(CANCER RESEARCH 52, 5353-5358, October I, I992|

Differential Inhibition of the Epidermal Growth Factor-, Platelet-derived GrowthFactor-, and Protein Kinase C-mediated Signal Transduction Pathways by the

Staurosporine Derivative CGP 41251Elisabeth Andrejauskas-Buchdunger1 and Urs Regenass

Oncology and Virology Research Department, Pharmaceuticals Division, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland

ABSTRACT

The microbial alkaloid Staurosporine is a potent but nonselectiveinhibitor of protein kinases. The derivative CGP 41251 has been shownto exert a high degree of selectivity for inhibition of protein kinase Cactivity. Both compounds are powerful inhibitors of proliferation of bothnormal and transformed cells in vitro and exert antitumor efficacy invivo. In this work we have studied the mode of action of these compounds by analyzing their effects on early events in the induction ofproliferation by different growth stimuli. Both drugs blocked the phor-bol ester-induced expression of the c-fos proto-oncogene. The effect of

CGP 41251 was reversible, since its removal led to a normal expressionof c-fos mRNA in response to phorbol 12-myristate 13-acetate. Submi-

cromolar concentrations of CGP 41251 and Staurosporine directly inhibited both the platelet-derived growth factor (PDGF) receptor auto-phosphorylation and the c-fos mRNA expression induced by PDGFstimulation of intact BALB/c 3T3 cells. In contrast, ligand-induced

epidermal growth factor receptor autokinase activity in A431 carcinomacells and epidermal growth factor-dependent c-fos mRNA expression

were relatively insensitive to inhibition by CGP 41251. Staurosporinesuppressed signal generation by the epidermal growth factor receptor byreducing overall levels of the receptor. We conclude that CGP 41251 isa potent reversible inhibitor of protein kinase C and PDGF-mediated

signal transduction. It inhibits the kinase activity of both protein kinaseC and the PDGF receptor tyrosine kinase and the subsequent signalingcascade. The broad inhibition of kinases by Staurosporine is also reflected at the cellular level and might contribute to the high toxicity ofthis compound, in comparison to CGP 41251.

INTRODUCTION

Protein phosphorylation has long been recognized as a crucial event in the regulation of differentiation and proliferation.Phosphorylation is catalyzed by protein kinases, which are classified into serine/threonine kinases and tyrosine kinases.Among the serine/threonine kinases, PKC2 has been shown to

play a key role in the signal transduction pathway elicted by avariety of extracellular stimuli, such as growth factors, hormones, and neurotransmitters (1). Furthermore, PKC has beendescribed as the major receptor for tumor-promoting phorbolesters, which directly activate the enzyme and stimulate cellproliferation by mimicking the natural ligand diacylglycerol (2).The finding that a large number of receptors for growth factors,such as EGF, fibroblast growth factor, insulin, PDGF, andproducts of oncogenes have protein tyrosine kinase activity emphasizes the important role that protein phosphorylation playsin the cell (3).

Received 5/1/92; accepted 7/17/92.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Ciba-Geigy Ltd., Oncology and Virology Research Department, K-125.416, CH-4002 Basel, Switzerland.

2 The abbreviations used are: PKC, protein kinase C; EGF, epidermal growthfactor; PDGF, platelet-derived growth factor; DMEM, Dulbecco's minimal essential medium; PCS, fetal calf serum; PMA, phorbol 12-myristate 13-acetate; SDS,sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; SSC, standardsaline citrate.

Investigations of the intracellular pathways by which extracellular stimuli activate cellular proliferation have shown thatligand stimulation of both the PDGF receptor and the EGFreceptor induces a cascade of biochemical events including activation of phospholipase C, which catalyzes the hydrolysisof phosphatidylinositol 4,5-bisphosphate to 1,2-diacylglyceroland inositol 1,4,5-trisphosphate. These products function in theactivation of PKC and in mobilizing calcium from intracellularstores (4). Evidence for a direct biochemical link between ligandstimulation of the PDGF receptor or the EGF receptor tyrosinekinase and the activation of the phosphoinositide-dependentsignaling pathway has been provided by the finding that phospholipase C-7 associates with, and is a substrate for, both receptor tyrosine kinases (5-7). A common event that occursearly following the activation of growth factor receptors is theinduction of "immediate early response genes," including the

proto-oncogenes c-fos and c-myc (8, 9). Thus, monitoring thelevel of expression of these genes offers a convenient methodfor the analysis of physiological and pharmacological interactions in living cells.

The role of protein phosphorylation in the generation andtransduction of signals in the cell has made protein kinasestargets for specific inhibitors with potential use in proliferativediseases like cancer. In addition, such inhibitors should helpelucidate the physiological roles of the various protein kinasesand clarify the interconnection of signal transduction pathways.Staurosporine has been identified as a potent in vitro inhibitorfor both serine/threonine and tyrosine protein kinases (10, 11).Recent studies have indicated that Staurosporine can act as akinase inhibitor in various cellular systems (12-15). AlthoughStaurosporine is highly toxic in vivo, it showed antitumor activity at tolerated doses (16). Recently we have described a derivative of Staurosporine (CGP 41251) which in vitro showsincreased specificity for PKC inhibition. It inhibits phorbolester-induced cellular events, such as H2O2 release from humanmonocytes (16) and protein phosphorylation in human neutro-phils (17). In contrast to Staurosporine, the compound was ableto completely inhibit tumor growth at well tolerated doses (16).

The present paper compares the effects of Staurosporine andCGP 41251 on signal transduction pathways activated by EGFor PDGF or by direct stimulation of PKC by phorbol ester.These data are of importance for validating inhibition of kinases and signal transduction as an antiproliferative principle atthe cellular level.

MATERIALS AND METHODS

Cells and Cell Culture Conditions. The EGF-dependent BALB/MKmouse epidermal keratinocytes were kindly provided by Dr. S. Aaron-son, National Cancer Institute, Bethesda, MD (18). Cells were grown incalcium-free DM EM/Ham's F12 medium (1/1, v/v) supplemented with

5% PCS and EGF (5 ng/ml; BiomédicalTechnologies Inc., Stoughton,MA). For experiments in which starvation of cells was required,BALB/MK cells were grown to confluency in medium containing PCSand EGF, followed by removal of EGF from the culture medium for 2days. Cells were then treated with different concentrations of drug for

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90 min prior to Stimulation with EGF or PMA (Sigma, St. Louis, MO).BALB/c mouse 3T3 cells were obtained from Dr. C. Stiles (Dana Farber Cancer Institute, Boston, MA). Cells were grown in DMEM supplemented with 10% bovine calf serum (Hyclone, Logan, UT). Quiescent BALB/c 3T3 cultures were obtained by growing the cells toconfluency within 5-7 days of plating, without medium change. Cellswere washed and DMEM containing l mg/ml bovine serum albuminwas added during treatment with the drug(s) and PDGF (humanB-chain homodimer; Bissendorf Biochemicals, Hannover, Germany).A431 human epithelial carcinoma cells were obtained from the European Collection of Animal Cell Cultures (Portón Down Salisbury, UK)and were cultured in DMEM supplemented with 10% FCS. Cells weregrown to 80% confluency and then starved for 24 h in DMEM containing 0.5% FCS. Compounds (in 1% final dimethylsulfoxide concentration) were added 90 min prior to stimulation with EGF.

Isolation of Cytoplasmic RNA and Northern Blot Analysis. TotalRNA was isolated according to the method described by Chomczynskiand Sacchi (19). Equal amounts (10-20 ng) of RNA samples werefractionated on a formaldehyde-agarose gel, transferred onto nitrocellulose filters by vacuum blotting, and hybridized to a random-primedprobe. For the detection of c-fos mRNA a 1-kilobase ft/I fragment of\-fos was used (20). Hybridization was carried out at 45°Covernight in

50% (v/v) formamide, 5x SSC (1x SSC = 0. 15 MNaCl, 0.0 15 Msodiumcitrate, pH 7.0), 5x Denhardt's solution (ix Denhardt's = 0.02% each

of bovine serum albumin, Ficoll, and polyvinylpyrrolidone), 5 mMEDTA, 10 min piperazine-AVV-bis(2-ethanesulfonic acid), pH 6.4, 200f¿g/mlsalmon sperm DNA, 100 jig/ml heparin, 0.2% SDS, with 3 x IO6cpm/ml random-primed probe (specific activity, IO9 cpm/jig DNA).Filters were washed with 2x SSC/0.1% SDS at 45°Cfor 30 min andwith O.lx SSC/0.1% SDS at 52°Cfor 15 min. Kodak XAR films and

intensifying screens were used for autoradiography. Equal loading ofRNA was verified by staining of the gels with ethidium bromide.

Western Blot Analysis. Cells were washed with ice-cold phosphate-buffered saline containing 200 MMNa3VO4 and were lysed in 0.8 ml oflysis buffer [50 HIM Tris-HCl, pH 7.5, 5 mm ethylene glycol bis-(i8-aminoethyl etherJ-AWV^-tetraacetic acid, 1% Triton X-100, 150mM NaCl, 1 mM phenylmethylsulfonylfluoride, 80 Mg/ml aprotinin, 50Mg/ml leupeptin, 200 UMNa3VO4] at 4°Cfor 10 min. Lysates were

cleared by 10-min centrifugation at 10,000 x g and were corrected forequal protein content. Protein determination was carried out accordingto the method of Bradford (21). Samples were subjected to 7.5% SDS-PAGE after boiling in SDS sample buffer. The separated proteins werethen transferred onto Immobilon-P membranes (Millipore, Bedford,MA), which were incubated with either rabbit antiserum to the humanEGF receptor (Cambridge Research Biochemicals, Cambridge, UK),rabbit peptide antiserum to the murine PDGF receptor (22), or mousemonoclonal antibody against phosphotyrosine (obtained from Dr. T.Roberts, Dana Farber Cancer Institute, Boston, MA). Bound antibodieswere detected using 125I-Protein A (Amersham, UK) and autoradiog

raphy.In Vitro Assay of PDGF-stimulated Tyrosine Kinase Activity.

PDGF receptor was immunoprecipitated from BALB/c 3T3 cell extracts with rabbit antiserum to the murine PDGF receptor (UpstateBiotechnology, Inc., Lake Placid, NY) for 2 h on ice. ProteinA-Sepharose beads were used to collect the antigen-antibody complexes. The immunoprecipitates were washed twice with TNET (50 HIMTris, pH 7.5, 140 mMNaCl, 5 mM EDTA, 1% Triton X-100), once withTNE (50 HIMTris, pH 7.5, 140 mMEDTA), and once with kinase buffer(20 HIMTris, pH 7.5,10 mM MgCl2). After stimulation with PDGF (50ng/ml) for 10 min at 4°C,different concentrations of drug were added

to the reaction mixture. PDGF receptor kinase activity was determinedby incubation with 10 ¿iCi[7-32P]-ATP and l UMATP for 10 min at4°C.Immune complexes were separated by SDS-PAGE on 7.5% gels.

Dried gels were exposed to Kodak XAR films, with intensifyingscreens.

RESULTS

Inhibition of PMA-induced c-fos mRNA Expression. Stimulation of quiescent cells with growth factors and phorbol ester

tumor promoters leads to a rapid and transient induction ofc-fos proto-oncogene expression (8). To analyze the effects ofstaurosporine and CGP 41251 on PKC-mediated c-fos expression, quiescent BALB/MK cells were pretreated with differentconcentrations of these compounds for 90 min prior to stimulation with PMA. A strong increase in c-fos mRNA levels wasdetectable in control cells within 30 min of PMA stimulation(Fig. \A). Staurosporine strongly inhibited the induction ofc-fos mRNA at concentrations of >1 UM.Treatment with CGP41251 resulted in a reduction in PMA-induced c-fos mRNAlevels at >10 UM. This approximately 10-fold difference in

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c-fos mRNA. A, quiescent EGF-starved BALB/MK cells were pretreated for 90min with control medium (lanes I and 2), with staurosporine at 10 MM(lane 3) orl UM(lane 4), or with CGP 41251 at 10 MM(lane 5) or 1 MM(lane 6) beforestimulation with PMA (100 ng/ml) for 30 min (lanes 2-6). B, reversibility ofinhibition of PMA-induced c-fos expression. Quiescent BALB/MK cells werepretreated for 90 min with control medium (lanes 1-4), 1 MMstaurosporine (lanes5-8), or 10 MMCGP 41251 (lanes 9 and 10) before intensive washing with freshmedium (lanes 2, 4, 6, 7, 8, and II»and stimulation with PMA for 30 min (lanes3-10). Lanes 7 and 8, cells were washed free of compound and incubated in freshmedium for l h and 3 h, respectively, before treatment with PMA for 30 min.Total cellular RNA was analyzed by the Northern blot technique, using a \-fosDNA probe.

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potency of staurosporine and CGP 41251 in inhibiting a PKC-mediated cellular function correlates with the slightly reducedpotency of CGP 41251 in inhibiting PKC enzyme activity (16).

Reversibility of Inhibition of PMA-induced c-fos Expression.To determine whether the effect of staurosporine and CGP41251 in inhibiting PMA-induced c-fos expression was reversible, BALB/MK cells were first treated with 1MMstaurosporineor 10 MMCGP 41251 for 90 min. The compounds were thenremoved by intensive washing of the cells with fresh medium,and PMA was added for 30 min. As shown in Fig. 1Ä,additionof fresh medium to untreated control cells resulted in an induction of c-fos mRNA which was less pronounced than the induction by PMA. The inhibition of PMA-induced c-fosexpressionby CGP 41251 was completely reversible. Removal of the drugand subsequent exposure to PMA caused an increase in c-fosmRNA levels comparable to that in control cells where noinhibitor had been added (Fig. IB). In contrast, the inhibition ofPMA-induced c-fos expression by staurosporine was irreversible. No increase in c-fos mRNA levels in PMA-treated cellswas detectable even 3 h after removal of the drug.

Inhibition of PDGF-stimulated c-fos Expression. The addition of PDGF to quiescent cells causes the stimulation of various responses, including enhanced expression of c-fos (9). Ithas been reported that the PDGF-induced c-fos expression ismediated via activation of PKC (23). As shown in Fig. 2, PDGFelicted substantial increases in c-fos mRNA levels in quiescentBALB/c 3T3 cells after 30 min of stimulation. There was noinduction of c-fos mRNA by PDGF when the cells were pre-treated with staurosporine or with CGP 41251 at concentrations of >0.1 MMfor 90 min. The compounds alone did notaffect c-fos expression (data not shown). These results can beexplained by inhibition of PKC activity, but they do not rule outthe possibility that the drugs are acting more upstream in thesignal transduction cascade.

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rylation by CGP 41251 and staurosporine in intact cells. Confluent, quiescent,BALB/c 3T3 cells were incubated with control medium (lanes I and 2), 1 MM(lane3).0.\ MM(/<"!<••<).0.01 MM(lane5),or0.001 w(lane6)CGP41251,or 1 nM(lane7), 0.1 MM(lane 8), or 0.01 MM(lane 9) staurosporine for 90 min prior to stimulation with PDGF (50 ng/ml) for 10 min (lanes 2-9). Whole-cell lysates werecorrected for protein content and analyzed by electrophoresis on 7.5% SDS-polyacrylamide gels. After transfer onto Immobilon-P membranes, immunoblotanalysis was performed using antiphosphotyrosine antibodies 1.1) and anti-PDGFreceptor antiserum (B).

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c-fos mRNA induced by PDGF. Confluent, quiescent, BALB/c 3T3 cells wereincubated for 90 min with control medium (lanes 1 and 2), 1 MM(lane 3). 0.1 MM(lane 4), or 0.01 MM(lane 5) staurosporine, or 10MM(lane 6), l UM(/</«<•7), 0.1 IHM(lane 8), or 0.01 MM(lane 9) CGP 41251. After Stimulation with PDGF ( 10 ng/ml)for 30 min (lanes 2-9), samples of total RNA were analyzed by the Northern blottechnique, using the \-fos probe.

Inhibition of Ligand-stimulated PDGF Receptor Autophos-phorylation in Intact Cells. The PDGF receptor tyrosine ki-nase becomes activated in response to PDGF, and a specific setof intracellular proteins, including the receptor itself, are phos-phorylated on tyrosine residues (for review, see Ref. 24). Thefollowing experimental approach was used to explore the possibility that staurosporine and CGP 41251 inhibited the PDGF-induced c-fos expression through a direct inhibitory effect onthe PDGF receptor tyrosine kinase activity. The compoundswere added to quiescent BALB/c 3T3 cells 90 min prior tostimulation by PDGF. Cells were lysed, analyzed by SDS-PAGE, and immmunoblotted with either antiphosphotyrosineor anti-PDGF receptor antibodies. Fig. ÃŒAdemonstrates thatPDGF stimulated a profound increase in tyrosine phosphory-lation of a protein with a molecular weight of 185,000, whichcorresponds to the size of the PDGF receptor. The protein bandwas identified as the PDGF receptor by demonstration of im-munoprecipitation with anti-PDGF receptor antibodies inother experiments (data not shown). Treatment of the cells withstaurosporine or CGP 41251 caused a concentration-dependentinhibition of ligand-stimulated PDGF receptor tyrosine phos-phorylation, with a maximal inhibitory effect at 1 MM.Immu-noblotting experiments with anti-PDGF receptor antibodiesshowed that CGP 41251 did not significantly affect overalllevels of the PDGF receptor (Fig. 3B). A slight decrease inPDGF receptor content was observed in cells treated with staurosporine. Taken together, these data indicate that inhibition ofthe PDGF receptor tyrosine kinase activity may contribute to

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the inhibitory effects of both compounds on PDGF-inducedc-fos mRNA expression.

In Vitro Inhibition of PDGF Receptor Autophosphorylation.Direct inhibition of PDGF receptor autophosphorylation wasanalyzed in PDGF receptor immunoprecipitates from BALB/c3T3 cells. Addition of CG P 41251 and staurosporine to theimmune complexes inhibited libami stimulated PDGF receptorautophosphorylation in a dose-dependent manner (Fig. 4).There was no increase in PDGF-stimulated phosphorylation ofthe receptor when the immunoprecipitates were treated withCGP 41251 or with staurosporine at concentrations of >0.1 MMand >0.01 MM,respectively.

Effects on EGF-induced c-fos Expression. As with PDGF,the early events induced by EGF include induction of the c-fosgene in various cell lines (8, 25-27). In order to investigate theeffects of staurosporine and CGP 41251 on EGF-induced c-fosexpression, quiescent BALB/MK cells were incubated with theinhibitors for 90 min prior to stimulation with EGF. A stronginduction of c-fos expression by EGF was observed in controlcells (Fig. 5). Expression of c-fos, however, was inhibited in cellspretreated with staurosporine at >1 MM(Fig. 5A). In contrast,CGP 41251 did not significantly affect EGF-induced c-fos expression even when used at concentrations up to 100 MM(Fig.5B). These data, together with the observation that CGP 41251inhibits PMA-induced c-fos expression (Fig. \A), support thehypothesis that the EGF-induced c-fos expression is not mediated via PKC. Similar results were obtained with BALB/c 3T3cells, where PMA- but not EGF-induced c-fos expression wasinhibited by CGP 41251 (data not shown).

Effects on Ligand-stimulated EGF Receptor Autophosphorylation in Intact Cells. To test the possibility that inhibition ofthe EGF receptor tyrosine kinase activity could account for theinhibitory effect of staurosporine on EGF-induced c-fos expression, growth-arrested A431 cells were incubated with the inhibitor for 90 min prior to stimulation with EGF. Cell lysates werethen separated on denaturing polyacrylamide gels and immu-noblotted with either antiphosphotyrosine or anti-EGF receptor antibodies. As shown in Fig. 6A, stimulation of control cellswith EGF resulted in tyrosine phosphorylation of a protein

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CGP 41251 and staurosporine. Lysates from BALB/c 3T3 cells which had beentreated in the absence (lane 1) or presence of PDGF (50 ng/ml) (lanes 2-9) for 10min were immunoprecipitated with rabbit polyclonal anti-PDGF receptor antibodies. Immunoprecipitates were stimulated with PDGF (50 ng/ml) for 10 min at4°C(lanes 2-9) prior to addition of control medium (lanes I and 2) or theindicated concentrations of CGP 41251 (lanes 3-6) or staurosporine (lanes 7-9).After 10 min at 4°C,10 mCi of [-y-32P]-ATP and 1 MMATP were added andfurther incubated for 10 min at 4°C.Samples were separated by SDS-PAGE.

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expression in BALB/MK cells. Cells were treated for 90 min with control medium(lanes I and 6 \nA and B), staurosporine (A), or CGP 41251 (B) at concentrationsof 100 MM(lanes 2 and 7), 10 MM(lanes 3 and 8), I JIM(lanes 4 and 9), or 0.1 MM(lanes 5 and 10). Levels of c-fos mRNA were determined 30 min after stimulationwith EGF (10 ng/ml) (lanes 6-10).

migrating at a molecular weight of about 180,000, most likelyreflecting ligand-induced autophosphorylation of the EGF receptor. Pretreatment of A431 cells with staurosporine at concentrations of >10 MMstrongly reduced the increase in EGF-stimulated protein tyrosine phosphorylation. In contrast, only aslight decrease in EGF receptor tyrosine phoshorylation wasobserved when cells were treated with 100 MMCGP 41251 before stimulation with EGF. An alternative assay for EGF receptor autophosphorylation using immunoprecipitation withanti-EGF receptor antibodies, followed by immunoblottingwith antiphosphotyrosine antibodies, gave identical results(data not shown). To examine whether the inhibition of EGFreceptor tyrosine phosphorylation by staurosporine could beexplained by reduction of overall levels of the EGF receptor,immunoblotting experiments with anti-EGF receptor antibodies were carried out. As shown in Fig. 6B, staurosporine (>10MM)caused a strong decrease in EGF receptor levels, in contrastto CGP 41251, which had only a slight effect on EGF receptorlevels when used at 100 MM.These data indicate that the EGFreceptor tyrosine kinase in intact cells is relatively insensitive toinhibition by CGP 41251, whereas the effects of staurosporineon the EGF signaling pathway can be attributed at least partially to reduced EGF receptor content in staurosporine-treatedcells.

DISCUSSION

Staurosporine is a powerful antiproliferative compoundwhich has been shown to inhibit the activity of a variety of

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phorylation. Serum-starved A431 cells were incubated for 90 min with controlmedium (lanes I and 2), CGP 41251 at 100 MM(lane 3) or 10 MM(lane 4), orstaurosporine at 100 MM(lane 5), 10 MM(lane 6), 1 MM(lane 7), or 0.1 MM(lane A)prior to stimulation with EGF (100 ng/ml) for 10 min (lanes 2-8). Equal amountsof protein of cell lysates were analyzed by Western blot using antiphosphotyrosineantibodies (A) and anti-EGF receptor antiserum (B).

protein kinases in a rather nonselective manner (10, 11). During the search for a selective PKC inhibitor, we identified astaurosporine analogue (CGP 41251) which is more selective inthe inhibition of PKC than is the parent compound. In vivo,CGP 41251 is better tolerated by mice and shows an increasedtherapeutic window for tumor growth inhibition, in comparisonto staurosporine (16). In the current study we have examinedthe action of staurosporine and CGP 41251 on signal transduc-tion via EGF-, PDGF-, and PKC-mediated pathways.

While the ligand-induced activation of the EGF receptor ty-rosine kinase, as well as the EGF-dependent c-fos mRNA expression, was resistant to inhibition by CGP 41251, the PDGFpathway was very sensitive to inhibition by the compound. Sub-micromolar concentrations of CGP 41251 directly inhibitedPDGF receptor autophosphorylation and c-fos mRNA expression induced by PDGF stimulation of intact BALB/c 3T3 cells.The finding that CGP 41251 did not affect EGF-induced c-fosexpression indicates that the compound does not induce generaldegradation of mRNAs. Since CGP 41251 did not affect thelevel of the PDGF or the EGF receptor, the preferential inhibitory activity of the compound toward signal output fromligand-activated PDGF receptor most likely reflects a highersensitivity of the PDGF receptor tyrosine kinase to this drug.These results also indicate that permeation problems of thecompound within the cell do not account for the lack of effectson EGF-mediated cellular events. Staurosporine suppressedsignal generation by both the PDGF receptor and the EGF

receptor. It should be noted, however, that ligand-induced

PDGF receptor autophosphorylation was almost completelyinhibited at 10~7 M staurosporine, whereas ligand-dependent

EGF receptor autophosphorylation was strongly affected onlyat concentrations of >10~5 M. A 100-fold higher sensitivity of

the PDGF receptor tyrosine kinase to staurosporine, comparedto the EGF receptor tyrosine kinase, has previously been reported (28). Since staurosporine is a kinase inhibitor with awide specificity which also effectively inhibits the EGF receptortyrosine kinase in vitro (16, 29), the inhibition of EGF receptorautophosphorylation and EGF-induced c-fos mRNA expres

sion in intact cells could be explained by a direct effect on theEGF receptor kinase activity. The observation that staurosporine at 1 MMreduces c-fos mRNA expression but at that concentration does not strongly affect EGF receptor autophosphorylation or EGF receptor levels can most likely be explained bythe fact that staurosporine is a potent but nonselective drug,which might inhibit other protein kinases involved in the induction of c-fos mRNA expression. However, the most likelyexplanation for the inhibitory effect of staurosporine on EGF-mediated signal output at concentrations of >10 MMis the drug-induced reduction in overall levels of the EGF receptor.Reduced levels of the EGF receptor in cells exposed to staurosporine are most probably due to toxic effects of the drug athigh concentrations, which could affect either degradation orsynthesis of the receptor. In contrast, at concentrations whereligand-induced PDGF receptor autophosphorylation was almost completely inhibited by staurosporine (<10~7 M) there

was only a small effect on PDGF receptor levels, indicating thatstaurosporine is acting as a direct inhibitor of the PDGF receptor kinase. Direct inhibition of PDGF receptor tyrosine kinaseactivity in vitro by staurosporine and CGP 41251 was demonstrated in PDGF receptor immunoprecipitates from BALB/c3T3 cells.

Previous studies have demonstrated that CGP 41251 inhibitsPKC activity in vitro (16) and PKC-mediated protein phosphor-ylation in intact cells (17). Our finding that CGP 41251 revers-ibly inhibits PMA-induced c-fos mRNA expression inBALB/MK cells but does not affect EGF-stimulated c-fosmRNA levels suggests the existence of a second pathway leading to c-fos mRNA expression, which bypasses PKC. Thesedata substantiate previous findings demonstrating that EGFinduces mitogenesis and c-fos mRNA expression in 3T3 cellsbut does not activate PKC (30, 31). Similarly, EGF has beenshown to stimulate c-myc mRNA expression in human fibro-blasts without activation of phosphatidylinositol turnover (32).

In vivo, staurosporine and CGP 41251 have been shown toexhibit antitumor activity (16). The increased therapeutic window of CGP 41251, compared to staurosporine, can most likelybe explained by the increased specificity of CGP 41251 for alimited number of kinases, in particular PKC. Our finding thatCGP 41251, in addition to inhibiting PKC, is a very potentinhibitor of PDGF receptor kinase-mediated cellular eventsraises the possibility that a synergism between inhibition ofPKC and PDGF receptor kinase activity might play an important role in modulating cellular growth, in vitro and in vivo.

PDGF is a ubiquitous growth factor which plays an important role in normal growth and development, and it has beensuggested that it is also a mediator of pathological cell growth,e.g., carcinogenesis as well as disorders of vascular smoothmuscle cells, such as atherosclerosis (for review, see Ref.33). PDGF was found to be involved in intimai accumulationand proliferation of smooth muscle cells responsible for the

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occlusive lesions of atherosclerosis (34). Potential clinical applications of PDG F receptor kinase inhibitors would, therefore,include treatment of tumors as well as nonmalignant prolifer-ative disorders. Inhibitory effects on PDGF receptor kinaseactivity have been described not only for compounds of thestaurosporine class (28, 35) but also for a series of synthetic,low molecular weight inhibitors called tyrphostins (36). Thesecompounds have previously been described as inhibitors ofEGF receptor kinase activity (37-39). In addition to their lowselectivity within the protein tyrosine kinase class, it has recently been reported that tyrphostin is not a competitive inhibitor of EGF receptor tyrosine kinase in intact cells and that itprobably inhibits tyrosine kinase activity by an indirect mechanism (40).

In summary, we have described a reversible protein kinaseinhibitor which, within the serine/threonine kinase family,shows a certain degree of selectivity for PKC and which inaddition is a potent inhibitor of PDGF receptor tyrosine kinaseand the subsequent signaling cascade. The evaluation of additional derivatives of staurosporine at the enzyme and cellularlevel, as described in this paper, should help to identify thesignal transduction pathways which are of importance in theregulation of tumor growth.

ACKNOWLEDGMENTS

We thank C. Koelbing and V. Rigo for excellent technical assistance,Dr. C-H. Heldin for the gift of antiserum to the PDGF receptor, andDr. N. Hynes and Dr. N. Lydon for critical reading of the manuscript.

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