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Negative Growth Regulation of SK-N-MC Cells by bFGF Defines a Growth Factor-sensitive Point in G2

2000; Elsevier BV; Volume: 275; Issue: 25 Linguagem: Inglês

10.1074/jbc.m001764200

1083-351X

Veronique A. J. Smits, Maartje A. van Peer, Marieke Essers, Rob Klompmaker, Gert Rijksen, René H. Medema,

Virus-based gene therapy research

Basic fibroblast growth factor (bFGF) has been shown to induce growth inhibition of the neuroepithelioma cell line SK-N-MC. Here we show that this growth inhibition occurs in G2. We show that bFGF is active on these cells during S and early G2 phase. Therefore, this constitutes a rather unusual mechanism of growth inhibition, because it is generally believed that cells become refractory to extracellular signals after passage through the restriction point. We show that bFGF treatment inhibits Tyr-15 dephosphorylation of cdc2 and prevents activation of Cdc25C, similar to what is seen upon activation of the G2DNA damage checkpoint. Interestingly, both DNA damage- and bFGF-induced effects on cdc2 phosphorylation are reverted by caffeine. To confirm the involvement of similar pathways induced by bFGF and DNA damage, we generated tetracycline-regulatable SK-N-MC clones expressing Cdc25C-S216A. Expression of this Cdc25C mutant can revert the bFGF-induced effects on cdc2 phosphorylation and can rescue cells from the block in G2 imposed by bFGF. Taken together, these data define a growth factor-sensitive point in G2 that most likely involves regulation of Cdc25C phosphorylation. Basic fibroblast growth factor (bFGF) has been shown to induce growth inhibition of the neuroepithelioma cell line SK-N-MC. Here we show that this growth inhibition occurs in G2. We show that bFGF is active on these cells during S and early G2 phase. Therefore, this constitutes a rather unusual mechanism of growth inhibition, because it is generally believed that cells become refractory to extracellular signals after passage through the restriction point. We show that bFGF treatment inhibits Tyr-15 dephosphorylation of cdc2 and prevents activation of Cdc25C, similar to what is seen upon activation of the G2DNA damage checkpoint. Interestingly, both DNA damage- and bFGF-induced effects on cdc2 phosphorylation are reverted by caffeine. To confirm the involvement of similar pathways induced by bFGF and DNA damage, we generated tetracycline-regulatable SK-N-MC clones expressing Cdc25C-S216A. Expression of this Cdc25C mutant can revert the bFGF-induced effects on cdc2 phosphorylation and can rescue cells from the block in G2 imposed by bFGF. Taken together, these data define a growth factor-sensitive point in G2 that most likely involves regulation of Cdc25C phosphorylation. basic fibroblast growth factor polyacrylamide 5-bromo-2′-deoxyuridine mitogen-activated protein kinase Cells can respond to a variety of extracellular signals, which together dictate cellular behavior including the decision to proliferate, differentiate, or undergo apoptosis (1.Beijersbergen R.L. Bernards R. Biochim. Biophys. Acta. 1996; 1287: 103-120Crossref PubMed Scopus (180) Google Scholar). Cell proliferation is controlled by multiple growth-regulatory pathways that act together to ensure proper cell division. At the late G1restriction point the cell weighs the activity of positive and negative regulatory signals. After passage through the restriction point, mitogenic growth factors are no longer required for cells to complete division, and cells become refractory to growth-inhibitory signals (2.Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1848) Google Scholar, 3.Laiho M. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massague J. Cell. 1990; 62: 175-185Abstract Full Text PDF PubMed Scopus (676) Google Scholar, 4.Albers M.W. Williams R.T. Brown E.J. Tanaka A. Hall F.L. Schreiber S.L. J. Biol. Chem. 1993; 268: 22825-22829Abstract Full Text PDF PubMed Google Scholar). Instead, cells come to rely upon the intrinsic regulators of the cell cycle machinery for orderly progression through the remainder of the cell cycle (2.Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1848) Google Scholar).Orderly progression through the mammalian cell cycle is dependent on the timed activation of cyclin-dependent kinases (5.Sherr C.J. Cell. 1993; 73: 1059-1065Abstract Full Text PDF PubMed Scopus (1986) Google Scholar). Each cell cycle phase is characterized by the presence of distinct cyclin-cyclin-dependent kinase complexes (6.Nurse P. Cell. 1994; 79: 547-550Abstract Full Text PDF PubMed Scopus (511) Google Scholar). Cell cycle control by checkpoints functions through interference with activation of these complexes. For example, for the onset of mitosis the activation of cyclin B-cdc2 complexes is required (7.Dunphy W.G. Trends Cell Biol. 1994; 4: 202-207Abstract Full Text PDF PubMed Scopus (249) Google Scholar), whereas activation of the G2 DNA damage checkpoint results in inhibition of these complexes, leading to an arrest in G2phase progression (8.Kharbanda S. Saleem A. Datta R. Yuan Z.M. Weichselbaum R. Kufe D. Cancer Res. 1994; 54: 1412-1414PubMed Google Scholar).Cyclin B is first synthesized during S phase, and cyclin B-cdc2 complexes continue to accumulate throughout G2. These complexes are held in an inactive state by phosphorylation of cdc2 at Thr-14 and Tyr-15, which is mediated by the Wee1 protein kinases (7.Dunphy W.G. Trends Cell Biol. 1994; 4: 202-207Abstract Full Text PDF PubMed Scopus (249) Google Scholar,9.King R.W. Jackson P.K. Kirschner M.W. Cell. 1994; 79: 563-571Abstract Full Text PDF PubMed Scopus (684) Google Scholar). At the end of G2, abrupt dephosphorylation of this site by the phosphatase Cdc25C triggers cdc2 activation (9.King R.W. Jackson P.K. Kirschner M.W. Cell. 1994; 79: 563-571Abstract Full Text PDF PubMed Scopus (684) Google Scholar). Cyclin B-cdc2 then phosphorylates and thereby further activates Cdc25C, which induces the full activation of cdc2 by forming a positive feedback loop by mutual activation (10.Hoffman I. Clarke P.R. Marcote M.J. Karsenti E. Draetta G. EMBO J. 1993; 12: 53-63Crossref PubMed Scopus (562) Google Scholar, 11.Izumi T. Maller J.L. Mol. Biol. Cell. 1993; 4: 1337-1350Crossref PubMed Scopus (251) Google Scholar). At the same time, certain sites within the cytoplasmic retention signal in the N terminus of cyclin B are phosphorylated, which allows translocation to the nucleus (12.Pines J. Hunter T. EMBO J. 1994; 13: 3772-3781Crossref PubMed Scopus (224) Google Scholar, 13.Li J. Meyer A.N. Donoghue D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 502-507Crossref PubMed Scopus (176) Google Scholar). Upon nuclear translocation, the cyclin B-cdc2 complex can phosphorylate critical substrates required for the initiation of mitosis. Completion of mitosis in turn depends on the ubiquitin-mediated degradation of cyclin B at the metaphase/anaphase transition (14.Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (490) Google Scholar).We have investigated the mechanism of growth inhibition by bFGF1 in SK-N-MC cells, neuroepithelioma cells of embryonic neuroectodermal origin. We find that addition of bFGF delays cell cycle progression through the G2 phase of the cell cycle rather than during G1. This bFGF-induced delay in G2 progression resembles the G2 arrest seen after DNA damage, indicating the existence of a growth factor-sensitive point in G2.DISCUSSIONIn this study we demonstrate that bFGF can induce growth inhibition of SK-N-MC cells by a delay in G2 phase progression, a mechanism that appears similar to the G2 DNA damage checkpoint. We showed that treatment with bFGF increased the doubling time of SK-N-MC cells from ∼20 to ∼30 h, and we demonstrated that cells are delayed in G2/M by about 8 h. This indicates that the major, if not only, growth inhibitory effect of bFGF on SK-N-MC cells is exerted in the G2 phase and makes it unlikely that bFGF affects other cell cycle phases. This is remarkable because it is generally believed that extracellular factors can influence cell cycle progression during G1 and that cells become refractory to growth inhibitory signals after passage through the restriction point (2.Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1848) Google Scholar). Our results clearly indicate that cells can be inhibited by growth inhibitory signals after passage through the restriction point, because the addition of bFGF to cells that are in S phase still results in a delay in G2/M progression of at least 8 h. Indeed, the addition of bFGF up to a point late in G2 still efficiently inhibited mitotic entry (data not shown).In addition to the results obtained with bFGF, stimulation of the Ret signaling pathway using a chimeric human epidermal growth factor receptor-Ret chimera receptor in SK-N-MC cells resulted in growth inhibition that was associated with a delay in G2 phase progression, similar to what is seen after bFGF treatment. 2V. A. J. Smits, M. A. van Peer, M. A. G. Essers, R. Klompmaker, G. Rijksen, and R. H. Medema, manuscript in preparation. These results indicate that this response is not restricted to a single growth factor receptor. Indeed, others have reported minor effects on G2progression by epidermal growth factor. Treatment of Hela and A431 cells with epidermal growth factor resulted in a short delay (1–2 h) in mitotic entry coupled with a delay in activation of cyclin B-cdc2 complexes (29.Kinzel V. Kaszkin M. Blume A. Richards J. Cancer Res. 1990; 50: 7932-7936PubMed Google Scholar). However, in these cells no overall growth inhibition was observed with epidermal growth factor, in contrast to what we see with bFGF in SK-N-MC cells. Therefore, this is the first example of overall growth inhibition by an extracellular factor that appears to be confined to regulation of the G2/M transition. Nevertheless, the data obtained with epidermal growth factor in Hela and A431 cells suggest that negative growth regulation in G2 by extracellular factors might be a more general phenomenon. In addition to the extensively studied G1restriction point, growth regulation in G2 may therefore play an important role in the proliferation of some cells.It will be of interest to study the signaling molecules in the pathway by which bFGF and Ret induce growth inhibition of SK-N-MC cells. It should be noted that van Puijenbroek et al. (18.van Puijenbroek A.A.F.L. van Weering D.H.L. van den Brink C.E. Bos J.L. van der Saag P.T. de Laat S.W. den Hertog J. Oncogene. 1997; 14: 1147-1157Crossref PubMed Scopus (34) Google Scholar) showed that growth inhibition by bFGF and Ret in these SK-N-MC cells is associated with sustained MAPK activation, whereas platelet-derived growth factor, which does not induce growth inhibition, also activates MAPK but gives rise to only a very transient activation of MAPK in these cells. Several studies support a role for MAPK in regulating progression through G2/M (30.Abrieu A. Fisher D. Simon M.N. Doree M. Picard A. EMBO J. 1997; 16: 6407-6413Crossref PubMed Scopus (72) Google Scholar, 31.Tamemoto H. Kadowaki T. Tobe K. Ueki K. Izumi T. Chatani Y. Kohno M. Kasuga M. Yazaki Y. Akanuma Y. J. Biol. Chem. 1992; 267: 20293-20297Abstract Full Text PDF PubMed Google Scholar), but other studies also suggest the involvement of other signaling molecules, for example, protein kinase C (32.Barth H. Kinzel V. Exp. Cell Res. 1994; 212: 383-388Crossref PubMed Scopus (23) Google Scholar, 33.Hofmann J. O'Conner P.M. Jackman J. Schubert C. Ueberall F. Kohn K.W. Grunicke H. Biochem. Biophys. Res. Com. 1994; 199: 937-943Crossref PubMed Scopus (35) Google Scholar, 34.Livneh E. Fishman D.D. Eur. J. Biochem. 1997; 248: 1-9Crossref PubMed Scopus (207) Google Scholar, 35.Thompson L.J. Fields A.P. J. Biol. Chem. 1996; 271: 15045-15053Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, using pharmacological inhibitors of MAPK, protein kinase C, phosphatidylinositol 3-kinase, and p38 MAPK we have been unable to obtain evidence for a possible involvement of any of these signaling molecules in the observed bFGF-induced growth inhibition (data not shown).A possible mediator of the G2 arrest induced by bFGF is the cyclin-dependent kinase inhibitor p21 waf1. This p53-regulated protein has been described as inhibiting kinase activity of the cyclins E, A, and B, resulting in an arrest in both the G1 and G2phases of the cell cycle (36.Medema R.H. Klompmaker R. Smits V.A.J. Rijksen R. Oncogene. 1998; 16: 431-441Crossref PubMed Scopus (151) Google Scholar). SK-N-MC cells do not express functional p53 (37.Moll U.M. Ostermeyer A.G. Haladay R. Winkfield B. Frazier M. Zambetti G. Mol. Cell. Biol. 1996; 16: 1126-1137Crossref PubMed Scopus (251) Google Scholar), but activation of p21 waf1 expression can also occur independently of p53 (38.Datto M.B. Li Y. Panus J.F. Howe D.J. Xiong Y. Wang X.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5545-5549Crossref PubMed Scopus (851) Google Scholar, 39.Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (888) Google Scholar). However, up-regulation of p21 waf1 could not be observed after bFGF treatment (data not shown), indicating that the inhibition of cyclin B kinase activity by bFGF must be due to another mechanism.Because Tyr-15 dephosphorylation is a critical step in the activation of the cyclin B-cdc2 complex, we investigated the effect of bFGF on this dephosphorylation event. We found that cdc2-Tyr-15 dephosphorylation, as well as activation of the Cdc25C phosphatase responsible for this dephosphorylation, was inhibited by bFGF. DNA damage also results in the inhibition of cdc2-Tyr-15 dephosphorylation by blocking activation of Cdc25C, thereby leading to an arrest in G2 (8.Kharbanda S. Saleem A. Datta R. Yuan Z.M. Weichselbaum R. Kufe D. Cancer Res. 1994; 54: 1412-1414PubMed Google Scholar). Interestingly, caffeine could revert both DNA damage- and bFGF-induced inhibition of cdc2-Tyr-15 dephosphorylation, indicating once more that these two pathways are remarkably similar. Moreover, our finding that caffeine can revert the effect of bFGF on cdc2 dephosphorylation makes it unlikely that the inhibition of cdc2 activation by bFGF is due to an arrest of cell cycle progression at an early point in G2, prior to cdc2 activation. Thus, our data indicate that bFGF directly interferes with the activation of cyclin B-cdc2 to inhibit cell proliferation.Our results suggested that bFGF would interfere with cdc2-Tyr-15 dephosphorylation via inhibition of Cdc25C. The inhibition of cdc2 activation induced by DNA damage results from Chk1-mediated phosphorylation of the Ser-216 site of Cdc25C, leading to the inability of Cdc25C to activate cyclin B-cdc2 complexes (23.Furnari B. Rhind N. Russell P. Science. 1997; 277: 1495-1497Crossref PubMed Scopus (473) Google Scholar, 24.Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1178) Google Scholar, 25.Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1118) Google Scholar, 26.Lopez-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). We showed that expression of Cdc25C mutated on Ser-216 was able to rescue the bFGF-induced inhibition of cdc2-Tyr-15 dephosphorylation (Fig. 5) and restored the cyclin B-cdc2 kinase activity (data not shown). Expression of this mutant, in contrast to wild type Cdc25C, resulted in a rescue of the bFGF-induced G2 delay as demonstrated by a BrdUrd pulse-chase experiment (Fig. 5). This demonstrates that regulation of Cdc25C phosphorylation is a critical factor in the bFGF-induced growth inhibition of SK-N-MC cells. Because we find a partial reversion, we cannot rule out other effects of bFGF during G2/M. Indeed, using a BrdUrd pulse-chase assay we showed that bFGF induced an ∼8 h delay in G2 progression (Fig. 1 C), whereas the mitotic entry was only inhibited for 4–6 h (Fig. 2), which could indicate that bFGF has additional effects on events later in mitosis.Besides Chk1-mediated inhibition of Cdc25C, other pathways are activated after DNA damage to enforce a delay in G2. For example, p53-regulated p21 expression results in a sustained G2 arrest in response to DNA damage (27.Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2517) Google Scholar), whereas inhibition of the nuclear translocation of cyclin B by DNA damage blocks the access of cyclin B to its mitotic substrates (28.Jin P. Hardy S. Morgan D.O. J. Cell Biol. 1998; 141: 875-885Crossref PubMed Scopus (259) Google Scholar). We found that expression of Cdc25C-S216A was unable to revert the inhibition of cdc2-Tyr-15 dephosphorylation that occurs in response to the DNA-damaging agent adriamycin (Fig. 5 B), consistent with the existence of parallel pathways. Thus, although certain similarities were observed between the DNA damage response and the effects of bFGF described here, the efficient rescue of bFGF-induced G2arrest seen in cells expressing Cdc25C-S216A mutant indicates that the pathways affected by bFGF are not as diverse as those activated after DNA damage.A potentially interesting link between bFGF-induced growth inhibition in G2 and the DNA damage checkpoint is suggested by studies on the radioprotective effect of bFGF. It has been reported that exogenous bFGF can protect cells from the lethal effects of ionizing radiation (40.Haimovitz-Friedman A. Vlodavsky I. Chaudhuri A. Witte L. Fuks Z. Cancer Res. 1991; 51: 2552-2558PubMed Google Scholar, 41.Fuks Z. Persaud R.S. Alfieri A. McLoughin M. Ehleiter D. Schwartz J.L. Seddon A.P. Cordon-Cardo C. Haimovitz-Friedman A. Cancer Res. 1994; 54: 2582-2590PubMed Google Scholar). This radioprotective effect of bFGF is correlated with a pronounced increase in the duration of the G2 arrest after irradiation (42.Jung M. Kern F.G. Jorgensen T.J. McLeskey S.W. Blair O.C. Dritschilo A. Cancer Res. 1994; 54: 5194-6197PubMed Google Scholar). Interestingly, bFGF expression has been reported to be present in abundant amounts in primary brain tumors, which are known for their poor responsiveness to radiation therapy (43.Takahashi J.A. Mori H. Fukumoto M. Igarashi K. Jaye M. Oda Y. Kikuchi H. Hatanaka M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5710-5714Crossref PubMed Scopus (247) Google Scholar). Given the effects of bFGF that we describe here, it is a distinct possibility that tumors producing high levels of bFGF respond so poorly to radiotherapy because of the suggested resemblance between DNA damage and bFGF in regulating cdc2 activity. Cells can respond to a variety of extracellular signals, which together dictate cellular behavior including the decision to proliferate, differentiate, or undergo apoptosis (1.Beijersbergen R.L. Bernards R. Biochim. Biophys. Acta. 1996; 1287: 103-120Crossref PubMed Scopus (180) Google Scholar). Cell proliferation is controlled by multiple growth-regulatory pathways that act together to ensure proper cell division. At the late G1restriction point the cell weighs the activity of positive and negative regulatory signals. After passage through the restriction point, mitogenic growth factors are no longer required for cells to complete division, and cells become refractory to growth-inhibitory signals (2.Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1848) Google Scholar, 3.Laiho M. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massague J. Cell. 1990; 62: 175-185Abstract Full Text PDF PubMed Scopus (676) Google Scholar, 4.Albers M.W. Williams R.T. Brown E.J. Tanaka A. Hall F.L. Schreiber S.L. J. Biol. Chem. 1993; 268: 22825-22829Abstract Full Text PDF PubMed Google Scholar). Instead, cells come to rely upon the intrinsic regulators of the cell cycle machinery for orderly progression through the remainder of the cell cycle (2.Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1848) Google Scholar). Orderly progression through the mammalian cell cycle is dependent on the timed activation of cyclin-dependent kinases (5.Sherr C.J. Cell. 1993; 73: 1059-1065Abstract Full Text PDF PubMed Scopus (1986) Google Scholar). Each cell cycle phase is characterized by the presence of distinct cyclin-cyclin-dependent kinase complexes (6.Nurse P. Cell. 1994; 79: 547-550Abstract Full Text PDF PubMed Scopus (511) Google Scholar). Cell cycle control by checkpoints functions through interference with activation of these complexes. For example, for the onset of mitosis the activation of cyclin B-cdc2 complexes is required (7.Dunphy W.G. Trends Cell Biol. 1994; 4: 202-207Abstract Full Text PDF PubMed Scopus (249) Google Scholar), whereas activation of the G2 DNA damage checkpoint results in inhibition of these complexes, leading to an arrest in G2phase progression (8.Kharbanda S. Saleem A. Datta R. Yuan Z.M. Weichselbaum R. Kufe D. Cancer Res. 1994; 54: 1412-1414PubMed Google Scholar). Cyclin B is first synthesized during S phase, and cyclin B-cdc2 complexes continue to accumulate throughout G2. These complexes are held in an inactive state by phosphorylation of cdc2 at Thr-14 and Tyr-15, which is mediated by the Wee1 protein kinases (7.Dunphy W.G. Trends Cell Biol. 1994; 4: 202-207Abstract Full Text PDF PubMed Scopus (249) Google Scholar,9.King R.W. Jackson P.K. Kirschner M.W. Cell. 1994; 79: 563-571Abstract Full Text PDF PubMed Scopus (684) Google Scholar). At the end of G2, abrupt dephosphorylation of this site by the phosphatase Cdc25C triggers cdc2 activation (9.King R.W. Jackson P.K. Kirschner M.W. Cell. 1994; 79: 563-571Abstract Full Text PDF PubMed Scopus (684) Google Scholar). Cyclin B-cdc2 then phosphorylates and thereby further activates Cdc25C, which induces the full activation of cdc2 by forming a positive feedback loop by mutual activation (10.Hoffman I. Clarke P.R. Marcote M.J. Karsenti E. Draetta G. EMBO J. 1993; 12: 53-63Crossref PubMed Scopus (562) Google Scholar, 11.Izumi T. Maller J.L. Mol. Biol. Cell. 1993; 4: 1337-1350Crossref PubMed Scopus (251) Google Scholar). At the same time, certain sites within the cytoplasmic retention signal in the N terminus of cyclin B are phosphorylated, which allows translocation to the nucleus (12.Pines J. Hunter T. EMBO J. 1994; 13: 3772-3781Crossref PubMed Scopus (224) Google Scholar, 13.Li J. Meyer A.N. Donoghue D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 502-507Crossref PubMed Scopus (176) Google Scholar). Upon nuclear translocation, the cyclin B-cdc2 complex can phosphorylate critical substrates required for the initiation of mitosis. Completion of mitosis in turn depends on the ubiquitin-mediated degradation of cyclin B at the metaphase/anaphase transition (14.Pines J. Biochem. J. 1995; 308: 697-711Crossref PubMed Scopus (490) Google Scholar). We have investigated the mechanism of growth inhibition by bFGF1 in SK-N-MC cells, neuroepithelioma cells of embryonic neuroectodermal origin. We find that addition of bFGF delays cell cycle progression through the G2 phase of the cell cycle rather than during G1. This bFGF-induced delay in G2 progression resembles the G2 arrest seen after DNA damage, indicating the existence of a growth factor-sensitive point in G2. DISCUSSIONIn this study we demonstrate that bFGF can induce growth inhibition of SK-N-MC cells by a delay in G2 phase progression, a mechanism that appears similar to the G2 DNA damage checkpoint. We showed that treatment with bFGF increased the doubling time of SK-N-MC cells from ∼20 to ∼30 h, and we demonstrated that cells are delayed in G2/M by about 8 h. This indicates that the major, if not only, growth inhibitory effect of bFGF on SK-N-MC cells is exerted in the G2 phase and makes it unlikely that bFGF affects other cell cycle phases. This is remarkable because it is generally believed that extracellular factors can influence cell cycle progression during G1 and that cells become refractory to growth inhibitory signals after passage through the restriction point (2.Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1848) Google Scholar). Our results clearly indicate that cells can be inhibited by growth inhibitory signals after passage through the restriction point, because the addition of bFGF to cells that are in S phase still results in a delay in G2/M progression of at least 8 h. Indeed, the addition of bFGF up to a point late in G2 still efficiently inhibited mitotic entry (data not shown).In addition to the results obtained with bFGF, stimulation of the Ret signaling pathway using a chimeric human epidermal growth factor receptor-Ret chimera receptor in SK-N-MC cells resulted in growth inhibition that was associated with a delay in G2 phase progression, similar to what is seen after bFGF treatment. 2V. A. J. Smits, M. A. van Peer, M. A. G. Essers, R. Klompmaker, G. Rijksen, and R. H. Medema, manuscript in preparation. These results indicate that this response is not restricted to a single growth factor receptor. Indeed, others have reported minor effects on G2progression by epidermal growth factor. Treatment of Hela and A431 cells with epidermal growth factor resulted in a short delay (1–2 h) in mitotic entry coupled with a delay in activation of cyclin B-cdc2 complexes (29.Kinzel V. Kaszkin M. Blume A. Richards J. Cancer Res. 1990; 50: 7932-7936PubMed Google Scholar). However, in these cells no overall growth inhibition was observed with epidermal growth factor, in contrast to what we see with bFGF in SK-N-MC cells. Therefore, this is the first example of overall growth inhibition by an extracellular factor that appears to be confined to regulation of the G2/M transition. Nevertheless, the data obtained with epidermal growth factor in Hela and A431 cells suggest that negative growth regulation in G2 by extracellular factors might be a more general phenomenon. In addition to the extensively studied G1restriction point, growth regulation in G2 may therefore play an important role in the proliferation of some cells.It will be of interest to study the signaling molecules in the pathway by which bFGF and Ret induce growth inhibition of SK-N-MC cells. It should be noted that van Puijenbroek et al. (18.van Puijenbroek A.A.F.L. van Weering D.H.L. van den Brink C.E. Bos J.L. van der Saag P.T. de Laat S.W. den Hertog J. Oncogene. 1997; 14: 1147-1157Crossref PubMed Scopus (34) Google Scholar) showed that growth inhibition by bFGF and Ret in these SK-N-MC cells is associated with sustained MAPK activation, whereas platelet-derived growth factor, which does not induce growth inhibition, also activates MAPK but gives rise to only a very transient activation of MAPK in these cells. Several studies support a role for MAPK in regulating progression through G2/M (30.Abrieu A. Fisher D. Simon M.N. Doree M. Picard A. EMBO J. 1997; 16: 6407-6413Crossref PubMed Scopus (72) Google Scholar, 31.Tamemoto H. Kadowaki T. Tobe K. Ueki K. Izumi T. Chatani Y. Kohno M. Kasuga M. Yazaki Y. Akanuma Y. J. Biol. Chem. 1992; 267: 20293-20297Abstract Full Text PDF PubMed Google Scholar), but other studies also suggest the involvement of other signaling molecules, for example, protein kinase C (32.Barth H. Kinzel V. Exp. Cell Res. 1994; 212: 383-388Crossref PubMed Scopus (23) Google Scholar, 33.Hofmann J. O'Conner P.M. Jackman J. Schubert C. Ueberall F. Kohn K.W. Grunicke H. Biochem. Biophys. Res. Com. 1994; 199: 937-943Crossref PubMed Scopus (35) Google Scholar, 34.Livneh E. Fishman D.D. Eur. J. Biochem. 1997; 248: 1-9Crossref PubMed Scopus (207) Google Scholar, 35.Thompson L.J. Fields A.P. J. Biol. Chem. 1996; 271: 15045-15053Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, using pharmacological inhibitors of MAPK, protein kinase C, phosphatidylinositol 3-kinase, and p38 MAPK we have been unable to obtain evidence for a possible involvement of any of these signaling molecules in the observed bFGF-induced growth inhibition (data not shown).A possible mediator of the G2 arrest induced by bFGF is the cyclin-dependent kinase inhibitor p21 waf1. This p53-regulated protein has been described as inhibiting kinase activity of the cyclins E, A, and B, resulting in an arrest in both the G1 and G2phases of the cell cycle (36.Medema R.H. Klompmaker R. Smits V.A.J. Rijksen R. Oncogene. 1998; 16: 431-441Crossref PubMed Scopus (151) Google Scholar). SK-N-MC cells do not express functional p53 (37.Moll U.M. Ostermeyer A.G. Haladay R. Winkfield B. Frazier M. Zambetti G. Mol. Cell. Biol. 1996; 16: 1126-1137Crossref PubMed Scopus (251) Google Scholar), but activation of p21 waf1 expression can also occur independently of p53 (38.Datto M.B. Li Y. Panus J.F. Howe D.J. Xiong Y. Wang X.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5545-5549Crossref PubMed Scopus (851) Google Scholar, 39.Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (888) Google Scholar). However, up-regulation of p21 waf1 could not be observed after bFGF treatment (data not shown), indicating that the inhibition of cyclin B kinase activity by bFGF must be due to another mechanism.Because Tyr-15 dephosphorylation is a critical step in the activation of the cyclin B-cdc2 complex, we investigated the effect of bFGF on this dephosphorylation event. We found that cdc2-Tyr-15 dephosphorylation, as well as activation of the Cdc25C phosphatase responsible for this dephosphorylation, was inhibited by bFGF. DNA damage also results in the inhibition of cdc2-Tyr-15 dephosphorylation by blocking activation of Cdc25C, thereby leading to an arrest in G2 (8.Kharbanda S. Saleem A. Datta R. Yuan Z.M. Weichselbaum R. Kufe D. Cancer Res. 1994; 54: 1412-1414PubMed Google Scholar). Interestingly, caffeine could revert both DNA damage- and bFGF-induced inhibition of cdc2-Tyr-15 dephosphorylation, indicating once more that these two pathways are remarkably similar. Moreover, our finding that caffeine can revert the effect of bFGF on cdc2 dephosphorylation makes it unlikely that the inhibition of cdc2 activation by bFGF is due to an arrest of cell cycle progression at an early point in G2, prior to cdc2 activation. Thus, our data indicate that bFGF directly interferes with the activation of cyclin B-cdc2 to inhibit cell proliferation.Our results suggested that bFGF would interfere with cdc2-Tyr-15 dephosphorylation via inhibition of Cdc25C. The inhibition of cdc2 activation induced by DNA damage results from Chk1-mediated phosphorylation of the Ser-216 site of Cdc25C, leading to the inability of Cdc25C to activate cyclin B-cdc2 complexes (23.Furnari B. Rhind N. Russell P. Science. 1997; 277: 1495-1497Crossref PubMed Scopus (473) Google Scholar, 24.Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1178) Google Scholar, 25.Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1118) Google Scholar, 26.Lopez-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). We showed that expression of Cdc25C mutated on Ser-216 was able to rescue the bFGF-induced inhibition of cdc2-Tyr-15 dephosphorylation (Fig. 5) and restored the cyclin B-cdc2 kinase activity (data not shown). Expression of this mutant, in contrast to wild type Cdc25C, resulted in a rescue of the bFGF-induced G2 delay as demonstrated by a BrdUrd pulse-chase experiment (Fig. 5). This demonstrates that regulation of Cdc25C phosphorylation is a critical factor in the bFGF-induced growth inhibition of SK-N-MC cells. Because we find a partial reversion, we cannot rule out other effects of bFGF during G2/M. Indeed, using a BrdUrd pulse-chase assay we showed that bFGF induced an ∼8 h delay in G2 progression (Fig. 1 C), whereas the mitotic entry was only inhibited for 4–6 h (Fig. 2), which could indicate that bFGF has additional effects on events later in mitosis.Besides Chk1-mediated inhibition of Cdc25C, other pathways are activated after DNA damage to enforce a delay in G2. For example, p53-regulated p21 expression results in a sustained G2 arrest in response to DNA damage (27.Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2517) Google Scholar), whereas inhibition of the nuclear translocation of cyclin B by DNA damage blocks the access of cyclin B to its mitotic substrates (28.Jin P. Hardy S. Morgan D.O. J. Cell Biol. 1998; 141: 875-885Crossref PubMed Scopus (259) Google Scholar). We found that expression of Cdc25C-S216A was unable to revert the inhibition of cdc2-Tyr-15 dephosphorylation that occurs in response to the DNA-damaging agent adriamycin (Fig. 5 B), consistent with the existence of parallel pathways. Thus, although certain similarities were observed between the DNA damage response and the effects of bFGF described here, the efficient rescue of bFGF-induced G2arrest seen in cells expressing Cdc25C-S216A mutant indicates that the pathways affected by bFGF are not as diverse as those activated after DNA damage.A potentially interesting link between bFGF-induced growth inhibition in G2 and the DNA damage checkpoint is suggested by studies on the radioprotective effect of bFGF. It has been reported that exogenous bFGF can protect cells from the lethal effects of ionizing radiation (40.Haimovitz-Friedman A. Vlodavsky I. Chaudhuri A. Witte L. Fuks Z. Cancer Res. 1991; 51: 2552-2558PubMed Google Scholar, 41.Fuks Z. Persaud R.S. Alfieri A. McLoughin M. Ehleiter D. Schwartz J.L. Seddon A.P. Cordon-Cardo C. Haimovitz-Friedman A. Cancer Res. 1994; 54: 2582-2590PubMed Google Scholar). This radioprotective effect of bFGF is correlated with a pronounced increase in the duration of the G2 arrest after irradiation (42.Jung M. Kern F.G. Jorgensen T.J. McLeskey S.W. Blair O.C. Dritschilo A. Cancer Res. 1994; 54: 5194-6197PubMed Google Scholar). Interestingly, bFGF expression has been reported to be present in abundant amounts in primary brain tumors, which are known for their poor responsiveness to radiation therapy (43.Takahashi J.A. Mori H. Fukumoto M. Igarashi K. Jaye M. Oda Y. Kikuchi H. Hatanaka M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5710-5714Crossref PubMed Scopus (247) Google Scholar). Given the effects of bFGF that we describe here, it is a distinct possibility that tumors producing high levels of bFGF respond so poorly to radiotherapy because of the suggested resemblance between DNA damage and bFGF in regulating cdc2 activity. In this study we demonstrate that bFGF can induce growth inhibition of SK-N-MC cells by a delay in G2 phase progression, a mechanism that appears similar to the G2 DNA damage checkpoint. We showed that treatment with bFGF increased the doubling time of SK-N-MC cells from ∼20 to ∼30 h, and we demonstrated that cells are delayed in G2/M by about 8 h. This indicates that the major, if not only, growth inhibitory effect of bFGF on SK-N-MC cells is exerted in the G2 phase and makes it unlikely that bFGF affects other cell cycle phases. This is remarkable because it is generally believed that extracellular factors can influence cell cycle progression during G1 and that cells become refractory to growth inhibitory signals after passage through the restriction point (2.Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1848) Google Scholar). Our results clearly indicate that cells can be inhibited by growth inhibitory signals after passage through the restriction point, because the addition of bFGF to cells that are in S phase still results in a delay in G2/M progression of at least 8 h. Indeed, the addition of bFGF up to a point late in G2 still efficiently inhibited mitotic entry (data not shown). In addition to the results obtained with bFGF, stimulation of the Ret signaling pathway using a chimeric human epidermal growth factor receptor-Ret chimera receptor in SK-N-MC cells resulted in growth inhibition that was associated with a delay in G2 phase progression, similar to what is seen after bFGF treatment. 2V. A. J. Smits, M. A. van Peer, M. A. G. Essers, R. Klompmaker, G. Rijksen, and R. H. Medema, manuscript in preparation. These results indicate that this response is not restricted to a single growth factor receptor. Indeed, others have reported minor effects on G2progression by epidermal growth factor. Treatment of Hela and A431 cells with epidermal growth factor resulted in a short delay (1–2 h) in mitotic entry coupled with a delay in activation of cyclin B-cdc2 complexes (29.Kinzel V. Kaszkin M. Blume A. Richards J. Cancer Res. 1990; 50: 7932-7936PubMed Google Scholar). However, in these cells no overall growth inhibition was observed with epidermal growth factor, in contrast to what we see with bFGF in SK-N-MC cells. Therefore, this is the first example of overall growth inhibition by an extracellular factor that appears to be confined to regulation of the G2/M transition. Nevertheless, the data obtained with epidermal growth factor in Hela and A431 cells suggest that negative growth regulation in G2 by extracellular factors might be a more general phenomenon. In addition to the extensively studied G1restriction point, growth regulation in G2 may therefore play an important role in the proliferation of some cells. It will be of interest to study the signaling molecules in the pathway by which bFGF and Ret induce growth inhibition of SK-N-MC cells. It should be noted that van Puijenbroek et al. (18.van Puijenbroek A.A.F.L. van Weering D.H.L. van den Brink C.E. Bos J.L. van der Saag P.T. de Laat S.W. den Hertog J. Oncogene. 1997; 14: 1147-1157Crossref PubMed Scopus (34) Google Scholar) showed that growth inhibition by bFGF and Ret in these SK-N-MC cells is associated with sustained MAPK activation, whereas platelet-derived growth factor, which does not induce growth inhibition, also activates MAPK but gives rise to only a very transient activation of MAPK in these cells. Several studies support a role for MAPK in regulating progression through G2/M (30.Abrieu A. Fisher D. Simon M.N. Doree M. Picard A. EMBO J. 1997; 16: 6407-6413Crossref PubMed Scopus (72) Google Scholar, 31.Tamemoto H. Kadowaki T. Tobe K. Ueki K. Izumi T. Chatani Y. Kohno M. Kasuga M. Yazaki Y. Akanuma Y. J. Biol. Chem. 1992; 267: 20293-20297Abstract Full Text PDF PubMed Google Scholar), but other studies also suggest the involvement of other signaling molecules, for example, protein kinase C (32.Barth H. Kinzel V. Exp. Cell Res. 1994; 212: 383-388Crossref PubMed Scopus (23) Google Scholar, 33.Hofmann J. O'Conner P.M. Jackman J. Schubert C. Ueberall F. Kohn K.W. Grunicke H. Biochem. Biophys. Res. Com. 1994; 199: 937-943Crossref PubMed Scopus (35) Google Scholar, 34.Livneh E. Fishman D.D. Eur. J. Biochem. 1997; 248: 1-9Crossref PubMed Scopus (207) Google Scholar, 35.Thompson L.J. Fields A.P. J. Biol. Chem. 1996; 271: 15045-15053Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, using pharmacological inhibitors of MAPK, protein kinase C, phosphatidylinositol 3-kinase, and p38 MAPK we have been unable to obtain evidence for a possible involvement of any of these signaling molecules in the observed bFGF-induced growth inhibition (data not shown). A possible mediator of the G2 arrest induced by bFGF is the cyclin-dependent kinase inhibitor p21 waf1. This p53-regulated protein has been described as inhibiting kinase activity of the cyclins E, A, and B, resulting in an arrest in both the G1 and G2phases of the cell cycle (36.Medema R.H. Klompmaker R. Smits V.A.J. Rijksen R. Oncogene. 1998; 16: 431-441Crossref PubMed Scopus (151) Google Scholar). SK-N-MC cells do not express functional p53 (37.Moll U.M. Ostermeyer A.G. Haladay R. Winkfield B. Frazier M. Zambetti G. Mol. Cell. Biol. 1996; 16: 1126-1137Crossref PubMed Scopus (251) Google Scholar), but activation of p21 waf1 expression can also occur independently of p53 (38.Datto M.B. Li Y. Panus J.F. Howe D.J. Xiong Y. Wang X.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5545-5549Crossref PubMed Scopus (851) Google Scholar, 39.Reynisdottir I. Polyak K. Iavarone A. Massague J. Genes Dev. 1995; 9: 1831-1845Crossref PubMed Scopus (888) Google Scholar). However, up-regulation of p21 waf1 could not be observed after bFGF treatment (data not shown), indicating that the inhibition of cyclin B kinase activity by bFGF must be due to another mechanism. Because Tyr-15 dephosphorylation is a critical step in the activation of the cyclin B-cdc2 complex, we investigated the effect of bFGF on this dephosphorylation event. We found that cdc2-Tyr-15 dephosphorylation, as well as activation of the Cdc25C phosphatase responsible for this dephosphorylation, was inhibited by bFGF. DNA damage also results in the inhibition of cdc2-Tyr-15 dephosphorylation by blocking activation of Cdc25C, thereby leading to an arrest in G2 (8.Kharbanda S. Saleem A. Datta R. Yuan Z.M. Weichselbaum R. Kufe D. Cancer Res. 1994; 54: 1412-1414PubMed Google Scholar). Interestingly, caffeine could revert both DNA damage- and bFGF-induced inhibition of cdc2-Tyr-15 dephosphorylation, indicating once more that these two pathways are remarkably similar. Moreover, our finding that caffeine can revert the effect of bFGF on cdc2 dephosphorylation makes it unlikely that the inhibition of cdc2 activation by bFGF is due to an arrest of cell cycle progression at an early point in G2, prior to cdc2 activation. Thus, our data indicate that bFGF directly interferes with the activation of cyclin B-cdc2 to inhibit cell proliferation. Our results suggested that bFGF would interfere with cdc2-Tyr-15 dephosphorylation via inhibition of Cdc25C. The inhibition of cdc2 activation induced by DNA damage results from Chk1-mediated phosphorylation of the Ser-216 site of Cdc25C, leading to the inability of Cdc25C to activate cyclin B-cdc2 complexes (23.Furnari B. Rhind N. Russell P. Science. 1997; 277: 1495-1497Crossref PubMed Scopus (473) Google Scholar, 24.Peng C.Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1178) Google Scholar, 25.Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1118) Google Scholar, 26.Lopez-Girona A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar). We showed that expression of Cdc25C mutated on Ser-216 was able to rescue the bFGF-induced inhibition of cdc2-Tyr-15 dephosphorylation (Fig. 5) and restored the cyclin B-cdc2 kinase activity (data not shown). Expression of this mutant, in contrast to wild type Cdc25C, resulted in a rescue of the bFGF-induced G2 delay as demonstrated by a BrdUrd pulse-chase experiment (Fig. 5). This demonstrates that regulation of Cdc25C phosphorylation is a critical factor in the bFGF-induced growth inhibition of SK-N-MC cells. Because we find a partial reversion, we cannot rule out other effects of bFGF during G2/M. Indeed, using a BrdUrd pulse-chase assay we showed that bFGF induced an ∼8 h delay in G2 progression (Fig. 1 C), whereas the mitotic entry was only inhibited for 4–6 h (Fig. 2), which could indicate that bFGF has additional effects on events later in mitosis. Besides Chk1-mediated inhibition of Cdc25C, other pathways are activated after DNA damage to enforce a delay in G2. For example, p53-regulated p21 expression results in a sustained G2 arrest in response to DNA damage (27.Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2517) Google Scholar), whereas inhibition of the nuclear translocation of cyclin B by DNA damage blocks the access of cyclin B to its mitotic substrates (28.Jin P. Hardy S. Morgan D.O. J. Cell Biol. 1998; 141: 875-885Crossref PubMed Scopus (259) Google Scholar). We found that expression of Cdc25C-S216A was unable to revert the inhibition of cdc2-Tyr-15 dephosphorylation that occurs in response to the DNA-damaging agent adriamycin (Fig. 5 B), consistent with the existence of parallel pathways. Thus, although certain similarities were observed between the DNA damage response and the effects of bFGF described here, the efficient rescue of bFGF-induced G2arrest seen in cells expressing Cdc25C-S216A mutant indicates that the pathways affected by bFGF are not as diverse as those activated after DNA damage. A potentially interesting link between bFGF-induced growth inhibition in G2 and the DNA damage checkpoint is suggested by studies on the radioprotective effect of bFGF. It has been reported that exogenous bFGF can protect cells from the lethal effects of ionizing radiation (40.Haimovitz-Friedman A. Vlodavsky I. Chaudhuri A. Witte L. Fuks Z. Cancer Res. 1991; 51: 2552-2558PubMed Google Scholar, 41.Fuks Z. Persaud R.S. Alfieri A. McLoughin M. Ehleiter D. Schwartz J.L. Seddon A.P. Cordon-Cardo C. Haimovitz-Friedman A. Cancer Res. 1994; 54: 2582-2590PubMed Google Scholar). This radioprotective effect of bFGF is correlated with a pronounced increase in the duration of the G2 arrest after irradiation (42.Jung M. Kern F.G. Jorgensen T.J. McLeskey S.W. Blair O.C. Dritschilo A. Cancer Res. 1994; 54: 5194-6197PubMed Google Scholar). Interestingly, bFGF expression has been reported to be present in abundant amounts in primary brain tumors, which are known for their poor responsiveness to radiation therapy (43.Takahashi J.A. Mori H. Fukumoto M. Igarashi K. Jaye M. Oda Y. Kikuchi H. Hatanaka M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5710-5714Crossref PubMed Scopus (247) Google Scholar). Given the effects of bFGF that we describe here, it is a distinct possibility that tumors producing high levels of bFGF respond so poorly to radiotherapy because of the suggested resemblance between DNA damage and bFGF in regulating cdc2 activity. We thank Dr. Piwnica-Worms for providing the Cdc25C plasmids. We thank the members of the Bos laboratory for helpful discussions, technical assistance, and reagents. We also thank the other members of the Jordan laboratory for critical discussions.