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Mol Cancer Ther. 2004;3:205-213
© 2004 American Association for Cancer Research

AG490 inhibits G1-S traverse in BALB/c-3T3 cells following either mitogenic stimulation or exogenous expression of E2F-1

Jason Savell1, Yihong Ma1, Kristin S. Morrow1, Richard Jove1, Nancy Olashaw1, Pope L. Moseley2, W. Douglas Cress1 and Walker Wharton1,2

1 Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL and 2 Department of Internal Medicine, Health Sciences Center, University of New Mexico, Albuquerque, NM

Requests for Reprints:Walker Wharton, Department of Internal Medicine, 5ACC, University of New Mexico Health Sciences Center, Albuquerque, NM 87131. Phone: (505) 272-9897; Fax: (505) 272-9912. E-mail: wwharton{at}salud.unm.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
AG490, a member of the tryphostin family of protein kinase inhibitors, repressed G0-G1 traverse in BALB/c-3T3 cells. While the early induction of STAT activity was repressed by AG490, extracellular signal-regulated kinase (ERK) activation was unaffected and a pattern of gene expression suggested that cells exited G0 in the presence of the inhibitor. Although AG490 did not alter the induction of cyclin D1 protein, neither cyclin D1- nor cyclin D3-associated kinase activity was observed in growth-inhibited cells. Surprisingly, p130 was partially phosphorylated, and E2F3A protein was expressed in mitogen-stimulated AG490-treated cells despite the lack of cyclin D-associated kinase activity. These data suggest that AG490 inhibits a cellular pathway required for mid-G0-G1 traverse that is located after the induction of early processes potentially mediated by E2F (although independent of cyclin D-associated kinase activity) but before the late G1 increase in E2F-dependent transcription. Infection of AG490-treated cells with an E2F-1 adenovirus caused the induction of cyclin A, but could not overcome the drug-induced cell cycle arrest that was coincident with the repression of cyclin-dependent kinase 2 (cdk2)-associated kinase activation. We conclude that cdk2-associated kinase activity is modulated by a cellular process repressed by AG490. Furthermore, this cdk2-associated kinase activity is required for G0-G1 traverse in some role other than the regulation of E2F-dependent transcription.

Key Words: AG490 • E2F • p130 • cyclins


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A fundamental understanding of the basic regulatory processes that control G0-G1 traverse in mitogen-stimulated quiescent cells is beginning to emerge. G1-specific cyclin-dependent kinases (cdks) phosphorylate retinoblastoma (RB) protein and related family members (p130 and p107), leading to the transcriptional activation of a set of E2F/RB-regulated genes the products of which are required for the initiation of DNA synthesis (1, 2). The central importance of these pathways is underscored by the observation that their disruption through the constitutive activation of positive elements or the repression of negative regulators is critical to the genesis and maintenance of a variety of human tumors (3).

A series of cellular processes acts synergistically to regulate the activity of cyclin/cdk complexes. Cyclin D1 mRNA and protein are low in quiescent cells and are induced within 3–6 h of mitogen addition (4, 5). Cyclin D1 protein synthesized early in the cell cycle binds cdk4/6, but is maintained as a catalytically inactive complex by the action of p27Kip1, a potent inhibitor of cdk activity (6, 7). Levels of p27Kip1 steadily fall after the mitogenic stimulation of quiescent cells due to a combination of translational repression (8, 9) and enhanced degradation (10, 11), leading to the activation of cyclin D1-associated kinase activity and a partial phosphorylation of RB (12). The subsequent low-level increase in E2F-dependent transcriptional activity leads to the expression of cyclin E and cdk2 and increases in cdk2-associated kinase activity, resulting in the complete activation of E2F-dependent genes including cyclin A (12, 13).

The E2F family of proteins contains at least seven members that individually bind to either DP1 or DP2 to form E2F DNA-binding complexes (1, 2). Members of the E2F family are expressed in a distinct manner depending on the cell type or proliferative state, suggesting that they might well play specific roles in the regulation of cell cycle traverse. E2F-3B, E2F-4, and E2F-5 are expressed in quiescent cells where they bind members of the RB family of pocket proteins to form complexes that function as transcriptional repressors (14–16). E2F-1, E2F-2, and E2F-3A are absent in quiescent cells and are induced in parallel with cyclin E and cdk2 in late G0-G1 (14, 17, 18). Complexes containing individual E2F proteins have differential sequence specificity and preferentially induce subsets of E2F-dependent genes following exogenous expression in cultured cells (19–21). The mitogenic activities of E2F family members differ as well. Exogenous expression of E2F-1, E2F-2, or E2F-3 in quiescent cells induces entry into S phase even in the absence of mitogens, whereas under identical conditions, E2F-4 and E2F-5 lack mitogenic activity (20). Taken together, these results suggest that individual E2F proteins play distinct biological roles, although the molecular basis for these differences remains elusive.

AG490 is a member of the tryphostin family of tyrosine kinase inhibitors (22). The most-studied targets of AG490 are the Janus kinases (JAKs), a family of tyrosine kinases that phosphorylate and thus activate the STAT transcription factors (23). AG490 inhibits the proliferation of several cell types including leukemia cells and fibroblasts (22, 24, 25). To provide a molecular basis for the antiproliferative actions of AG490, we examined its effects on several cell cycle-related parameters in BALB/c-3T3 mouse fibroblasts. We found that AG490 prevented quiescent cells from entering S phase when added to cultures at the same time as or 8 h after mitogenic stimulation. Although AG490 repressed STAT activation, events associated with the traverse of early G0-G1 were unaffected (e.g., the up-regulation of cyclin D1). In contrast, stimulation of cells in the presence of AG490 blocked events that normally occurred in mid G0-G1 (D cyclin/cdk4 activation) and late G0-G1 (E2F-dependent cyclin A expression) probably due to the stabilization of p27Kip1. Infection of quiescent, AG490-treated cells with an adenovirus encoding E2F-1 resulted in the expression of cyclin A; however, cyclin A/cdk2 complexes were not active, and cells did not enter S phase. These findings demonstrate that AG490 prevents the passage of cells into late G0-G1 by inhibiting processes required for the activation of cdk4 and cdk2 and that E2F activation is insufficient for S phase entry.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
Stock cultures of BALB/c-3T3 cells were grown in alpha MEM containing 10% FCS, 50 µg/ml streptomycin, and 50 units/ml penicillin, as previously described (26). Cells to be used in individual experiments were seeded into 100-mm plates at an initial density of 5–7.5 x 105 cells/dish. Cultures were fed 3 days later, and the cells were used after a further 5- to 6-day incubation (i.e., 2–3 days after undergoing density-dependent growth arrest). Cells were stimulated by refeeding with fresh medium containing 10–20 ng/ml platelet-derived growth factor (PDGF) in the presence or absence of 5% serum. Cells that had been pretreated with AG490 received fresh AG490 at the time of stimulation.

Flow Cytometry
Cells were detached from plates by incubation at 37°C in 4 ml PBS containing 0.125% trypsin and 0.5 mM EDTA. Suspended cells were added to an equal volume of medium containing 10% serum (to neutralize the action of the trypsin), centrifuged, and resuspended in 1 ml PBS. Four milliliters of 95% ethanol were slowly added as the cell slurry was continuously vortexed, and samples were stored at 4°C for a minimum of 24 h. Fixed cells were centrifuged, and the pellets were resuspended in PBTB (PBS containing 0.1% Tween 20 and 0.05% bovine serum albumin) containing 10 µg/ml RNase A and 50 µg/ml propidium iodide. Following an overnight incubation at 4°C, cell cycle distributions were determined using a Becton Dickinson FACScan.

Western Blots
Culture dishes were cooled on ice, rinsed twice with cold PBS, scraped into 1 ml PBS, and pelleted in a microfuge. The supernatant was aspirated, and samples were frozen in a dry ice/methanol bath and stored at –80°C. Pellets were then thawed in immunoprecipitation (IP) buffer [50 mM HEPES (pH 7.2), 250 mM NaCl, 0.1% Tween 20, 1 mM DTT, 1 mM EGTA, 1 mM EDTA, 0.1 mM orthovanadate, 0.5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 2.5 µg/ml leupeptin], and supernatants were cleared by a 5-min centrifugation in a microfuge. Aliquots containing 40–80 µg of cellular protein were fractionated by discontinuous SDS-PAGE and transferred to nitrocellulose. Blots were blocked by incubation in PBST (PBS with 0.1% Tween 20) containing 5% dry milk, and PBST containing the primary antibody was added. Following a 1- to 2-h incubation at room temperature, the blots were rinsed in several changes of PBST and incubated for 30 min in PBST containing the secondary antibody. Following a second series of washes, complexes were visualized using enhanced chemiluminescence (ECL). Equal protein loading was verified by staining either the nitrocellulose blot used in the Western analysis or a parallel PAGE gel.

Cyclin-Dependent Kinase Assay
Cell extracts were prepared as described above for the Western analysis. Cyclin-specific antiserum was added to 350 µl of IP buffer containing 80 µg of cellular protein, and the mixture was rocked at 4°C for 2 h. Protein A-agarose beads were then added, and the samples were mixed for an additional hour. Immune complexes were pelleted, washed twice in IP buffer, and once in kinase buffer [50 mM HEPES (pH 7.5), 10 mM MgCl2, 5 mM MnCl2, 10 mM DTT], and resuspended in 10 µl kinase buffer containing 10 µCi 32PO4-ATP, 10 µM ATP, and 1 µg GST-RB for cyclin D or histone H1 for cyclin A or cyclin E assays, and incubated at 30°C for 15 min. Proteins were separated on SDS-polyacrylamide gels, and phosphorylated substrates were visualized by autoradiography.

Electrophoretic Mobility Shift Assays
STAT DNA-binding activity was measured in nuclear extracts as outlined earlier (27). Cell monolayers were rinsed first with PBS, and subsequently with 2 ml hypotonic buffer [20 mM HEPES (pH 7.9), 1 mM EDTA, and 1 mM EGTA]. Cells were then lysed directly on the plate by the addition of 500 µl hypotonic buffer containing 0.2% NP40. Nuclei were pelleted in a microfuge, resuspended in 50 µl hypertonic buffer [420 mM NaCl, 20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 20% glycerol], and rotated at 4°C for 30 min. Following a 20-min centrifugation in a microfuge, extracts were stored at –80°C. Aliquots containing 15 µg of protein were incubated in hybridization buffer [10 mM HEPES (pH 7.9), 10% glycerol, 1 mM DTT, 100 ng/ml salmon sperm DNA, 500 ng/ml bovine serum albumin] containing 0.1 ng 32PO4-labeled synthetic DNA fragment corresponding to the high-affinity M67 variant of the SIE (28) for 30 min at 30°C. Complexes were separated on a 5% polyacrylamide gel (29:1 acrylamide/bisacrylamide) containing 2.5% glycerol in Tris-borate EDTA and visualized by autoradiography.

Adenovirus Infection
Adenoviruses containing either no exogenous insert or a gene encoding human E2F-1 were obtained from Dr. Timothy Kowalik (University of Massachusetts). NIH 3T3 cells were maintained essentially as described above, except that a growth arrest was induced by incubation in medium supplemented with 0.5% serum. Aliquots of virus sufficient to give a final multiplicity of infection of 10 (as measured by plaque assay) were added to cells in a 100-mm dish in 3 ml of medium containing 0.5% serum. Following a 90-min incubation to allow for virus attachment, 7 ml of medium (also supplemented with 0.5% serum) were added, and the cells were harvested at 48 h.

Materials
Culture medium, FCS, and antibiotics were purchased from Life Technologies (Carlsbad, CA), while PDFG-BB was from BioSource (Camarillo, CA). All buffers, detergents, and salts were from Sigma (St. Louis, MO). AG490 was obtained from Calbiochem (San Diego, CA). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA; cyclin D1, cyclin E, and p107), or Transduction Labs (Franklin Lakes, NJ; cyclin D3 and p130). The cyclin A antibody was a generous gift from Dr. E. B. Leof, while the p27Kip1 and cyclin D1 antibody used in the kinase assays were produced locally against his-fusion proteins.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of Mitogenesis by AG490
The effects of AG490 on the stimulation of G0-G1 traverse in density-arrested BALB/c-3T3 cells are shown in Fig. 1. Before mitogenic stimulation, over 90% of the cells contained a 2N content of DNA. Eighteen hours after the addition of medium supplemented with 10 ng/ml PDGF and 5% serum, approximately 50% of the cells had exited G0-G1 and initiated DNA synthesis. Addition of AG490 together with the mitogens inhibited S phase entry in a dose-dependent manner, with complete inhibition observed at 50 µM.



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  		Figure 1. Inhibition of G0-G1 traverse by AG490. Quiescent, density-arrested BALB/c-3T3 cells were stimulated by the addition of fresh medium supplemented with 10 ng/ml PDGF and 10% serum in the presence of different concentrations of AG490. After an 18-h incubation, cultures were harvested and fixed, and the percentage of cells remaining in G0-G1 was determined by flow cytometry. Q, quiescent.

 
Inhibition of STAT Activation by AG490
Mitogen-induced increases in JAK-dependent kinase activity mediate an increase in STAT-dependent DNA-binding activity by phosphorylating STAT proteins on a conserved tyrosine residue, leading to dimerization and nuclear localization (29, 30). The effects of the JAK inhibitor AG490 on the induction of STAT activity in PDGF-treated BALB/c-3T3 cells are shown in Fig. 2A. As assessed by electrophoretic mobility shift assay (EMSA), density-arrested cells contained very low basal levels of binding activity. Exposure of cells to PDGF for 30 min caused a marked induction of multiple STAT complexes. As shown by our lab (31), as well as others (28), these complexes correspond to STAT3 homodimers (upper band), STAT1 homodimers (lower band), and STAT1/STAT3 heterodimers (intermediate band). Addition of 50 or 100 µM AG490 together with PDGF not only caused a marked repression of the induction of all three forms of STAT DNA-binding complexes, but actually lowered levels of complexes below what had been seen in control cells.



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  		Figure 2. Alterations in PDGF-induced early events by AG490. A, quiescent, BALB/c-3T3 cells were stimulated with fresh medium containing 20 ng/ml PDGF either alone or in combination with AG490. After a 30-min incubation, cells were harvested, nuclear extracts were prepared, and STAT-dependent DNA-binding activity was determined by EMSAs. Q, quiescent. B, quiescent cells were harvested either at quiescence, after exposure to 50 µM AG490, or 5 min following the addition of PDGF to control or AG490-treated cells. Cellular proteins were separated by PAGE, and levels of activated ERKs were determined by Western analysis using a phospho-ERK-specific antibody. Pilot experiments indicated that AG490 did not cause alterations in cellular levels of either STAT or ERK proteins over a time period corresponding to the incubation periods used in these experiments.

 
AG490 did not prevent the activation of all PDGF-induced second messenger systems, as shown in Fig. 2B. Neither quiescent BALB/c-3T3 cells nor cells treated with 50 µM AG490 contained activated extracellular signal-regulated kinases (ERKs), as measured on Western blots using an antibody that recognizes ERK isoforms phosphorylated on threonine 202 and tyrosine 204. Five minutes after the addition of PDGF to quiescent cells, there was a dramatic increase in the phosphorylation of a Mr 42,000 protein corresponding to ERK2 and a somewhat lower, although substantial, phosphorylation of a Mr 44,000 protein corresponding to ERK1. The addition of AG490 together with PDGF, a condition that completely prevented STAT activation, did not inhibit either the extent or pattern of the PDGF-mediated induction of ERK activity. Thus, the activation of ERKs by PDGF is independent of the AG490-repressed events that are involved in STAT activation (potentially JAK family kinases), indicating that the multiple second messenger pathways invoked following addition of PDGF are differentially sensitive to the actions of the inhibitor.

Differential Effects of AG490 on Cell Cycle-Dependent Gene Expression
The expression of genes induced during the traverse of late G0-G1 was markedly repressed in AG490-treated cells, as shown in Fig. 3. Quiescent, density-arrested BALB/c-3T3 cells were stimulated by the addition of medium supplemented with PDGF and serum together with increasing concentrations of AG490. Eighteen hours later, cellular extracts were prepared, and the expression of two proteins induced in late G1 was determined by Western analysis. As shown in Fig. 3A, quiescent cells did not express detectable levels of p107, while 18 h following mitogenic stimulation, protein levels were markedly increased. Addition of AG490 at the time of mitogenic stimulation caused a dose-dependent decrease in the levels of p107 protein that, in general, reflected the decrease in mitogenesis shown in Fig. 1. A small decrease in the expression of p107 was observed at 10 µM AG490, with essentially a complete loss of expression seen at 50 µM, a concentration that fully repressed cell cycle traverse. The expression of cyclin A protein in duplicate samples is shown in Fig. 3B. A marked induction of cyclin A protein was observed in stimulated cultures. The co-addition of AG490 caused a progressive loss of cyclin A expression that paralleled the decrease in both mitogenesis and the expression of p107. Loss of cyclin A protein was also accompanied by loss of cyclin A-associated kinase activity (Fig. 3C). The repression of both cyclin A and p107 protein expression in mitogen-stimulated AG490-treated cells was paralleled by a decrease in the expression of the corresponding mRNAs (data not shown).



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  		Figure 3. Repression of late G0-G1 events in AG490-treated cultures. Quiescent BALB/c-3T3 cells were stimulated with fresh medium supplemented with 10 ng/ml PDGF, 5% serum, and the indicated concentrations of AG490. Following an 18-h incubation, cultures were harvested, and protein levels of p107 (A) and cyclin A (B) were determined by Western blots. The histone kinase activity in cyclin A immunoprecipitates was also determined in parallel samples (C). Q, quiescent.

 
The results shown in Fig. 3 suggest that AG490 blocks cellular proliferation at a point before the induction of late G1 genes (induced 12–15 h after addition of mitogens). However, it was possible that, despite the marked decrease in PDGF-induced STAT induction in the presence of AG490, growth-inhibited cells retained the ability to progress through some early portions of G0-G1 in the presence of the drug. To determine whether this was the case, quiescent BALB/c-3T3 cells were stimulated with PDGF and serum and, at various times following the addition of mitogens, AG490 was added to a final concentration of 50 µM. The percentage of cells in S phase 18 h following stimulation was determined by flow cytometry. As shown in Fig. 4A, over 90% of the density-arrested population of cells contained a 2N content of DNA. Following the addition of mitogens, approximately 70% of the cells were stimulated to enter S phase. The addition of AG490 at the time of stimulation completely repressed G0-G1 traverse, similar to the results shown above in Fig. 1. When AG490 was added at times up to 8 h following stimulation with PDGF and serum, there was an inhibition of proliferation similar to what was observed when the drug was added at 0 h. Cyclin A protein levels in cells where AG490 was added at various times after stimulation are shown in Fig. 4B. Levels of cyclin A protein were low in samples prepared from quiescent cells and were induced following the addition of PDGF and serum in the absence of AG490. The coaddition of the inhibitor at 0 h repressed the expression of cyclin A, similar to what was shown above (Fig. 3B). Addition of AG490 as late as 8 h following stimulation repressed cyclin A expression to an extent identical to that seen when the drug was added at 0 time.



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  		Figure 4. Growth inhibitory properties of AG490. Quiescent cultures of BALB/c-3T3 cells were stimulated with fresh medium supplemented with 10 ng/ml PDGF and 5% serum at 0 h. AG490 was added to some cultures at the indicated times to a final concentration of 50 µM, and all plates were harvested at 18 h. Some cultures were fixed, and DNA distributions were determined by flow cytometry (A). Other cultures were used for determination of cyclin A protein levels by Western analysis (B). Q, quiescent.

 
Previous studies suggest that STAT activity is required for G0-G1 cell cycle traverse (32). We have shown that STAT activity returns to basal levels within 4 h after stimulation of quiescent BALB/c-3T3 cells with PDGF (31). Thus, the data presented above indicate that AG490 blocks S phase entry and cyclin A and p107 expression at times when STATs are not active (i.e., when added to cells at times after 4 h), suggesting that AG490 modulates additional cell cycle-regulatory processes. For this reason, we next investigated mid-G0-G1 processes that could potentially be targeted by AG490 in a manner to induce a cell cycle arrest.

Inhibition of cdk4 Activity by AG490
While the levels of cyclin D3 protein are relatively constant in BALB/c-3T3 cells during G0-G1 traverse (33), cyclin D1 protein expression has been reported to be low in quiescent cells and to be induced 3–6 h following the addition of medium containing PDGF (4, 5). As shown in Fig. 5A, we also found that levels of cyclin D1 protein were low in quiescent cells and were increased 5- to 6-fold 9 h after the addition of medium containing PDGF and serum. The addition of AG490 at the time of mitogenic stimulation did not repress the induction of cyclin D1, even at a concentration of 50 µM, where mitogenesis was completely inhibited. Thus, AG490 did not alter the induction of a gene expressed during early G0-G1 traverse.



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  		Figure 5. Regulation of cyclin D-associated kinase activity in AG490-treated cells. A and B, quiescent, density-arrested BALB/c-3T3 cells were stimulated at 0 h with fresh medium containing 10 ng/ml PDGF, 5% serum (CS), and the indicated concentrations of AG490. Following a 12-h incubation, cells were harvested, and cyclin D1 (A) and p27Kip1 (B) protein levels were determined by Western analysis. C and D, quiescent cells and cells stimulated for 12 h with PDGF and serum, either alone or together with 50 µM AG490, were harvested, and RB kinase activity in cyclin D1 (C) and cyclin D3 (D) immunoprecipitates was measured. Q, quiescent.

 
p27Kip1 inhibits the activity of several classes of cyclin/cdk complexes (6, 7) and is thought to specifically regulate the ability of cells to enter and exit quiescence (34, 35). We therefore determined whether the growth inhibition mediated by AG490 was accompanied by alterations in p27Kip1 metabolism. As shown in Fig. 5B, p27Kip1 levels were elevated in quiescent cells and fell by over 90% 12 h following stimulation with PDGF and serum, as we and others have reported (7, 36). The addition of AG490 caused a dose-dependent inhibition of p27Kip1 repression in mitogen-stimulated cells, suggesting that the growth inhibition might be mediated by a failure to induce cyclin D-associated kinase activity. To determine whether this was the case, cyclin D1-associated kinase activity was measured in control and AG490-treated cultures, as shown in Fig. 5C. A very low level of kinase activity was observed in cyclin D1 immunoprecipitates isolated from quiescent cells. Twelve hours following the addition of medium containing PDGF and serum, there was a 3- to 4-fold increase in kinase activity, similar to the extent of the induction previously observed (5, 37). The addition of 50 µM AG490 together with the mitogens caused a complete repression of the activation of the RB kinase activity associated with cyclin D1 complexes. Essentially the same pattern of induction and inhibition of cyclin D3-associated kinase activity was observed in AG490-treated cells, as shown in Fig. 5D.

Effects of AG490 on E2F
It has generally been thought that the induction of E2F-dependent transcriptional cascades is initiated by the activation of cyclin D-associated kinase activity in a manner mediated by the subsequent phosphorylation of members of the RB family of proteins (12). p130 is present in a hypophosphorylated form in quiescent cells, where it functions as a repressor of E2F-mediated transcription (38). As cells progress through G0-G1, p130 is progressively phosphorylated by a process that involves cyclin D3-associated kinase activity (39), leading to a loss of the transcriptional repression of a subset of E2F-dependent genes. The effects of AG490 on the phosphorylation of p130 in mitogen-stimulated BALB/c-3T3 cells are shown in Fig. 6A. In quiescent cultures, p130 was present in a rapidly migrating, hypophosphorylated form. Fifteen hours following stimulation in the absence of AG490, the levels of p130 protein had markedly decreased, and all of the remaining p130 accumulated in a hyperphosphorylated form. The addition of AG490 together with PDGF and serum largely prevented the loss of p130 protein as well as the shift to the hyperphosphorylated form, although an intermediate form was evident. It can be concluded from these results that a p130 kinase activity responsible for the conversion of p130 from the hypophosphorylated form to the intermediate form is present in AG490-treated growth-arrested cells, even in the absence of cyclin D-associated kinase activity.



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  		Figure 6. Modulation of E2F expression in AG490-treated cells. A, BALB/c-3T3 cells were left quiescent (Q) or stimulated for 18 h with 10 ng/ml PDGF, 5% serum, and the indicated concentrations of AG490. The levels and electrophoretic mobilities of p130 were determined by Western blots. B and C, cells were left quiescent (Q) or stimulated with PDGF and serum for 18 h in the presence or absence of 50 µM AG490. E2F-3A or E2F-1 protein levels were determined by Western blots.

 
Two forms of E2F-3 are expressed in cultured fibroblasts, E2F-3A, which has been reported to be absent in quiescent cells and induced following mitogenic stimulation, and E2F-3B that is expressed throughout the cell cycle (14, 15). As shown in Fig. 6B, quiescent BALB/c-3T3 cells did not contain detectable levels of E2F-3A protein. Nine hours following the addition of medium supplemented with PDGF and serum, there was a marked increase in the expression of E2F-3A protein, which appeared to migrate as a closely spaced doublet. The addition of 50 µM AG490 at the time of mitogenic stimulation caused approximately a 30% reduction in the total levels of E2F-3A. Therefore, while the alterations in the levels of the E2F-3 proteins in mitogen-stimulated cells were somewhat modulated in the presence of AG490, the magnitude of the effects was markedly less than the repression of E2F-dependent late G1 gene products (cyclin A and p107) in AG490-treated cultures. In contrast to the case with E2F-3A, the late G0-G1 induction of E2F-1 protein was completely repressed by the coaddition of AG490 (Fig. 6C). These findings concur with previous studies showing that the induction of E2F-3A is myc-dependent and occurs early in G0-G1, whereas the induction of E2F-1 is E2F-dependent and occurs in late G0-G1 (17, 40).

Inhibition of E2F-Mediated Cell Cycle Traverse by AG490
The data presented in Fig. 6C suggest that the failure of mitogen-stimulated AG490-treated cells to initiate DNA synthesis was not due to a failure to form E2F-3A and E2F-4 DNA-binding complexes that are free of repressive RB family members, although the E2F-dependent gene products cyclin A and p107 were not induced. To determine if the forced expression of late G0-G1 E2F-dependent genes could bypass the mid-G0-G1 growth arrest imposed by AG490, we investigated whether the stimulation of DNA synthesis driven by exogenous expression of E2F family members in the absence of mitogens is also inhibited by the tryphostin. In these experiments, quiescent NIH 3T3 cells were infected with an adenovirus encoding E2F-1. These experiments were performed using NIH 3T3 cells, because we found that BALB/c-3T3 cells are unable to be successfully infected with adenovirus. As shown in Fig. 7, approximately 90% of the quiescent population contained a 2N content of DNA. Addition of medium containing 10% serum induced a marked entry of cells into S phase that was abolished by coaddition of AG490, an effect identical to that seen in BALB/c-3T3 cells (Fig. 1). Infection of cells with the control adenovirus alone or together with AG490 did not induce cells to initiate DNA synthesis. Expression of E2F-1 caused essentially the entire population of cells to exit G0 and enter S phase. The addition of AG490 at the time of infection dramatically inhibited the mitogenic response following E2F-1 expression to the same extent as the inhibition of the initiation of DNA synthesis seen in serum-treated cells.



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  		Figure 7. Inhibition of the initiation of DNA synthesis by AG490 in cells infected with an E2F-1 adenovirus. NIH 3T3 cells were harvested at quiescence or 24 h following stimulation with medium containing 10% serum alone or together with 50 µM AG490. In addition, cells were harvested 48 h following infection with a control adenovirus or a virus directing the expression of E2F-1 in either the presence or absence of AG490. Fixed cells were stained, and DNA distributions were determined by flow cytometry.

 
The cellular lesion responsible for the failure of E2F-1-infected cells to enter S phase in the presence of AG490 was characterized in the studies shown in Fig. 8. In all of these experiments, quiescent cultures were stimulated with fresh medium containing 10% serum or were infected with the E2F-1 adenovirus in either the presence or absence of 50 µM AG490. As shown in Fig. 8A, E2F-1 protein was undetectable in quiescent cells and was markedly induced after infection with the adenoviral construct. Although E2F-1 is also induced by serum (see Fig. 6), its levels in serum-stimulated cells are much lower than those in infected cells and thus are not detectable at the level of exposure used for quantitation of E2F-1 in infected cells. Addition of AG490 together with the virus caused a detectable increase in E2F-1 protein levels over that observed in cells infected in the absence of the inhibitor. Expression of cyclin A, a late G0-G1 E2F-dependent gene, is shown in Fig. 8B. Cyclin A levels were low in quiescent cultures and were markedly increased following either the addition of serum or infection with the E2F-1 adenovirus. Treatment of infected cells with AG490 further increased cyclin A expression. Taken together, the results shown in the first two panels of Fig. 8 indicate that the lack of entry into S phase in cells infected with the E2F-1 virus in the presence of AG490 is not due to a failure to induce exogenous expression of E2F-1 or the induction of downstream E2F-dependent genes. Instead, as shown in Fig. 8C, the AG490-mediated G1 arrest is due to a repression of the activation of cyclin A-associated kinase activity. While quiescent cells lacked kinase activity, large increases were seen following stimulation with serum or exogenous expression of E2F-1 under control conditions. In contrast, treatment of virally infected cells with AG490 largely prevented the induction of cyclin A-associated kinase activity.



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  		Figure 8. Induction of E2F-1-dependent events in the presence of AG490. NIH 3T3 cells were harvested at quiescence, after stimulation with10% serum, and after infection with an adenovirus directing the exogenous expression of E2F-1 under control conditions or in the presence of 50 µM AG490. Levels of E2F-1 (A) and cyclin A (B) were determined by Western blotting. C, cdk2-associated activity was measured by in vitro kinase assay using histone H1 as substrate.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The initiation of G0-G1 traverse in cultured fibroblasts is regulated by a cascade of second messenger pathways initiated by the binding of primary mitogens such as PDGF to their cognate receptors (41, 42). Multiple second messengers act in a manner that has been postulated to be combinatorial (43) to activate the transcription of a large family of Immediate Early Genes the induction of which is independent of new protein synthesis and its activity is required for exit from G0 (44, 45). In a similar manner, the biological processes regulating late G0-G1 traverse are beginning to be understood in more detail. Cyclin D1-associated kinase activity, induced in BALB/c-3T3 cells approximately 9 h after the mitogenic stimulation of quiescent cultures, phosphorylates RB at a number of serine/threonine residues, resulting in the initial release of E2F from repressive complexes and the formation of transcriptionally active E2F/DP-1 dimers (1, 2). Subsequent E2F-dependent events result in the expression of a large family of genes required for G1-S traverse. In contrast, the cellular or molecular regulatory events that control traverse through mid-G0-G1 are largely unknown. It is known that EGF and insulin-like growth factors mediate traverse of PDGF-treated BALB/c-3T3 cells into S phase (46). However, the relevant second messenger systems and cellular responses induced by these ligands remain undescribed.

One approach toward the eventual elucidation of these potentially important regulatory networks is the description of pharmacological agents that arrest cells at a point between the induction of early events (e.g., the expression of Immediate Early Genes) and the relatively late induction of cyclin/cdk-associated kinase activity. In this paper, we describe the growth inhibitory properties of the protein kinase inhibitor, AG490. Addition of AG490 to mitogen-stimulated BALB/c-3T3 cells represses a cellular pathway(s) required for traverse of mid G0-G1, even if late events are induced following the exogenous expression of E2F-1.

Signaling mediated by the PDGF receptor was not totally abolished by AG490 based on the ligand-dependent activation of the ERK cascade. In contrast, PDGF-mediated activation of all forms of STAT-dependent DNA-binding activity was abolished. These results were somewhat unexpected because it had been reported that PDGF receptors directly phosphorylate STAT1, while STAT3 phosphorylation is dependent on an intermediary kinase, presumably a member of the JAK family (47). Several methodological differences potentially explain this discrepancy. In the experiments reported here, endogenous PDGF receptors were stimulated in a biologically relevant target cell, as opposed to the activation of a transfected receptor construct in a human tumor-derived line. In addition, we attempted to probe activation of STAT DNA-binding complexes in an in vivo context. Although there are confounding variables inherent to approaches involving the utilization of either an in vitro kinase assay to measure physiologically relevant substrates or the addition of pharmacological agents to inhibit metabolic pathways, we can conclude that under the conditions described here, STAT1 does not appear to serve as a direct substrate for activated PDGF receptors. The precise role that JAK family members might play in this process will require a further molecular analysis. We have found previously (31, 32) that Src activity is also required for PDGF-mediated increases in STAT binding activity. It is therefore possible that multiple PDGF-induced second messenger systems converge to induce STAT activity or that Src and JAKs cooperate in a single linear pathway leading to STAT phosphorylation.

Despite the lack of a PDGF-mediated induction of STAT activity, by several criteria, it appears that mitogen-stimulated cells exit G0 in the presence of AG490. E2F-3A, a mid G0-G1 gene (14), is induced in the presence of the kinase inhibitor. In addition, we observed an increased phosphorylation of p130 in mitogen-stimulated AG490-treated cells despite the complete repression of cyclin D-associated kinase activity in growth-arrested cultures. We have previously determined that cyclin D3 complexes preferentially phosphorylate p130 (33). However, based on the discrete forms of phosphorylated p130 that sequentially accumulate during G0-G1 traverse, it appears that multiple cell cycle-dependent kinase activities target p130, although the specific residues targeted by different kinases or the functional consequences of modification at individual sites remain unknown. Even in the absence of cyclin D-dependent phosphorylation of p130, we observed an induction of E2F-3A protein in AG490-treated cells. These data directly suggest that the regulation of the induction of E2F-dependent transcriptional activity is more complex than had initially been thought and involves initiating events that are both dependent on and independent of cyclin D-associated kinase activity.

The mitogen-induced fall in p27Kip1 protein levels, mediated by a combination of translational repression (8, 9) and induced instability (10, 11), was repressed in the presence of AG490, potentially mediating the lack of cyclin D-associated kinase and the failure of cells to initiate DNA synthesis in the presence of the inhibitor. While it has been postulated that p27Kip1 enhances complex formation between cyclin D and cdk4 as well as activation of cyclin D complexes (48), it has been suggested that all cyclin D complexes associated with p27Kip1 are inactive (6). Adding more complexity, it appears that only a subset of cyclin D complexes free of p27Kip1 are active (6), suggesting the roles of multiple regulatory pathways potentially targeted by AG490. However, cellular pathways other than the potential regulation of cyclin D-associated kinase activity by p27Kip1 must be involved in the growth inhibitory activity of AG490, because bypassing the functions of cyclin D by the exogenous expression of E2F-1 did not allow AG490-treated cultures to initiate DNA synthesis.

E2F-1-dependent DNA-binding activity is down-regulated by cyclin A/cdk2-associated kinase activity, reflecting a putative negative feedback loop in which a process linked to entry into S phase represses events that mediate late G0-G1 traverse (49). Consistent with this hypothesis, we observed a paradoxical increase in cyclin A and E2F-1 expression, likely mediated by enhanced E2F-1 DNA-binding activity, in virus-infected AG490-treated cells. We propose that the lack of cdk2-associated kinase activity under these conditions leads to the loss of the negative feedback loop and the marked enhancement of the expression of E2F-dependent genes.

Several important conclusions can be drawn from the characterization of the AG490-induced cell cycle arrest. We have presented data suggesting that an induction of E2F-3 and E2F-4 D protein (which can be observed in the absence of cyclin D-associated kinase activity) is not sufficient for a complete induction of E2F-dependent gene transcription. Further downstream AG490-sensitive pathways, potentially involving the actions of E2F-1, are required for expression of genes such as those encoding cyclin A and p107. The differences in the actions of members of the E2F family have been suggested by results obtained in a CASTing analysis (21) as well as studies using cells infected with adenoviruses expressing specific E2F family members (20). In addition, we suggest that cdk2-associated kinase activity is required for processes critical to the initiation of DNA synthesis independent of its role in the regulation of E2F expression and activation. This conclusion is consistent with our earlier observation of the roscovitine-mediated repression of mitogenesis induced by DNA tumor viruses (35), although somewhat divergent from the observation that a radiation-induced checkpoint, presumably mediated by the repression of cdk2 activity by p21Cip1, could be overcome by the exogenous expression of E2F-1 (50). The reasons for these differences are not clear.

In summary, we propose that a cellular pathway required for mid G0-G1 traverse in mitogen-stimulated BALB/c-3T3 cells is targeted by AG490. Even in the presence of a marked overexpression of E2F-1 protein, DNA-binding, and transcriptional activity, the AG490-repressed pathway is limiting for both activation of cdk2 and entry into S phase.


   Acknowledgments
 
We thank Dr. Edward B. Leof and Dr. Timothy Kowalik for generously providing cyclin A antibody and the E2F-1 adenovirus, respectively. We also acknowledge the helpful service of the Molecular Imaging and Flow Cytometry Core Laboratories at the Moffitt Cancer Center.


   Footnotes
 
NIH Grant CA55652 (R. Jove) and NCI Grant CA78214 (W.D. Cress).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/15/03; revised 10/22/03; accepted 11/ 3/03.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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