Proc. NaiL Acad. Sci. USA Vol. 74, No. 2, pp. 666-670, February 1977 Cell Biology
Role of surface modulating assemblies in growth control of normal and transformed fibroblasts (Rous sarcoma virus/concanavalin A/microtubules/cell cycle)
DONALD A. MCCLAIN, PETER D'EUSTACHIO, AND GERALD M. EDELMAN The Rockefeller University, New York, N.Y. 10021
Contributed by Gerald M. Edelman, December 9,1976
in the lymphocyte. Individual lymphocytes are recruited into the S phase of the cell cycle at different times after exposure to Con A, consistent with stochastic models (4) of the cell cycle. Colchicine appears to block this early recruitment event (3). High doses of Con A induce anchorage modulation of surface receptors and inhibit mitogenesis but do not interfere with the primary growth signal (5). Additional evidence for a role of the SMA in growth control has come from the observation that it-is a direct or indirect target of the sarc gene product of Rous sarcoma virus (RSV) in chick embryo fibroblasts (6). By using temperature-sensitive mutants of RSV, it was found that expression of the sarc gene product resulted in rapid loss of microfilament and MT structure, accompanied by--loss of anchorage modulation, andf owed by loss of normal growth control. In view of the observations on lymphocytes, it was of particular interest to determine the relationship between the effects of the sarc gene product and those of Con A and colchicine in this less heterogeneous cell population. It was found that for. both normal and virally transformed fibroblasts, there appears to be an intimate relationship between the state of the SMA and the regulation of cell growth. MATERIALS AND METHODS Chick embryo fibroblasts (CEF) were maintained in culture and transformed with RSV (SR-A strain, or the tsNY68 mutant of this strain) (7) as described previously (6). To study cellular morphology, cells grown on 18 mm square coverslips were treated and examined in situ (6). Serum starvation was achieved by replacing growth medium with serum-free medium containing 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. To inhibit protein synthesis, cells were incubated in the presence of 0.1 mM puromycin (dihydrochloride salt, Sigma). Ninety percent of protein synthesis was inhibited within 15 min of adding the drug tocultures of growing cells. Inhibition was reversed by removing the drug. The rate of DNA synthesis was estimated from the incorporation of [3H]thymidine (Schwarz/Mann) as detected by autoradiographic analysis of cells grown on coverslips or by bulk counts of acid-precipitable material (3). In vitro DNA replication was assayed as described (8). The morphology of cellular MT was assessed by immunofluorescence microscopy (6). RESULTS Perturbation of the SMA and Growth Control in Normal Fibroblasts. CEF were arrested in the G1 phase of the cell cycle by incubation in serum-free medium (9). Addition of serum to these starved fibroblasts (Fig. 1) resulted in a wave of DNA synthesis beginning at about 8 hr. Addition of colchicine to a final concentration of 1 WM at the same time as the serum delayed the entry of cells into S phase and reduced the number
ABSTRACT Cellular microtubules, microfilaments, and surface receptors have been postulated to form a surface modulating assembly that regulates surface receptor mobility and cell growth. To test this hypothesis, we examined three agents known to affect cell growth [colchicine, concanavalin A (Con A), and the src gene product of Rous sarcoma virus] for their effects on chick embryo fibroblasts. Individual cells from serum-starved normal fibroblast populations became committed to enter S phase at various times over a 12 hr period after exposure to serum. Colchicine and other microtubule-disrupting agents blocked entry into S phase at a point close to the commitinent point for each cell. The lectin Con A also blocked entry into the S phase when present in doses sufficient to modulate surface receptor mobility. In contrast, succinyl-Con A, which does not induce surface modulation, had no effect. Both Con A and colchicine blocked the appearance of cytopsmic factors capable of stimulating DNA replication in a cellfr system. To study endogenous effects on the surface modulating assembly, we infected fibroblasts with a Rous sarcoma virus (thNY68) having a temperature-sensitive mutation in the transforming (src) gene. We have previously shown that microtubular and microfilamentous structures of the surface modulating assembly are direct or indirect targets of the src gene product with consequent reduction in the capacity of Con A to induce surface modulation. TsNY68-infected fibroblasts shifted to the nonpermissive temperature acquired normal microtubular morpholoky more rapidly (2 hr) than cells grown at the permissive temperature in the presence of protein synthesis inhibitors (7.5 hr) This suggests that the src gene product acts directly on the surface modulating assembly rather than via the nucleus or at the level of protein synthesis. Furthermore, "transformation" of the surface modulating assembly was partly blocked by treatment of the infected cells with Con A but not succinyl-Con A. Both Con A and colchicine inhibited entry into the S phase following a shift from -nonpermissive to permissive growth conditions. All of these observations are in accord with the hypothesis that the surface modulating assembly acts as a signal regulator in growth control. It has been suggested that submembranous arrays of microtubules (MT), microfilaments, and associated contractile and membrane proteins form a surface modulating assembly (SMA) that controls the mobility of cell surface- receptors (1, 2) and also regulates signals from the cell surface to the cell interior (1, 3). In support of this idea, local crosslinkage of cell surface glycoproteins by lectins induces a propagated restriction in the lateral mobility of a variety of cell surface receptors (anchorage modulation) that depends on cytoplasmic MT (1, 2). Moreover, the state of the SMA (particularly, of its MT components) appears to be causally related to early events in growth simulation or inhibition. This is shown by the effects of the lectin concanavalin A (Con A) and MT-disrupting agents on mitogenesis Abbreviations: MT, microtubules; SMA, surface modulating assembly; Con A, concanavalin A; RSV, Rous sarcoma virus; CEF, chick embryo fibroblasts. 666
Cell Biology: McClain et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
667
Table 1. Inhibition by Con A and colchicine of serum-induced DNA synthesis and of appearance of DNA replication activity in serum-starved normal CEF In vitro DNA
Incorporation of replication [3H]thymidine (pmol of
TiM
fW)
The
(hr)
FIG. 1 (left). Inhibition of DNA synthesis by colchicine after release of serum starvation in CEF. Cells (2 X 105) were seeded onto coverslips in 2 ml of medium with serum. After the cells settled, the coverslips were transferred to serum-free medium. After 60 hr, 0.22 ml of fetal calf serum was added (time 0 on abscissa) (0). Parallel cultures were also made 1 uM in colchicine (0). At various times, 6 ACi of [3H]thymidine were added to the cultures; after 2 hr the cultures were processed for autoradiography (3). FIG. 2 (right). Effect of colchicine on commitment to serum stimulation. Serum-starved cells on coverslips (see Fig. 1) were placed in medium containing 10% fetal calf serum at time 0. At the times indicated, coverslips were washed with buffered saline and put in serum-free medium (0-0), colchicine was added to 1 AM (O- -0) or both n---- -0). At 12 hr, the cultures received 6 ACi of [3H]thymidine, and at 13 hr they were processed for autoradiography (3).
into cells
dTTP/ml
Condition
(cpm)
of extract)
Cells, no serum Cells + serum Cells + serum + colchicine (1 ,M) Cells + serum + Con A (30mg/ml) Succinyl-Con A (100 .g/ml)
1,900 29,900
265 413
14,500
230
10,700 32,600
280
Cells (106) in 60 mm dishes were serum-starved for 48 hr. Serum was added to 10% concomitantly with Con A or colchicine, and 5 mCi of [3H]thymidine was added 12 hr later. At 13 hr, incorporation of counts into acid-rrecipitable material was determined for duplicate cultures. For the in vitro assay, cell-free extracts prepared 9 hr after serum addition were incubated with frog spleen nuclei and labeled nucleotide triphosphates for 1 hr at 370 (8). Results are the means of duplicate determinations for both 20 and 40 gl of extract.
-
of cells entering S phase. Similar results were obtained with 3T3 fibroblasts. The amount of label incorporated per cell in S phase (estimated from grain counts of autoradiograms) was not affected by colchicine, and thus the effect of the drug did not appear to be due to inhibition of [3H]thymidine transport. Lumicolchicine, which does not dissociate MT, did not inhibit the entry of the CEF into S phase. Other agents that disrupt MT-podophyllotoxin and vinblastine-had the same inhibitory effect at 1 tiM as iolchicine. The effect of colchicine was reversible: CEF exposed to the drug but then washed free of it responded to serum as well as untreated cells. The kinetics of inhibition by colchicine closely paralleled the kinetics of commitment to serum stimulation. It is convenient to define the point at which a cell will go on to replicate even after the growth stimulus is removed as the commitment point (3). To study the kinetics of commitment, CEF that had been arrested by serum starvation were exposed to serum. At various times thereafter, the serum was removed. Twelve hours after the initial exposure to serum, the number of cells in S phase was determined. In accord with previous observations (9), committed CEF began appearing soon after serum was added and the number of these cells increased for a period of at least 12 hr (Fig. 2). If, after adding serum, colchicine was added at various times, reduced numbers of cells in S phase were observed 12 hr after the serum addition (Fig. 2). Less inhibition was seen the later the colchicine was added. When serum was removed at the same time that colchicine was added, the same amount of inhibition was observed as a function of time as with serum removal or colchicine addition alone. This suggests that both of these treatments of serum-stimulated CEF were affecting the same subpopulations of cells at any given time. Another exogenous agent that appears to affect MT is the lectin Con A, and this agent also inhibited growth of CEF after release from serum starvation (Table 1). The lectin was added mouse
concomitantly with the serum in doses that inhibit receptor mobility (6). Autoradiographic analysis confirmed that the inhibition ofgrowth reflected a restriction of the number of cells entering S phase. In contrast, dimeric succinyl-Con A (10) did not inhibit growth. Because the lectin and its derivative have similar binding specificities, the lack of inhibition by succinyl-Con A suggests that the inhibition by Con A is not due to competition for, or interference with, the binding of serum growth factors to the fibroblast surface. It is notable that succinyl-Con A stimulates lymphocyte growth, but unlike Con A, shows no inhibition of mitogenesis or of cell surface receptor mobility (10). Both Con A and colchicine also inhibited the appearance of cytoplasmic factors that stimulate the replication of nucleochromatin in vitro (8) (Table 1). The level of these factors was low in extracts of resting, serum-starved CEF. With serum stimulation, the activity began increasing shortly before the CEF entered S phase; in the presence of Con A or colchicine no such increase was seen. Endogenous and Exogenous Perturbation of the SMA in Transformed Fibroblasts. Additional evidence for the hypothesis that the SMA is involved in the regulation of cell commitment and division has been obtained using endogenous alteration of cells by viral transforming genes such as the sarc gene of RSV. We have previously examined the relationship between expression of the gene and the state of the SMA. CEF infected with tsNY68 virus, a strain of RSV which has a temperature-sensitive mutation in its sarc gene, are phenotypically transformed when grown at 370, but become phenotypically normal when shifted to 410. We have shown that cells grown at 370 do not contain organized cytoplasmic MT, but acquire these structures within 2 hr of being shifted to 410. Conversely, the organized MT disappear within 2 hr of a shift from 410 to 370 (6). We wished to show that the effect of the src gene product on the SMA is cytoplasmic and is not related to the control of protein synthesis or nuclear events. This demonstration would facilitate investigation of the relationship be-
668
Cell Biology: McClain et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
A
B
7
C
807
~60\CI
40
C20-
0
2
4
6
8
10
12
0
2
4
6
8
0
12
2
0
6
4
Time (hr) FIG. 3. Effect of inhibition of protein synthesis on MT patterns at permissive and nonpermissive temperatures. (A) CEF infected with tsNY68 virus and grown on coverslips for 12 hr at 370 were either incubated at 37° in the presence of 0.1 mM puromycin (@) or shifted to 410 (0). At each time point shown, slips-were fixed, stained, and scored for MT morphology. "Normal" CEF exhibited radial arrays of organized MT. "Transformed" CEF were diffusively stained (6). (B) CEF infected with SR-A virus and grown at 37 (0) or 41° (-) were incubated further at the same temperature in the presence of puromycin and scored for MT. (C) CEF infected with tsNY68 virus and grown at 410 for 36 hr were shifted to 370 in the presence (v) or absence (v) of puromycin, incubated for various periods, and scored for MT.
tween the effects of the src gene product and those of colchicine and Con A. The relative effects of growth at 410 and of growth at 370 were therefore examined in the presence of protein synthesis inhibitors. If the src product itself were not acting on the SMA, but rather at a stage prior to translation to induce the appearance of a separate SMA effector, then a shift to 410 or the inhibition of protein synthesis would both prevent new synthesis of that effector. In both cases, the return of the SMA to the normal phenotype would result from the natural decay of the effector substance, and the cells should revert to the normal phenotype after the temperature shift at the same rate as after blocking protein synthesis. In contrast, if the sarc gene product acted directly on the SMA, a shift to 410 should inactivate the effector itself and cause a more rapid loss of the transformed phenotype. CEP infected with mutant (tsNY68) or wild-type (SR-A) virus were grown at 370 or 410 in the presence or absence of puromycin and scored for microtubular morphology by indirect immunofluorescence (Fig. 3). When CEF infected with mutant virus and grown at 370 were shifted to 410, organized MT appeared in most of the cells within- 2.5 hr (Fig. 3A). If these cells were maintained at 370 and puromycin was added, however, less than 25% of the cells had organized MT after 2.5 hr. Approximately 7.5 hr incubation with the drug was needed for the process to go to completion, consistent with the observations of others (11) on RSV-transformed rat kidney cells. CEF infected with wild-type SR-A virus and grown at either temperature lacked organized MT (6). Incubation of these cells in the presence of puromycin led to a slow increase in the fraction of cells containing organized MT (Fig. 3B). The rates of appearance of such cells at the two temperatures were indistinguishable from each other and from the rate observed for mutant cells at 370 (Fig. 3A). When cells infected with mutant virus and grown at 410 were shifted to 370, organized MT disappeared from most of- the cells in less than 1 hr (Fig. 3C). When puromycin was added to the culture medium at the time of the shift to 370 however, no change in the MT pattern could be detected even - after a 4 hr incubation at 370. All of the data support the conclusion that the src gene product itself interacts with components of the SMA, and that it does not exert its effects in the
nucleus or at some stage of the control of protein synthesis. it is possible that the endogenous agent, the sarc product, acts ultimately on the same cytoplasmic structure as the exogenous agent Con A. If so, the addition of Con A might be expected to alter the expression of the transformed phenotype. When CEF infected with the mutant virus and grown at 410 were shifted to 370 in the presence of Con A, the fraction of cells whose MT became disordered was reduced (Fig. 4). This inhibitory effect was not observed in cells treated with succinyl-Con A, nor was it observed in cells treated with Con A in the presence of the competitive inhibitor a-methyl-D-mannoside. We next determined whether Con A and colchicine would also block induction by the sarc product of factors leading to unregulated DNA replication. CEF were infected with tsNY68, grown at 410, arrested by serum starvation, and tested for entry into S phase after a shift-down in the presence or absence of Con A, colchicine, and serum (Table 2). Although the CEF that were shifted to 370 without serum showed relatively lower levels of 60
u
40 I-
W
O
20
a13
11
33
100
pg/ml FIG. 4. Inhibition by Con A of the conversion of cytoplasmic MT to a "transformed" pattern. CEF infected with tsNY68 virus and grown on coverslips for 36 hr at 410 were incubated for 15 min at 410 in the presence of various amounts of Con A (0) or succinyl-Con A (0), incubated with the lectin at 370 for 1 hr, and then scored for MT morphology. The frequency of diffusely stained cells in a population grown continuously at 410 (no lectin) is shown by the dashed line
Cell Biology: McClain et al. Table 2. Effects of Con A and colchicine on growth of synchronized tsNY68-infected cells
Proc. Natl. Acad. Sci. USA 74 (1977)
669
to block entry of neuroblastoma cells into S phase (13) and to block the serum-induced increase of ornithine decarboxylase activity seen in the G1 phase (14). All of these results suggest that Peak [3H]- % Inhibi- % Inhibithe effect is a general one acting in a variety of cells. thymidine tion by tion by Several lines of evidence suggest that the inhibition by Con incorporation Con A colchicine A of the entry into S phase is related to the modulation of surCondition (30 ipg/ml) (1 /.M) (cpm) face receptor mobility: (i) Con A, but not succinyl-Con A, inhibits both fibroblast mitogenesis (Table 1) and surface receptor Cells grown at 410, mobility (6); (ii) Con A inhibits lymphocyte mitogenesis with 53 serum added 7640 47 the same dose-response curve as it inhibits receptor mobility Cells shifted to 370, (5); and (iii) succinyl-Con A shows no high dose inhibition of serum added 1900 59 68 lymphocyte mitogenesis and no modulation of receptor moCells shifted to 370, bility (10). no serum added 840 54 100 These observations are in accord with those on transformed cells. In particular, expression of the src gene of RSV leads to Cells (3 X 105) infected with tsNY68 and plated in 60 mm dishes alterations in the SMA of chick embryo fibroblasts (6). In cells were grown for 24 hr at 410 in complete medium, and then for 48 hr infected with a RSV carrying a temperature-sensitive sarc at 410 in serum-free medium. Calf serum (10%) was then added or the mutation (tsNY68) (7), alterations in the structure of the SMA, cells were shifted to 370 or both. Con A or colchicine was added at the time of the shift. At 4 % hr intervals, incorporation of [3H~thymidine and anchorage modulating functions are seen withiSn into acid-precipitable material was measured (see Table 1). Cells at of a shift from the nonpermissive to the permissive temperature 410 without serum incorporated 430 cpm into acid-precipitable ma(6). We have now shown that a shift up to the nonpermissive terial. Peak DNA synthesis was observed at 12-13 hr at 410 and at of a cell transformed by tsNY68 virus results in temperature 21-22 hr at 370. a much more rapid return to the normal phenotype than does blocking protein synthesis at the permissive temperature. DNA synthesis than those with serum, the response was reThe sarc gene product couldcaffect the SMA either by reguproducible, confirming the previous observations of Bell et al. the synthesis of SMA components at the levels of tranlating (12). The cells that were shifted to 370 with serum showed a or translation or by interacting directly with SMA scription diminished entry into S phase that was delayed by approxiin the cytoplasm (6). In the first case, infected ficomponents mately 9 hr as compared with the cells arrested and then broblasts shifted to restrictive conditions (inactivating all sac stimulated with serum at 410. This delay is probably an effect gene product in the cell) should acquire ordered microtubules of the src gene product. It appears not to be due simply to slower no more rapidly than cells grown under permissive conditions metabolism at 370, because the response of normal CEF to in the absence of protein synthesis (preventing synthesis of new serum at 370 (Fig. 1) is kinetically similar to that of the ts NY68src gene product). We observed a large difference in rate after infected cells at 41°. The key observation is that when Con A the two treatments, however. This result is therefore most was added to the cells at the time of the temperature shift at a readily explained by the hypothesis that the sarc product acts concentration that stabilized the untransformed MT morpost-translationally as a cytoplasmic effector leading either phology (Fig. 4) it also inhibited the entry into S phase (Table or indirectly to an altered SMA. directly 2). Colchicine had a similar inhibitory effect. Thus, both agents If the sarc product, Con A, and colchicine act at the level of that affect the SMA block the induction by sarc of unregulated the SMA, then these agents should interact at both the morDNA synthesis. phological and functional levels. We have found several such interactions (Fig. 4 and Table 2). Con A stabilized the unDISCUSSION transformed microtubular staining pattern of tsNY68-infected The effects of Con A and colchicine on both anchorage modcells, and Con A blocked the entry of serum-starved tsNY68ulation and lymphocyte mitogenesis have prompted us to hyinfected cells into S phase after a 41° to 370 temperature shift. pothesize that the SMA may be a regulator of mitogenic signals Neither of these effects was induced by succinyl-Con A which from the cell surface (1, 3). In this paper, we have shown that is incapable of inducing surface modulation. Furthermore, like Con A, colchicine, and the product of the RSV transforming colchicine, expression of the src gene itself both disrupts migene (src) all perturb both the SMA and the cell cycle of ficrotubular morphology and interferes with serum stimulation broblasts in a fashion consistent with this hypothesis. (Table 2). We hypothesize that the SMA may serve as a-mulThe data on serum-stimulated normal fibroblasts resemble tistate integrator of the varied signals that affect cell growth and those previously obtained with Con A-stimulated lymphocytes. divisionii7TfeFochastic properties of commitment may be reFibroblasts, like Iymphocytes, were stimulated asynchronously, lated to the capacity of the SMA to exist in multiple states. The i.e., the longer a population of CEF was exposed to serum, the sarc product may represent a decontrolled or altered form f a larger the fraction of cells recruited into a determinate of phase normal regulator of this system. of the cell cycle leading to the S phase (3, 9). Smith and Martin It is necessary to be cautious in relating biochemical events (4) have suggested that the entry into a determinate phase of directly to morphological changes. This is emphasized by the G1 is probabilistic, and the asynchrony we have observed in observation that both the src gene and colchicine treatment commitment could reflect the same transition from the inderesult in dispersion of the microtubular patterns. Nevertheless, terminate ("A") state to the determinate ("B") phase postulated a tentative order of events is suggested by our data. A diagram by these authors. showing a possible place for the SMA in the order of events Colchicine appears to inhibit an early event in the entry of leading to stimulation, is given in Fig. 5. The first microtubular fibroblasts from the arrested G1 or Go phase into the S phase. event is sensitive to colchicine and it may represent the "start" The kinetics of the colchicine inhibition (Fig. 2) suggest that, or commitment point. The second event is defined by Con as in the lymphocyte (3), the drug acts to block mitogenesis at A-induced anchorage modulation and growth inhibition (2, 5, or near the crucial commitment point. Colchicine is also known 6). The sarc gene product may affect both events, blocking
670
Proc. Nati. Acdd. Sci. USA 74 (1977)
Cell Biology: McClain et al. "1B PHASE"
"A STATE" SRC PRODUCT SERU14 OR
EXTERNAL *MEMBRAE
FIRST
GROWTH STIM4ULUS
EVENT
EVENT
MT EVENT MT ~~~SECOND WITH INDUCTION OF * GROWTH SIGNAL
Disorganized MT (via colchicine) interfere with transmission of signal arc or
INDUCTION OF
REPLICATIVE MACHINERY
DIVISION AND DIVISION
Con A modulates and inhibits expression of growth signal
FIG. 5. Proposed scheme for early events related to commitment. MT, microtubules. The position of entry into "B" phase (4) is not known. The src product acts ultimately on the SMA; this effect has been assayed by observing changes in the microtubular component of the SMA, but data available at present do not allow the direct target of the src product to be identified.
transmission of an outside signal but inducing a growth signal related to the second event. Although, at this time, this scheme is defined only in terms of correlations, it provides a convenient
framework for future tests of the SMA hypothesis. grateful to Dr. H. Hanafusa for his generous gift of the vihis advice on their propagation in culture. This work was -supported in part by USPHS Grants AI-11378, AM-04256, and AI-09273. We
are
ruses used in this study and for
1. Edelman, G. M. (1976) Science 192; 218-226. 2. Yahara, I. & Edelman, G. M. (1975) Exp. Cell Res. 91, 125142. 3. Gunther, G. R., Wang, J. L. & Edelman, G. M. (1976) Exp. Cell Res. 98, 15-22. 4. Smith, J. A. & Martin, L. (1973) Proc. Natl. Acad. Sci. USA 70, 1263-1267. 5. McClain, D. A. & Edelman, G. M. (1976) J. Exp. Med. 144, 1494-1508. 6. Edelman, G. M. & Yahara, I. (1976) Proc. Natl. Acad. Sci. USA 73,2047-2051. 7. Kawai, S. & Hanafusa, H. (1971) Virology 46,470-479. 8. jazwinski, S. M., Wang, J. L. & Edelman, G. M. (1976) Proc. Natl. Acad. Sci. USA 73,2231-2235. 9. Temin, H. M. (1971) J. Cell. Physiol. 78, 161-170. 10. Gunther, G, R., Wang, J. L., Yahara, I., Cunningham, B. A. & Edelman, G. M. (1973) Proc. Natl. Acad. Sci. USA 70, 10121016. 11. Ash, J. F., Vogt, P. K. & Singer, S. J. (1976) Proc. Natl. Acad. Sci.
USA 73,3603-3607. 12. Bell, J. G., Wyke, J. A. & Macpherson, I. A. (1975) J. Gen. Virol. 27, 127-134. 13. Baker, M. E. (1976) Nature 282,785-786. 14. Chen, K., Heller, J. & Canellakis, E. S. (1976) Biocherr. Biophys. Res. Commun. 68,401-408.
Role of surface modulating assemblies in growth control of normal and transformed fibroblasts (Rous sarcoma virus/concanavalin A/microtubules/cell cycle)
DONALD A. MCCLAIN, PETER D'EUSTACHIO, AND GERALD M. EDELMAN The Rockefeller University, New York, N.Y. 10021
Contributed by Gerald M. Edelman, December 9,1976
in the lymphocyte. Individual lymphocytes are recruited into the S phase of the cell cycle at different times after exposure to Con A, consistent with stochastic models (4) of the cell cycle. Colchicine appears to block this early recruitment event (3). High doses of Con A induce anchorage modulation of surface receptors and inhibit mitogenesis but do not interfere with the primary growth signal (5). Additional evidence for a role of the SMA in growth control has come from the observation that it-is a direct or indirect target of the sarc gene product of Rous sarcoma virus (RSV) in chick embryo fibroblasts (6). By using temperature-sensitive mutants of RSV, it was found that expression of the sarc gene product resulted in rapid loss of microfilament and MT structure, accompanied by--loss of anchorage modulation, andf owed by loss of normal growth control. In view of the observations on lymphocytes, it was of particular interest to determine the relationship between the effects of the sarc gene product and those of Con A and colchicine in this less heterogeneous cell population. It was found that for. both normal and virally transformed fibroblasts, there appears to be an intimate relationship between the state of the SMA and the regulation of cell growth. MATERIALS AND METHODS Chick embryo fibroblasts (CEF) were maintained in culture and transformed with RSV (SR-A strain, or the tsNY68 mutant of this strain) (7) as described previously (6). To study cellular morphology, cells grown on 18 mm square coverslips were treated and examined in situ (6). Serum starvation was achieved by replacing growth medium with serum-free medium containing 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. To inhibit protein synthesis, cells were incubated in the presence of 0.1 mM puromycin (dihydrochloride salt, Sigma). Ninety percent of protein synthesis was inhibited within 15 min of adding the drug tocultures of growing cells. Inhibition was reversed by removing the drug. The rate of DNA synthesis was estimated from the incorporation of [3H]thymidine (Schwarz/Mann) as detected by autoradiographic analysis of cells grown on coverslips or by bulk counts of acid-precipitable material (3). In vitro DNA replication was assayed as described (8). The morphology of cellular MT was assessed by immunofluorescence microscopy (6). RESULTS Perturbation of the SMA and Growth Control in Normal Fibroblasts. CEF were arrested in the G1 phase of the cell cycle by incubation in serum-free medium (9). Addition of serum to these starved fibroblasts (Fig. 1) resulted in a wave of DNA synthesis beginning at about 8 hr. Addition of colchicine to a final concentration of 1 WM at the same time as the serum delayed the entry of cells into S phase and reduced the number
ABSTRACT Cellular microtubules, microfilaments, and surface receptors have been postulated to form a surface modulating assembly that regulates surface receptor mobility and cell growth. To test this hypothesis, we examined three agents known to affect cell growth [colchicine, concanavalin A (Con A), and the src gene product of Rous sarcoma virus] for their effects on chick embryo fibroblasts. Individual cells from serum-starved normal fibroblast populations became committed to enter S phase at various times over a 12 hr period after exposure to serum. Colchicine and other microtubule-disrupting agents blocked entry into S phase at a point close to the commitinent point for each cell. The lectin Con A also blocked entry into the S phase when present in doses sufficient to modulate surface receptor mobility. In contrast, succinyl-Con A, which does not induce surface modulation, had no effect. Both Con A and colchicine blocked the appearance of cytopsmic factors capable of stimulating DNA replication in a cellfr system. To study endogenous effects on the surface modulating assembly, we infected fibroblasts with a Rous sarcoma virus (thNY68) having a temperature-sensitive mutation in the transforming (src) gene. We have previously shown that microtubular and microfilamentous structures of the surface modulating assembly are direct or indirect targets of the src gene product with consequent reduction in the capacity of Con A to induce surface modulation. TsNY68-infected fibroblasts shifted to the nonpermissive temperature acquired normal microtubular morpholoky more rapidly (2 hr) than cells grown at the permissive temperature in the presence of protein synthesis inhibitors (7.5 hr) This suggests that the src gene product acts directly on the surface modulating assembly rather than via the nucleus or at the level of protein synthesis. Furthermore, "transformation" of the surface modulating assembly was partly blocked by treatment of the infected cells with Con A but not succinyl-Con A. Both Con A and colchicine inhibited entry into the S phase following a shift from -nonpermissive to permissive growth conditions. All of these observations are in accord with the hypothesis that the surface modulating assembly acts as a signal regulator in growth control. It has been suggested that submembranous arrays of microtubules (MT), microfilaments, and associated contractile and membrane proteins form a surface modulating assembly (SMA) that controls the mobility of cell surface- receptors (1, 2) and also regulates signals from the cell surface to the cell interior (1, 3). In support of this idea, local crosslinkage of cell surface glycoproteins by lectins induces a propagated restriction in the lateral mobility of a variety of cell surface receptors (anchorage modulation) that depends on cytoplasmic MT (1, 2). Moreover, the state of the SMA (particularly, of its MT components) appears to be causally related to early events in growth simulation or inhibition. This is shown by the effects of the lectin concanavalin A (Con A) and MT-disrupting agents on mitogenesis Abbreviations: MT, microtubules; SMA, surface modulating assembly; Con A, concanavalin A; RSV, Rous sarcoma virus; CEF, chick embryo fibroblasts. 666
Cell Biology: McClain et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
667
Table 1. Inhibition by Con A and colchicine of serum-induced DNA synthesis and of appearance of DNA replication activity in serum-starved normal CEF In vitro DNA
Incorporation of replication [3H]thymidine (pmol of
TiM
fW)
The
(hr)
FIG. 1 (left). Inhibition of DNA synthesis by colchicine after release of serum starvation in CEF. Cells (2 X 105) were seeded onto coverslips in 2 ml of medium with serum. After the cells settled, the coverslips were transferred to serum-free medium. After 60 hr, 0.22 ml of fetal calf serum was added (time 0 on abscissa) (0). Parallel cultures were also made 1 uM in colchicine (0). At various times, 6 ACi of [3H]thymidine were added to the cultures; after 2 hr the cultures were processed for autoradiography (3). FIG. 2 (right). Effect of colchicine on commitment to serum stimulation. Serum-starved cells on coverslips (see Fig. 1) were placed in medium containing 10% fetal calf serum at time 0. At the times indicated, coverslips were washed with buffered saline and put in serum-free medium (0-0), colchicine was added to 1 AM (O- -0) or both n---- -0). At 12 hr, the cultures received 6 ACi of [3H]thymidine, and at 13 hr they were processed for autoradiography (3).
into cells
dTTP/ml
Condition
(cpm)
of extract)
Cells, no serum Cells + serum Cells + serum + colchicine (1 ,M) Cells + serum + Con A (30mg/ml) Succinyl-Con A (100 .g/ml)
1,900 29,900
265 413
14,500
230
10,700 32,600
280
Cells (106) in 60 mm dishes were serum-starved for 48 hr. Serum was added to 10% concomitantly with Con A or colchicine, and 5 mCi of [3H]thymidine was added 12 hr later. At 13 hr, incorporation of counts into acid-rrecipitable material was determined for duplicate cultures. For the in vitro assay, cell-free extracts prepared 9 hr after serum addition were incubated with frog spleen nuclei and labeled nucleotide triphosphates for 1 hr at 370 (8). Results are the means of duplicate determinations for both 20 and 40 gl of extract.
-
of cells entering S phase. Similar results were obtained with 3T3 fibroblasts. The amount of label incorporated per cell in S phase (estimated from grain counts of autoradiograms) was not affected by colchicine, and thus the effect of the drug did not appear to be due to inhibition of [3H]thymidine transport. Lumicolchicine, which does not dissociate MT, did not inhibit the entry of the CEF into S phase. Other agents that disrupt MT-podophyllotoxin and vinblastine-had the same inhibitory effect at 1 tiM as iolchicine. The effect of colchicine was reversible: CEF exposed to the drug but then washed free of it responded to serum as well as untreated cells. The kinetics of inhibition by colchicine closely paralleled the kinetics of commitment to serum stimulation. It is convenient to define the point at which a cell will go on to replicate even after the growth stimulus is removed as the commitment point (3). To study the kinetics of commitment, CEF that had been arrested by serum starvation were exposed to serum. At various times thereafter, the serum was removed. Twelve hours after the initial exposure to serum, the number of cells in S phase was determined. In accord with previous observations (9), committed CEF began appearing soon after serum was added and the number of these cells increased for a period of at least 12 hr (Fig. 2). If, after adding serum, colchicine was added at various times, reduced numbers of cells in S phase were observed 12 hr after the serum addition (Fig. 2). Less inhibition was seen the later the colchicine was added. When serum was removed at the same time that colchicine was added, the same amount of inhibition was observed as a function of time as with serum removal or colchicine addition alone. This suggests that both of these treatments of serum-stimulated CEF were affecting the same subpopulations of cells at any given time. Another exogenous agent that appears to affect MT is the lectin Con A, and this agent also inhibited growth of CEF after release from serum starvation (Table 1). The lectin was added mouse
concomitantly with the serum in doses that inhibit receptor mobility (6). Autoradiographic analysis confirmed that the inhibition ofgrowth reflected a restriction of the number of cells entering S phase. In contrast, dimeric succinyl-Con A (10) did not inhibit growth. Because the lectin and its derivative have similar binding specificities, the lack of inhibition by succinyl-Con A suggests that the inhibition by Con A is not due to competition for, or interference with, the binding of serum growth factors to the fibroblast surface. It is notable that succinyl-Con A stimulates lymphocyte growth, but unlike Con A, shows no inhibition of mitogenesis or of cell surface receptor mobility (10). Both Con A and colchicine also inhibited the appearance of cytoplasmic factors that stimulate the replication of nucleochromatin in vitro (8) (Table 1). The level of these factors was low in extracts of resting, serum-starved CEF. With serum stimulation, the activity began increasing shortly before the CEF entered S phase; in the presence of Con A or colchicine no such increase was seen. Endogenous and Exogenous Perturbation of the SMA in Transformed Fibroblasts. Additional evidence for the hypothesis that the SMA is involved in the regulation of cell commitment and division has been obtained using endogenous alteration of cells by viral transforming genes such as the sarc gene of RSV. We have previously examined the relationship between expression of the gene and the state of the SMA. CEF infected with tsNY68 virus, a strain of RSV which has a temperature-sensitive mutation in its sarc gene, are phenotypically transformed when grown at 370, but become phenotypically normal when shifted to 410. We have shown that cells grown at 370 do not contain organized cytoplasmic MT, but acquire these structures within 2 hr of being shifted to 410. Conversely, the organized MT disappear within 2 hr of a shift from 410 to 370 (6). We wished to show that the effect of the src gene product on the SMA is cytoplasmic and is not related to the control of protein synthesis or nuclear events. This demonstration would facilitate investigation of the relationship be-
668
Cell Biology: McClain et al.
Proc. Natl. Acad. Sci. USA 74 (1977)
A
B
7
C
807
~60\CI
40
C20-
0
2
4
6
8
10
12
0
2
4
6
8
0
12
2
0
6
4
Time (hr) FIG. 3. Effect of inhibition of protein synthesis on MT patterns at permissive and nonpermissive temperatures. (A) CEF infected with tsNY68 virus and grown on coverslips for 12 hr at 370 were either incubated at 37° in the presence of 0.1 mM puromycin (@) or shifted to 410 (0). At each time point shown, slips-were fixed, stained, and scored for MT morphology. "Normal" CEF exhibited radial arrays of organized MT. "Transformed" CEF were diffusively stained (6). (B) CEF infected with SR-A virus and grown at 37 (0) or 41° (-) were incubated further at the same temperature in the presence of puromycin and scored for MT. (C) CEF infected with tsNY68 virus and grown at 410 for 36 hr were shifted to 370 in the presence (v) or absence (v) of puromycin, incubated for various periods, and scored for MT.
tween the effects of the src gene product and those of colchicine and Con A. The relative effects of growth at 410 and of growth at 370 were therefore examined in the presence of protein synthesis inhibitors. If the src product itself were not acting on the SMA, but rather at a stage prior to translation to induce the appearance of a separate SMA effector, then a shift to 410 or the inhibition of protein synthesis would both prevent new synthesis of that effector. In both cases, the return of the SMA to the normal phenotype would result from the natural decay of the effector substance, and the cells should revert to the normal phenotype after the temperature shift at the same rate as after blocking protein synthesis. In contrast, if the sarc gene product acted directly on the SMA, a shift to 410 should inactivate the effector itself and cause a more rapid loss of the transformed phenotype. CEP infected with mutant (tsNY68) or wild-type (SR-A) virus were grown at 370 or 410 in the presence or absence of puromycin and scored for microtubular morphology by indirect immunofluorescence (Fig. 3). When CEF infected with mutant virus and grown at 370 were shifted to 410, organized MT appeared in most of the cells within- 2.5 hr (Fig. 3A). If these cells were maintained at 370 and puromycin was added, however, less than 25% of the cells had organized MT after 2.5 hr. Approximately 7.5 hr incubation with the drug was needed for the process to go to completion, consistent with the observations of others (11) on RSV-transformed rat kidney cells. CEF infected with wild-type SR-A virus and grown at either temperature lacked organized MT (6). Incubation of these cells in the presence of puromycin led to a slow increase in the fraction of cells containing organized MT (Fig. 3B). The rates of appearance of such cells at the two temperatures were indistinguishable from each other and from the rate observed for mutant cells at 370 (Fig. 3A). When cells infected with mutant virus and grown at 410 were shifted to 370, organized MT disappeared from most of- the cells in less than 1 hr (Fig. 3C). When puromycin was added to the culture medium at the time of the shift to 370 however, no change in the MT pattern could be detected even - after a 4 hr incubation at 370. All of the data support the conclusion that the src gene product itself interacts with components of the SMA, and that it does not exert its effects in the
nucleus or at some stage of the control of protein synthesis. it is possible that the endogenous agent, the sarc product, acts ultimately on the same cytoplasmic structure as the exogenous agent Con A. If so, the addition of Con A might be expected to alter the expression of the transformed phenotype. When CEF infected with the mutant virus and grown at 410 were shifted to 370 in the presence of Con A, the fraction of cells whose MT became disordered was reduced (Fig. 4). This inhibitory effect was not observed in cells treated with succinyl-Con A, nor was it observed in cells treated with Con A in the presence of the competitive inhibitor a-methyl-D-mannoside. We next determined whether Con A and colchicine would also block induction by the sarc product of factors leading to unregulated DNA replication. CEF were infected with tsNY68, grown at 410, arrested by serum starvation, and tested for entry into S phase after a shift-down in the presence or absence of Con A, colchicine, and serum (Table 2). Although the CEF that were shifted to 370 without serum showed relatively lower levels of 60
u
40 I-
W
O
20
a13
11
33
100
pg/ml FIG. 4. Inhibition by Con A of the conversion of cytoplasmic MT to a "transformed" pattern. CEF infected with tsNY68 virus and grown on coverslips for 36 hr at 410 were incubated for 15 min at 410 in the presence of various amounts of Con A (0) or succinyl-Con A (0), incubated with the lectin at 370 for 1 hr, and then scored for MT morphology. The frequency of diffusely stained cells in a population grown continuously at 410 (no lectin) is shown by the dashed line
Cell Biology: McClain et al. Table 2. Effects of Con A and colchicine on growth of synchronized tsNY68-infected cells
Proc. Natl. Acad. Sci. USA 74 (1977)
669
to block entry of neuroblastoma cells into S phase (13) and to block the serum-induced increase of ornithine decarboxylase activity seen in the G1 phase (14). All of these results suggest that Peak [3H]- % Inhibi- % Inhibithe effect is a general one acting in a variety of cells. thymidine tion by tion by Several lines of evidence suggest that the inhibition by Con incorporation Con A colchicine A of the entry into S phase is related to the modulation of surCondition (30 ipg/ml) (1 /.M) (cpm) face receptor mobility: (i) Con A, but not succinyl-Con A, inhibits both fibroblast mitogenesis (Table 1) and surface receptor Cells grown at 410, mobility (6); (ii) Con A inhibits lymphocyte mitogenesis with 53 serum added 7640 47 the same dose-response curve as it inhibits receptor mobility Cells shifted to 370, (5); and (iii) succinyl-Con A shows no high dose inhibition of serum added 1900 59 68 lymphocyte mitogenesis and no modulation of receptor moCells shifted to 370, bility (10). no serum added 840 54 100 These observations are in accord with those on transformed cells. In particular, expression of the src gene of RSV leads to Cells (3 X 105) infected with tsNY68 and plated in 60 mm dishes alterations in the SMA of chick embryo fibroblasts (6). In cells were grown for 24 hr at 410 in complete medium, and then for 48 hr infected with a RSV carrying a temperature-sensitive sarc at 410 in serum-free medium. Calf serum (10%) was then added or the mutation (tsNY68) (7), alterations in the structure of the SMA, cells were shifted to 370 or both. Con A or colchicine was added at the time of the shift. At 4 % hr intervals, incorporation of [3H~thymidine and anchorage modulating functions are seen withiSn into acid-precipitable material was measured (see Table 1). Cells at of a shift from the nonpermissive to the permissive temperature 410 without serum incorporated 430 cpm into acid-precipitable ma(6). We have now shown that a shift up to the nonpermissive terial. Peak DNA synthesis was observed at 12-13 hr at 410 and at of a cell transformed by tsNY68 virus results in temperature 21-22 hr at 370. a much more rapid return to the normal phenotype than does blocking protein synthesis at the permissive temperature. DNA synthesis than those with serum, the response was reThe sarc gene product couldcaffect the SMA either by reguproducible, confirming the previous observations of Bell et al. the synthesis of SMA components at the levels of tranlating (12). The cells that were shifted to 370 with serum showed a or translation or by interacting directly with SMA scription diminished entry into S phase that was delayed by approxiin the cytoplasm (6). In the first case, infected ficomponents mately 9 hr as compared with the cells arrested and then broblasts shifted to restrictive conditions (inactivating all sac stimulated with serum at 410. This delay is probably an effect gene product in the cell) should acquire ordered microtubules of the src gene product. It appears not to be due simply to slower no more rapidly than cells grown under permissive conditions metabolism at 370, because the response of normal CEF to in the absence of protein synthesis (preventing synthesis of new serum at 370 (Fig. 1) is kinetically similar to that of the ts NY68src gene product). We observed a large difference in rate after infected cells at 41°. The key observation is that when Con A the two treatments, however. This result is therefore most was added to the cells at the time of the temperature shift at a readily explained by the hypothesis that the sarc product acts concentration that stabilized the untransformed MT morpost-translationally as a cytoplasmic effector leading either phology (Fig. 4) it also inhibited the entry into S phase (Table or indirectly to an altered SMA. directly 2). Colchicine had a similar inhibitory effect. Thus, both agents If the sarc product, Con A, and colchicine act at the level of that affect the SMA block the induction by sarc of unregulated the SMA, then these agents should interact at both the morDNA synthesis. phological and functional levels. We have found several such interactions (Fig. 4 and Table 2). Con A stabilized the unDISCUSSION transformed microtubular staining pattern of tsNY68-infected The effects of Con A and colchicine on both anchorage modcells, and Con A blocked the entry of serum-starved tsNY68ulation and lymphocyte mitogenesis have prompted us to hyinfected cells into S phase after a 41° to 370 temperature shift. pothesize that the SMA may be a regulator of mitogenic signals Neither of these effects was induced by succinyl-Con A which from the cell surface (1, 3). In this paper, we have shown that is incapable of inducing surface modulation. Furthermore, like Con A, colchicine, and the product of the RSV transforming colchicine, expression of the src gene itself both disrupts migene (src) all perturb both the SMA and the cell cycle of ficrotubular morphology and interferes with serum stimulation broblasts in a fashion consistent with this hypothesis. (Table 2). We hypothesize that the SMA may serve as a-mulThe data on serum-stimulated normal fibroblasts resemble tistate integrator of the varied signals that affect cell growth and those previously obtained with Con A-stimulated lymphocytes. divisionii7TfeFochastic properties of commitment may be reFibroblasts, like Iymphocytes, were stimulated asynchronously, lated to the capacity of the SMA to exist in multiple states. The i.e., the longer a population of CEF was exposed to serum, the sarc product may represent a decontrolled or altered form f a larger the fraction of cells recruited into a determinate of phase normal regulator of this system. of the cell cycle leading to the S phase (3, 9). Smith and Martin It is necessary to be cautious in relating biochemical events (4) have suggested that the entry into a determinate phase of directly to morphological changes. This is emphasized by the G1 is probabilistic, and the asynchrony we have observed in observation that both the src gene and colchicine treatment commitment could reflect the same transition from the inderesult in dispersion of the microtubular patterns. Nevertheless, terminate ("A") state to the determinate ("B") phase postulated a tentative order of events is suggested by our data. A diagram by these authors. showing a possible place for the SMA in the order of events Colchicine appears to inhibit an early event in the entry of leading to stimulation, is given in Fig. 5. The first microtubular fibroblasts from the arrested G1 or Go phase into the S phase. event is sensitive to colchicine and it may represent the "start" The kinetics of the colchicine inhibition (Fig. 2) suggest that, or commitment point. The second event is defined by Con as in the lymphocyte (3), the drug acts to block mitogenesis at A-induced anchorage modulation and growth inhibition (2, 5, or near the crucial commitment point. Colchicine is also known 6). The sarc gene product may affect both events, blocking
670
Proc. Nati. Acdd. Sci. USA 74 (1977)
Cell Biology: McClain et al. "1B PHASE"
"A STATE" SRC PRODUCT SERU14 OR
EXTERNAL *MEMBRAE
FIRST
GROWTH STIM4ULUS
EVENT
EVENT
MT EVENT MT ~~~SECOND WITH INDUCTION OF * GROWTH SIGNAL
Disorganized MT (via colchicine) interfere with transmission of signal arc or
INDUCTION OF
REPLICATIVE MACHINERY
DIVISION AND DIVISION
Con A modulates and inhibits expression of growth signal
FIG. 5. Proposed scheme for early events related to commitment. MT, microtubules. The position of entry into "B" phase (4) is not known. The src product acts ultimately on the SMA; this effect has been assayed by observing changes in the microtubular component of the SMA, but data available at present do not allow the direct target of the src product to be identified.
transmission of an outside signal but inducing a growth signal related to the second event. Although, at this time, this scheme is defined only in terms of correlations, it provides a convenient
framework for future tests of the SMA hypothesis. grateful to Dr. H. Hanafusa for his generous gift of the vihis advice on their propagation in culture. This work was -supported in part by USPHS Grants AI-11378, AM-04256, and AI-09273. We
are
ruses used in this study and for
1. Edelman, G. M. (1976) Science 192; 218-226. 2. Yahara, I. & Edelman, G. M. (1975) Exp. Cell Res. 91, 125142. 3. Gunther, G. R., Wang, J. L. & Edelman, G. M. (1976) Exp. Cell Res. 98, 15-22. 4. Smith, J. A. & Martin, L. (1973) Proc. Natl. Acad. Sci. USA 70, 1263-1267. 5. McClain, D. A. & Edelman, G. M. (1976) J. Exp. Med. 144, 1494-1508. 6. Edelman, G. M. & Yahara, I. (1976) Proc. Natl. Acad. Sci. USA 73,2047-2051. 7. Kawai, S. & Hanafusa, H. (1971) Virology 46,470-479. 8. jazwinski, S. M., Wang, J. L. & Edelman, G. M. (1976) Proc. Natl. Acad. Sci. USA 73,2231-2235. 9. Temin, H. M. (1971) J. Cell. Physiol. 78, 161-170. 10. Gunther, G, R., Wang, J. L., Yahara, I., Cunningham, B. A. & Edelman, G. M. (1973) Proc. Natl. Acad. Sci. USA 70, 10121016. 11. Ash, J. F., Vogt, P. K. & Singer, S. J. (1976) Proc. Natl. Acad. Sci.
USA 73,3603-3607. 12. Bell, J. G., Wyke, J. A. & Macpherson, I. A. (1975) J. Gen. Virol. 27, 127-134. 13. Baker, M. E. (1976) Nature 282,785-786. 14. Chen, K., Heller, J. & Canellakis, E. S. (1976) Biocherr. Biophys. Res. Commun. 68,401-408.
