Microinjection offos-Specific Antibodies Blocks DNA Synthesis in ...

MOLECULAR AND CELLULAR BiOLOGY, Apr. 1988, p. 1670-1676

Vol. 8, No. 4

0270-7306/88/041670-07$02.00/0 Copyright © 1988, American Society for Microbiology

Microinjection of fos-Specific Antibodies Blocks DNA Synthesis in Fibroblast Cells KARL T. RIABOWOL,1* ROBERT J. VOSATKA,2t EDWARD B. ZIFF,2 NED J. LAMB,' AND JAMES R. FERAMISCO' Cold Spring Harbor Laboratory, P. 0. Box 100, Cold Spring Harbor, New York 11724,1 and Department of Biochemistry and Kaplan Cancer Center, New York University Medical Center, New York, New York 100162 Received 29 September 1987/Accepted 22 December 1987 Transcription of the protooncogene c-fos is increased >10-fold within minutes of treatment of fibroblasts with serum or purified growth factors. Recent experiments with mouse 3T3 cell lines containing inducible fos antisense RNA constructs have shown that induced fos antisense RNA transcripts cause either a marked inhibition of growth in continuously proliferating cells or, conversely, a minimal effect except during the transition from a quiescent (Go) state into the cell cycle. Since intracellular production of large amounts of antisense RNA does not completely block gene expression, we microinjected affinity-purified antibodies raised against fos to determine whether and when during the cell cycle c-fos expression was required for cell proliferation. Using this independent method, we found that microinjected fos antibodies efficiently blocked serum-stimulated DNA synthesis when injected up to 6 to 8 h after serum stimulation of quiescent REF-52 fibroblasts. Furthermore, whenfos antibodies were injected into asynchronously growing cells, a consistently greater number of cells was prevented from synthesizing DNA than when cells were injected with nonspecific immunoglobulins. Thus, whereas the activity of c-fos may be necessary for transition of fibroblasts from Go to G1 of the cell cycle, its function is also required during the early G, portion of the cell cycle to allow subsequent DNA synthesis.

The fos oncogene was first identified as the transforming gene of the FBJ murine osteosarcoma virus (7) and has since been sequenced in its proviral and cellular forms. The c-fos gene encodes a protein of 380 amino acids (26) which localizes in the nucleus (3) and is highly phosphorylated, resulting in proteins with apparent molecular weights ranging from 53,000 to 68,000 when separated by sodium dodecyl sulfate-polyacrylamide gels (1). Due to rapid transcriptional induction and accumulation of c-fos protein after mitogen stimulation of quiescent fibroblasts (9, 13, 19), the DNAbinding properties of the protein (5, 23), and the reported ability of v-fos of transactivate transcription from heterologous promoter sequences (24), it has been suggested that c-fos serves a role in coupling external stimuli such as mitogens to long-term transcriptional responses, leading to cell proliferation. In an effort to determine whether the function offos was required for cell growth, we isolated lines of rat fibroblast cells transfected with a glucocorticoidinducible fos antisense RNA construct (Riabowol et al., manuscript in preparation). We found, as others have reported (20), that high levels of fos antisense RNA reduced the level of c-fos protein when serum was added to quiescent cells, and that fewer cells were able to incorporate [3H] thymidine in the presence of dexamethasone and serum compared with untransfected cells. In some of our cell lines, however, production offos antisense RNA did not correlate well with reduced levels of fos protein or with a decreased rate of logarithmic growth as previously reported (11). To determine directly whether the function of c-fos was required for growth of fibroblasts by an independent method, we introduced polyclonal, affinity-purified antibodies di-

rected against the fos protein into both quiescent (serumdeprived) and logarithmically growing cells by needle micro-

injection. MATERIALS AND METHODS

Cells. Rat embryo fibroblast (REF-52) cells (14) were cultured in Dulbecco modified Eagle medium (DMEM) containing 15% fetal calf serum (FCS). Before microinjection, cells were made quiescent by incubation in DMEM containing 0.1% FCS for 24 h. Cells were induced to enter the cell cycle (before or after injection) by the addition of DMEM containing 15% FCS and 2 ,uCi of [3H]thymidine per ml. For microinjection of antibodies into asynchronously growing cultures, cells were plated and grown for 48 h before microinjection, at which point 2 ,uCi of [3H]thymidine per ml was added as a bolus. Synchronization of cells by double thymidine block was done by growing monolayers alternately in the presence or absence of 2.5 mM thymidine (16 h with, 10 h without, and 16 h with thymidine) in DMEM containing 15% FCS and releasing them from the block by incubation in DMEM containing 15% FCS. Antigen. The bacterially produced trp-fos fusion protein used for immunization and affinity purification of antibodies consists of a 262-base-pair PstI fragment of v-fos (FBJ murine osteosarcoma virus), which is conserved between the viral and cellularfos proteins, fused to PstI-linearized plasmid PATH11, which contains coding sequences for the bacterial trpE protein (R. J. Vosatka, A. HermanowskiVosatka, and E. B. Ziff, manuscript in preparation). Maintenance of the reading frame of the construct was verified by DNA sequencing of relevant portions of the construct. Bacterial production of the fusion protein was stimulated by

* Corresponding author. t Present address: Department of Pediatrics, Mount Sinai Hospital, New York, NY 10029.

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REQUIREMENT OF c-fos PROTEIN FOR DNA SYNTHESIS

the addition of 10 ,ug of indole acrylic acid (Sigma Chemical Co.) per ml to log-phase bacterial cultures growing in M-9 medium containing 100 jxg of ampicillin per ml. After incubation for 12 h at 37°C, cells were collected by centrifugation and suspended in 50 mM Tris (pH 7.5)-0.5 mM EDTA-0.3 M NaCl. Lysozyme was added to a final concentration of 1 mg/ml, and the solution was stirred on ice for 15 min. Lysis of bacteria was accomplished by addition of 4% Nonidet P-40 to a final concentration of 0.2%. An equal volume of 1.5 M NaCl containing 12 mM MgCl2 was added, followed by the addition of DNase I (Sigma) to 2 ,ug/ml, and the solution was stirred on ice for 1 h. The solution was centrifuged at 4°C for 15 min at 4,000 x g, and the insoluble pellet containing fusion protein was collected, washed, boiled in Laemmli sample buffer, and electrophoresed through preparative 10% polyacrylamide gels. The fusion protein was identified by brief staining with Coomassie brilliant blue, excised, electroeluted, and dialyzed extensively against 0.1 M 3-(N-morpholino)propanesulfonic acid (pH 7.0) for coupling or against phosphate-buffered saline (PBS) for immunization. Antibodies. Female New Zealand White rabbits were initially injected subcutaneously with 100 ,ug of trp-fos protein mixed 1:1 with complete Freund adjuvant. After the first injection, animals were boosted every 4 weeks with 100 ,ug of fusion protein diluted with an equal volume of incomplete Freund adjuvant and bled 7 days after each booster injection. Antiserum (10 ml) was precipitated by the dropwise addition of saturated (NH4)2SO4 to a final concentration of 38% and stirred at 4°C for 6 to 12 h. The pellet obtained by centrifugation at 10,000 x g for 15 min, which contained immunoglobulins, was suspended in 5 ml of PBS and dialyzed at 4°C against several changes of PBS. The dialysate was repeatedly passed over a 1-ml Affi-Gel 10 column containing purified trpE protein covalently bound to the matrix according to the instructions of the manufacturer (Bio-Rad Laboratories). The flow-through fraction was then passed over a similarly prepared column containing bound trp-fos fusion protein four times. The column was sequentially washed with 5 ml each of PBS, 0.1 M NaHCO3 (pH 7.0), and 0.1 M NaHCO3 (pH 7.0) containing 0.5 M NaCl and eluted directly into 2 M Tris (pH 8.0) by the addition of 0.1 M glycine (pH 2.5). Fractions containing immunoglobulins, as visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue, were pooled and concentrated to S mg of protein per ml in 0.5x PBS by centrifugation in Centricon 30 microconcentrators (Amicon Corp.) at 4°C. Microinjection. Microinjection into the cytoplasm of cells was carried out as previously described (6). Except where indicated, control and fos antibodies were injected into subconfluent cells at a concentration of 5 mg/ml. Immunochemistry. After growth for 24 h in medium containing 2 ,uCi of [3H]thymidine per ml, cells were fixed by sequential immersion for 10 min at room temperature in PBS containing 3.7% formaldehyde, 0.5% Triton X-100, and 0.05% Tween 20. Injected cells were identified by incubation with biotinylated goat anti-rabbit antibodies (Cooper Biochemical, Inc.; 1:200 dilution in PBS containing 1% bovine serum albumin for 30 min at 37°C), followed by strep-avidinconjugated horseradish peroxidase (same conditions as above) and development in 4-chloro-1-napthol-H202 as recommended by the supplier (Bio-Rad). Cells were then photographed with Kodak Techinal Pan film and prepared for emulsion autoradiography as described previously (6, 28). For determination of the levels of c-fos protein in the nuclei

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of cells previously injected with antibodies, affinity-purified fos antibody was biotinylated as suggested by the manufacturer (Vector Laboratories Inc.) and used for indirect immunofluorescence analysis (28). RESULTS Figure 1A shows a Coomassie brilliant blue-stained sodium dodecyl sulfate-polyacrylamide gel of the anti-fos (lane f) and control (lane c) antibodies used in this study. Figure 1B shows the nuclear staining typical of fos (27), observed when serum-deprived (upper) or serum-stimulated (lower) fibroblasts were fixed, incubated with this antibody, and processed for indirect immunofluorescence analysis. Figure 1C shows the results of using this antibody for immunoprecipitation of cell lysates from serum-deprived (lanes b and d) or serum-stimulated (lanes a and c) rat fibroblast cells. Control lane e shows the cell lysate from serum-stimulated fibroblasts immunoprecipitated with preimmune serum. When the fos antibody was used to immunoprecipitate cell lysates from fibroblasts under nondenaturing conditions, an additional band was detected with an Mr of 39,000, which has been previously shown to complex with both viral and cellularfos proteins (4; data not shown). In addition, preliminary observations indicate that microinjection of fos antibodies into RS2 cells (a cell line derived by transformation of rat 208F cells with FBJ murine osteosarcoma virus) results in morphological reversion these cells 24 h after injection

(unpublished observation). Microinjection into quiescent cells. Figure 2 shows the effect of injecting anti-fos and control antibodies into REF52 cells. Cells made quiescent (Go) by incubation in medium containing 0.1% FCS for 24 h were injected with either anti-fos or control immunoglobulins 2 h before refeeding with complete medium containing 15% FCS and 2 ,uCi of [3H]thymidine per ml. After 24 h in this medium, cells were fixed and stained for immunoglobulins by using conjugated horseradish peroxidase. This technique allows unequivocal identification of cells which have been microinjected, and thus there is no uncertainty as to whether subsequent cellular effects are a consequence of the injected antibody. Both anti-fos (Fig. 2A) and control (Fig. 2C) immunoglobulins were maintained in the cytoplasm of injected cells for this time period. Upon processing for emulsion autoradiography, it is clear that antibodies directed against fos prevented the incorporation of [3H]thymidine (Fig. 2B), whereas control immunoglobulins at the same concentration did not (Fig. 2D) in the majority cells injected. When cells were injected with anti-fos antibodies 8 h after the addition of complete medium, approximately 60% of injected cells were able to incorporate [3H]thymidine (Fig. 2E). These cells normally undergo DNA synthesis in response to serum between 15 and 18 h after stimulation with approximately 80% synchrony. Results of injecting control orfos (Fig. 2F) antibodies were indistinguishable when cells were injected 12 or more h after the addition of complete medium. To more precisely estimate the time during which the function of c-fos is required, cells were injected at various times after refeeding with complete medium (Fig. 3). Whereas injection of control immunoglobulins uniformly resulted in only a 6 to 12% decrease in the number of cells incorporating [3H]thymidine over a 24-h period, injection of anti-fos antibodies had markedly different effects on DNA synthesis in these experiments. Injection of anti-fos antibodies at 2 h before to 4 h after serum stimulation prevented 90% of the injected cells from incorporating [3H]thymidine,

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RIABOWOL ET AL.

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FIG. 1. Characterization of affinity-purified fos antibodies. (A) Lane f shows fos affinity-purified polyclonal antibody stained with Coomassie blue dye after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lane c is control antibody prepared from the same antiserum by depletion offos antibodies. A second control antibody from preimmune rabbit antiserum was also used and gave similar results. (B) Nuclear fluorescence pattern typical of fos (20, 25, 27) when the affinity-purified fos antibody was used to stain serum-arrested (upper panel) or serum-stimulated (lower panel) fibroblasts fixed as outlined in Materials and Methods. After incubation of fixed cells with anti-fos antibodies and incubation with rhodamine isothiocyanate-conjugated goat anti-rabbit immunoglobulin G, fields of cells were photographed with the same exposure times to allow direct comparison. Cells fixed and stained for c-fos after growth for 48 h in medium containing 0.1% FCS showed no detectable nuclear staining. Arrows in panel C show the major isoforms of c-fos recognized when fos antibodies were used to immunoprecipitate identical numbers of counts from [35S]methionine-labeled cell lysates of serum-stimulated (lanes a and c) or serum-arrested (lanes b and d) fibroblasts. The band with an Mr of approximately 45,000 is probably actin, whereas bands at Mr 45,000 to 50,000 likely represent vimentin since they are also present when immunoprecipitating with preimmune serum (lane e). For immunoprecipitation, quiescent fibroblasts were incubated in methionine-free medium containing 200 ,uCi of [35S]methionine per ml in the absence or presence of 15% dialyzed FCS for 90 min and lysed in boiling sodium dodecyl sulfate-gel electrophoresis sample buffer. Immunoprecipitation in antibody excess and fluorography were performed as previously described (28).

whereas injection of control immunoglobulins had little or no effect. Injection of anti-fos 8 to 14 h after stimulation resulted in considerably less inhibition of DNA synthesis within the 24-h time period. These data suggest that expression of c-fos provides a function necessary for traverse of the early G, portion of the cell cycle. Microinjection into logarithmically growing cells. To further test the idea that the function of c-fos is required for traverse of the cell cycle, anti-fos antibodies were injected into REF-52 cells growing asynchronously in complete medium (Fig. 4). After microinjection and growth for 24 h in the presence of 2 ,uCi of [3H]thymidine in 5 separate trials, 45 + 9% (mean ± standard deviation) of cells injected with anti-fos antibodies incorporated [3H]thymidine, whereas 86 8% of cells injected with control immunoglobulins incorporated [3H]thymidine. Since few, if any, cells would be in the Go phase of the cell cycle during logarithmic growth, this observation strongly supports the idea that c-fos expression is required during the cell cycle to enable progression into DNA synthesis, distinct from activities required for transition from a quiescent Go state induced by serum deprivation. Microinjection into cells synchronized by thymidine block. ±

Cells synchronized at the G1-S interface of the cell cycle released from thymidine block by the addition of DMEM containing 15% FCS and cultured 9 h before microinjection of control and anti-fos antibodies. After injection, cells were grown in the presence of FCS and [3Hlthymidine for 24 h, fixed, stained for immunoglobulins, and processed for emulsion autoradiography. Table 1 shows the results of four separate experiments. Again, most cells injected with control immunoglobulins synthesized DNA, whereas only 39 + 8% of cells injected with anti-fos antibodies were able to do so. Microinjection into different lines of fibroblast cells. Response to mitogens (21), cell density, and microinjected proteins (6) varies widely between different established and primary cell types. To determine whether the effects of injecting anti-fos antibody into REF-52 cells were common to other cell types, the effect of anti-fos injections on serum-stimulated DNA synthesis was determined with other cell types. Anti-fos immunoglobulin G (but not control immunoglobulins) similarly inhibited DNA synthesis induced by serum in rat 208F cells (22) and primary human and NIH 3T3 fibroblasts (data not shown). It would therefore were

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b-~~~~~~~~~FIG. 2. Microinjection of antibodies. At various times after injection of antibodies, cells were fixed, incubated with biotinylated goat anti-rabbit immunoglobulin G, washed with PBS, and incubated with strep-avidin-conjugated horseradish peroxidase. Staining was then completed as outlined in Materials and Methods. Stained fields of cells were photographed on Kodak Technical Pan film and processed for emulsion autoradiography as described previously (6). Panels A to D show two fields of cells fixed after injection with fos (A and B) or control (C and D) antibodies 2 h before the addition of 15% FCS and 2 FCi of [3H]thymidine per ml for 24 h. Panels A and C show HRP staining, whereas panels B and D show the corresponding fields of cells after emulsion autoradiography. Panels E and F show fields of cells microinjected with fos antibodies 8 and 12 h respectively, after the addition of FCS and [3H]thymidine. Results shown in Fig. 2 through 5 were obtained by injecting control and fos-specific antibodies at a concentration of 5 mg/ml. Injection of fos antibodies 2 h before the addition of medium containing 155 FCS at concentrations of 2.5, 0.5, and 0.1 mg/ml inhibited synthesis 92, 26, and 2% as well, respectively, as when used at S mg/ml.

appear that expression of fos is a common requirement for growth in several different fibroblast types. Determination of levels of c-fos in the nuclei of cells microinjected with antibodies. To determine the effect of antibody injection on levels of c-fos in the nucleus, quiescent fibro-

blasts were injected with control or anti-fos antibodies, refed with medium containing 15% FCS, and fixed 90 min later as described in Materials and Methods. After processing for indirect immunofluorescence analysis, cells injected withfos antibody (Fig. 5B) showed no detectable nuclear fluores-

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RIABOWOL ET AL.

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FIG. 3. Time course injections of anti-fos and control antibodies. Fibroblasts grown for 24 h in medium containing 0.1% FCS were injected before or after the addition of 15% FCS and 2 ,uCi of [3H]thymidine per ml as indicated on the abscissa. Fields of 25 to 40 cells were injected for each time point in the three separate experiments. Thus, each time point represents data from 75 to 120 injected cells. Cells which received antibody were identified by staining for immunoglobulins with horseradish peroxidase, and the percent incorporating [3H]thymidine was determined after emulsion autoradiography. The inset panel shows a time course of [3H] thymidine incorporation after the addition of serum to quiescent REF-52 cells under the conditions used in these studies. cence, whereas cells injected with control antibody (Fig. 5A) had levels of nuclear fluorescence comparable to those in uninjected cells.

DISCUSSION The observation that microinjection of anti-fos and control immunoglobulins differentially affects the ability of cells to synthesize DNA depending upon the time elapsed from serum stimulation rules out nonspecific or cytotoxic effects of anti-fos antibody microinjection. In contrast to previous reports of partial inhibition of logarithmic cell growth (11) or blockage of only serum-stimulated DNA synthesis (20) by intracellular production of fos antisense RNA, this study indicates a specific requirement of fos activity to allow traverse of the cell cycle. Microinjection of anti-fos antibodies prevented DNA synthesis in both serum-stimulated and asynchronous paradigms. From these results we cannot determine whether the large percentage of cells prevented from synthesizing DNA by injection of quiescent cells is a result of preventing entry into the cell cycle (Go to Gl) or of blocking subsequent transit through the early portion of G1. However, since a large percentage of cells was prevented from synthesizing DNA when injected during logarithmic growth and after release from thymidine block, any cells able to enter the cell cycle from a quiescent state would subsequently be blocked in early Gl. Our data indicate that the activity of c-fos is required for progression through the cell cycle. However, a previous study in which inducible production of fos antisense RNA was used to decrease production of c-fos protein (20) showed

FIG. 4. Microinjection into asynchronously growing cells. REF52 cells were plated and grown for 48 h in DMEM containing 15% FCS. Microinjection of control (A) and fos (B) antibodies and subsequent procedures were carried out as described in the legend to Fig. 3.

that entry into the cell cycle from quiescence was inhibited by production offos antisense RNA but that antisense RNA had little effect upon logarithmically growing cells. Since antisense RNA does not completely block gene expression (12, 20; P. Hooper and J. Coffin, personal communication) and antisense RNA should have little effect upon c-fos protein once synthesized, it is reasonable to assume that sufficient c-fos protein exists under these conditions to allow progression through the cell cycle. This assumption is further supported by the observation that sufficient c-fos protein exists in the cell to complex with the fos-associated 39-kilodalton protein for several hours after serum stimulaTABLE 1. Effect of antibody microinjection upon DNA synthesis in cells synchronized by thymidine block Control immunoglobulin

No.

injected 31 38 44 32

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26 34 38 29

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injected after serum stimulation, there are two populations of fos, one already in the nucleus and a second in the cytoplasm representing newly synthesized c-fos protein. Since the antibodies (injected into the cytoplasm) do not appear to freely enter the nucleus (Fig. 2A and C), we might expect that in these experiments the injected antibodies are predominantly capable of inhibiting the function of newly synthesized c-fos protein. It therefore seems that the latter experiments indicate the requirement for newly synthesized fos in the process of cell proliferation. It cannot be ruled out, however, that the Kd for antibody-fos binding is lower than that for c-fos with its proposed chromatin-binding sites (23). This could result in depletion of fos protein previously localized in the nucleus by cytoplasmic injection of anti-fos immunoglobulins. Quiescent REF-52 cells require 15 to 18 h to initiate the S phase of the cell cycle after the addition of serum. Since anti-fos antibody blocks 50% of cells from incorporating [3H]thymidine when injected 7 to 8 h after serum addition but has little effect when injected >12 h after the addition of complete medium, it appears that expression of c-fos is not required for events of late G1 leading to DNA synthesis. It

FIG. 5. Nuclear fluorescence of cells injected with antibodies. REF-52 cells grown in medium containing 0.1% FCS for 24 h were injected with control (A) orfos (B) antibodies 2 h before the addition of 15% FCS. After 90 min cells were fixed and sequentially incubated with rhodamine isothiocyanate-conjugated goat anti-rabbit immunoglobulin G, biotinylated rabbit anti-fos antibody, and strepavidin-conjugated fluorescein isothiocyanate. Microinjected cells are readily identified at both R fluorescein isothiocyanate and fluorescein isothiocyanate wavelengths. Fields of cells shown above were photographed at the fluorescein isothiocyanate emission wavelength to allow simultaneous visualization of fos nuclear fluorescence and cytoplasmic fluorescence, which identifies injected cells.

tion of quiescent fibroblasts (Vosatka et al., manuscript in preparation). Thus, the high levels of c-fos induction seen when cells are treated with mitogens (9, 10, 13. 19), viral proteins (29), ras proteins (25), drugs (17), or agents that induce differentiation (21) or are mechanically perturbed (27) may reflect a role in signal transduction. This role could be distinct from that fulfilled by low levels of c-fos protein present throughout the cell cycle (2). The effect of these preparations of antibodies on serumstimulated cell growth was determined by microinjection into the cytoplasm, where fos is initially produced as a translation product. We assume that the antibodies in the cytoplasm would bind to newly synthesized fos protein and thereby prevent it from functioning in the nucleus. This assumption would be valid for the experiments in which anti-fos antibodies were injected before serum stimulation. In other experiments in which anti-fos antibodies were

should be noted that similar results have been obtained by injecting monoclonal antibodies directed against the transformation-related protein p53 (16) and the ras protein (18). Antibodies directed against p53 blocked serum-stimulated DNA synthesis when injected up to 2 h after serum addition (16) but had no effect upon progression of several cell types from mitosis to the S phase (15). In addition, time course analysis of injection of ras antibody 259 was shown to efficiently block entry into the S phase of NIH-3T3 cells when the antibody was injected up to 6 h after the addition of serum to quiescent cells. Thus, p53, ras, and fos expression seem to be required for cells to initiate DNA synthesis from a resting Go state and, in the case of fos, also during logarithmic growth. The observation that microinjection of transforming ras protein induced c-fos expression, but that injection of ras antibody 259 did not completely block serum-induced expression of c-fos (25), again suggests that there are multiple, partially overlapping pathways involved in the activation of c-fos expression. The major protein species recognized by the fos antibody have Mrs similar to those previously described for the c-fos protein (Fig. 1C) (23). Weaker bands with Mrs of 45,000 to 50,000 are also visible in all lanes including the control (Fig. 1C, lane e), indicating that these bands likely represent the abundant proteins actin (Mr, 45,000) and vimentin (Mr, 50,000 to 55,000). However, it is possible that the antibody might also recognize small amounts of previously described fos-related antigens (8). It is thus formally possible that injected cells are prevented from synthesizing DNA due to interaction of the fos antibody with small amounts of proteins which share antigenic determinants with c-fos. We are now examining this possibility. This study, while demonstrating a requirement for fos activity during early G, before the S phase, leaves it exact role unknown. Many observations are suggestive of a role for c-fos in the control of gene expression after exposure to external stimuli. Our observations are not inconsistent with this idea and furthur suggest that considerably lower levels of fos are required to allow cells to initiate the S phase for 6 to 8 h after serum stimulation, a period of time much greater than the 1 to 2 h of very high levels of c-fos after the addition of serum. We are now in the process of examining cellular proteins for which intracellular levels are affected by the expression of c-fos protein.

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ACKNOWLEDGMENTS We thank J. D. Watson for support of this work, P. Renna for expert photographic assistance, and M. Szadkowski for preparation of the manuscript. This work was supported by Public Health Service grants from the National Institutes of Health (to E.B.Z. and J.R.F.). R.J.V. was a fellow of the National Institutes of Health MSTP and the New York University MSTP. K.T.R. was supported by a Public Health Service training fellowship from the National Institutes of Health and by an ACS institutional grant. N.J.L. was supported by an American Heart Association fellowship (Nassau county chapter).

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15. 16.

LITERATURE CITED 1. Barber, J. R., and I. M. Verma. 1987. Modification of fos proteins: phosphorylation of c-fos, but not v-fos is stimulated by 12-tetradecanoyl-phorbol-13-acetate and serum. Mol. Cell. Biol. 7:2201-2211. 2. Bravo, R., J. Burckhardt, T. Curran, and R. Muller. 1986. Expression of c-fos in NIH3T3 cells is very low but inducible throughout the cell cycle. EMBO J. 5:695-700. 3. Curran, T., A. D. Miller, L. Zokas and I. M. Verma. 1984. Viral and cellular fos proteins: a comparative analysis. Cell 36:259268. 4. 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. 5. Distel, R. J., H. S. Ro, B. S. Rosen, D. L. Groves, and B. M. Spiegelman. 1987. Nucleoprotein complexes that affect gene expression in adipocyte differentiation: direct participation of c-fos. Cell 49:835-844. 6. Feramisco, J. R., M. Gross, T. Kamata, M. Rosenberg, and R. W. Sweet. 1984. Microinjection of the oncogene form of the human H-ras (T24) protein results in rapid proliferation of quiescent cells. Cell 38:109-117. 7. Finkel, M. P., B. 0. Biskis, and P. B. Jinkins. 1966. Virus induction of osteosarcomas in mice. Science 151:698-701. 8. Franza, B. R., L. C. Sambucetti, D. R. Cohen, and T. Curran. 1987. Analysis of Fos protein complexes and Fos-related antigens by high-resolution two-dimensional gel electrophoresis. Oncogene 1:213-221. 9. 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. 10. Hayes, T. E., A. M. Kitchen, and B. H. Cochran. 1987. Inducible binding of a factor to the c-fos regulatory region. Proc. Natl. Acad. Sci. USA 84:1272-1276. 11. Holt, J. T., T. Venkat-Gopal, A. D. Moulton, and A. W. Nienhuis. 1986. Inducible production of c-fos antisense RNA inhibits 3T3 cell proliferation. Proc. Natl. Acad. Sci. USA 83:4794-4798. 12. Knecht, D. A., and W. F. Loomis. 1987. Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum. Science 236:1081-1086. 13. Kruiger, W., J. A. Cooper, T. Hunter, and I. M. Verma. 1984.

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18. 19. 20. 21.

22. 23. 24. 25.

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