and -independent Pathways in p21Cip1/Waf1 Induction

Ras- and Mitogen-activated Protein Kinase Kinase-dependentand -independent Pathways in p21Cip1/Waf1 Induction byFibroblast Growth Factor-2, Platelet-derived GrowthFactor, and Transforming Growth Factor-b11

Laura Kivinen and Marikki Laiho2

Haartman Institute, Department of Virology, University of Helsinki, FIN-00014 Helsinki, Finland

Abstractp21Waf1/Cip1 (hereafter referred to as p21) is up-regulated in differentiating and DNA-damaged cells,but it is also up-regulated by serum and growthfactors. We show here that fibroblast growth factor-2(FGF-2), platelet-derived growth factor (PDGF), andtransforming growth factor-b1 (TGF-b1) all induce p21expression in mouse fibroblasts, but with markedlydifferent kinetics. We link their effect on p21 to Rasand mitogen-activated protein kinase kinase-1(/2)[MEK1(/2)]-regulated pathways using either a specificMEK1(/2) inhibitor (PD 098059) or cells expressingconditionally activated Ras or dominant negative Ras.We demonstrate that p21 induction by PDGF and TGF-b1 requires MEK1(/2) and, additionally, that the TGF-b1effect on p21 depends on Ras, whereas the PDGFeffect does not. In contrast, FGF-2 regulation of p21 islargely independent of MEK and Ras. However, PD098059 efficiently inhibited S-phase entry of quiescentcells induced by either FGF-2 or PDGF, suggestingseparate signaling pathways for FGF-2 in induction ofp21 and in S-phase entry. The results suggest differentbut partly overlapping signaling pathways in growthfactor regulation of p21.

IntroductionGrowth factor receptor binding activates multiple intracellu-lar pathways; the appropriate integration of these pathwaysin proliferation, differentiation, and growth arrest are crucialfor the function of a cell. The final outcome of growth factorsignaling is the result of complicated interplay between ac-tivated pathways and feedback loops, and it is also depend-ent on the amplitude and duration of the signals as well as

type of cell involved. Ras protein is an important mediator ofgrowth factor signals (reviewed in Ref. 1). Its activation toGTP-bound form is induced by various extracellular stimuliincluding EGF,3 PDGF, FGF, and NGF via activated tyrosinekinase receptors as well as by TGF-b and interleukins (re-viewed in Ref. 2). Ras generates diverse cellular outcomes.Its essential role in eukaryotic cell growth has emerged fromstudies in which neutralizing Ras-antibodies or microinjec-tion of dominant negative Ras into mouse fibroblasts preventthe S-phase entry of the cells by serum stimulation (3, 4).Furthermore, proliferation and transformation of mouse fi-broblasts by tyrosine kinase oncogenes such as src, fms,and fes are blocked by dominant negative Ras (4). In mousepheochromocytoma PC-12 cells, activated Ras induces dif-ferentiation in the absence of extracellular differentiation sig-nals, whereas dominant negative Ras is sufficient to preventdifferentiation of these cells by NGF or FGF (reviewed in Ref.5). In addition to proliferation and differentiation signals, highamounts of Ras lead to senescence in primary cells viainduction of growth-inhibitory proteins like p53, p16Ink4a, andp21Cip1/Waf1 (hereafter referred to as p21; Ref. 6).

Ras activates a multitude of downstream pathways. Thebest characterized of these is Raf and MAPK (also calledERK) pathway. Like Ras, MAPK activation is essential forfibroblast proliferation (7), and most growth factors activatingRas also phosphorylate and activate MAPKs (reviewed inRef. 8). In many cell types, activation of Ras leads to consti-tutive activation of MAPKs (9), whereas dominant negativeRas blocks MAPK phosphorylation by various extracellularsignals such as NGF, FGF, EGF, and 12-O-tetradecanoyl-phorbol-13-acetate (10–12); PDGF (13, 14); and TGF-b (15).However, cell type differences in Ras regulation of MAPKphosphorylation, at least by 12-O-tetradecanoylphorbol-13-acetate and EGF, have been observed (13, 14). Activation ofMEK leads to growth factor-independent proliferation andtransformation of NIH 3T3 cells, whereas dominant negativeMEK mutant restores normal growth of Ras-transformedcells (9). Furthermore, similar to dominant negative Ras,dominant negative MEK prevents neuronal differentiation ofPC-12 cells, whereas constitutively active MEK induces dif-ferentiation of these cells in the absence of growth factorstimulation (9). Although these results imply a close linkReceived 3/26/99; revised 6/25/99; accepted 7/27/99.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.1 Supported by the Academy of Finland, the Finnish Cancer Organiza-tions, the Emil Aaltonen Foundation, the Finnish Medical Society Duo-decim, the Instrumentarium Scientific Fund, and Biocentrum Helsinki.2 To whom requests for reprints should be addressed, at Haartman Insti-tute, Department of Virology, University of Helsinki, P.O. Box 21, Haart-maninkatu 3, FIN-00014 Helsinki, Finland. Phone: 358-9-1912 6509; Fax:358-9-1912 6491; E-mail: [email protected].

3 The abbreviations used are: EGF, epidermal growth factor; PDGF, plate-let-derived growth factor; FGF, fibroblast growth factor; NGF, nervegrowth factor; TGF-b, transforming growth factor-b; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK,MAPK kinase; cdk, cyclin-dependent kinase; IPTG, isopropyl-b-D-thiogal-actoside; BrdUrd, 5-bromo-29-deoxyuridine; NBCS, newborn calf serum;GAPDH, glyceraldehyde phosphate dehydrogenase.

621Vol. 10, 621–628, September 1999 Cell Growth & Differentiation

between Ras and MAPK pathway in cell proliferation anddifferentiation, additional pathways activated by Ras exist.MAPK-independent pathways, including guanine-nucleotideexchange factors Rho, its homologue Rac1, and Ral, havebeen characterized in Ras-mediated cellular transformation(16, 17). In addition, other candidates for Ras effector func-tions, including phosphatidyl inositol-3-OH kinase (18), GAPproteins, RalGDS, AF6, and Rin1 (reviewed in Ref. 19), havebeen described. These results suggest several Ras-inducedpathways in different cellular responses. Furthermore, evi-dence of Ras-independent activation of MAPK pathway byprotein kinase C exists (20, 21).

p21 cyclin kinase inhibitor is up-regulated in response togrowth arresting and differentiation signals (22, 23), and itmediates cell cycle arrest by inhibiting cdk activity (24). Be-sides inhibiting the cell cycle machinery, p21 also functionsas an assembly factor for cdk/cyclin complexes (25, 26).Thus, p21 action may be required for coordinated cell cycleprogression. Indeed, p21 cyclin kinase inhibitor is induced byserum (27) and growth factors, including NGF (28, 29), EGF(30), TGF-b (31), PDGF, and FGF (32–34), as well as by theRas (6, 35), Raf (36, 37), and MAPK (38) pathways. Rho, onthe other hand, negatively controls Ras-mediated p21 induc-tion (39). Evoking p21 action by both positive and negativegrowth-regulatory signals may, thus, serve to control the cellcycle in various cellular contexts. In addition, the durationand magnitude of p21 induction may determine the cellularresponse. Here, we have addressed the involvement of Rasand MAPK pathways in p21 induction by FGF-2, PDGF, andTGF-b1, with the use of conditional expression of activatedor dominant negative Ras and MEK1(/2) inhibitor PD 098059.

ResultsEffects of TGF-b1, FGF-2, and PDGF on p21 Expression.We and others have recently demonstrated that Ras, Raf,and MEK (6, 35–38) induce p21 by both transcriptional andposttranscriptional means. In addition, PDGF, FGF, EGF, andTGF-b induce p21 expression in mouse fibroblasts andHaCaT cells (31, 32). Because several of the growth factorsare known to use Ras-mediated intracellular pathways forsignaling, we wanted to evaluate the involvement of Ras andMEK/MAPK pathway in the growth factor action leading top21 induction. NIH 3T3 cells were treated with increasingconcentrations of FGF-2, PDGF, and TGF-b1 in low serumand incubated for 12 h. As shown by Western blot analysis,p21 protein was induced by all tested growth factors in adose-dependent manner (3.0-fold induction by 7 ng/mlFGF-2, 5.5-fold by 10 ng/ml PDGF, and 4.2-fold by 250 pM

TGF-b1; Fig. 1). Analysis of the same gels by anti-phospho-ERK1/2 antibodies showed also dose-dependent inductionof active, phosphorylated ERK1 and ERK2 by FGF-2, PDGF,and TGF-b1 (Fig. 1). To test whether a p21-related inhibitor,p27Kip1 (40), is affected by the growth factors, we reprobedfilters used for p21-probing with anti-p27Kip1 antibodies.However, the levels of p27Kip1 were unaffected by the growthfactors (Fig. 1). Even sample loading was confirmed by rep-robing the filters with an anti-ERK2 antibody, which showedinvariant levels of ERK2 (see Figs. 1, 2, 4, and 5).

Kinetics of p21 Induction and ERK1/2 Phosphorylationby Growth Factors. To analyze the kinetics of p21 induc-tion, we treated starved NIH 3T3 cells with FGF-2, PDGF,and TGF-b1 for various times in low serum. The induction ofp21 was most rapid by FGF-2, being detectable as early as30 min after growth factor stimulation and peaking at 2 h(1.6- and 4.8-fold inductions; Fig. 2A). The PDGF effect onp21 was also rapid, with the induction peaking at 2 h (2.5-foldinduction; Fig. 2B). However, comparison of the p21 levelsshowed that, after 2 h, the levels of FGF-2- and PDGF-induced p21 gradually started to decline (Fig. 2, A and B). Incontrast to the effect by FGF-2 and PDGF, the TGF-b1-induction of p21 was significantly slower reaching maximallevels only 12 h after growth factor addition (2.1-fold; Fig.2C). Longer starvation of the cells was observed to decrease

Fig. 1. p21 induction by FGF-2, PDGF, and TGF-b1. NIH 3T3 cells werestarved in 0.2% NBCS for 4 h, after which increasing amounts of FGF-2(A), PDGF (B), or TGF-b1 (C) were added, and the cells were incubated for12 h further. Cell lysates were prepared and analyzed by 12.5% SDS-PAGE, followed by immunoblotting with anti-p21, anti-p27Kip1, anti-phos-pho-ERK1/2, and anti-ERK2 antibodies, as indicated. The protein levelswere quantitated, and p27Kip1 and ERK2 levels were used as a loadingcontrols for p21 and phospho-ERK1/2, respectively. Fold inductions ofp21 as compared with nontreated cells starved for 16 h are shown.

622 p21Cip1/Waf1 Induction by Growth Factors

the basal p21 levels (data not shown), thereby affecting themagnitude of p21 induction by the growth factors. Therefore,because the control cells were starved for 16 h in Fig. 1 ascompared to 4 h in Fig. 2, the decrease in basal p21 levelyielded an apparently greater p21 induction by, e.g., PDGF inFig. 1B versus Fig. 2B (5.5-fold by 12 h as compared with2.5-fold by 2 h). Induction of ERK1/2 phosphorylation byFGF-2 and PDGF was maximal 30 min after growth factortreatment, followed by a decrease 2 h after FGF-2 and 6 hafter PDGF addition (Fig. 2, A and B). Similar to the inductionof p21, the induction of ERK1/2 phosphorylation by TGF-b1was slower than that by FGF-2 or PDGF peaking at 12 h (Fig.2C). Again, there was no variation in p27Kip1 or total ERK2levels.

Requirement of MEK1(/2)-dependent Pathways inGrowth Factor Regulation of p21. The involvement ofMAPK pathway in p21 induction by FGF-2, PDGF, andTGF-b1 was studied by pretreating the cells with MEK1(/2)inhibitor PD 098059 (80 mM) for 4 h in low serum followed byaddition of growth factors and analysis of p21 levels and

ERK1/2 phosphorylation by Western blotting at differenttimepoints. PD 098059 was found to inhibit phosphorylationof ERK1/2 by FGF-2 by 58% 0.5 h after the growth factoraddition, with prominent inhibition at later timepoints (72 and59% inhibition at 2 and 6 h, respectively; Fig. 2A). However,the induction of p21 by FGF-2 was unaffected, although thelevel of phospho-ERK1/2 was decreased (Fig. 2A). Similarresults were obtained in cells incubated with PD 098059 andincreasing concentrations of FGF-2 for 12 h (data notshown). This further confirmed that FGF-2 at concentrations3–7 ng/ml increased p21 independently of MEK1(/2). PD098059, which efficiently inhibited PDGF-stimulated ERK1/2phosphorylation (64 and 85% inhibition by 30 min and 2 h,respectively; Fig. 2B), also inhibited fully the PDGF inductionof p21 (Fig. 2B). TGF-b1 induction of phospho-ERK1/2 wasclearly reduced by the MEK1(/2) inhibitor at all timepoints,and at the same time, TGF-b1 induction of p21 after 12 htreatment was abolished (Fig. 2C). The results suggest thatactive MEK1(/2) is required for induction of p21 by PDGF,and TGF-b1, whereas the induction by FGF-2 seems tolargely use MEK-independent pathways. Unexpectedly, si-multaneous treatment of the cells with PD 098059 andTGF-b1 for 2 h repeatedly induced p21 by 2-fold (Fig. 2C),whereas at the same timepoint, PD 098059 alone had noeffect (data not shown). This amplified p21 induction by PD098059 in cells treated for 2 h with TGF-b1 may imply MEK1(/2)-dependent negative regulation of p21 in response to TGF-b1. Reprobing of the filters used for detection of p21 andphospho-ERK1/2 showed no significant change in the levelsof either p27Kip1 or total ERK2 (Fig. 2).

To investigate whether the growth factor effects are ex-erted via regulation of p21 mRNA, we cultured NIH 3T3 cellsin low serum in the presence of TGF-b1, FGF-2, and PDGFwith or without PD 098059 followed by Northern blotting.Incubation of the cells for 1 h with FGF-2 or PDGF inducedp21 mRNA by 2.8- and 2.5-fold, respectively, whereasTGF-b1 had no effect (Fig. 3A). PD 098059 decreased thep21 mRNA induction by FGF-2 and PDGF by 50 and 30%,respectively. In contrast, p21 mRNA induction was absent incells incubated with the growth factors for 16 h (Fig. 3B),suggesting a transient effect by the growth factors. Theresults indicate that the increase in p21 protein by FGF-2 orPDGF involves an increase in the amount of p21 mRNA,whereas TGF-b1 may act by enhancing translation and/orprotein stabilization. However, as shown in Fig. 2A, down-regulation of FGF-2-induced p21 mRNA by PD 098059 doesnot lead to a corresponding decrease in p21 protein level.This would suggest that FGF-2 may increase p21 also bymeans other than affecting its mRNA expression. Further-more, although no evidence exists of major MEK1(/2)-de-pendent p21 regulation by FGF-2 at the protein level, p21mRNA induction by FGF-2 seems to be partially underMEK1(/2) control.

Effects of Oncogenic Ras on p21 Induction by FGF-2,PDGF, and TGF-b1. To study the possible modulation ofp21 induction by Ras, we used NIH 3T3 cells stably trans-fected with lactose analogue (IPTG) inducible Ha-Ras(Val-12)expression vector (ras8 cells). Ras induction is detectablewithin 6 h after IPTG treatment, being maximal by 16 h (35).

Fig. 2. Kinetics and MEK1(/2) dependency of p21 induction. NIH 3T3cells were pretreated in low serum with or without PD 098059 (80 mM) for4 h, after which FGF-2 (5 ng/ml; A), PDGF (10 ng/ml; B), or TGF-b1 (250pM; C) were added for the indicated times. Cell lysates were preparedfollowed by protein separation in 12.5% SDS-PAGE and immunoblottingwith anti-p21, anti-p27Kip1, anti-phospho-ERK1/2, and anti-ERK2 anti-bodies as indicated. p21 levels were normalized against p27Kip1 levelsand the fold induction as compared with nontreated cells starved for 4 hare shown below.

623Cell Growth & Differentiation

Due to basal expression of Ha-Ras(Val-12) in uninducedcells, the levels of p21 and phospho-ERK1/2 were constitu-tively higher in ras8 cells as compared with parental NIH 3T3cells (data not shown). However, induction of Ras expression(7-fold induction by IPTG; Fig. 4A), accompanies induction ofERK1/2 phosphorylation (mean induction of ERK1/2 phos-phorylation, 1.9-fold; Fig. 4) and p21 expression (mean in-duction of p21, 3.2-fold; in Fig. 4). The induction of Rasexpression was analyzed in each experiment and was foundto be similar in all growth factor-treated cells (data notshown): Ras expression in FGF-2-treated cells is shown (Fig.4A). All growth factors stimulated phosphorylation of ERK1/2in the Ras-expressing cells (Fig. 4), and a synergistic effecton p21 expression was observed by FGF-2 and PDGF (Fig.4, A and B). The effect was detected 2 h after FGF-2 andPDGF addition, being maximal at 6 h, indicating prolongedinduction of p21 expression and ERK1/2 phosphorylation inRas-expressing cells as compared with NIH 3T3 cells (Fig. 4,A and B). In FGF-2-treated Ras-expressing cells, phospho-rylation of ERK1/2 was sustained for 12 h (7.9-fold increase;Fig. 4A), whereas in PDGF-treated cells, phospho-ERK1/2levels decreased to the basal levels by 12 h (Fig. 4B). InFGF-2- and PDGF-treated Ras-expressing cells; however,the synergistic effect on p21 disappeared by 12 h (Fig. 4, Aand B). In contrast to the kinetics seen in NIH 3T3 cells,TGF-b1 treatment of Ras-expressing cells increased phos-phorylation of ERK1/2 2 h after growth factor addition (Fig.4C). Similarly, p21 was induced by TGF-b1 with somewhatfaster kinetics starting at 6 h in Ras-expressing cells ascompared with NIH 3T3 cells (Fig. 4C). However, p21 was notfurther increased by TGF-b1 in Ras-expressing cells at anytimepoint (Fig. 4C). The specificity of p21- and phospho-ERK1/2 effects in Ras-expressing cells were confirmed by

analyzing the levels of p27Kip1 (data not shown) and ERK2(Fig. 4), which were unaffected by Ha-Ras or the growthfactors at all timepoints. We, therefore, suggest that, be-cause FGF-2 and PDGF synergistically affect p21 levels inRas-expressing cells, they may activate also other, Ras-independent pathways for induction of p21. TGF-b1, in con-trast, seems to use a Ras-activated pathway because it isunable to further induce p21 in Ras-expressing cells.

Effects of Dominant Negative Ras Expression on p21Induction by the Growth Factors. To further investigatethe role of Ras in p21 induction by the growth factors, weused a stable NIH 3T3 clone conditionally expressing dom-inant negative Ras(Asn-17) mutant. Induction of Ras(Asn-17)expression after a 30-h treatment with IPTG was prominent(mean induction, 5.5-fold; Fig. 5), and was not affected by thegrowth factors. Expression of Ras(Asn-17) was able to abol-ish the induction of p21 by TGF-b1, with concomitant inhi-bition of ERK1/2 phosphorylation by 47% (Fig. 5). Inductionof p21 by FGF-2 was slightly attenuated (33%) whereasphospho-ERK1/2 was reduced by 66%. However, there wasno effect of Ras(Asn-17) expression on p21 induction byPDGF, although phosphorylation of ERK1/2 was greatly re-duced (75%; Fig. 5). The levels of p27Kip1 (data not shown)

Fig. 3. FGF-2 and PDGF induce p21 mRNA levels. A and B, Northernblotting analyses of p21 mRNA levels. A, NIH 3T3 cells were pretreated inlow serum with or without PD 098059 (80 mM) for 4 h followed by incu-bation with FGF-2 (5 ng/ml), PDGF (10 ng/ml), or TGF-b1 (250 pM) for 1 h.B, NIH 3T3 cells were cultured in low serum in the absence (Lanes 2) orpresence (Lanes 1) of 50 mM PD 098059 and TGF-b1 (250 pM), FGF-2 (7ng/ml), or PDGF (10 ng/ml) for 16 h. Poly(A)1 mRNAs were prepared, andNorthern blotting analyses were performed using 32P-labeled mouse p21and GAPDH cDNAs as probes. The signals were quantitated by Phos-phoImager analyzer and normalized against GAPDH. Fold induction ofp21 mRNA as compared with nontreated controls are shown below.

Fig. 4. Effect of Ha-Ras expression on p21 regulation by growth factors.ras8 cells were cultured with or without IPTG (10 mM) for 8–12 h to induceRas expression. The cells were transferred to low serum for 4 h, followedby culturing the cells for indicated times with FGF-2 (5 ng/ml; A), PDGF (10ng/ml; B), or TGF-b1 (250 pM; C) in the presence or absence of IPTG.Immunoblotting was performed with anti-p21, anti-phospho-ERK1/2, an-ti-ERK2, and anti-Ras antibodies as indicated. Fold inductions of p21 ascompared with non-IPTG-treated cells grown in low serum for 6 h areshown below.


and ERK2 (Fig. 5) analyzed from the same filters remainedunchanged by Ras(Asn-17) expression or the growth factortreatments. These results implicate that Ras function is re-quired for p21 induction by TGF-b1, whereas PDGF, and, inlarge part, also FGF-2 activate pathways not involving Ras.

MEK1(/2) Inhibitor Prevents S-Phase Entry Induced byFGF-2 and PDGF. To correlate the p21 regulation to the cellcycle effects of TGF-b1, FGF-2, and PDGF in NIH 3T3 cells,we analyzed DNA replication of the cells and cell cycle dis-tribution by BrdUrd incorporation and flow cytometry of cellscultured in low serum with or without growth factors and PD098059. In low serum, only 1% of the cells were replicatingDNA (Fig. 6), and a 12-h incubation with TGF-b1 (250 pM)was unable to induce S-phase entry of the cells (1.6% rep-lication; Fig. 6). In contrast, treatment with FGF-2 or PDGF

increased the amount of S-phase cells to 23 and 18%,respectively (Fig. 6). Similar results were obtained by flowcytometry (Table 1). Simultaneous treatment of the cells withPD 098059 prevented the S-phase entry of the cells byFGF-2 and PDGF as shown by BrdUrd incorporation (89 and72% inhibition of DNA replication, respectively), with similarresults obtained by fluorescence-activated cell sorting anal-ysis (64 and 62% inhibition, respectively; Table 1). The re-sults, therefore, indicate that inhibition of MEK1(/2)-activatedpathway(s) significantly decreases the mitogenic effects ofboth FGF-2 and PDGF, regardless of differences in the levelof p21 or pathways required for its induction.

DiscussionWe show here that FGF-2, PDGF, and TGF-b1 induce p21expression in NIH 3T3 cells, which is consistent with resultsobtained by using other cell types (31, 32, 34, 41). Our resultsindicate, however, that pathways leading to p21 induction bythese growth factors vary, as suggested by their requirementof Ras and MEK1(/2) and by their different kinetics. In NIH3T3 cells, by using a MEK1(/2) inhibitor and conditional ex-pression of Ha-Ras or dominant negative Ras(Asn-17), p21induction by FGF-2 was shown to be mainly MEK1(/2) inde-pendent, whereas PDGF-regulation of p21 was MEK1(/2)dependent and, like FGF-2-regulation, independent of Ras(Fig. 7). In contrast, TGF-b1 regulation of p21 expression wasfound to require both MEK1(/2) and Ras (Fig. 7). However,based on these results, we cannot exclude that also FGF-2and PDGF could use intact Ras and MEK signaling pathwaysfor p21 induction. In addition, in differing cellular contexts,the signaling pathways used by the growth factors may vary.Reprobing of all filters used for detection of p21 with anti-p27Kip1 antibodies demonstrated that p27Kip1 expressionwas unaffected by the growth factors, MEK1(/2) inhibitor,Ha-Ras, or dominant negative Ras as well as their combina-tion and confirmed the specificity of the observed p21 effect.

FGF-2, at the doses used, was found to function largely ina manner independent of Ras and MEK1(/2) and to rapidlyinduce p21 in NIH 3T3 cells (Fig. 7). However, we cannotexclude that residual MEK1(/2) activity stimulated by FGF-2exists, which leads to partial p21 induction. Still, p21 induc-tion by FGF-2 was not down-regulated by PD 098059, al-though the MEK1(/2) inhibitor inhibited ERK1/2 phosphoryl-ation and reversed S-phase entry of the cells. Similarly,dominant negative Ras decreased p21 induction by FGF-2somewhat, but not significantly. Thus, we cannot fully ex-clude the utilization of MEK/Ras pathway by FGF-2 in p21regulation, but based on the studies here, neither MEK norRas seems to be exclusively required. Previous studies showthat FGF activates both Ras- and MAPK-dependent as wellas -independent pathways in execution of its various cellularactions, suggesting that diverse activated pathways specifydifferent outcomes in the cell. Indicating the complexity ofthe FGF signaling, activation of prolactin promoter by FGF isa Ras-independent but MAPK-dependent event (42),whereas transcriptional activation of cyclic AMP-responsiveelement-binding protein by FGF requires Ras but not MAPK(43). In primary endothelial cells FGF has been shown toinduce p21 transiently in a MEK1(/2)-dependent manner fol-

Fig. 5. Effects of dominant negative Ras(Asn-17) expression on p21 bygrowth factors. Conditionally Ras(Asn-17) expressing NIH 3T3 cells weretreated with 10 mM IPTG for 30–48 h to induce Ras(Asn-17) expression,and the cells were transferred to low serum. After a 4-h starvation, FGF-2(5 ng/ml) or PDGF (10 ng/ml) were added for 2 h or TGF-b1 (250 pM) for12 h, followed by lysis, protein separation in 12.5% SDS-PAGE andimmunoblotting with anti-p21, anti-phospho-ERK1/2, anti-ERK2, and an-ti-Ras antibodies as indicated. Cells cultured in low serum for 6 h in theabsence of IPTG were used as a control to determine the fold induction ofp21.

Fig. 6. DNA replication induced by growth factors is modulated by PD098059. NIH 3T3 cells were cultured in low serum for 4 h, followed byaddition of TGF-b1 (250 pM), FGF-2 (7 ng/ml), or PDGF (10 ng/ml) in thepresence (1, ■) or absence (2, h) of 50 mM PD 098059, and the cells wereincubated for 12 h. For the last hour of the incubation, BrdUrd was added,after which the cells were fixed and immunostained with anti-BrdUrdantibody. Columns, mean fraction of anti-BrdUrd-stained nuclei as com-pared with all nuclei was determined from five independent experiments;bars, SD. c, control cells.


lowed by S-phase entry (34). The differing results on require-ments of MEK1(/2) action in p21 induction by FGF-2 may bedue to differences in cell type and/or differences in cellsurface receptors present (33).

PDGF signaling activates Ras (reviewed in Ref. 2) andERK1/2 (44). Our results show, however, that, in NIH 3T3cells, p21 induction by PDGF occurs mainly independently ofRas but through MEK1(/2)-dependent pathway(s) (Fig. 7).Additionally, because dominant negative Ras did not affectp21 induction by PDGF, although ERK1/2 phosphorylationwas markedly reduced, PDGF seems to act also independ-ently of ERK1/2.

TGF-b1 induction of p21 in quiescent NIH 3T3 cells oc-curred relatively slowly and, thus, differed from that by othergrowth factors (Fig. 2). Slow ERK1/2 phosphorylation andlate induction of p21 by TGF-b1 suggest that they are notdirect targets of TGF-b1 but that TGF-b1 may recruit otherpathways that in turn activate MEK1(/2) to induce p21. Asimilar delayed p21 induction by TGF-b has been reported inHaCaT keratinocytes and in epithelial cells growth-inhibitedby TGF-b (41, 45). Surprisingly, at early timepoints, MEK1(/2)inhibitor augmented the TGF-b1 effect (Fig. 2C), suggestingthat MEK1(/2) may also act in negative regulation of p21upon TGF-b1 treatment. In addition, negative feedback

loops along the MAPK pathway are known to exist, e.g., PD098059 enhances Raf activity by various extracellular stimuli(46), which could lead to additional p21 induction.

TGF-b1 was found to elicit its effects on p21 through bothRas and MEK1(/2) (Fig. 7), whereas requirement of ERK1/2by TGF-b1 is not conclusive from these experiments. TheTGF-b effects on MAPK pathway appear to be cell typespecific. In rat hepatic stellate cells TGF-b induces a rapidactivation of Ras, Raf, MEK1, and ERK1/2 (47), whereas inSwiss 3T3 cells, there is no ERK activation by TGF-b (48). Inepithelial cells sensitive to growth inhibition by TGF-b, TGF-b

activates Ras (49) and ERK1 (50) as well as induces p21 andp27 cyclin kinase inhibitors (41). The TGF-b effects on ERK1,p21 and p27 are abolished by dominant negative Ras ex-pressed in these cells (15, 41). The results on epithelial cellssupport our data on Ras dependency of p21 induction byTGF-b, whereas the MEK1(/2) dependency by TGF-b1 ap-pears to be a novel finding. Unlike its behavior in epithelialcells, TGF-b did not regulate p27 levels in NIH 3T3 cells (Figs.1C and 2C).

p21 mRNA levels were increased shortly after FGF-2 andPDGF treatments in NIH 3T3 cells, similarly to other studies(32, 34). TGF-b1 was unable to induce p21 mRNA in NIH 3T3cells, although in HaCaT keratinocytes, it induces p21 atleast partially through transcriptional activation (31). Cell typedifferences may explain these conflicting results. However,several studies support posttranscriptional regulation of p21by growth factors and serum (27, 34, 35, 51).

Whereas p21 regulation by FGF-2 and PDGF is accompa-nied by S-phase entry of starved NIH 3T3 cells, the late p21induction by TGF-b1 appears not to be coupled with cellcycle progression, because TGF-b1 is unable to induce S-phase entry of quiescent NIH 3T3 cells. Several studies havedemonstrated transient p21 induction by either FGF-2 orPDGF at both protein and mRNA levels, which is accompa-nied by cell cycle entry of quiescent cells (27, 32, 34),whereas a sustained p21 induction takes place in growth-arrested cells (32–34). For example, in breast cancer cellsgrowth arrested by FGF, a sustained p21 induction is de-tected, which is lacking in a breast cancer cell line resistantto growth inhibition (33). Thus, S-phase entry is probablyfeasible due to the transient nature of p21 expression byFGF-2 and PDGF found also here. Inhibition of MEK actionprevented the S-phase entry by both PDGF and FGF-2,although the MEK-inhibition was unable to down-regulateFGF-2-induced p21. This suggests separate pathways forFGF-2 in regulation of cell cycle progression and p21 induc-tion. It is possible that the transient induction of p21 by

Table 1 Cell cycle distribution of growth factor-treated NIH 3T3 cells in the presence and absence of PD 098059a

ControlTGF-b1 FGF-2 PDGF

2PD98059 1PD98059 2PD98059 1PD98059 2PD98059 1PD98059

G1 85 81 85 77 80 76 83S 2 3 3 14 5 13 5G2/M 13 16 12 9 16 11 12

a Starved NIH 3T3 cells were treated with TGF-b1 (250 pM), FGF-2 (7 ng/ml), or PDGF (10 ng/ml) in the presence or absence of 50 mM PD 098059 for 12 hfollowed by flow cytometry.

Fig. 7. Growth factor-activated pathways involved in p21 induction. Onthe basis of these results, p21 induction by FGF-2 occurs mainly inde-pendent of MEK1(/2) and Ras. PDGF, in contrast, requires MEK1(/2) toinduce p21, whereas TGF-b1 uses both Ras- and MEK1(/2)-dependentpathways.


growth-stimulating factors serves as a regulatory step in cellcycle progression, e.g., by modulating the assembly of cdk4/cyclin D complexes (25, 26), and that S-phase entry occurswhen the amount of active cdk/cyclin complexes exceedsthe amount of their inhibitors, such as p21.

Materials and MethodsCells and Cell Culture. Mouse NIH 3T3 fibroblasts (ATCC CRL 1658,Rockville, MD) were maintained in DMEM containing 10% NBCS (LifeTechnologies, Inc., Grand Island, NY). Conditionally Ha-Ras(Val-12) ex-pressing NIH 3T3 cells have been described previously (52). For condi-tional expression of dominant negative Ras, Ras(Asn-17) kindly providedby Dr. L. A. Feig (Tufts University, Boston, MA) was cloned into pSVlacO-vector [pSVlacO-Ras(Asn-17)], and NIH 3T3 cells were transfected bycalcium phosphate precipitation with equal amount of pHbINLSneo andpSVlacO-Ras(Asn-17) plasmids followed by selection with 0.6 mg/mlG418 and ringcloning of single-cell colonies. All NIH 3T3 transfectantswere maintained in DMEM containing 10% NBCS supplemented with 0.4mg/ml G418. Ha-Ras(Val-12) and Ras(Asn-17) expression was induced byaddition of 10 mM lactose analogue IPTG (Promega, Madison, WI) to theculture medium.

Immunoblotting Analyses. Preparation of cell lysates for immuno-blotting analyses has been described previously (53). Bio-Rad DC proteinassay was used to determine protein concentration of the lysates, and 150mg of protein were electrophoretically resolved in 12.5% SDS-PAGE andtransferred to Immobilon P membranes (Millipore, Bedford, MA). Mousep21 was detected with anti-p21 antibody (13436E; PharMingen), p27 withanti-p27Kip1 antibody (K25020; Transduction Laboratories, Lexington,KY), ERK2 with anti-ERK2 antibody (E16220; Transduction Laboratories),and Ras with anti-Ras antibody (Abx1; Oncogene Science, Cambridge,MA). For detection of phosphorylated ERK1 and ERK2, phosphospecificanti-MAPK antibody (V667A; Promega) were used. Bound immunoglobu-lins were detected with horseradish peroxidase-conjugated antimouse(for p27, ERK2, and Ras) and antirabbit IgG (for p21 in Figs. 2, 6, and 7;Dako, Glostrup, Denmark), or with biotinylated antirabbit IgG (for phos-pho-ERK1/2 and for p21 in Fig. 1; Dako) followed by biotin-streptavidin-conjugated horseradish peroxidase (Amersham, Buckinghamshire, UnitedKingdom). Final detection step was performed by chemiluminescence(ECL; Amersham). The quantitation of the signals was carried out usingBio-Rad MultiAnalyst Version 1.0.

mRNA Isolation and Hybridization. Poly(A)1 mRNAs were prepared,and Northern blotting was performed as described previously (52). Nitro-cellulose filters were probed with 32P-labeled mouse p21 cDNA (from Dr.Bert Vogelstein, Johns Hopkins University, Baltimore, MD), followed byprobing with GAPDH cDNA to confirm equal loading. Signals were quan-titated by Fujifilm BAS 2500 PhosphoImager analyzer and MacBas Ver-sion 2.5 program and, thereafter, normalized against GAPDH.

Cell Cycle Analyses. DNA replication of the cells was analyzed by5-BrdUrd incorporation. Growth factors were added in the presence orabsence of PD 098059 (50 mM) to cells precultured for 4 h in DMEMcontaining 0.2% NBCS. BrdUrd (50 mM; Sigma Chemical Co., St. Louis,MO) was added as indicated for the last hour of the 12-h incubation withgrowth factors and MEK1(/2) inhibitor, after which the cells were fixed with3.5% paraformaldehyde for 20 min, permeabilized with 0.5% NP40 for 5min, and treated with 1.5 N HCl for 15 min to denaturate DNA. BrdUrdimmunofluorescence staining was subsequently performed as describedpreviously (54). For fluorescence-activated cell sorting analysis of differentcell cycle phases, cells were precultured in DMEM supplemented with0.2% NBCS followed by growth factor and MEK1(/2) inhibitor (50 mM)treatments for 12 h. After trypsinization cells were fixed, stained withpropidium iodide, and analyzed by flow cytometry as described previously(54).

Chemicals. MEK1(/2) inhibitor PD 098059, FGF-2, and PDGF wereobtained from Calbiochem (La Jolla, CA). TGF-b1 was purified fromhuman platelets.

AcknowledgmentsWe thank L. A. Feig for Ha-Ras(Asn-17) cDNA and Bert Vogelstein formouse p21 cDNA. J. Eriksson is kindly acknowledged for good technicaladvice and reagents for ERK1/2 and MEK inhibitor experiments.

References1. Marshall, C. J. Specificity of receptor tyrosine kinase signalling: tran-sient versus sustained extracellular signal-regulated kinase activation.Cell, 80: 179–185, 1995.

2. Satoh, T., Nakafuku, M., and Kaziro, Y. Function of Ras as a molecularswitch in signal transduction. J. Biol. Chem., 267: 24149–24152, 1992.

3. Mulcahy, L. S., Smith, M. R., and Stacey, D. W. Requirement for Rasproto-oncogene function during serum-stimulated growth of NIH 3T3cells. Nature (Lond.), 313: 241–243, 1985.

4. Stacey, D. W., Roudebush, M., Day, R., Mosser, S. D., Gibbs, J. B., andFeig, L. A. Dominant inhibitory Ras mutants demonstrate the requirementfor Ras activity in the action of tyrosine kinase oncogenes. Oncogene, 6:2297–2304, 1991.

5. Chao, M. V. Growth factor signaling: where is the specificity? Cell, 68:995–997, 1992.

6. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D., and Lowe, S. W.Oncogenic Ras provokes premature cell senescence associated withaccumulation of p53 and p16INK4a. Cell, 88: 593–602, 1997.

7. Pages, G., Lenormand, P., L’Allemain, G., Chambard, J-C., Meloche,S., and Pouyssegur, J. Mitogen-activated protein kinase p42mapk andp44mapk are required for fibroblast proliferation. Proc. Natl. Acad. Sci.USA, 90: 8319–8323, 1993.

8. Hunter, T. Oncoprotein networks. Cell, 88: 333–346, 1997.

9. Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. Activation ofMAP kinase kinase is necessary and sufficient for PC12 differentiation andfor transformation of NIH3T3 cells. Cell, 77: 841–852, 1994.

10. Thomas, S. M., DeMarco, M., D’Arcangelo, G., Halegoua, S., andBrugge, J. S. Ras is essential for nerve growth factor- and phorbolester-induced tyrosine phosphorylation of MAP kinases. Cell, 68: 1031–1040, 1992.

11. Wood, K. W., Sarnecki, C., Roberts, T. M., and Blenis, J. Ras medi-ates nerve growth factor receptor modulation of three signal-transducingprotein kinases: MAP kinase, Raf-1 and RSK. Cell, 68: 1041–1050, 1992.

12. Gardner, A. M., and Johnson, G. L. Fibroblast growth factor-2 sup-pression of tumor necrosis factor a-mediated apoptosis requires ras andthe activation of mitogen-activated protein kinase. J. Biol. Chem., 271:14560–14566, 1996.

13. de Vries-Smits, A. M. M., Burgering, B. M. T., Leevers, S. J., Marshall,C. J., and Bos, J. L. Involvement of p21 ras in activation of extracellularsignal-regulated kinase 2. Nature (Lond.), 357: 602–604, 1992.

14. Burgering, B. M. T., de Vries-Smits, A. M. M., Medema, R. H., vanWeeren, P. C., Tertoolen, L. G. J., and Bos, J. L. Epidermal growth factorinduces phosphorylation of extracellular signal-regulated kinase 2 viamultiple pathways. Mol. Cell. Biol., 13: 7248–7256, 1993.

15. Hartsough, M. T., Frey, R. S., Zipfel, P. A., Buard, A., Cook, S. J.,McCormick, F., and Mulder, K. M. Altered transforming growth factor b

signaling in epithelial cells when Ras activation is blocked. J. Biol. Chem.,271: 22368–22375, 1996.

16. Khosravi-Far, R., White, M. A., Westwick, J. K., Solski, P. A., Chrza-nowska-Wodnicka, M., Van Aelst, L., Wigler, M. H., and Der, C. J. Onco-genic Ras activation of Raf/mitogen-activated protein kinase-independentpathways is sufficient to cause tumorigenic transformation. Mol. Cell.Biol., 16: 3923–3933, 1996.

17. Urano, T., Emkey, R., and Feig, L. A. Ral-GTPases mediate a distinctdownstream signaling pathway from Ras that facilitates cellular transfor-mation. EMBO J., 15: 810–816, 1996.

18. Rodriguez-Viciana, P., Warne, P. H., Dhand, R., Vanhaesebroeck, B.,Gout, I., Fry, M., Waterfield, M. D., and Downward, J. Phosphatidylinosi-tol-3-OH kinase as a direct target of Ras. Nature (Lond.), 370: 527–532,1994.

19. Campbell, S. L., Khosravi-Far, R., Rossman, K. L., Clark, G. J., andDer, C. J. Increasing complexity of Ras signaling. Oncogene, 17: 1395–1413, 1998.

20. Hawes, B. E., van Biesen, T., Koch, W. J., Luttrell, L. M., and Lefkow-itz, R. J. Distinct pathways of Gi- and Gq-mediated mitogen-activatedprotein kinase activation. J. Biol. Chem., 270: 17148–17153, 1995.


21. Ueda, Y., Hirai, S-I., Osada, S-I., Suzuki, A., Mizuno, K., and Ohno, S.Protein kinase C delta activates the MEK-ERK pathway in a mannerindependent of Ras and dependent on Raf. J. Biol. Chem., 271: 23512–23519, 1996.

22. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R.,Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Vogelstein, B.WAF1, a potential mediator of p53 tumor suppression. Cell, 75: 817–825,1993.

23. Parker, S. B., Eichele, G., Zhang, P., Rawls, A., Sands, A. T., Bradley,A., Olson, E. N., Harper, J. W., and Elledge, S. J. p53-independentexpression of p21Cip1 in muscle and other terminally differentiating cells.Science (Washington DC), 267: 1024–1027, 1995.

24. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J.The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell, 75: 805–816, 1993.

25. LaBaer, J., Garrett, M. D., Stevenson, L. F., Slingerland, J. M.,Sandhu, C., Chou, H. S., Fattaey, A., and Harlow, E. New functionalactivities for the p21 family of CDK inhibitors. Genes Dev., 11: 847–862,1997.

26. Cheng, M., Olivier, P., Diehl, J. A., Fero, M., Roussel, M. F., Roberts,J. M., and Sherr, C. J. The p21Cip1 and p27Kip1 CDK “inhibitors” areessential activators of cyclin D-dependent kinases in murine fibroblasts.EMBO J., 18: 1571–1583, 1999.

27. Macleod, K. F., Sherry, N., Hannon, G., Beach, D., Tokino, T., Kinzler,K., Vogelstein, B., and Jacks, T. p53-dependent and independent expres-sion of p21 during cell growth, differentiation, and DNA damage. GenesDev., 9: 935–944, 1995.

28. Yan, G-Z., and Ziff, E. B. Nerve growth factor induces transcription ofthe p21WAF1/CIP1 and cyclin D1 genes in PC12 cells by activating theSp1 transcription factor. J. Neurosci., 17: 6122–6132, 1997.

29. Pumiglia, K. M., and Decker, S. J. Cell cycle arrest mediated by theMEK/mitogen-activated protein kinase pathway. Proc. Natl. Acad. Sci.USA, 94: 448–452, 1997.

30. Fan, Z., Lu, Y., Wu, X., DeBlasio, A., Koff, A., and Mendelsohn, J.Prolonged induction of p21Cip1/Waf1/CDK2/PCNA complex by epider-mal growth factor receptor activation mediates ligand-induced A431 cellgrowth inhibition. J. Cell Biol., 131: 235–242, 1995.

31. Datto, M. B., Yu, Y., and Wang, X-F. Functional analysis of thetransforming growth factor b responsive elements in the WAF1/Cip1/p21promoter. J. Biol. Chem., 270: 28623–28628, 1995.

32. Michieli, P., Chedid, M., Lin, D., Pierce, J. H., Mercer, W. E., and Givol,D. Induction of WAF1/CIP1 by a p53-independent pathway. Cancer Res.,54: 3391–3395, 1994.

33. Johnson, M. R., Valentine, C., Basilico, C., and Mansukhani, A. FGFsignaling activates STAT1 and p21 and inhibits the estrogen response andproliferation of MCF-7 cells. Oncogene, 16: 2647–2656, 1998.

34. Zezula, J., Sexl, V., Hutter, C., Karel, A., Schutz, W., and Freissmuth,M. The cyclin-dependent kinase inhibitor p21Cip1 mediates the growthinhibitory effect of phorbol esters in human venous endothelial cells.J. Biol. Chem., 272: 29967–29974, 1997.

35. Kivinen, L., Tsubari, M., Haapajarvi, T., Datto, M. B., Wang, X-F., andLaiho, M. Ras induces p21Cip1/Waf1 cyclin kinase inhibitor transcription-ally through Sp1-binding sites. Oncogene, in press, 1999.

36. Woods, D., Parry, D., Cherwinski, H., Bosch, E., Lees, E., and Mc-Mahon, M. Raf-induced proliferation or cell cycle arrest is determined bythe level of Raf-activity with arrest mediated by p21Cip1. Mol. Cell. Biol.,17: 5598–5611, 1997.

37. Sewing, A., Wiseman, B., Lloyd, A., and Land, H. High-intensity Rafsignal causes cell cycle arrest mediated by p21Cip1. Mol. Cell. Biol., 17:5588–5597, 1997.

38. Liu, Y., Martindale, J. L., Gorospe, M., and Holbrook, N. J. Regulationof p21WAF1/CIP1 expression through mitogen-activated protein kinasesignaling pathway. Cancer Res., 56: 31–35, 1996.

39. Olson, M. F., Paterson, H. F., and Marshall, C. J. Signals from Ras andRho GTPases interact to regulate expression of p21Waf1/Cip1. Nature(Lond.), 394: 295–299, 1998.

40. Polyak, K., Lee, M. H., Erdjument-Bromage, H., Koff, A., Roberts,J. M., Tempst, P., and Massague, J. Cloning of p27Kip1, a cyclin-de-pendent kinase inhibitor and a potential mediator of extracellular antimi-togenic signals. Cell, 78: 59–66, 1994.

41. Yue, J., Buard, A., and Mulder, K. M. Blockade in TGF b 3 up-regulation of p27Kip1 and p21Cip1 by expression of RasN17 in epithelialcells. Oncogene, 17: 47–55, 1998.

42. Schweppe, R. E., Frazer-Abel, A. A., Gutierrez-Hartmann, A., andBradford, A. P. Functional components of fibroblast growth factor (FGF)signal transduction in pituitary cells. J. Biol. Chem., 272: 30852–30859,1997.

43. Tan, Y., Rouse, J., Zhang, A., Cariati, S., Cohen, P., and Comb, M. J.FGF and stress regulate CREB and ATF-1 via a pathway involving p38MAP kinase and MAPKAP kinase-2. EMBO J., 15: 4629–4642, 1996.

44. Lubinus, M., Meier, K. E., Smith, E. A., Gause, K. C., LeRoy, E. C., andTrojanowska, M. Independent effects of platelet-derived growth factorisoforms on mitogen-activated protein kinase activation and mitogenesisin human dermal fibroblasts. J. Biol. Chem., 269: 9822–9825, 1994.

45. Reynisdottir, I., Polyak, K., Iavarone, A., and Massague, J. Kip/Cipand Ink4 Cdk inhibitors cooperate to induce cell cycle arrest in responseto TGF-b. Genes Dev., 9: 1831–1845, 1995.

46. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R.PD 098059 is a specific inhibitor of the activation of mitogen-activatedprotein kinase kinase in vitro and in vivo. J. Biol. Chem., 270: 27489–27494, 1995.

47. Reimann, T., Hempel, U., Krautwald, S., Axmann, A., Scheibe, R.,Seidel, D., and Wenzel, K-W. Transforming growth factor-b induces ac-tivation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells. FEBSLett., 403: 57–60, 1997.

48. Chatani, Y., Tanimura, S., Miyoshi, N., Hattori, A., Sato, M., andKohno, M. Cell type-specific modulation of cell growth by transforminggrowth factor b1 does not correlate with mitogen-activated protein kinaseactivation. J. Biol. Chem., 270: 30686–30692, 1995.

49. Mulder, K. M., and Morris, S. L. Activation of p21ras by transforminggrowth factor b in epithelial cells. J. Biol. Chem., 267: 5029–5031, 1992.

50. Hartsough, M. T., and Mulder, K. M. Transforming growth factor b

activation of p44mapk in proliferating cultures of epithelial cells. J. Biol.Chem., 270: 7117–7124, 1995.

51. Gorospe, M., Wang, X., and Holbrook, N. J. p53-dependent elevationof p21Waf1 expression by UV light is mediated through mRNA stabiliza-tion and involves a vanadate-sensitive regulatory system. Mol. Cell. Biol.,18: 1400–1407, 1998.

52. Kivinen, L., Tiihonen, E., Haapajarvi, T., and Laiho, M. p21ras-medi-ated decrease of the retinoblastoma protein in fibroblasts occurs throughgrowth factor-dependent mechanisms. Cell Growth Differ., 7: 1705–1712,1996.

53. Kivinen, L., Pitkanen, K., and Laiho, M. Human retinoblastoma geneproduct prevents c-Ha-ras oncogene mediated cellular transformation ofmouse fibroblasts. Oncogene, 8: 2703–2711, 1993.

54. Haapajarvi, T., Kivinen, L., Pitkanen, K., and Laiho, M. Cell cycledependent effects of u.v.-radiation on p53 expression and retinoblastomaprotein phosphorylation. Oncogene, 11: 151–159, 1995.


and -independent Pathways in p21Cip1/Waf1 Induction

Documents

Transcript of and -independent Pathways in p21Cip1/Waf1 Induction