Jul 17, 1986 - Microinjection of p2l' induced c-fos protein accumulation in three types of 3T3 cells ... such as those related to growth factors and their receptors.
MOLECULAR AND CELLULAR BIOLOGY, Jan. 1987, p. 523-527 0270-7306/87/010523-05$02.00/0 Copyright © 1987, American Society for Microbiology
Vol. 7, No. 1
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Microinjection of Transforming ras Protein Induces c-fos Expression DENNIS W. STACEY,* THOMAS WATSON, HSIANG-FU KUNG,t AND TOM CURRAN Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 Received 17 July 1986/Accepted 24 September 1986
Microinjection of p2l' induced c-fos protein accumulation in three types of 3T3 cells. The induction was rapid and efficient and persisted for many hours. In addition, anti-ras antibody dramatically reduced c-fos accumulation after serum stimulation of injected cells. However, cells which expressed p2l1 continuously did not maintain a high level of c-fos expression.
entry into the cell cycle when introduced into NIH 3T3 cells by microinjection (8, 23). These effects, however, require at least 12 h. Furthermore, the requirement for p21ras is known to occur late in mitogenic stimulation, just before the initiation of DNA synthesis (16). In this study, we provide evidence to demonstrate a relationship between the activity of p2lras and the induction of c-fos expression. This observation indicates that p2lras might function during the early stage as well as the late stages of mitogenic stimulation. Previously, it was shown that p2lras purified from Escherichia coli is biologically active after microinjection into cultured cells. Recipient NIH 3T3 cells become morphologically transformed for at least 24 h after p21ras injection and are induced to initiate a cycle of DNA synthesis in the absence of added growth factors (23). In this study, p2lras was prepared from bacterial cells expressing the BALB murine sarcoma virus Ha-ras gene as well as those expressing the normal cellular Ha-ras homolog (14). These proteins, prepared in soluble form in the absence of other contaminating or solubilizing proteins (3.0 mg/ml), were microinjected into the cytoplasm of NIH 3T3 cells which had been rendered quiescent by culturing in 0.5% calf serum for 24 h. At various times after injection, the cells were fixed and stained for c-fos protein by using afos-specific peptide antibody (5). The c-fos protein began to accumulate within the nuclei of ras-injected cells between 1 and 2 h after injection. Similar results were obtained when c-fos induction was assayed by using fos-specific tumor-bearing rat antiserum (4) (data not shown). By comparison, calf serum treatment routinely induces fos protein accumulation within 1 h after addition (17). The brief delay observed after ras injection in comparison with that of serum induction might result from the time required for modification of the bacterial protein. In addition to the rapid induction of c-fos expression, viral p2lras was also observed to produce high levels of c-fos protein accumulation, which persisted for at least 22 h after injection. In Fig. 1, a comparison has been made between the expression of c-fos 3 h after serum addition and that which occurred 4, 10, and 22 h after viral p2lras injection. The p21ras-injected cells were maintained in 0.5% serum-containing medium after injection. In all cases, the injected protein resulted in levels of c-fos protein expression at least as great as those resulting from serum addition. This was the case even when cells injected with p2lras 22 h previously were compared
A number of studies have implicated the cellular homologs (proto-oncogenes) of retroviral oncogenes in the control of cell proliferation and differentiation. The proto-oncogenes c-fos, c-myc, and c-myb, which encode nuclear proteins, are induced after the addition of serum to quiescent cells (10, 11, 12, 17). Although the biological functions of these proteins are not known, c-fos expression is the earliest known transcriptional response to mitogenic stimulation (10, 17). The protein product of the c-ras proto-oncogene (p2lras, which is associated with the plasma membrane) is required for progression through the cell cycle (16). Recent evidence suggests that p2lras may be involved in a mitogenic signal transduction mechanism involving other proto-oncogenes, such as those related to growth factors and their receptors (22). The c-fos gene is expressed at relatively low levels in the majority of cell types, including fibroblasts. However, treatment of fibroblasts with polypeptide growth factors and other agents leads to a dramatic but transient induction (10, 12, 17). In serum-deprived fibroblasts, activation of c-fos transcription occurs within 5 min of adding serum or growth factors, such as platelet-derived growth factor (10). Subsequently, there is an accumulation of mature mRNA which reaches peak levels at 30 to 45 min poststimulation (17). The c-fos protein is also synthesized soon after stimulation with serum (12, 17). Maximal nuclear fluorescence with a fosspecific antibody is detected at 1 to 2 h postinduction, is reduced at about 4 h, and is undetectable at 20 h (17). The c-fos protein is normally transported to the nucleus and extensively modified within 15 min (3, 15, 17). Similar profiles of c-fos induction have been obtained after stimulation of cells with many different mitogenic agents (2). Since microinjection of p21ras induces DNA synthesis in mouse NIH 3T3 cells (23), we wished to determine whether introduction of p2lras would also activate c-fos expression. The c-ras genes can be converted to transforming oncogenes by a single point mutation (19, 20, 24). The resulting protein is able to induce morphological transformation and * Corresponding author. t Present address: Laboratory of Biochemical Physiology, Biological Response Modifiers Program, Division of Cancer Treatment, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701.
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On the basis of these p21ras injection experiments, it was clear that a connection existed between ras activity within the cell and c-fos production. It was therefore possible that c-ras proteins played a role in the normal induction of c-fos accumulation after serum stimulation. To test this possibility, a monoclonal antibody known to neutralize p2lras activity was used. Previous studies have established the neutralizing potential and specificity of action of the antibody Y13-259, originally prepared by Furth et al. (9). For example, the injected antibody has been shown to neutralize the activity of purified coinjected p2lras and to abolish the transformed phenotype of ras-transformed cells (13). This antibody inhibits the initiation of the S-phase in NIH 3T3 cells unless a viral ras gene mutant has been previously introduced into the cells which do not bind the antibody (18). To analyze the participation of cellular ras in the serum induction of c-fos, three types of quiescent 3T3 cells received an injection of antibody 259 or non-neutralizing control antibody 238. Serum (20%) was added to the injected
FIG. 1. Induction of c-fos protein by viral p2lras protein at various times after injection. NIH 3T3 cells were cultured for 24 h in 0.5% calf serum-containing medium. Serum (20%) was added to the culture (A) 3 h before fixation and stained with an affinity-purified antibody against fos protein. Viral p2lras protein (3.0 mg/ml) was prepared as described previously (14) and microinjected into cells which were fixed at 4 h (B), 10 h (C), or 22 h (D) and stained for c-fos protein. Untreated serum-deprived cells were also stained for c-fos protein (E). Stained cells were exposed for equivalent lengths of time on Kodak Tri-X (Eastman Kodak Co., Rochester, N.Y.) film and printed under identical conditions on medium-contrast (Kodabromide FIll) paper.
with cells stained soon after serum addition, when c-fos protein accumulation was near its peak. To establish the specificity of p2lras induction of c-fos accumulation, the activity of viral p2lras was compared with that of bacterially expressed cellular p2lras. In several separate injection experiments, viral and cellular p2lras at 3.0 and 0.3 mg/ml were injected into NIH, Swiss, or BALB 3T3 cells. Injected cells were fixed and stained for c-fos protein after 10 to 18 h. In all cases, the greatest accumulation of c-fos was associated with the higher concentrations of the transforming viral protein. The higher concentration (3.0 mglml) of cellular p2lras led to c-fos accumulation in some cases, but the levels observed were no greater than those produced by injection of 1/10 the concentration (0.3 mg/ml) of viral p21as (Fig. 2). This result is entirely consistent with the activities of the two forms of the protein in inducing cellular transformation and entry into the cell cycle (23). Cellular p21as is known to induce both effects, but only at high concentrations relative to that required by transforming viral p21as (23). The induction of c-fos by p2lras, therefore, mimics the relative abilities of these proteins to induce cellular transformation. No accumulation of c-fos above a background level was ever observed with 0.3 mg of cellular p21ras per ml or with 3.0 mg of the unrelated protein bovine serum albumin or interleukin II per ml.
C FIG. 2. Comparison of c-fos induction by viral and cellular p2lras injections. Serum-deprived NIH 3T3 cells received injection of viral p2lras at 3.0 mg/ml (A) or 0.3 mg/ml (B) or received injections of cellular p2lras at 3.0 mg/ml (C). At 10 h after injection, cells were fixed and stained for c-fos protein. Separate plates were photographed and printed under identical conditions (as described in the legend to Fig. 1). Most cells in the right half of the panel were injected while cells at the left were uninjected. Cells injected with 0.3 mg of c-ras protein per ml did not accumulate identifiable c-fos protein (not shown).
VOL. 7, 1987
FIG. 3. Inhibition of c-fos protein accumulation by anti-ras antibody. These BALB 3T3 cells were serum deprived for 24 h. Anti-ras antibody (10 mg/ml) was injected into the cells in the left half of panel B, and the cells were cultured for another 18 h before addition of serum (20%) to cells in panels B and C. At 80 min after serum addition, all cells were fixed, stained for c-fos, and photographed under fluorescence optics. Panel A indicates the background staining of cells which had not been induced by serum, while panel C indicates the level of staining by cells after serum induction. Cells at the left of panel B, which are nearly equivalent in fluorescent appearance to uninduced cells, are those which received anti-ras antibody injection before serum induction. Uninjected cells
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cultures, and the cells were fixed and stained forfos protein at a variety of times (from 45 min to 18 h) after serum addition. In no case did the non-neutralizing control antibody interfere with fos protein accumulation (data not shown). Neutralizing antibody 259, however, inhibited c-fos accumulation in NIH, Swiss, and BALB 3T3 cell types at all times tested. While inhibition was unmistakable, it was clear that the antibody was unable to block c-fos protein accumulation totally. Similar observations were made when purified porcine platelet-derived growth factor (Bethesda Research Laboratories, Gaithersburg, Md.) was used in the place of serum to induce c-fos expression. The most dramatic inhibitions were observed in BALB 3T3 cells 80 min after serum addition, at approximately the time c-fos protein concentration was approaching its highest level. In this case, antibodyinjected cells often contained c-fos protein levels barely distinguishable from that of uninduced control cells (Fig. 3). To eliminate the possibility that the physical process of microinjection had been inhibitory, cells received antibody injection but were not induced with serum until 18 h thereafter. The antibody is known to remain active in cells for this length of time (13). Any adverse affect of microinjection, however, would have disappeared after several hours. Inhibition of c-fos protein accumulation was indistinguishable in cells injected just before or 18 h before serum addition, indicating that the inhibitions were not the result of physical damage to the cells during microinjection (data not shown). These data indicate that c-ras proteins may play an important role in the induction of c-fos but leave uncertain what that role is. Inhibition offos protein accumulation after inoculation of p21ras antibody was often only partial. Thus, it is possible that the antibody was not able to completely inhibit c-ras activity and that the low level of ras activity in injected cells might then support a reduced rate of c-fos protein synthesis. However, c-fos expression can be induced in other cell types by multiple biochemical pathways, some of which may not require the c-ras function (3, 15). The incomplete inhibition of c-fos accumulation by injected anti-ras antibody might, therefore, be due to the action of such a ras-independent pathway in 3T3 cells. To assess a possible role for p2lras in the processes involved in c-fos induction, we examined the activation of c-fos expression in Ha-ras-transformed NIH 3T3 cells compared with that in nontransformed NIH 3T3 cells after serum stimulation. In both cell types, little or no c-fos protein was detected in cells maintained in either 0.5 or 10% serum (Fig. 4, lanes 1, 3, 5, and 7). However, after the addition of 20% serum to serum-deprived cultures, a similar c-fos induction was obtained in normal and transformed cells (Fig. 4, lanes 2 and 6). Only a very small increase in c-fos protein synthesis was observed after the addition of serum to cultures maintained in 10% serum (Fig. 4, lanes 4 and 8). The c-fos protein was detected as a band of approximately 62 kilodaltons, which is the size of the highly modified fos protein that is synthesized after serum induction (2, 17). Thus, continuous expression of the transforming ras protein did not maintain elevated levels of c-fos expression and did not affect c-fos induction by serum. Similarly, by using the immunofluorescence assay, the c-fos protein was only detected in rastransformed cells after serum induction. This induction was inhibited by microinjected anti-ras antibodv, as observed in at the right of panel B accumulated expectedly higni levels of c-fos protein. Under these conditions, the antibody inhibited c-fos protein accumulations with high efficiency.
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that transformation by viral oncogenes related to a growth factor or membrane-bound growth factor receptors (6, 7, 21, 25) requires p2lras activity within the cell (16). From these studies, it is apparent that p2lras activity is related to the induction of at least one member of the nuclear protooncogene family. This observation supports the idea that mitogenic stimulation is controlled by a series of interactions involving a group of genes which includes many of the known proto-oncogenes. Recent results from Bar-Sagi and Feramisco (1) demonstrate rapid (within 30 min to 1 h) effects of p21as on membrane ruffling and pinocytosis in injected cells. In the case of the transforming ras protein, these effects persist for more than 15 h, which is similar to the induction time of the c-fos protein. When these observations and the results of this study are considered, it appears that p2lras can activate cellular processes which lead to rapid alterations in cell surface activities and to the generation of a second messenger that ultimately acts within the nucleus to promote changes in gene expression. We thank E. P. Reddy and J. I. Morgan to helpful reviews of this manuscript.
FIG. 4. Synthesis of c-fos protein in ras-transformed and in normal NIH 3T3 cells. NIH 3T3 cells transformed by the viral Ha-ras gene (lanes 1 to 4) and normal NIH 3T3 cells (lanes 5 to 8) were maintained in either 0.5% serum (lanes 1, 2, 5, 6) or 10% serum (lanes 3, 4, 7, 8) for 24 h. For 30 min before labeling, fresh serum (20%) was added to determine c-fos protein synthesis in serumstimulated cells (lanes 2, 4, 6, and 8) compared with nonstimulated cells (lanes 1, 3, 5, and 7). In each case, cells were incubated with 300 ,uCi of [35S]methionine (=800 ,uCi/mmol; Amersham Corp., Arlington Heights, Ill.) per ml for a 30-min pulse-labeling period. Preparation of cell lysates, immunoprecipitation with fos-specific peptide antibodies, sodium dodecylsulfate-polyacrylamide gel electrophoresis, fluorography, and autoradiography were performed exactly as described previously (17). The numbers on the left indicate the positions of the 14C-methylated marker proteins (Amersham). The position of the highly modified form of the c-fos protein (p62c-fOs) present in serum-stimulated cells is indicated.
nontransformed cells. Furthermore, microinjection of p21ras induced c-fos expression in the ras-transformed cells (data not shown). While it is clear that injected viral p2lras efficiently induced c-fos for an extended period of time, the labeling studies indicate that c-fos protein levels eventually returned to uninduced levels in cells persistently exposed to high levels of viral p2lras. Furthermore, while cellular p2lras appeared to play an important role in c-fos induction, constitutive high levels of viral p2lras did not inhibit a normal serum induction. These observations indicate that the control of c-fos expression involves a combination of ras-
dependent and ras-independent pathways. Long-term maintenance of c-fos expression depends upon factors independent of high levels of ras expression. These results emphasize the importance of cellular p2lras in the control of proliferation. In addition to the known requirement for cellular p2lras late in the G1 phase of the cell cycle, this study suggests a role for cellular p2lras early in the Go phase. While there is no known substitute for p2lras activity late in the G1 phase of normal cells (16), it appears that the function of p2lras in c-fos induction might also be performed by other biochemical pathways. In addition, this study extends the list of proto-oncogenes and viral oncogenes related to p2lras activity. Previous studies have shown
LITERATURE CITED 1. Bar-Sagi, D., and J. R. Feramisco. 1986. Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins. Science 223:1061-1068. 2. Curran, T., R. Bravo, and R. Muller. 1985. Transient induction of c-fos and c-myc is an immediate consequence of growth factor stimulation. Cancer Surv. 4:655-681. 3. Curran, T., and J. I. Morgan. 1985. Superinduction of c-fos by nerve growth factor in the presence of peripherally active benzodiazepines. Science 229:1265-1268. 4. Curran, T., and N. M. Teich. 1982. Identification of a 39,000 dalton protein in cells transformed by the FBJ osteosarcoma virus. Virology 116:221-235. 5. Curran, T., C. Van Beveren, N. Ling, and I. M. Verma. 1985. Viral and cellular fos proteins are complexed with a 39,000dalton cellular protein. Mol. Cell. Biol. 5:167-172. 6. Doolittle, R. F., M. W. Hunkapiller, L. E. Hood, S. G. Devare, K. C. Robbins, S. A. Aaronson, and H. N. Antoniades. 1983. Simian sarcoma virus oncogene, v-sis, is derived from the gene (genes) encoding platelet-derived growth factor. Science 221: 275-277. 7. Downward, J., Y. Yarden, E. Mayes, G. Scrace, N. Totty, P. Stockwell, A. Ulirich, J. Schlessinger, and M. D. Waterfield. 1984. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequence. Nature (London) 307: 521-527. 8. Feramisco, J. R., M. Gross, T. Kamata, M. Rosenberg, and R. W. Sweet. 1984. Microinjection of the oncogene form of the human H-ras (T-24) protein results in rapid proliferation of quiescent cells. Cell 38:109-117. 9. Furth, M. E., L. J. Davis, B. Fleurdelys, and E. M. Scolnick. 1982. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J. Virol. 43:294-304. 10. Greenberg, M. E., and E. B. Ziff. 1984. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature (London) 311:433-438. 11. Kelly, K., B. H. Cochran, C. D. Stiles, and P. Leder. 1983. Cell-specific regulation of the myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell 35:603-610. 12. Kruier, W., J. A. Cooper, T. Hunter, and I. M. Verma. 1984. Platelet-derived growth factor induces rapid transient expression of the c-fos gene and protein. Nature (London) 312: 711-716. 13. Kung, H.-F., M. R. Smith, E. Bekesi, V. Manne, and D. W. Stacey. 1986. Reversal of transformed phenotype by monoclonal antibodies against Ha-ras p21 proteins. Exp. Cell Res. 162:
VOL. 7, 1987 363-371. 14. Manne, V., E. Bekesi, and H.-F. Kung. 1985. Ha-ras proteins exhibit GTPase activity: point mutations that activate Ha-ras gene products result in decreased GTPase activity. Proc. Natl. Acad. Sci. USA 82:376-380. 15. Morgan, J. I., and T. Curran. 1986. Role of ion flux in the control of c-fos expression. Nature (London) 322:552-555. 16. Mulcahy, L. S., M. R. Smith, and D. W. Stacey. 1985. Requirement for ras proto-oncogene function during serum-stimulated growth of NIH3T3 cells. Nature (London) 313:241-243. 17. Muller, R., R. Bravo, J. Burckhardt, and T. Curran. 1984. Induction of c-fos gene and protein by growth factors precedes activation of c-myc. Nature (London) 312:716-720. 18. Papageorge, A. G., B. M. Willumsen, M. Johnsen, H.-F. Kung, D. W. Stacey, W. C. Vass, and D. R. Lowy. 1986. A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene. Mol. Cell. Biol. 6:1843-1846. 19. Reddy, E. P., R. K. Reynolds, E. Santos, and M. Barbacid. 1982. A point mutation is responsible for the aquisition of transforming properties by the T-24 human bladder carcinoma oncogen. Nature (London) 300:149-152. 20. Santos, E., S. R. Tronick, S. A. Aaronson, S. Pulciani, and M.
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21.
22.
23. 24.
25.
527
Barbacid. 1982. T-24 human bladder carcinoma oncogene is an activated form of the normal homologue of BALB and HarveyMSV transforming gene. Nature (London) 298:343-347. Sherr, C. J., C. W. Rettenmier, R. Sacca, M. F. Roussel, A. T. Look, and E. R. Stanley. 1985. The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF-1. Cell 41:665-676. Smith, M. R., S. J. DeGudicibus, and D. W. Stacey. 1986. Requirement for c-ras proteins during viral oncogene transformation. Nature (London) 320:540-543. Stacey, D. W., and H.-F. Kung. 1984. Transformation of NIH3T3 cells by microinjection of Ha-ras p21 proteins. Nature (London) 310:508-511. Tabin, C. J., S. M. Bradley, C. I. Bargmann, R. A. Weinberg, A. G. Papageorge, E. M. Scolnick, R. Dahr, D. R. Lowy, and E. H. Chang. 1982. Mechanism of activation of a human oncogene. Nature (London) 300:143-149. Waterfield, M. D., G. T. Scrace, N. Whittle, P. Stroobant, A. Johnsson, A. Wasteson, B. Westermark, C.-H. Heldin, J. S. Huang, and T. F. Deuel. 1983. Platelet-derived growth factor is structurally related to the putative transforming protein p28sis of simian sarcoma virus. Nature (London) 304:35-39.
