JOURNAL OF CELLULAR PHYSIOLOGY 141:535-542 (1989)

Cell Cycle Dependent Growth Factor Regulation of Gene Expression

CLAUDIA J. MORGAN AND W. J. PLEDGER* Department of Cell Biology, Vanderbilt University School of Medicine, Nashville,

Tennessee 37232

The expression of the proto-oncogenes c-fo3 and c-niyc is a rapid response of Go-arrested fibroblasts to serum and peptide growth factors; however, the role of the c-fos and c-myc gene products in subsequent cell cycle transit is not under- stood. We examined the expression of c-fos and c-myc mKNA in Balbic 3T3 murine fibroblasts in response to platelet-derived growth factor (PDGF) and plate- let-poor plasma, using arrest points associated with density dependent growth inhibition or metabolic inhibition to synchronize cells in S phase of the cell cycle. The expression of c-fos and c-myc mRNA in Balbic 3T3 cells was differentially regulated with respect to growth factor dependence and cell cycle dependence. c-fos expression was induced in the presence of PDGF and was unaffected by plasma. The induction of c-fos expression in response to PDGF was cell cycle independent, occurring in cells transiting S phase and G, as well as in G,, arrest. In contrast, c-myc expression was both growth factor and cell cycle dependent. In Go arrested cells, c-myc expression was PDGF-dependent and plasma-inde- pendent, and PDGF was required for maintenance of elevated c-myc levels dur- ing G, transit. In cells transiting S phase, c-myc mRNA was induced in response to PDGF, but was also plasma-dependent in S phase cells that had been ”primed” by exposure to PDGF during S phase.

The regulation of growth by polypeptide growth fac- tors involves rapid changes in the expression of a num- ber of “immediate early genes” (Lau et al., 1987), in- cluding the proto-oncogenes c-fos and c-myc (Kelley et al., 1983; Cochran et al., 1983) and many others such as JE, KC, and JB, whose gene products are relatively uncharacterized. The function of these gene products in the cellular events that regulate the proliferative cycle is not understood. It is not known whether these genes are directly involved only in the modulation of G, traverse or in processes occuring during other phases of the cell cycle. Some of these gene products may be in- volved in intracellular communication.

The mechanism of regulation of the expression of these putative regulatory genes is speculative. The ki- netics of expression of c-fos and c-myc in arrested and cycling cells in response to serum and polypeptide growth factors have been described by several investi- gators (Bravo et al., 1985, 1986, 1987; Dean et al., 1986; Muller et al., 1984; Rollins et al., 1987). In gen- eral, stimulation of Go arrested cells with serum or growth factors results in elevated c-fos levels by ap- proximately 15 minutes after stimulation. c-fos mRNA is maximal at approximately 30 minutes after stimu- lation and declines to an undetectable level by 1-2 hours, even with continued stimulation. c-myc mRNA is elevated within 1 hour after serum or growth factor addition, is maximal a t approximately two hours, and declines to an intermediate level which is maintained throughout the cell cycle in the presence of serum. The responsiveness of these genes to induction by serum or growth factors during cell cycle transit is less well un-

derstood. Bravo et al. (1986) observed serum- inducibility of both c-fos and c-myc a t various times during G, and in cells synchronized in S phase by treat- ment with hydroxyurea. Alternatively, Rollins and co- workers (1987) found that c-myc was induced by PDGF in cycling cells preferentially over arrested cells, but that cycling cells were unable to respond to PDGF with increased expression of c-fos, which they found to be induced exclusively in Go arrested cells. Most of these studies have focused on Go arrested cells, heteroge- neously growing populations, or exponentially growing populations as they leave the cell cycle and enter Go arrest. The interpretation of the data obtained from these studies may be limited by ambiguities associated with population heterogeneity and assessment of the effect of cell cycle status on responsiveness.

The cell cycle parameters of the Balbic 3T3 murine fibroblast cell line have been defined to the extent that these cells can be synchronized a t numerous points in the cell cycle by experimental manipulation of growth factors, nutrients and metabolic inhibitors. Density-ar- rested Balb/c 3T3 cells become competent to proliferate after transient treatment with a competence factor such as platelet-derived growth factor (PDGF) (Pledger et al., 1978). These cells do not enter the cell cycle, but remain 12 hours from S phase. Competent cells are stimulated to proceed through G, to DNA synthesis by

Received June 12, 1989; accepted August 9, 1989 *To whom reprint requestskorrespondence should be addressed.

0 1989 ALAN R. LISS, INC

536 MORGAN AND PLEDGER

factors in platelet-poor plasma, the fluid portion of whole blood (Russell et al., 1984). The 12 hour GI phase is subdivided by plasma-dependent arrest points and nutritional arrest points (Leof et al., 1982a, 1983; Par- dee, 1984; Stiles et al., 1979). Once cells have entered S phase, the remainder of the cell cycle can be completed in the absence of supplemental growth factors (Whar- ton, 1983). In addition, cells can be synchronized in S phase by a variety of reversible inhibitors of DNA syn- thesis, such as hydroxyurea (Bravo et al., 1986), aphid- icolin (Heintz et al., 19821, and methotrexate (Scher et al., 1979).

These arrest points can be used to design protocols to compare the regulation of gene expression during dif- ferent phases of the cell cycle. In the studies described here, we have examined the expression of the proto- oncogenes c-fos and c-myc in response to PDGF and plasma in cells arrested in Go, undergoing synchronous transit through G, from Go arrest, and during S phase transition. We found that PDGF-induced expression of c-fos mRNA was not cell cycle dependent and was un- affected by plasma factors. In contrast, increased ex- pression of c-myc mRNA was not only induced by PDGF but was also in part regulated by plasma and was cell cycle dependent. The novel observation of the regulation of c-myc expression by plasma occurred only in S phase cells that were exposed to PDGF when they were in S phase.

METHODS Cell culture

Balbic 3T3 cells (clone A311 were grown in 10% calf serum-supplemented Dulbecco-Vogt modified Eagle's medium containing 4 mM glutamine, 50 U/ml penicil- lin, and 50 pgiml streptomycin (DME) in a humidified 5% CO, atmosphere. PDGF and dialyzed plasma were prepared as described (Herman et al., 1985). Unless otherwise specified, PDGF was used at 100 Uiml and all plasma preparations were exhaustively dialyzed. For density arrest, cells were grown to confluence in serum-containing medium and used 2-3 days after growth cessation.

S phase arrest Density-arrested cells were treated for 18 hours with

DME containing PDGF, 5% platelet-poor plasma, and 0.2 pM methotrexate (MTX, Sigma). Alternatively, density-arrested cells were made competent by culture for 6 hours with PDGF in DME containing 0.25% plate- let-poor plasma. Medium was then replaced with DME lacking PDGF, containing 5% plasma and 0.2 pM methotrexate for 18 hours. Cells were released from arrest by replacing the medium with MTX-free me- dium containing 106 nM hypoxanthine and 16 nM thy- midine, as described by Scher e t al., (1979).

RNA preparation and analysis Total cellular RNA was prepared by LiCl precipita-

tion of guanidine thiocyanate extracts, as described by Cathala et al. (1983). Twenty micrograms total RNA was separated by electrophoresis on 1.2% or 1.5% aga- rose-2.2 M formaldehyde gels and transfered to nitro- cellulose filters (Nitroplus 2000, MCI). Filters were baked in vacuo at 80°C, prehybridized in 50% forma- mide, 5 x SSC, 5 x Denhardt's solution, 100 pgiml de-

PDQF PPP TPA EQF 7-m-

1 2 3 4 6 6 7 8910111213

A

B

Fig. 1. c-fos expression in Go arrested cells stimulated by PDGF, TPA, and EGF. Density-arrested cells were treated with medium con- taining 0.25% plasma and PDGF (lanes 2 4 1 , TPA (160 nM, lanes 8-10), or EGF (50 ngiml, lanes 11-13), or with medium containing 5% plasma alone (lanes 5-7) for 0.5 hour (lanes 2, 5, 8, 111, 1 hour (lanes 3, 6, 9, 12), or 2 hours (lanes 4, 7, 10, 13). Lane 1: Untreated cells. A Northern blot analysis of c-fos mRNA. B: Corresponding RNA de- tected by ethidium bromide stain.

natured salmon sperm DNA at 42°C for a t least 4 hours and hybridized in 50% formamide, 5 x SSC, 1 x Den- hardt's solution, 100pgiml denatured salmon sperm DNA, 10% dextran sulfate, and denatured probe at 42°C for 24-48 hours. Probes were labeled by nick translation as described (Maniatis, et al., 19821 to spe- cific activities of 1-3 x lo8 cpmipg. Filters were washed to 0.5 x SSC at 42°C and autoradiographed a t - 70°C using intensifying screens. Autoradiographs were analyzed using an LKB Ultroscan XL laser den- sitometer. Where indicated, c-fos and c-myc signals were quantitated by normalizing to the signal for the constitutively expressed mRNA encoding cyclophilin detected by the 1B15 probe (Danielson et al., 1988).

Probes Probes included v-fos, the 1 kb PstI fragment of the

plasmid pfosl (Curran et al., 1982); c-myc, the 4.8 kb BamHIiXbaI fragment or the 1 kb SacI/XbaI fragment of pSV-c-myc-I containing the second and third exons of the mouse gene (Land et al., 1983); glyceraldehyde- 3-phosphate dehydrogenase, the 1 kb PstI fragment from pGAD-28, containing the chicken glyceraldehyde- 3-phosphate dehydrogenase cDNA (Dugiaczyk et al., 1983); and 1B15, the plasmid SP65-1B15, containing a 680 bp cDNA clone of rat cyclophilin mRNA (Danielson et al., 1988).

RESULTS Expression of c-fos mRNA in arrested and

cycling 3T3 cells Density-arrested Balbic 3T3 cells require only tran-

sient exposure to PDGF to become competent to enter the cell cycle (Pledger et al., 1978). The effect of PDGF on early gene expression was determined by northern blotting of total cellular RNA from quiescent and PDGF-treated cells. The effect of PDGF on c-fos expres- sion in synchronized cells was relatively unambiguous. We present these data on c-fos expression first as a means of defining the system and as a basis for com- parison with the complex pattern of regulation of c-myc expression described below. As shown in Figure 1, den-

CELL CYCLE DEPENDENT GENE EXPRESSION

GO S PHASEIG2 I 1’ 0 2 1 4 ’ 10 ‘ TIME FROM 8 PHASE

b RELEASE (HR)

537

0-fom + ?

QSPD I,

Fig. 2. c-fos expression in S phase cells stimulated by PDGF. Den- sity-arrested cells and cells released from S phase arrest into 5% plasma for 0, 2, 4, or 10 hours were treated with PDGF (50 U/ml) for 0.5 or 2 hours. Northern blots of total RNA were probed for c-fos and

sity-arrested cells did not express detectable c-fos mRNA. Addition of 5% platelet-poor plasma, which does not cause arrested cells to enter the cell cycle in the absence of PDGF, did not stimulate c-fos accumu- lation. PDGF induced rapid transient accumulation of c-fos mRNA, which declined to an undetectable level by one hour after growth factor addition. The increase in c-fos mRNA in response to PDGF did not require the presence of platelet-poor plasma at concentrations re- quired to promote cell cycle traverse, indicating that c-fos expression was a Go-associated event independent of progression into G,.

The tumor promoter 12-O-tetradecanoyl-phorbol-13- acetate (TPA) can replace PDGF in stimulating DNA synthesis and cell division in the presence of plasma (Frantz et al., 1979), although the mechanism of TPA- induced mitogenesis may differ from that of PDGF (Collins and Rozengurt, 1984; Coughlin et al., 1985; Olashaw et al., 1986). Addition of TPA to density-ar- rested cells induced c-fos mRNA accumulation compa- rable to that stimulated by PDGF (Fig. 1, lanes 8-10). Epidermal growth factor (EGF) is not a potent mitogen in density-arrested BALBic-3T3 cells, but a t a concen- tration of 50 ngiml stimulated a small induction of c-fos mRNA (25% of PDGF response, Fig. 1, lanes 11-13). The accumulation and decline in c-fos mRNA followed a similar time course in Balbic 3T3 cells regardless of the growth factor used to elicit expression.

Other investigators (Bravo et al., 1987; Rollins et al., 1987) have shown that cells growing exponentially in serum-containing medium do not express detectable c- fos mRNA. Some discrepancy exists in their observa- tions regarding the inducibility of c-fos mRNA by se- rum or PDGF during the cell cycle. To determine whether c-fos mRNA could be induced in homogeneous populations of cells a t points in the cell cycle other than Go, density-arrested Balbic 3T3 cells were treated with PDGF and serum in the presence of 0.2 FM methotrex- ate (MTX) for 18 hours. This treatment causes cells to become reversibly arrested in early S phase (Scher et al., 1979). A 3 hour pulse with tritiated thymidine im- mediately after release from the MTX inhibition re- sulted in 95% to 100% nuclear labeling indicating the cells where arrested in S phase. In agreement with the observations of Scher e t al. (1979), treatment of cells with colchicine after removal of MTX confirmed that

glyceraldehyde-3-phosphate dehydrogenase (GSPD) mRNA. G3PD mRNA was very low in Go arrested cells but was indicative of the relative amount of RNA in cycling cells. Arrowheads indicate cross- hybridization with the 18s and 28s ribosomal subunits.

mitosis occurred between 10 and 15 hours after MTX was removed (data not shown).

Cells arrested a t S phase were released from MTX and cultured in medium containing 5% plasma with or without PDGF. No c-fos mRNA was detected in cells immediately after release from the MTX block in 5% plasma (Fig. 2). Moreover, when MTX-inhibited cells were allowed to complete traverse through S phase and G, in 5% plasma, c-fos mRNA did not accumulate dur- ing 25 hours after MTX removal. Addition of PDGF to the culture at several timepoints after MTX removal consistently stimulated c-fos expression by 30 minutes (Fig. 2); as in Go arrested cells, the response was tran- sient, and c-fos mRNA decreased to an undetectable level by 2 hours after PDGF addition.

Expression of c - m y mRNA in Go arrested cells Other investigators (Bravo et al., 1987; Dean et al.,

1986; Rollins et al., 1987) have shown that arrested cells stimulated to proliferate express increased c-myc mRNA within one hour after stimulation. The accumu- lation of c-myc mRNA is maximal at 2 hours after stim- ulation and decreases thereafter to an intermediate level, which persists for a t least 18 hours (Dean et al., 1986). This persistent level of c-myc RNA is dependent on the continued presence of PDGF as illustrated by the following experiments. The c-myc responses were normalized to mRNA encoding cyclophilin as detected by the 1B15 probe (Danielson et al., 1988) which we found to be a superior constituatively expressed gene standard.

Density-arrested Balbic 3T3 cells can be stimulated to undergo DNA synthesis and cell division by addition of PDGF and platelet-poor plasma either together or sequentially. When density-arrested cells were given medium containing PDGF together with 5% plasma, the pattern of c-myc mRNA expression was similar to that observed by others (Fig. 3). c-myc mRNA was stimulated from an undetectable level to maximal by 2 hours after growth factor addition. By 12 hours after stimulation, c-myc mRNA declined to 20-25% of the peak value, and remained at that level for 30 hours after stimulation. In contrast, when cells were made competent by a 6 hour exposure to PDGF, then allowed to proceed through the cell cycle in PDGF-free medium containing 5% plasma, c-myc mRNA declined to an un-

538 MORGAN AND PLEDGER

0.10 \o-------o 0.00 1.. - -

0 6 12 18 24 30 36

TIME (hours)

1BW b

Fig. 3. c-myc expression in cells stimulated to proliferate from Go arrest. Density arrested cells were stimulated with medium contain- ing 5% plasma and PDGF for 30 hours (-0-1 or with medium contain- ing 0.2575 plasma and PDGF for 6 hours followed by 5 8 plasma- containing medium for 30 hours (-*-). The zero time was made from untreated quiescent cells. Upper panel: c-myc expression normalized to 1B15 mRNA. Lower panel: Corresponding northern blot analysis of cells stimulated with PDGF in a 6 hour pulse (left panel, -04 and in continuous incubation (right panel, -0-1. The left panel times were 9,2,6,12, and 30 hours; the right panel times were 0,2,6,12,16, and 36 hours.

detectable level between 12 and 26 hours after PDGF addition.

These observations suggest that the continued pres- ence of PDGF is required for the persistence of elevated c-myc mRNA during the cell cycle, and that a sustained increase in c-myc expression is not needed for cells t o complete DNA synthesis and cell division. To define regulatory events involved in the accumulation and persistence of c-myc mRNA during the cell cycle, we compared the expression of c-myc mRNA in cells stim- ulated with PDGF and plasma to enter the cell cycle from Go arrest, and in cells synchronized by reversible arrest in S phase.

Expression of c-myc mRNA in S phase and G, Balbic 3T3 cells were arrested in early S phase by

combined treatment with PDGF, 5% plasma, and MTX for 18 hours. The level of c-myc mRNA in cells at arrest in S phase was elevated in comparison to Go cells, and was approximately equivalent to the level of c-myc mRNA in cells 12 hours after stimulation from Go (Fig. 4). When the MTX-containing medium was removed and replaced with medium containing 5% plasma alone, there was a gradual accumulation of c-myc mRNA which reached a steady state at approximately 12 hours after release from the MTX block (Fig. 4B). The induction of c-myc expression in Figure 4 is con- sistent with the observations of Bravo et al. (1986), who demonstrated serum-inducibility of both c-fos and c- myc mRNA in cells arrested in S phase by treatment with hydroxyurea. Addition of PDGF or TPA at several

times after release did not stimulate additional c-myc mRNA accumulation over plasma alone (data not shown). When cells were released from the MTX inhi- bition into 0.25% plasma, c-myc mRNA accumulated after a delay, approaching the level in 5% plasma by twenty hours. Our results (data not shown) indicate that addition of PDGF to cells released in 0.25% plasma did not increase c-myc mRNA to the level of cells released from the MTX block in 5% plasma. These data suggest that the cells arrested in S phase, unlike density-arrested cells, could induce c-myc in response to plasma.

By replacing serum with a competence factor, PDGF, and platelet-poor plasma, we were able apparently t o dissociate the regulation of expression of the two onco- genes in S phase cells: c-fos expression was induced by PDGF and not by plasma, and c-myc expression was induced by plasma and not by PDGF. However, be- cause cultures in Figure 4 had been brought to S phase arrest by treatment with MTX in the presence of PDGF and plasma, cells were exposed to residual levels of PDGF in the medium while in early S phase. The ap- parent PDGF-independent, plasma-dependent re- sponse during S phase and G, could, in part, reflect the effect of exposure to PDGF at the G,!S boundary or in early S phase. In order to clarify the induction of c-myc expression by plasma and the cells apparent refractory response to PDGF in S phase, the following experiment was performed. Cells were made competent by incuba- tion with PDGF in 0.25% plasma for 6 hours, then transfered to fresh medium containing 5% plasma and methotrexate for 18 hours. This treatment resembles that in Figure 4 except that cells reach the arrest point in early S phase in the absence of PDGF in the me- dium. As shown in Figure 5B, when MTX was removed and cells were allowed to transit S phase in 5% plasma, c-myc mRNA was very low, and did not increase throughout 20 hours. However, the addition of PDGF to the plasma-containing medium resulted in a sub- stantial increase in c-myc mRNA, reaching a peak level by 6 hours after PDGF addition. By 12 hours, c-myc mRNA had declined by approximately one-half and remained at that level for a t least 8 hours. These data clearly indicate that PDGF can induce c-myc in S phase cells. The plasma-dependent induction of c-myc in S phase cells was dependent on a n initial S phase exposure to PDGF.

Dependence of c-myc expression in S phase on exposure to PDGF

It was apparent from these observations that 1) PDGF was able to induce c-myc mRNA accumulation in S phase cells, and 2) cells that had been brought to S phase in PDGF-containing medium accumulated c- myc mRNA in a plasma-dependent manner in the ab- sence of PDGF when allowed to complete traverse through S phase and G,. We were intrested in deter- mining whether a transient exposure to PDGF of cells arrested in S phase (as in Fig. 5) in the absence of PDGF would restore the ability of these cells to express increased c-myc mRNA in response to plasma during transit through S phase. Density-arrested cells were treated with PDGF for 6 hours, then allowed to traverse G, and accumulate in S phase by incubation in medium with 5% plasma and MTX for 18 hours. This

CELL CYCLE DEPENDENT GENE EXPRESSION 539

1.20

U l - 0.80 m ..- 3 2

0.40

0.00

1 0.80 7

1 1 0.00 0 4 8 12 16 20

TIME (hours) Fig. 4. c-myc expression in cells transiting G,IG, (A) and S phase/(=, (B). A: Density-arrested cells were treated with medium containing 5% plasma and PDGF. Zero time was untreated density-arrested cells. B: Cells were arrested in S phase in the continuous presence of PDGF. At time 0, S phase transit was initiated by replacing the medium with

medium containing 0.25% plasma (-0-1 or 5% plasma (-o-). Zero time represents cells arrested as described in MTX. Data points were ob- tained by northern blot analysis of c-myc mRNA normalized to 1B15 mRNA.

ro c m c > 2 I 0

0.60 0.60 A B

A - 0.40

0.20 A 0.20

0.00 0.00 o 4 8 12 16 20 24 o 4 a 12 16 20

TIME (hours) Fig. 5. c-myc expression in cells transiting Go/G, (A) and S phaseiG, (B) following brief exposure to PDGF in Go. A: Density-arrested cells were incubated for 6 hours with medium containing 0.25% plasma and PDGF. Medium was removed and replaced with 5% plasma-con- taining medium for the remaining incubation. Zero time was from untreated density-arrested cells. B: Density-arrested cells were made

protocol inhibited greater than 95% of the cells in early S phase without PDGF present. PDGF was then added to the MTX-containing medium of half of the cultures for an additional 6 hours before all cultures were trans- fered to PDGF-free, MTX-free medium containing 5% plasma and allowed to complete the cell cycle. Figure 6 shows the increase in c-myc mRNA levels in response to plasma in the cells that were transiently exposed to PDGF during arrest in early S phase. As shown (t = 20 h), the c-myc mRNA level in cells arrested in early S phase in the absence of PDGF was that of Go cells. As observed previously (Fig. 5), cells that had been ar- rested in PDGF-free medium did not accumulate c-myc mRNA in response to plasma alone during arrest or after release from arrest. Addition of PDGF to MTX- arrested cells caused a substantial increase in c-myc mRNA within 2 hours, even though the cells remained

competent by a 6 hour incubation with PDGF in 0.25% plasma-con- taining medium and brought to arrest in early S phase in medium containing 5% plasma and MTX. At time 0, S phase transit was ini- tiated by replacing the medium with medium containing 5% plasma alone (-0-1 or with PDGF (A), The zero time was taken from the MTX arrested cells that had not received additional treatment.

arrested in MTX (t = 26 h). When the MTX-arrested, PDGF-treated cells were subsequently released into 5% plasma alone, c-myc mRNA continued to accumu- late, in the absence of PDGF. Readdition of PDGF to these cells after two hours in plasma did not result in any additional accumulation of c-myc mRNA over that in plasma alone (data not shown).

A number of control experiments were performed to confirm that these observations were associated with cell cycle transit rather than unrelated effects of MTX on gene expression. Cells arrested in early S phase by treatment with aphidicolin rather than MTX, showed similar effects on the regulation of c-myc expression (data not shown). Incubation of density-arrested non- stimulated cells in MTX did not alter the normal in- duction of c-myc expression by PDGF nor did the cells become responsive to exposure to plasma. Additionally,

540 MORGAN AN

PDGF/O.ZS%PPP 6%PPP/MTX S%PPP 0 - O h e - 3oh 30 - 6Oh

0.6 -1

0.4 1 s I

0 .6 2 20 24.6 28 30.6 32 37 60

TIME (hours) Fig. 6. c-myc expression in S phase cells exposed to PDGF during S phase arrest. Density-arrested cells were made competent by a 6 hour exposure to PDGF in 0.25% plasma (0-6 hj, then arrested in early S phase in 5% plasma and MTX 16-30 h). After 18 hours in MTX (t = 24 h, arrow), PDGF was added to the MTX-containing medium of one- half of the culture plates for an additional 6 hours. Timepoints were taken 0.5 hour and 2 hours after PDGF addition (t =24.5, 26 h). The zero time point was untreated density-arrested cells. All cultures were then allowed to transit S phase in medium containing 5% plasma lacking PDGF (30-50 h). The 20 hour point was from cells arrested in S phase in MTX as described in text. c-myc expression in PDGF-treated (hatched bars) and untreated (solid bars) cultures was normalized to 1B15 mRNA.

cultures were treated for 18 hours with medium con- taining PDGF, plasma, and MTX in the presence of a high concentration of thymidine, which circumvented the MTX block and allow cells to complete the cell cycle as a relatively heterogeneous population. When the medium containing MTX was replaced with 5% plasma-supplemented medium, no plasma-dependent c-myc accumulation occurred. These results are consis- tent with a cell cycle dependent ( S phase) effect of plasma on c-myc expression in PDGF-treated cultures.

DISCUSSION The expression of the c-fos and c-myc cellular onco-

genes has been tentatively associated with the estab- lishment of a growth response in quiescent murine fi- broblasts. Some of the experimental evidence supporting this association is summarized below. Both genes have been shown to be induced by mitogens such as PDGF and fibroblast growth factor, and not by pro- gression factors present in platelet-poor plasma (Bravo et al., 1985). When a confluent, quiescent monolayer of NIH 3T3 fibroblasts is “wounded’ by scraping a portion of the cells from the plate, the expression of c-fos is induced in cells lining the periphery of the wound. These cells proliferate in the presence of plasma, thus reforming a confluent cell monolayer (Verrier et al., 1986). Inhibiting the synthesis of c-fos by induction of the expression of antisense c-fos mRNA in quiescent NIH 3T3 fibroblasts inhibits PDGF- or fetal calf se-

D PLEDGER

rum stimulated DNA synthesis (Nishikura and Mur- ray,- 1987). Induction of the expression of an exoge- nously introduced c-myc DNA in Balbic 3T3 cells augments DNA synthesis in the absence of PDGF stim- ulation (Armelin et al., 1984). When purified c-myc protein is introduced into quiescent Swiss 3T3 fibro- blasts by microinjection, these cells acquire the ability to proliferate in the presence of platelet-poor plasma without added PDGF (Kaczmarek et al., 1985).

The nature of competence and the role of the c-fos and c-myc genes in its induction and maintenance is not well understood. Bravo et al. (1985) demonstrated that the competent state induced in Go arrested NIH 3T3 cells by PDGF persisted for considerably longer (t, 1 = 16 h) than the duration of stimulated expression of c-fos and c-myc, which implicates these genes in the establishment, but not the maintenance, of compe- tence. Scher et al. (1979) showed that competence for a second round of DNA synthesis and cell division could be induced in Balbic 3T3 cells transiting S phase by exposure to PDGF. However, Holt et al., (1986) re- ported that induction of antisense c-fos RNA tran- scripts inhibited the proliferation of cycling Swiss 3T3 cells, which would not be undergoing G,/G, transition. These ambiguities have prompted a number of inves- tigators t o examine more closely the expression of these oncogenes as a function of proliferation and cell cycle status.

In the experiments presented here, when Go arrested cells were stimulated to proliferate in PDGF and 5% platelet-poor plasma, c-fos mRNA increased within 30 minutes and returned to the basal, undetectable level by 1 hour after stimulation. Expression of c-myc mRNA was increased within 1 hour, maximal a t 2 hours, and declined between 6 and 12 hours after stimulation to an intermediate level, which was maintained through the remainder of the cycle. These results are consistent with those of Bravo et al. (1986) and Dean et al. (1986), both of whom also showed that removal of serum from growing cells results in a rapid (within 2 hours) de- crease in the level of c-myc mRNA. Our observation that c-myc mRNA declined to a n undetectable level in 5% plasma after removal of PDGF suggests that the serum component required for maintenance of elevated c-myc expression throughout the cell cycle is PDGF, despite the fact that cells will continue through DNA synthesis and mitosis in its absence.

Although c-fos and c-myc expression are associated with the stimulation of growth in Go arrested cells, the induction of these genes is not restricted to Go cells. c-fos mRNA was induced by PDGF in cells undergoing synchronous transit through S phase and G2, whether PDGF was presented in a 6 hour pulse or throughout the prereplicative cycle (Fig. 2 and data not shown). The induction of c-fos in response t o PDGF in S phase cells confirms that cells have functional PDGF recep- tors by early S phase. These results are in agreement with those of Bravo et al. (1986), who demonstrated the serum inducibility of c-fos in NIH 3T3 cells synchro- nized in S phase by treatment with hydroxyurea. In contrast, Stiles and coworkers (Rollins et al., 1987) re- cently showed that c-fos expression is not induced by PDGF in sparse Balbic 3T3 cells cultured in platelet- poor plasma, but is induced by PDGF in confluent cells. These data could be interpreted to indicate that c-fos

CELL CYCLE DEPENDENT GENE EXPRESSION 54 1

gene expression is induced only in Go arrested cells. An alternative interpretation suggested by Stiles and co- workers is that the expression of c-fos in 3T3 cells in response to PDGF may require an unidentified se- creted factor that is quickly accumulated in dense cells but may not be present in sufficient concentration in sparse cultures. In our data the induction of c-fos dem- onstrates that the PDGF signaling pathway is intact.

The regulation of c-myc mRNA expression in S phase cells appears to be considerably more complex than that of c-fos. When cells arrested in early S phase in the presence of PDGF were released from arrest into me- dium supplemented with plasma alone, c-myc mRNA increased in a plasma-dependent, PDGF-independent manner until mitosis, a t which point the net accumu- lation of c-myc mRNA ceased (Fig. 4). Addition of PDGF to the plasma-supplemented medium a t the point of release or at several timepoints thereafter did not stimulate c-myc expression over that induced by plasma. TPA also failed to increase c-myc expression over the plasma level (data not shown), confirming that the lack of response to PDGF is not due to a defect specific to the PDGF receptor signal pathway.

Bravo et al. (1986) demonstrated serum-inducibility of both c-fos and c-myc in cells undergoing synchronous transit through S phase after hydroxyurea treatment. Our data imply that in S phase, PDGF alone accounts for serum-induction of c-fos expression, in contrast to the plasma-specific induction of c-myc expression in PDGF-treated S phase cells (as discussed below). This is in apparent conflict with our observations described above, as well as those of Bravo et al., (1986) and Dean et al., (1986), which indicate that PDGF is needed to maintain elevated c-myc levels throughout the cell cy- cle. We reasoned that when cells were synchronized in early S phase by concurrent exposure to PDGF, plasma and MTX, the presence of PDGF in the later stages of the prereplicative cycle might influence the regulation of c-myc expression with plasma. Therefore cells were made competent by transient treatment with PDGF, then arrested in early S phase in the absence of PDGF. When these cells were released from arrest in 5% plasma, no c-myc mRNA accumulation occurred. Read- dition of PDGF at the time of release from MTX in- duced c-myc expression, which declined to a steady- state level, similar t o the plasma-dependent steady state level reached in the experiment shown in Figure 4, at mitosis (Fig. 5) . Transient exposure to PDGF at the S phase arrest point was sufficient to trigger sub- sequent plasma-dependent c-myc accumulation as cells completed S phase and G, in the absence of PDGF. Thus the plasma-dependent accumulation of c-myc mRNA during S phase and G, was dependent on expo- sure to PDGF prior to or after the release from the S phase block. This pattern of c-myc regulation is consis- tent with the observed difference in the persistence of c-myc mRNA in 3T3 cells proliferating in plasma-sup- plemented medium in the presence or absence of PDGF. As PDGF receptors reappear after initial down- regulation the PDGF present in the medium stimu- lates c-myc expression and “primes” cells to express c-myc mRNA in the presence of plasma as they traverse S phase and G,. The relative asynchrony of the cell population under these conditions results in the averaging of the c-myc signal over time, such that

it resembles a steady-state elevation of c-myc over the level in cells cultured in the absence of PDGF.

The molecular basis for the induction of plasma-re- sponsive c-myc expression by PDGF in S phase, but not in GI, is unclear. Measurement of the rate of c-myc mRNA turnover in the presence of actinomycin D re- vealed no significant difference in the stability of c-myc mRNA in cells transiting S phase in plasma or plasma and PDGF compared to cells transiting G, in plasma and PDGF (data not shown). Thus plasma does not ap- pear to enhance c-myc mRNA accumulation by affect- ing the decay rate of c-myc mRNA transcribed in re- sponse to PDGF.

A role for oncogene expression in response to PDGF and plasma in S phase is suggested by the work of Scher et al. (1979). These investigators showed that treatment of Balbic 3T3 cells with PDGF during S phase stimulated post-mitotic daughter cells to un- dergo a second round of DNA synthesis. The fraction of daughter cells entering S phase was dependent on the concentration of plasma present in the medium. While these authors did not determine at what point in the growth of the parent and daughter cells the plasma concentration was crucial, our results suggest that the plasma-dependent accumulation of c-myc in cells that have been primed by exposure to PDGF in S phase may be important in the stimulation of the post-mitotic daughter cells to undergo a second round of DNA syn- thesis.

ACKNOWLEDGMENTS We thank N. Olashaw, J. Holt, and E. Leof for re-

viewing this manuscript, and Mrs. Rebecca Koransky for preparing the manuscript.

This work was suppored by grant CA 42713 from the National Institutes of Health.

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