changes in cytoskeletal organization caused by serum withdrawal might be mediated via the activation of phosphatases, two phosphatase inhibitors were used. Neither okadaic acid (40#M) nor sodium orthovanadate (10/~M) prevented actin fibre disassembly in quiescent Balb/c-3T3 cells deprived of serum for 20 min. Fibronectin is an important extracellular matrix component which is present in serum, and functions in cell adherence to the substratum. We supplemented serum-free medium with fibronectin (5 #g/ml); however, this had no apparent effect on the morphological change quiescent Balb/c-3T3 cells underwent in 30 min in serum-free medium or on the disappearance of actin fibres.
Distribution of the time of occurrence of cell death in cell population In the present study we determined the death curve by time-lapse cinemicrography, photographing cultures of serum-deprived cells every 30 s for 3 h. By this high resolution technique, an initial short lag was evident after which the occurrence of cell deaths followed approximately first-order kinetics (Fig. 4). The data are not inconsistent with the Eyring-Stover formal-
~'IG. 1. Early cytoskeletal response of quiescent Balb/c-3T3 cells to serum deprivation. Cells were seeded on glass coverslips, 8000 cells/cm2, in )ulbecco's medium with 10% bovine serum. The cultures were refed 4 days later and used 3 days after the medium change. The cells were washed ,nce with warm Dulbecco's medium and incubated at 37°C for varying periods, after which the cultures were stained for F-actin. (A) 0 rain; (B) 10 nin; (C) 20 rain; (D) 30 rain. Arrows in B mark punctate actin, x 443.
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FIG. 2. Localization of F-actin, phosphotyrosine, and focal adhesions by cytoch~aistry and reflection interference microscopy in quiescent Balb/c-3T3 cells after serum removal. Glass coverslips were seeded at 3500 cells/cm 2, and quiescent cells were exposed to serum-free Dulbccco's medium for 0 rain (A,C,E) or 30 rain (B,D,F) following which they were double stained for F-actin (A,B) and phosphotyrosine (C,D) and visualized by fluorescence microscopy. The same fields were also visualized by reflection interference microscopy (E,F). In A,C and E an arrow marks a focal adhesion at the end of actin stress fibres. In B,D and F the arrowhead marks same position in the cell; open arrow in F (ce) marks a diffuse, grey-toned close contact, x 1216.
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FIc. 3. Localization of F-actin, phosphotyrosine and focal adhesions by cytochemistry and reflection interference microscopy in serum-deprived growing Balb/c-3T3 cells. Glass coverslips were seeded at 3500 cells/cm2 and 3 days later were exposed to serum-free Dulbecco's medium for 0 min (A,C,E) or 30 min (B,D,F) following which they were double stained for F-actin (A,B) and phosphotyrosine (C,D) and visualized by fluorescence microscopy. The same fields were also observed using reflection interference microscopy (E,F). In A,C and E arrows mark focal adhesions at the end of actin stress fibres. In B,D and F the arrowhead marks the same position in the cell. x 1216.
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FIG. 4. Cell death curve of quiescent Balb/c-3T3 cells in serum-free medium. Flasks 25 cm2 were seeded at 3500 cells/era: and allowed to grow to quiescence with one medium change, as described in Materials and Methods. Monolayers were washed once before addition of serum-free medium. Time-lapse photomicrographs were taken at 30-s intervals, and analysed for cell deaths/time interval. Two fields were analysed in one film (68 and 83 cells/field). O, Number of cells based on starting number less the number of deaths; II, number of cells based on the Eyring-Stover equation [14].
ism [14]. However, a more extensive study would be required to establish whether the dynamics of survival described by this formalism apply to serum-deprived quiescent Balb/c-3T3 cells. The Eyring-Stover formalism is based on the assumption that two reactions are operational, one leading to damage and death, and the other to repair of damage and survival. During the close to linear part of the curve, the time required for a 50% decrease in the calculated cell number was 70 min. This is consistent with the results based on neutral red assays, which gave a mean value of 60 min for the population half-life [2]. As has been pointed out before, the specific death rate varies from experiment to experiment [1,4].
The extent of protection of viable cells by survival factors is independent of time of incubation in serum-free medium During the initial period of 5 h after serum removal, an approximately constant fraction of the population of density-inhibited quiescent
z
0.1
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Hours before survival factor addition FIG. 5. Relationship between pre-incubation time in serum-free medium and the survival-enhancing activity of PDGF, bFGF, Br-cAMP, and TPA. Ninety-six-well plates were seeded at 3500 cells/cm2 in Dulbecco's medium with 10% bovine serum (BS) and allowed to grow to quiescence with one medium change, as described in Materials and Methods. After a wash with warm serum-free Dulbecco's medium, groups of cultures were incubated at 37°C for 0, 1, or 2 h in serum-free medium after which PDGF (5ng/ml), bFGF (20ng/ml), Br-cAMP (l.45mM), TPA (100nM), or serum (10%) was added. Survival was assayed by neutral red uptake after a total of 3 h of incubation (i.e. 0 + 3; 1 + 2; or 2 + l h ) . Mean results of four experiments with PDGF and Br-cAMP and of five experiments with bFGF and TPA. Neutral red uptake in controls incubated in serum-free medium for 3 h was 0.1615 O.D. unit or 17% of that in cells incubated with 10% bovine serum for this period.
cells enters the death process per unit time. We propose a model according to which entry into the death process is preceded by as yet unidentified initiating events and that certain growth factors and second messenger agonists block these initiating events. The model predicts that during the first several hours after serum deprivation the extent of protection of viable cells by survival factors should be independent of time of incubation in serum-free medium. To test the model we compared the protective effect of survival factors added immediately after serum
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removal with the extent of protection of remaining viable cells when a factor is added 1 or 2 h after serum withdrawal. The total period of incubation from the time of serum removal to the beginning of the viability assay was constant, i.e. 3 h, in all cases. The number of viable cells in density-inhibited quiescent cultures is stable after replacement of medium with fresh serum-containing medium [4] or single survival factors (unpublished observations). The results in Fig. 5 support the model. As expected, the number of viable cells declines when re-addition of serum to serum-deprived cells is delayed. Delayed addition of PDGF, bFGF, Br-cAMP, or TPA yielded a family of curves approximately paralleling the serum readdition curve (cf. Fig. 5). The survivalenhancing effects of the four factors used, expressed relative to the fraction of cells still surviving after 0-, 1-, or 2-h incubation of quiescent cells in serum-free medium, respectively, were as follows: PDGF: 69, 58, and 60; bFGF: 60, 60, and 62; Br-cAMP: 51, 65, and 50; TPA: 62, 53, and 59. The mean standard error of the determinations was 4 (range 3-8). These results show that there is no systematic change in the extent of protection when the addition of P D G F (5 ng/ml), b F G F (20 ng/ml),
Br-cAMP (1.45mM), or TPA (100nM) is delayed after removal of serum from the quiescent cells.
G-proteins and cell survival Pertussis toxin (100 ng/ml), which inhibits the action of several G-proteins (reviewed in [1517]), did not affect to a measurable extent the survival of quiescent cells in serum-free medium or in medium containing serum, PDGF, bFGF, Br-cAMP, or TPA (Table 1). Table 2 documents the expected result of enhancement by cholera toxin (5/zg/ml) of survival of quiescent cells in serum-free medium, as it is well known that cholera toxin activates a G-protein that stimulates adenylate cyclase (reviewed in [16,17]). Cholera toxin did not appear to have any major effects on the enhancement of cell survival by serum, PDGF, bFGF, Br-cAMP, or TPA. DISCUSSION Fibroblasts have well-developed actin fibres which play a role in the attachment of the cells to the substratum and in maintaining their extended form. When serum is withdrawn from the medium of quiescent density-inhibited murine Balb/c-3T3 cells, most cells in the
TABLE 1. EFFECTS OF PERTUSSIS TOXIN (PT) ON GROWTH FACTORAND SECOND MESSENGER AGONIST-STIMULATED SHORT-TERM (3-h) CELL SURVIVAL
Cell survival (% of 10% BS control*) Agents and concentrations
No PT
BS (10%) None PDGF (5 ng/ml) bFGF (20 ng/ml) Br-cAMP (1.45 mM) TPA (100 nM)
100 31 74 75 65 76
Pertussis toxin (100 ng/ml) % no PTt 90 27 73 64 66 64
90+ 15 89 4- 2 984- 16 86_+ 17 101 _+12 83__.21
* Neutral red uptake in 10% bovine serum (BS) controls was 0.736 O.D. unit. Pertussis toxin was added for 3 h prior to and also during growth factor treatment. Mean results of three experiments. "~__S.D.
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Cell death process initiation TABLE 2. EFFECTSOF CHOLERATOXIN (CT) ON GROWTHFACTORAND SECOND MESSENGERAGONIST-STIMULATEDSHORT-TERM (3-h) CELL SURVIVAL Cell survival (% of 10% BS control*)
Agents and concentrations
No CT
BS (10%) None P D G F (5 ng/ml) b F G F (20 ng/ml) Br-cAMP (1.45 mM) TPA (100 nM)
100 30 74 72 59 69
Choleratoxin (5 #g/ml) % no CTt 88 62 77 62 44 85
88_ 24 210_ 29 103-t- 2 87_ 9 75+ 1 124+ 10
* Neutral red uptake in 10% bovine serum (BS) controls was 0.682 O.D. unit. Cholera toxin was added for 3 h prior to and also during growth factor treatment. Mean results of two experiments. t _S.D.
culture undergo a rapid morphological change characterized by loss of actin fibres and focal contacts. The cells separate away from each other and show increased motility. These changes are apparent within 5-10 min of serum withdrawal and are essentially complete by 2030 min. In contrast, cell deaths in serumdeprived cultures are distributed over ~ 5 h and their occurrence follows approximately first-order kinetics ([2] and present results). The cell death process is characterized by vesiculation accompanied by shrinkage of the cell and the nucleus and it culminates in the disintegration of the cell ([2] and present results). Several observations speak against a direct relationship between the early morphologicalfunctional changes and the subsequent death of cells. First, non-confluent growing cultures show the early changes when serum is withdrawn, but the cells survive. Second, addition of appropriate growth factors or second messenger agonists to quiescent cells may not prevent the early changes and can indeed accentuate them and yet it protects cells against death. However, the possibility cannot be excluded that the skeletal rearrangement is a precondition of the onset of the cell death process.
Further studies are needed to determine the biochemical mechanism whereby serum deprivation rapidly leads to loss of actin fibres and focal adhesions. Although supplementation of serum-free medium with fibronectin did not prevent disruption of the actin cytoskeleton, it is possible that removal of serum impairs integrin adhesivity. Attempts to prevent actin fibre disassembly by the addition of the phosphatase inhibitors okadaic acid and sodium orthovanadate to serum-free medium were unsuccessful. The possibility that activation of a protease may play a part in this early response also merits exploration. Our evidence indicates that the onset of the death process can be prevented by growth factors or second messenger agonists independent of the time that has lapsed since serum deprivation. This was demonstrated by adding a growth factor or second messenger agonist at different times after serum withdrawal from the medium (cf. Fig. 5). We postulate that in cells of the quiescent population a death process initiating event takes place with approximately first-order kinetics following serum withdrawal and that the presence of an appropriate growth factor or second messenger agonist can block this event by activating one or more of several
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signal transduction pathways which converge on an as yet unidentified mechanism that is critical for the maintenance of cellular integrity. Attempts with pertussis toxin to obtain evidence for a role for G-proteins in the survival of cells mediated by P D G F , b F G F , Br-cAMP, or TPA have so far given negative results. It has been shown previously that treatment of Swiss 3T3 fibroblasts with pertussis toxin (5 ng/ml) has essentially no effect on the mitogenic responses of the cells to either P D G F or phorbol 12,13-dibutyrate although it completely blocks bombesin-stimulated D N A synthesis [18]. Thus, our results contribute further evidence that P D G F - or phorbol esteractivated pathways in 3T3 cells do not involve a pertussis-sensitive guanine nucleotide-binding protein. The complexity of the signal transduction mechanisms in growth factor action is illustrated by the findings that pertussis toxin blocks b F G F - or TPA-induced D N A synthesis in quiescent Balb/c-3T3 cells but does not inhibit PDGF-induced formation of inositol phosphates [19]. The fact that, following serum deprivation, the cell deaths in the population occur with approximately first-order kinetics presents an experimental challenge as within a given interval, such as 15 min, only a small fraction ( ,,, 16%) of the cells alive at the beginning of the interval would be undergoing the death process and they would presumably be doing so asynchronously. An approach to further mechanistic analysis would be to establish by time-lapse cinemicrography distinct morphological stages in the death process in individual cells and then conduct biochemical-microscopic analyses on single cells in different stages of the process. It is evident that cell growth factors can serve not only as mediators of cell cycle entry and progression, but also as mediators of cell survival. Their action in regulating cell cycling in most instances clearly involves one or more pathways by which a signal from membrane receptors occupied by the growth factor is transduced to the nucleus. Recent studies have shown that activation of signal transduction
pathways in a cell at risk of dying can prevent death [1,2]. Much remains to be learned about the physiological role o f growth factors as survival factors. Acknowledgements---We thank DR EUGENIA WANG for reading the manuscript and MRS Sopm~ NADELL for assistance in processing the manuscript. This work was supported by National Institutes of Health Research grant CA-18608.
REFERENCES I. Tamm I. and Kikuchi T. (1991) J. ceil. Physiol. 148, 85-95. 2. Tamm I., Kikuchi T. and Zychlinsky A. (1991) Proc. natn. Acad. Sci. U.S.A. 88, 3372-3376. 3. Scher C.D., Young S. A. and Locatell K. L. (1982) J. cell. PhysioL 113, 211-218. 4. Tamm I. and Kikuchi T. (1990) J. cell. PhysioL 143, 494-500. 5. Gillis S., Baker P. E., Ruscetti F. W. and Smith K. A. (1978) 3". exp. Med. 148, 1093-1098. 6. Martin D. P., Sehmidt R. E., DiStefano P. S., Lowry O. H., Carter J. G. and Johnson E. M. Jr (1988) J. cell. BioL 106, 829-844. 7. Nieto M. A., Gonzhlez A., L6pez-Rivas A., Diaz-Espada F. and Gambrn F. (1990) J. lmmun. 145, 1364-1368. 8. McConkey D. J., Hartzell P., Chow S. C., Orrenius S. and Jondal M. (1990) J. biol. Chem. 265, 3009-3011. 9. Williams G. T., Smith C. A., Spooncer E., Dexter T. M. and Taylor D. R. (1990) Nature 343, 76-79. 10. Borenfreund E. and Puerner J. A. (1985) J. Tiss. Cult. Met. 9, 7-9. 11. Izzard C. S. and Lochner L. R. (1976) J. CellSci. 21, 129-159. 12. Bockus B. J. and Stiles C. D. (1984) Expl Cell Res. 153, 186-197. 13. Herman B. and Pledger W. J. (1985) J. Cell Biol. 100, 1031-1040. 14. Eyring H. and Stover B. J. (1972) Proc. natn. Acad. Sci. U.S.A. 69, 3512-3515. 15. Ui Michio (1986) In Phosphoinositides and Receptor Mechanisms (Putney J. W., Ed.), pp. 163-165. Alan R. Liss, New York. 16. Neer E. J. and Clapham D. E. (1988) Nature 333, 129-134. 17. Birnbaumer L. (1990) FASEB J. 4, 3178-3188. 18. Letterio J. J., Shaun R. C. and Williams L. T. (1986) Science 234, 1117-1119. 19. Logan A. and Logan S. D. (1991) Cellular Signalling 3, 215-223.
DISSOCIATION SUBSEQUENT
0898-6568/92 $5.00 + 0.00 © 1992 Pergamon Press Ltd
BETWEEN EARLY LOSS OF ACTIN CELL DEATH IN SERUM-DEPRIVED BALB/C-3T3
FIBRES AND QUIESCENT
CELLS
IGOR TAMM, TOYOKO KIKUCHI, JAMES KRUEGER and JAMES S. MURPHY Cell Physiology and Virology Laboratory, The Rockefeller University, New York, NY 10021, U.S.A. (Received 25 March 1992; and accepted 22 June 1992)
A~tract--Serum withdrawal from either growing or quiescent Balb/c-3T3 murine fibroblasts causes a loss of F-actin fibres and focal adhesions within 30 min. Cells that are growing survive serum deprivation, whereas the great majority of density-arrested quiescent cells die during a period of up to 5 h from serum withdrawal. During this time an approximately constant fraction of the quiescent cell population dies per unit time. The population half-life is 60-70 min during this time. Addition of an appropriate cell growth factor or second messenger agonist at the time of serum withdrawal or within 2 h after serum withdrawal protects a similar fraction of viable cells. These findings suggest a model according to which withdrawal of serum (i.e. growth factors) initiates the death process in cells of the population with kinetics that approximate first-order kinetics. We postulate that appropriate growth factors or second messenger agonists block the initiating event that starts the cell death process. Key words: Actin fibres, vinculin, focal contacts, PDGF, bFGF, Br-cAMP, TPA.
second messenger agonists [1,2,4]. The evidence indicates that this can be achieved via a multiplicity of pathways and that different growth factors use distinct pathways which presumably converge on a mechanism critical for the maintenance of cellular integrity. Platelet-derived growth factor (PDGF) or 8bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP) can each activate one or more protein synthesis-independent pathways whereas basic fibroblast growth factor ( b F G F ) or 12-0tetradecanoyl phorbol-13-acetate (TPA) each activates an obligatorily protein synthesisdependent pathway as demonstrated in experiments with cycloheximide [2] and anisomycin (Tamm and Kikuchi, unpublished observations). Kinetic analysis has shown that the requirement for protein synthesis in the survival-mediating action of b F G F is similar during the first, second and third hours of incubation with b F G F [2]. This is consistent with the approximately first-order cell death kinetics, which indicate that during the first several hours o f serum deprivation, a constant fraction of the population enters the death
INTRODUCTION DENSXTY-rNmmTED quiescent Balb/c-3T3 murine fibroblasts are critically dependent for their survival on the presence o f growth factors (reviewed [1]). In their absence, most of the cells die within 5 h following a change to fresh serum-free Dulbecco's medium. The decrease in viable cells incubated in serum-free medium follows approximately first-order kinetics until only a residual fraction remains in steady state [2]. The death process in the individual cells is short (usually ~ 0.5 h) and is characterized by contraction and rounding of cells with vesiculation at the plasma membrane and loss of cytoplasmic material as vesicles are shed [2,3]. The death process can be prevented in the majority of cells by the addition to the serum-free medium of one or more growth factors or Abbreviations: bFGF--basic fibroblast growth factor; Br-cAMP--8-bromoadenosine3', Y-cyclicrnonophosphate; EGF---epidermal growth factor; IGF--insulin-like growth factor; PBS----phosphate-buffered saline; PDGF--platelet-derived growth factor; TPA--12-Otetradecanoyl phorbol-I3-acetate. 675
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process per unit time. I f a protective growth factor or second messenger agonist is removed after 5 h incubation of quiescent cells in serumfree medium, the protective effect is lost [1]. Thus, as with serum, continued presence of factors is necessary to maintain cellular integrity. That growth factors can function as cell survival factors has become apparent in several systems [5-9]. Murine 3T3 fibroblasts represent a widely used system for studies of signal transduction, regulation of gene expression, and the cell cycle. For serum-deprived quiescent cells to survive it is not necessary that all the processes required for activation of the cell cycle and progression to S-phase take place [1-4]. Thus, P D G F (competence factor) used alone or insulin-like growth factor (IGF) + epidermal growth factor (EGF) (progression factors) without P D G F have marked survival factor activity but are not sufficient to enable cells to enter S-phase. In the present studies we show that Balb/c-3T3 cells deprived of serum rapidly lose actin fibres and focal adhesions (adhesion plaques). This occurs over a period of 30 min. These striking changes precede cell death, but are not obligatorily linked to deaths which occur over a period of several hours with kinetics that approximate first-order kinetics. Evidence is presented that survival factors added at 0, 1 or 2 h after serum removal produce a protective effect of similar magnitude on the remaining living cells. We propose that activation of one or more appropriate signal transduction pathway(s) blocks the initiating event that starts the cell death process.
MATERIALS AND METHODS Materials
PDGF and EGF were obtained from Collaborative Research, Inc. (Bedford, MA, U.S.A.), bFGF from Biosource International (Westlake Village, CA, U.S.A.), and IGF-I from Amgen (Thousand Oaks, CA, U.S.A.). TPA, Br-cAMP, cholera toxin and sodium orthovanadate were purchased from Sigma Chemical Co. (St Louis, MO,
U.S.A.). Pertussis toxin was purchased from Calbiochem (San Diego, CA, U.S.A.). Affinity-isolated goat F(ab')z anti(mouse IgG) rhodamine B conjugate was obtained from Tago, Inc. (Burlingame, CA, U.S.A.), fluorescein phalIoidin from Molecular Probes (Eugene, OR, U.S.A.), and monoelonal anti-phosphotyrosine from Upstate Biological (Lake Placid, NY, U.S.A.). Fibronectin was obtained from the New York Blood Center and okadaic acid from Dr Stuart Decker.
Cell culture and survival assays
Baib/c-3T3 cells were obtained from Dr W. J. Pledger, Vanderbilt University, TN, U.S.A., and stock cultures passaged as previously described [4]. Cultures for experiments were seeded at a density of 3500 cells/cm 2 in 96-well or 6-well plates or in 25 cm' flasks. The growth medium was Dulbecco's modified Eagle's medium and supplemented with 10% bovine serum (Hyclone, UT, U.S.A.). After planting, cultures were incubated for 3 days at 37°C. The serum-containing medium was changed and incubation continued for four more days at which point they were used. Cultures were washed once with serum-free medium prior to the addition of survival factors. After varying periods, cell survival was assayed by neutral red uptake as previously described [1,4,10] or by Coulter count of trypsinized samples.
Time-lapse cinemicrography
Time-lapse cinemicrographic observations were made on cells grown to quiescence in 25-GaTI 2 flasks. The monolayers were washed once with serum-free medium before the addition of serum-free medium. Cultures were photographed every 30 s using a x 6.3 planar phase-contrast objective. Initial cell counts were performed on the image of the negative projected on a ground-glass plate. Rapid rounding in association with detachment of a cell was considered as the death end-point and the time was noted.
lmmunofluorescence Cells were grown and treated as described in the figure legends and then were fixed with 4 % formalin in phosphate-buffered saline (PBS) for 20 rain, treated with 0.50 Triton in PBS for ~ 5 min, and washed twice with PBS. They were stained for F-actin with FITC-phalloidin for 30 min at 4°C. For double staining, cells were incubated with the primary antibody, anti-phosphotyrosine, overnight at 4°C before the addition of the secondary antibody,
Cell death processinitiation rhodamine-conjugated anti-mouse IgG, and FITC-phalloidin for 2 h. After washing with PBS, the coverslips were mounted in PBS--glycerol, 1:1. The preparations were viewed and photographed using a x 63 objective with epifluorescenceor reflection interference optics.
RESULTS
Early morphological change following serum deprivation of quiescent Balb/c-3 T3 cells Time-lapse cinemicrographic observation of quiescent cultures (data not shown) reveals that most cells respond to serum deprivation within minutes by contraction of the cytoplasm, separation from neighbouring cells, and loss of the extended overall shape characteristic of fibroblasts. There does not appear to be any time relationship between these initial changes and the previously described death process characterized by vesiculation, cell shrinkage, and detachment [2,3].
Serum deprivation causes loss of actin fibres Withdrawal of serum from quiescent densityarrested Balb/c-3T3 cells leads to a rapid loss of actin fibres with a simultaneous marked increase in the punctate distribution of F-actin in the cytoplasm (Fig. 1). Some cells display a heavy concentration of punctate F-actin staining in the perinuclear region. F-actin in the cell cortex is increased in serum-deprived cells. These changes are complete by 20-30 min, by which time most cells have lost actin fibres. The F-actin fibres in apposed control cells sometimes appear to continue from one cell into another. This appearance is probably due to the fact that in each of the apposed cells, actin fibres are anchored in focal adhesions which may be aligned to fibronectin in the extracellular matrix. The concentration of F-actin in serum-deprived cells is heaviest at those cell corners or extensions that probably had a heavy concentration of focal adhesions before serum deprivation. Clusters of fine brightly stained spikes can be seen on the sides of some serum-deprived cells. CELLS 4:6-F
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Serum deprivation causes loss of focal adhesions (adhesion plaques) Actin fibres are anchored in adhesion plaques which form focal adhesions with the underlying substratum. Vinculin mediates binding of actin filaments to membranes. Because vinculin contains phosphotyrosine, immunocytochemical staining for phosphotyrosine provides a sensitive means for the detection of focal adhesions. Reflection interference microscopy can then be used to establish the presence of focal adhesions, close contacts, and areas of cell-substratum separation [11]. The effects of 30 min serum deprivation on actin fibres and focal adhesions in densityarrested quiescent Balb/c-3T3 cells are illustrated in Fig. 2. The 0-h control cells contain numerous well-formed actin fibres that in many instances span the cell (Fig. 2A). Along the cell periphery and also elsewhere, streaks of rhodamine anti-phosphotyrosine-stained structures are present in precisely the same orientation as actin fibres (Fig. 2C). In many instances location of the phosphotyrosine-containing structure coincides with the termination of an actin fibre. At such sites, reflection interference microscopy reveals the presence of focal adhesions (dark grey areas in Fig. 2E). After 30 min serum deprivation the actin fibres (Fig. 2B) have disappeared as have the associated phosphotyrosine-stained structures (Fig. 2D). Phosphotyrosine staining is limited to segments of the cell border and a diffusely stained mass in the centre of the cell. Reflection interference microscopy reveals a remarkable change in cell adherence to the substratum (Fig. 2F). No streak-shaped focal adhesions can be seen but there are extensive areas of close contacts. These findings emphasize the role that actin fibres and focal adhesions play in maintaining the fibroblast in an extended form in which it adheres strongly to the substratum and moves little. Loss of these structures, as through serum deprivation, then results in shape-change and an increase in motility. Growing Balb/c-3T3 cells undergo a similar set of changes when serum is withdrawn (Fig.
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3). Again, it can be seen that in 0-h control cells actin fibres (Fig. 3A) terminate in streak-shaped structures that can be stained with phosphotyrosine (Fig. 3C) and recognized as focal adhesions by reflection interference microscopy (Fig. 3E). The growing cells, too, lose actin fibres after serum withdrawal and F-actin assumes a punctate distribution with a heavy concentration in the perinuclear region or it relocates to the cell margin (Fig. 3B). In the serum-deprived cells, phosphotyrosine staining is heavy and diffuse over and also beyond the nucleus and in cell corners (Fig. 3D). Reflection interference microscopy (Fig. 3F) indicates the presence of close contacts in some of the cell corners. To summarize, serum-deprived cells, either quiescent or growing, rapidly lose focal adhesions and actin fibres and separate from each other. This process is complete within only 30 min of serum withdrawal. In contrast, the occurrence of cell death in the population of quiescent cells is distributed over a period of 5 h ([2] and present results); growing cells survive serum withdrawal.
Approaches to the mechanism of actin fibres dissolution in serum-deprived cells We determined that supplementation of serum-free medium with PDGF (5ng/ml), bFGF (20 ng/ml), IGF-I (40 ng/ml), Br-cAMP (1.45 mM), or TPA (100nM) did not prevent the disappearance of actin fibres, although, as previously reported [1,2,4], each of these factors has a marked cell survival-enhancing effect on quiescent Balb/c-3T3 cells that have been deprived of serum in the medium. Indeed, PDGF caused cell contraction over and above that caused by serum withdrawal, as did bFGF and Br-cAMP. TPA had a lesser contractionaccentuating effect and IGF-l-treated cells did not display such an effect. Actin fibres were rare in all cases. In these experiments cells were incubated in serum-free medium for 30 min and examined by phase-contrast and fluorescence microscopy after staining for F-actin. It has been shown previously that PDGF
itself causes disappearance of actin fibres in quiescent Balb/c-3T3 cells with redistribution of vinculin from focal adhesions to the cytoplasm around the nucleus where it displays a punctate distribution [12,13]. These effects of PDGF are dose dependent in the range 5-50ng/ml; at 3ng/ml it has little or no effect [12,13]. We therefore used PDGF at 3 ng/ml and found that at this relatively low concentration it partially protected actin fibres against dissolution in serum-free medium. This effect was somewhat enhanced by the simultaneous presence of EGF at 10ng/ml. As serum contains PDGF and EGF at low concentrations, it is likely that their presence is at least in part responsible for the ability of serum to maintain an actin cytoskeleton in cells. In these experiments, as previously, quiescent cells were incubated with various agents for 30 min and then examined. To investigate the possibility that the rapid changes in cytoskeletal organization caused by serum withdrawal might be mediated via the activation of phosphatases, two phosphatase inhibitors were used. Neither okadaic acid (40#M) nor sodium orthovanadate (10/~M) prevented actin fibre disassembly in quiescent Balb/c-3T3 cells deprived of serum for 20 min. Fibronectin is an important extracellular matrix component which is present in serum, and functions in cell adherence to the substratum. We supplemented serum-free medium with fibronectin (5 #g/ml); however, this had no apparent effect on the morphological change quiescent Balb/c-3T3 cells underwent in 30 min in serum-free medium or on the disappearance of actin fibres.
Distribution of the time of occurrence of cell death in cell population In the present study we determined the death curve by time-lapse cinemicrography, photographing cultures of serum-deprived cells every 30 s for 3 h. By this high resolution technique, an initial short lag was evident after which the occurrence of cell deaths followed approximately first-order kinetics (Fig. 4). The data are not inconsistent with the Eyring-Stover formal-
~'IG. 1. Early cytoskeletal response of quiescent Balb/c-3T3 cells to serum deprivation. Cells were seeded on glass coverslips, 8000 cells/cm2, in )ulbecco's medium with 10% bovine serum. The cultures were refed 4 days later and used 3 days after the medium change. The cells were washed ,nce with warm Dulbecco's medium and incubated at 37°C for varying periods, after which the cultures were stained for F-actin. (A) 0 rain; (B) 10 nin; (C) 20 rain; (D) 30 rain. Arrows in B mark punctate actin, x 443.
!
FIG. 2. Localization of F-actin, phosphotyrosine, and focal adhesions by cytoch~aistry and reflection interference microscopy in quiescent Balb/c-3T3 cells after serum removal. Glass coverslips were seeded at 3500 cells/cm 2, and quiescent cells were exposed to serum-free Dulbccco's medium for 0 rain (A,C,E) or 30 rain (B,D,F) following which they were double stained for F-actin (A,B) and phosphotyrosine (C,D) and visualized by fluorescence microscopy. The same fields were also visualized by reflection interference microscopy (E,F). In A,C and E an arrow marks a focal adhesion at the end of actin stress fibres. In B,D and F the arrowhead marks same position in the cell; open arrow in F (ce) marks a diffuse, grey-toned close contact, x 1216.
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FIc. 3. Localization of F-actin, phosphotyrosine and focal adhesions by cytochemistry and reflection interference microscopy in serum-deprived growing Balb/c-3T3 cells. Glass coverslips were seeded at 3500 cells/cm2 and 3 days later were exposed to serum-free Dulbecco's medium for 0 min (A,C,E) or 30 min (B,D,F) following which they were double stained for F-actin (A,B) and phosphotyrosine (C,D) and visualized by fluorescence microscopy. The same fields were also observed using reflection interference microscopy (E,F). In A,C and E arrows mark focal adhesions at the end of actin stress fibres. In B,D and F the arrowhead marks the same position in the cell. x 1216.
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Cell death process initiation
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683
0.8 10% BS - - a - POGF ~ bFGF
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0.8
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0.5
~ a. ~
0.4 0.3
~
0.2
Oo 10~ o
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I 120 Minutes
FIG. 4. Cell death curve of quiescent Balb/c-3T3 cells in serum-free medium. Flasks 25 cm2 were seeded at 3500 cells/era: and allowed to grow to quiescence with one medium change, as described in Materials and Methods. Monolayers were washed once before addition of serum-free medium. Time-lapse photomicrographs were taken at 30-s intervals, and analysed for cell deaths/time interval. Two fields were analysed in one film (68 and 83 cells/field). O, Number of cells based on starting number less the number of deaths; II, number of cells based on the Eyring-Stover equation [14].
ism [14]. However, a more extensive study would be required to establish whether the dynamics of survival described by this formalism apply to serum-deprived quiescent Balb/c-3T3 cells. The Eyring-Stover formalism is based on the assumption that two reactions are operational, one leading to damage and death, and the other to repair of damage and survival. During the close to linear part of the curve, the time required for a 50% decrease in the calculated cell number was 70 min. This is consistent with the results based on neutral red assays, which gave a mean value of 60 min for the population half-life [2]. As has been pointed out before, the specific death rate varies from experiment to experiment [1,4].
The extent of protection of viable cells by survival factors is independent of time of incubation in serum-free medium During the initial period of 5 h after serum removal, an approximately constant fraction of the population of density-inhibited quiescent
z
0.1
1
2
Hours before survival factor addition FIG. 5. Relationship between pre-incubation time in serum-free medium and the survival-enhancing activity of PDGF, bFGF, Br-cAMP, and TPA. Ninety-six-well plates were seeded at 3500 cells/cm2 in Dulbecco's medium with 10% bovine serum (BS) and allowed to grow to quiescence with one medium change, as described in Materials and Methods. After a wash with warm serum-free Dulbecco's medium, groups of cultures were incubated at 37°C for 0, 1, or 2 h in serum-free medium after which PDGF (5ng/ml), bFGF (20ng/ml), Br-cAMP (l.45mM), TPA (100nM), or serum (10%) was added. Survival was assayed by neutral red uptake after a total of 3 h of incubation (i.e. 0 + 3; 1 + 2; or 2 + l h ) . Mean results of four experiments with PDGF and Br-cAMP and of five experiments with bFGF and TPA. Neutral red uptake in controls incubated in serum-free medium for 3 h was 0.1615 O.D. unit or 17% of that in cells incubated with 10% bovine serum for this period.
cells enters the death process per unit time. We propose a model according to which entry into the death process is preceded by as yet unidentified initiating events and that certain growth factors and second messenger agonists block these initiating events. The model predicts that during the first several hours after serum deprivation the extent of protection of viable cells by survival factors should be independent of time of incubation in serum-free medium. To test the model we compared the protective effect of survival factors added immediately after serum
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removal with the extent of protection of remaining viable cells when a factor is added 1 or 2 h after serum withdrawal. The total period of incubation from the time of serum removal to the beginning of the viability assay was constant, i.e. 3 h, in all cases. The number of viable cells in density-inhibited quiescent cultures is stable after replacement of medium with fresh serum-containing medium [4] or single survival factors (unpublished observations). The results in Fig. 5 support the model. As expected, the number of viable cells declines when re-addition of serum to serum-deprived cells is delayed. Delayed addition of PDGF, bFGF, Br-cAMP, or TPA yielded a family of curves approximately paralleling the serum readdition curve (cf. Fig. 5). The survivalenhancing effects of the four factors used, expressed relative to the fraction of cells still surviving after 0-, 1-, or 2-h incubation of quiescent cells in serum-free medium, respectively, were as follows: PDGF: 69, 58, and 60; bFGF: 60, 60, and 62; Br-cAMP: 51, 65, and 50; TPA: 62, 53, and 59. The mean standard error of the determinations was 4 (range 3-8). These results show that there is no systematic change in the extent of protection when the addition of P D G F (5 ng/ml), b F G F (20 ng/ml),
Br-cAMP (1.45mM), or TPA (100nM) is delayed after removal of serum from the quiescent cells.
G-proteins and cell survival Pertussis toxin (100 ng/ml), which inhibits the action of several G-proteins (reviewed in [1517]), did not affect to a measurable extent the survival of quiescent cells in serum-free medium or in medium containing serum, PDGF, bFGF, Br-cAMP, or TPA (Table 1). Table 2 documents the expected result of enhancement by cholera toxin (5/zg/ml) of survival of quiescent cells in serum-free medium, as it is well known that cholera toxin activates a G-protein that stimulates adenylate cyclase (reviewed in [16,17]). Cholera toxin did not appear to have any major effects on the enhancement of cell survival by serum, PDGF, bFGF, Br-cAMP, or TPA. DISCUSSION Fibroblasts have well-developed actin fibres which play a role in the attachment of the cells to the substratum and in maintaining their extended form. When serum is withdrawn from the medium of quiescent density-inhibited murine Balb/c-3T3 cells, most cells in the
TABLE 1. EFFECTS OF PERTUSSIS TOXIN (PT) ON GROWTH FACTORAND SECOND MESSENGER AGONIST-STIMULATED SHORT-TERM (3-h) CELL SURVIVAL
Cell survival (% of 10% BS control*) Agents and concentrations
No PT
BS (10%) None PDGF (5 ng/ml) bFGF (20 ng/ml) Br-cAMP (1.45 mM) TPA (100 nM)
100 31 74 75 65 76
Pertussis toxin (100 ng/ml) % no PTt 90 27 73 64 66 64
90+ 15 89 4- 2 984- 16 86_+ 17 101 _+12 83__.21
* Neutral red uptake in 10% bovine serum (BS) controls was 0.736 O.D. unit. Pertussis toxin was added for 3 h prior to and also during growth factor treatment. Mean results of three experiments. "~__S.D.
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Cell death process initiation TABLE 2. EFFECTSOF CHOLERATOXIN (CT) ON GROWTHFACTORAND SECOND MESSENGERAGONIST-STIMULATEDSHORT-TERM (3-h) CELL SURVIVAL Cell survival (% of 10% BS control*)
Agents and concentrations
No CT
BS (10%) None P D G F (5 ng/ml) b F G F (20 ng/ml) Br-cAMP (1.45 mM) TPA (100 nM)
100 30 74 72 59 69
Choleratoxin (5 #g/ml) % no CTt 88 62 77 62 44 85
88_ 24 210_ 29 103-t- 2 87_ 9 75+ 1 124+ 10
* Neutral red uptake in 10% bovine serum (BS) controls was 0.682 O.D. unit. Cholera toxin was added for 3 h prior to and also during growth factor treatment. Mean results of two experiments. t _S.D.
culture undergo a rapid morphological change characterized by loss of actin fibres and focal contacts. The cells separate away from each other and show increased motility. These changes are apparent within 5-10 min of serum withdrawal and are essentially complete by 2030 min. In contrast, cell deaths in serumdeprived cultures are distributed over ~ 5 h and their occurrence follows approximately first-order kinetics ([2] and present results). The cell death process is characterized by vesiculation accompanied by shrinkage of the cell and the nucleus and it culminates in the disintegration of the cell ([2] and present results). Several observations speak against a direct relationship between the early morphologicalfunctional changes and the subsequent death of cells. First, non-confluent growing cultures show the early changes when serum is withdrawn, but the cells survive. Second, addition of appropriate growth factors or second messenger agonists to quiescent cells may not prevent the early changes and can indeed accentuate them and yet it protects cells against death. However, the possibility cannot be excluded that the skeletal rearrangement is a precondition of the onset of the cell death process.
Further studies are needed to determine the biochemical mechanism whereby serum deprivation rapidly leads to loss of actin fibres and focal adhesions. Although supplementation of serum-free medium with fibronectin did not prevent disruption of the actin cytoskeleton, it is possible that removal of serum impairs integrin adhesivity. Attempts to prevent actin fibre disassembly by the addition of the phosphatase inhibitors okadaic acid and sodium orthovanadate to serum-free medium were unsuccessful. The possibility that activation of a protease may play a part in this early response also merits exploration. Our evidence indicates that the onset of the death process can be prevented by growth factors or second messenger agonists independent of the time that has lapsed since serum deprivation. This was demonstrated by adding a growth factor or second messenger agonist at different times after serum withdrawal from the medium (cf. Fig. 5). We postulate that in cells of the quiescent population a death process initiating event takes place with approximately first-order kinetics following serum withdrawal and that the presence of an appropriate growth factor or second messenger agonist can block this event by activating one or more of several
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signal transduction pathways which converge on an as yet unidentified mechanism that is critical for the maintenance of cellular integrity. Attempts with pertussis toxin to obtain evidence for a role for G-proteins in the survival of cells mediated by P D G F , b F G F , Br-cAMP, or TPA have so far given negative results. It has been shown previously that treatment of Swiss 3T3 fibroblasts with pertussis toxin (5 ng/ml) has essentially no effect on the mitogenic responses of the cells to either P D G F or phorbol 12,13-dibutyrate although it completely blocks bombesin-stimulated D N A synthesis [18]. Thus, our results contribute further evidence that P D G F - or phorbol esteractivated pathways in 3T3 cells do not involve a pertussis-sensitive guanine nucleotide-binding protein. The complexity of the signal transduction mechanisms in growth factor action is illustrated by the findings that pertussis toxin blocks b F G F - or TPA-induced D N A synthesis in quiescent Balb/c-3T3 cells but does not inhibit PDGF-induced formation of inositol phosphates [19]. The fact that, following serum deprivation, the cell deaths in the population occur with approximately first-order kinetics presents an experimental challenge as within a given interval, such as 15 min, only a small fraction ( ,,, 16%) of the cells alive at the beginning of the interval would be undergoing the death process and they would presumably be doing so asynchronously. An approach to further mechanistic analysis would be to establish by time-lapse cinemicrography distinct morphological stages in the death process in individual cells and then conduct biochemical-microscopic analyses on single cells in different stages of the process. It is evident that cell growth factors can serve not only as mediators of cell cycle entry and progression, but also as mediators of cell survival. Their action in regulating cell cycling in most instances clearly involves one or more pathways by which a signal from membrane receptors occupied by the growth factor is transduced to the nucleus. Recent studies have shown that activation of signal transduction
pathways in a cell at risk of dying can prevent death [1,2]. Much remains to be learned about the physiological role o f growth factors as survival factors. Acknowledgements---We thank DR EUGENIA WANG for reading the manuscript and MRS Sopm~ NADELL for assistance in processing the manuscript. This work was supported by National Institutes of Health Research grant CA-18608.
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