asbestos causes stimulation of the extracellular signal ... · asbestos causes stimulation of the...

ICANCER RESEARCH 56. 5334-5338, December I. 19961

Advances in Brief

Asbestos Causes Stimulation of the Extracellular Signal-regulated Kinase 1Mitogen-activated Protein Kinase Cascade after Phosphorylation of theEpidermal Growth Factor Receptor'

Christine L. Zanella,2 James Posada, Thomas R. Tritton, and Brooke T. Mossman

Departments of Pathology (C. L Z, B. T. MI. Molecular Physiology & Biophysics (J. P.1, Pharmacology (T. R. TI, and Vermont Cancer Center (C. L Z, J. P., T. R. T.,B. T. MI, University of Vermont College ofMedicine. Burlington. Vermont 05405

Abstract

Asbestos fibers are human carcinogens with undefined mechanisms ofaction. In studies here, we examined signal transduction events induced byasbestos in target cells of mesothelioma and potential cell surface originsfor these cascades. Asbestos fibers, but not their nonfibrous analogues,induced protracted phosphorylation of the mitogen-activated protein

(MAP) kinases and extracellular signal-regulated kinases (ERK) 1 and 2,and increased kinase activity of ERK2. ERK1 and ERK2 phosphorylationand activity were initiated by addition of exogenous epidermal growthfactor (EGF) and transforming growth factor-a, but not by Isoforms ofplatelet-derived growth factor or insulin-like growth factor-i in mesothe

Halcells. MAP kinase activation by asbestos was attenuated by suramin,which inhibits growth factor receptor interactions, or tyrphostin AG 1478,a specific inhibitor of EGF receptor tyrosine kinase activity (IC@ 3 aM).Moreover, asbestos caused autophosphorylation of the EGF receptor, anevent triggering the ERK cascade. These studies are the first to establishthat a MAP kinase signal transduction pathway is initiated after phosphorylation of a peptide growth factor receptor following exposure to

asbestos fibers.

Introduction

Exposure to asbestos has been linked to the development of bothmalignant (lung cancer, mesothelioma) and nonmalignant (asbestosis)diseases, all of which involve a dysregulation of cell proliferation (1).Asbestos fibers cause persistent increases in mRNA levels of theproto-oncogenes c-jun and c-fos in target cells of disease in vitro andin vivo, as well as increases in DNA binding activity of the activatorprotein-I transcription factor (2). Because expression of both c-losand c-jun is required for transition through the Gl phase and entry intothe S phase of the cell cycle, and functional overexpression of c-juncan lead to increased cell replication and morphological transformation of tracheal epithelial cells (3), transcriptional activation of immediate early response proto-oncogenes by asbestos may be linked tochronic cell proliferation and carcinogenesis.

Other investigators have focused on DNA damage induced byasbestos fibers in vitro at cytotoxic concentrations, which can disruptthe mitotic spindle and may physically interact with chromosomes (4,5). We hypothesized here that interaction of fibers with cell surface

receptors and initiation of signal transduction cascades are more

Received 7/22/96; accepted 10/17/96.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I This research was supported in part by Grant ROl ES0006499 from the National

Institute of Environmental Health Sciences, Environmental Pathology Training GrantT3207122 from the National Institute of Environmental Health Sciences, and Grant ROlHL39 469 from the National Heart, Lung, and Blood Institute.

2 To whom requests for reprints should be addressed, at Department of Pathology,

Medical Alumni Building, University of Vermont College of Medicine, Burlington,Vermont 05405. Phone: (802) 656-0535; Fax: (802) 656-8892.

plausible scenarios for regulation of immediate-early gene expressionby asbestos fibers.

The MAPS kinases ERK1 and ERK2 are members of a family ofevolutionarily conserved enzymes involved in regulating the responseof eukaryotic cells to extracellular signals (6). Activation of the ERKsby asbestos may be critical to c-los and c-jun transcription, becauseupon phosphorylation, ERKs translocate to the nucleus to phosphorylate substrates, including the ternary complex factorlElk-l, which isinvolved in c-los transactivation (7). To examine whether asbestoscauses ERK phosphorylation, we used RPM cells, the progenitor celltype of malignant mesothelioma, and concentrations of asbestosknown to induce steady-state mRNA levels of c-fos and c-jun (2).

Materials and Methods

Cell CUltUreand Treatments. RPMcells were isolatedby gentle scrapingof the parietal pleura of Fischer 344 rats and propagated as described previously (2). Cells were maintained in DMEM/F-12 containing 10% fetal bovineserum,hydrocortisone(100 ng/ml),insulin(2.5 @g/ml),transferrin(2.5 @g/ml), and selenium (2.5 ng/ml). Twenty-four h before exposure to minerals,growth factors, or TPA, cells were switched to medium containing reducedserum (0.5% fetal bovine serum). Particulates were suspended in HBSS at 1mg/ml, triturated 8 times with a 22-gauge needle, and then added to medium

for final concentrations between 1.25and 25 @.&g/cm2of culture dish. Referencesamples of National Institute of Environmental Health Sciences-processed

crocidolite and chrysotile (Jeffery Mines, Montreal, Quebec, Canada) asbestosfibers were obtained from the Thermal Insulation Manufacturers AssociationFiber Repository (Liuleton, CO). Riebeckite and antigorite, the nonfibrous(<3:1 length to diameter ratio) chemically similar analogues ofcrocidolite andchrysotile asbestos, do not induce alterations in cell proliferation and were usedto determine the specificity of asbestos-induced ERK activation by fibers (8).TPA (Consolidated Midland, Brewster, NY) was added to culture medium at100ng/ml from a 1mglml stock solution in acetone (0.1% final concentration).EGF,PDGF-AA,PDGF-BB,TGF-a, andIGF-I werepurchasedfrom UnitedBiomedical Inc. (Lake Placid, NY).

MAP Kinase Western Blots. Assays were performed as described previously using polyclonal rabbit antibodies 1913.2 or 1913.3 raised to the carboxyl terminus of the Xenopus laevis M1 42,000 MAP kinase (peptide sequence KELIFEETARFQPGY;Refs. 9 and 10).Antibodies 1913.2and 1913.3recognize both the Mr 42,000 (ERK2) and the Mr 44'000 (ERK1) mammalianMAP kinases, as well as their phosphorylated forms, which have reducedmobility on SDS-polyacrylamide gels.

ERK2 in Vitro Kinase Assays. RPM cells were washed twice in ice-coldPBS and then lysed in cold NP4Oimmunoprecipitation buffer [1% NP-40, 10mM HEPES (pH 7.4), 2 nmi EDTA, 50 nmi NaC1, 50 mM NaF, 0.1% 2-mercaptoethanol, 1% aprotinin, 0.2 mrsisodium orthovanadate, and I mM phenylmethylsulfonyl fluoridej. Lysates were centrifuged at 14,000 rpm at 4°Cfor10 mm, and the supernatant was removed to a clean tube. ERK2 was immu

3 The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular

signal-regulated kinase; RPM, rat pleural mesothelial; TPA, 12-O-tetradecanoylphorbol13-acetate; EGF, epidermal growth factor; PDGF, platelet-derived growth factor, TGF,transforming growth factor, IGF, insulin-like growth factor; PKC, protein kinase C;EGF-R, EGF receptor.

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STIMULATION OF ERK MAP KINASE CASCADE BY ASBESTOS

noprecipitated using an anti-ERK2 (C-14) antibody (Santa Cruz) at a 1:100

dilution. The washed immunoprecipitates were incubated for 20 mm at 30°C

in kinase buffer [20 mist HEPES (pH 7.5), 2 mM 2-mercaptoethanol, 5 miii

MgC12]containing 5 p@Ci[y-32P]ATPand 5 p.g of GST-Myc (1—300)bacterialfusion protein or 10 p@gmyelin basic protein per reaction as a substrate forERK activity (11). Incorporation of 32P to the GST-Myc or myelin basicprotein substrate was visualized by autoradiography and quantitated on aBio-Rad phosphoimage analyzer.

EGF-R in VitroKinase Assays. Procedureswere modifiedfrom Sorokinet aL (12). RPM cells were washed twice in ice-cold PBS and then lysed incold Triton-X extraction buffer [1% Triton X-lOO,5 mistEDTA, 50 mMNaC1,50 mMNaF, 10nmiTris (pH 7.6), 1%aprotinin, 0.2 mMsodium orthovanadate,1 mr@iphenylmethylsulfonyl fluoride, and 0.5 @.tMmicrocystin]. Lysates werecentrifuged at 14,000rpm at 4°Cfor 10 mm, and the supematant was removedto a clean tube. EGF-R was immunoprecipitated using an anti-EGF-R antibody

from the IgG 151 AE4 hybridoma, a subclone of IgG 15 1 BH6. The washedimmunoprecipitates were incubated for 10 ruin at 30°C in 25 M1 of kinase

buffer plus 5 @Ci[y-32P]ATP. Incorporation of 32P to the EGF-R was visualized by autoradiography and quantitated on a phosphoimage analyzer. Westem blots were performed using anti-EGF-R antibody P1 (a gift from thelaboratory ofJ. J. M. Bergeron, McGill University, Montreal, Quebec, Canada)to confirm the presence of immunoprecipitated EGF-R in each sample.

Asbestos Fibers Cause a Persistent Phosphorylation of ERK1and ERK2 and Increased Activity of ERK2. Fig. 1A shows thetime course of ERK phosphorylation induced by crocidolite asbestos(5 @g./cm2), the most pathogenic type of asbestos in the causation of

mesothelioma (1), or by the phorbol ester tumor promoter TPA (100

Results

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Fig. I. Asbestos induces phosphorylation of ERK1 and ERK2 proteins as shown by Westem blot analysis (A) and increases in ERK2 activity (B—D).A, time course of ERK responsecomparing controls (0) with crocidolite asbestos (Croc.) at 5 @g/cm2and with TPA at 100 ng/ml. S. lane showing protein standards. B, ERK2 activity is increased with TPA or EGF,but not PDGF, stimulation at 45 ruin. C, ERK2 kinase activity is increased in a dose-dependent manner by both crocidolite and chrysotile asbestos (8 h incubation). D, ERK2 kinaseactivity is not increased above control values in response to nonfibrous particulates (riebeckite and antigorite at 1.25—10@zg/cm').

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STIMULATION OF ERK MAP KINASE CASCADE BY ASBESTOS

ng/ml). TPA is a PKC agonist known to activate MAP kinases (13)and is used here as a positive control (2). In contrast to untreated cellsat each time point, exposure of RPM cells to 100 ng/ml TPA for 1, 2,or 4 h results in activation of ERK1 and ERK2, as evidenced bydecreased mobility of the molecules indicative of phosphorylation (9,

10). These responses to TPA are diminished at 8 h and absent after24 h. The early and transient ERK response to TPA contrasts with themore protracted response to crocidolite asbestos, in which phosphorylation of the ERKs is first apparent at 4 h after exposure to asbestosand increases at 8 and 24 h (Fig. 1A, Lanes 8, ii, and 14). The patternand time frame of ERK responses to TPA or crocidolite correspond tothe kinetics of induction of c-los and c-jun transcription by theseagents (2).

To determine whether the ERKs are also catalytically activated byasbestos fibers, immune complex kinase assays were performed onERK2 immunoprecipitated from asbestos-treated RPM cells. ERK2kinase assays confirm that MAP kinase is enzymatically activated bycrocidolite and chrysotile asbestos (Fig. 1B—D).Activity was alsoincreased with TPA or EGF (but not PDGF-BB; Fig. 1B). Furthermore, at 24 h, ERK2 activity was increased by crocidolite asbestos butnot by its nonfibrous analogue riebeckite.

To examine the dose dependence of the ERK response in cells exposedto asbestos fibers, immune complex kinase assays were performed withRPM cells that had been treated for 1 h with TPA or EGF, or for 8 withcrocidolite or chrysotile asbestos (Fig. 1C). A dose-related increase inERK2 kinase activity was observed in cells treated with increasingconcentrations of crocidolite or chiysotile asbestos.

RPM cells also were exposed to crocidolite or chrysotile asbestos(each at 10 @g/cm2)and their nonfibrous analogues riebeckite andantigorite over a range of concentrations (1.25—10p@g/cm2;Fig. 1D).EGF (5 ng/ml) and chrysotile and crocidolite asbestos elevated thekinase activity of ERK2 as quantitated by phosphoimage analysis.However, the nonfibrous particles riebeckite and antigorite failed toincrease the ERK2 activity above control levels in untreated cells.These data support the importance of fibrous geometry in reactivity.

EGF and TGF-a Stimulate Prolonged Phosphorylation ofERK1 and ERK2 but PDGF Isoforms and IGF-I Do NoL Differential expression of growth factor receptors, including EGF-R, and theproduction of many growth factors have been reported in both normalmesothelial and transformed mesothelioma cells and in mesothelial cellsafter exposure to asbestos fibers in vitro (14—17). However, no experi

mental evidence to date has demonstrated a causal involvement of growthfactors or their receptors in cellular responses to asbestos. As EGFstimulates transcription of c-jun and c-los in other cells (18) through amechanism of action involving EGF-R dimerization and autophospho

rylation (19), we hypothesizedthat the EGF-R was a potential target sitefor asbestos-induced activation of the MAP kinase pathway.

Because RPM cells possess features of both epithelial and mesenchymal cells and have EGF, PDGF, and IGF receptors (14—17),wescreened EGF and other candidate growth factors for their ability toactivate the ERK proteins by initiating a signaling cascade at the RPMcell surface. RPM cells were exposed to receptor grade EGF at a rangeof concentrations and time points up to 8 h. TPA (100 ng/ml), as wellas all concentrations of EGF examined, caused band shifting of ERK1

and ERK2 at all time points. Fig. 2A shows the time course from 15mm to 2 h. At 15 mm, untreated cells that were handled in an identicalmanner demonstrate phosphorylation of the ERK proteins in all experiments. This response is observed consistently and is similar to theearly induction of c-los and c-jun observed in RPM cells at early timepoints (2). The response appears to be a nonspecific response tomechanical manipulations as has been observed by others (3).

We next examined RPM cells after exposure to TGF-a, a peptidethat is regulated differently than EGF but which interacts with the

A

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E@0 0 ,@0.251 5 16 30, o@@ 15 30, o@@@ 15 3O@@}TGF.a(nglml)@ TGF.u(n@ml)@Fig. 2. EGF (A) and TGF-a (B) cause protracted band shifts of the ERK1 and ERK2

proteins.

same cell surface receptor. RPM cells treated with TGF-a at a rangeof time points and concentrations exhibited ERK1 and ERK2 bandshifting at all doses and times examined (Fig. 2B). These resultsindicate that stimulation of the EGF-R with EGF or TGF-a phosphorylates and activates ERKs over an extended time course in a mannersimilar to asbestos.

PDGF is a dimeric molecule consisting of A and/or B polypeptidechains. All PDGF isoforms (PDGF AA, AB, and BB) and IGF-1 wereexamined here over a range of concentrations for time periods up to4 h. At times ranging from 30 mm to 4 h, neither control, PDGFtreated, nor IGF-1-treated cells exhibited ERK1 and ERK2 phosphorylation, whereas TPA-exposed cell groups showed ERK phosphorylation (data not shown). The lack of increase in ERK2 kinase activityexperiments using PDGF-BB (Fig. 1B) also indicates that PDGF doesnot induce an ERK response in RPM cells.

Blocking Growth Factor Receptors Inhibits the ERK Responseto Asbestos. To assess the causal role of cell surface receptors in ERKactivation by asbestos or EGF, we examined effects of surainin, a polysulfonated napthylurea hexaanion that has been shown to inhibit growthfactor receptor interactions at doses ranging from 106 to iO@ M innumerous other cell types (20). The coaddition of suramin prevented theinduction of ERK phosphorylation by crocidolite or EGF (Fig. 3A).Untreated cells (Fig. 3A, Lane 1) and cells treated with suramin (Fig. 3A,Lanes 2 and 3) did not show activation of ERKs. In contrast, cells

exposed for 8 or 24 h to EGF (Fig. 3A, Lane 4) or crocidolite (Fig. 3A,Lanes 6 and iO) exhibit ERK activation. These phosphorylation events

were not observedifsuramin was added with the stimulatingagents to theculture media (Fig. 3A, Lanes 5, 7-9, and 1i-i3). Suramin inhibits PKCactivity in cell lysate systems (20). Thus, to rule out possible artifacts ofPKC inhibition by suramin in the RPM cell system, the ERK response toTPA in the absence and presence of suramin was examined. At 1 h,activation of ERKs by TPA was not abolished even at highest concentrations of suramin (5 mrvi;Fig. 3B).

To examine whether the EGF-R is directly involved in ERK activation by asbestos, we used tyrphostin AG 1478, a specific inhibitorof the EGF-R tyrosine kinase (21). Fig. 4A shows that AG 1478 aloneat 0.1, 1, or 10 @LMhas no effect on the ERKs and does not inhibit the

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STIMULATION OF ERK MAP KINASE CASCADE BY ASBESTOS

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Fig. 3. A, suramin blocks ERK activation by EGF and crocidolite asbestos. B, suramindoes not block ERK phosphorylation by TPA.

activations of ERKs by TPA. By contrast, AG 1478 blocks activation

of the MAP kinases in response to EGF and crocidolite asbestos at 1and 8 h. These results were confirmed using immune complex kinaseassays, in which increases in ERK2 activity induced by EGF orcrocidolite asbestos (5 and 10 @g/cm2)were completely abolished inthe presence of AG 1478 (Fig. 4B, Lanes 7 and 8).

Asbestos Activates EGF-R Kinase Activity. On the basis of findings with suramin and AG 1478, we next examined the ability ofcrocidolite asbestos or EGF to modulate the in vitro kinase activity of theEGF-R as a possible site of initiation of signals leading to activation ofER@Ks(Fig. 5). In these experiments, the EGF-R was immunoprecipitatedafter addition of agents, and its kinase activity was evaluated by performing in vitro autophosphotylation assays with the receptor as substrate.Experiments were performed at 4°C(to slow the reaction rate at whichthe EGF-R autophosphorylates), as well as at 37°C.Asbestos was addedfor 2 h, because this is the minimal time to allow for significant numbersof fibers to settle onto and make contact with the cells. At 4°C,stimulation of RPM cells with EGF for 1 or 5 mm produced a large increasein EGF-R phosphorylation (Fig. 5, Lanes 4 and 5). At 37°C,this increasewas evident within 5 or 15 s (Fig. 5, Lanes 8 and 9), and phosphorylationof the receptor was diminished below untreated control levels at 2 h (Fig.5, Lane 10). At both 4°C and 37°C, asbestos at concentrations (5 @g/cm2)

inducing ERK phosphorylation and activity increased the phosphorylation of the EGF-R.

Discussion

Our studies indicate that the EGF-R may be a site at which asbestosfibers initiate the MAP kinase signaling cascade. Results here areconsistent with reports showing that in addition to specific ligand,various agents, including divalent cations and catiomc polypeptides,increase the tyrosine kinase activity of the EGF-R and cause aggregation of the EGF-R intracellular domain (22). The finding that theEGF-R is phosphorylated in RPM cells in response to asbestos is thefirst reported observation linking interaction of asbestos fibers with a

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growth factor receptoron the surfaceof a target cell of disease.Moreover,we demonstrate a functional response (i.e., activation of ERKs triggeredafter fiber-inducedphosphorylationofEGF-R) that may be critical to theadvent of abnormal cell proliferation and differentiation, features ofasbestos-induced diseases (1). Our studies with asbestos fibers supportother findings that ERKs can be phosphorylated and activated in responseto a number of external stimuli that promote growth and differentiation.These factors include ionizing radiation and 11202, which activate MAPkinases by the production of active oxygen species (23), as well as agentsthat induce oligomerization of receptor tyrosine kinases (19). Asbestosfibers, which generate active oxygen species from cells or by redoxreactions on the fiber surface (1), also may phosphoiylate the EGF-Rthrough an oxidant-dependent mechanism, as has been suggested inrecent studies using H2O2 (24). We are presently exploring these possibilities, as well as more protracted time courses of EGF-R phosphorylation by asbestos, using an EGF-R kinase activity assay.

The protracted activation of ERKs induced by asbestos may also beexplained by the direct interactions of asbestos fibers with cells inculture. After initial addition to medium, fibers continue to settle ontocells over a number of hours and may trigger sequential and additionalsignaling cascades. Because fibers are insoluble and not metabolized,they remain in contact with cells and thus serve as persistent sourcesof signals. In addition to signal transduction events that are mediatedthrough the EGF-R and phosphorylation of ERKs, it is possible thatasbestos may activate other ERK-independent pathways to provideadditional signaling stimuli to the nucleus (25).

AAG 1478

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Fig. 4. The EGF-R inhibitor AG 1478 inhibits activation of ERKs by EGF andcrocidolite asbestos in a dose-related manner but does not effect ERK activation by TPA.A, Western blot analyses. B, ERK2 immune complex kinase assays.

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STIMULATION OF ERK MAP KINASE CASCADE BY ASBESTOS

tics Program, Division of Cancer Treatment, National Cancer Institute, for

suramin; and J. Kessler, L. Sabens, and 1. Quinn at this institution for

assistance with the text and figures.

References

1. Mossman, B. T., Bignon, J., Corn, M., Seaton, A., and Gee, J. B. Asbestos: scientificdevelopments and implications for public policy. Science (Washington DC), 247:294—301, 1990.

2. Heintz, N. H., Janssen, Y. M. W., and Mossman, B. T. Persistent induction of c-fosand c-jun expression by asbestos. Proc. NatI. Acad. Sci. USA, 90: 3299—3303,1993.

3. Timblin,C. R., Janssen,Y. W. M., and Mossman,B. T. Transcriptionalactivationof theproto-oncogenec-jun by asbestos and H202 is directly related to increasedproliferationand transformationof trachealepithelialcells. Cancer Res., 55: 2723—2726,1995.

4. Yegles, M., Saint-Etienne, L., Renier, A., Janson, X., and Jaurand, M-C. Induction ofmetaphase and anaphase/telophase abnormalities by asbestos fibers in rat pleuralmesothelial cells in vitro. Am. J. Respir. Cell Mol. Biol., 9: 186—191,1993.

5. Hesterberg, T. W., and Barrett, J. C. Induction by asbestos fibers of anaphaseabnormalities: mechanisms for aneuploidy induction and possibly carcinogenesis.Carcinogenesis (Lond.), 6: 473—475, 1985.

6. Boulton, T. G., Yancopoulos, 0. D., Gregory, J. S., Slaughter, C., Moomaw, C., Hsu,J., and Cobb, M. H. An insulin-stimulated protein kinase similar to yeast kinasesinvolved in cell cycle control. Science (Washington DC), 249: 64—67, 1990.

7. Zinck, R., Hipskind, R. A., Pingoud, V., and Nordheim, A. c-fos transcriptionalactivation and repression correlate temporally with the phosphorylation status of TCF.EMBO J., 12: 2377—2387,1993.

8. Woodworth, C. D., Mossman, B. T., and Craighead, J. E. Induction of squamousmetaplasia in organ cultures of hamster trachea by naturally occurring and syntheticfibers. Cancer Res., 43: 4906—4912,1983.

9. Posada, J., and Cooper, J. A. Requirements for phosphorylation ofMAP kinase duringmeiosis in Xenopus oocytes. Science (Washington DC), 255: 212—215,1992.

10. Posada, J., Sanghera, J., Pelech, S., Aebersold, R., and Cooper, J. A. Tyrosinephosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts. Mol. Cell. Biol., 11: 2517—2528,1991.

11. Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C.,Curran, T., and Davis, R. J. Pro-Leu-SeriThr-Pro is a consensus primary sequence forsubstrate protein phosphorylation. Characterization of the phosphorylation of c-mycand c-jun proteins by an epidermal growth factor receptor threonine 669 proteinkinase. J. Biol. Chem., 266: 15277—15285,1991.

12. Sorokin, A., Lemmon, M. A., Ullrich, A., and Schlessinger, J. Stabilization of anactive dimeric form of the epidermal growth factor receptor by introduction of aninter-receptor disulfide bond. J. Biol. Chem., 269: 9752—9759,1994.

13. de Vries-Smits, A. M., Burgering, B. M., Leevers, S. J., Marshall, C. J., and Bos, J. L.Involvement of p2lras in activation of extracellular signal-regulated kinase 2. Nature(Lond.), 357: 602—604, 1992.

14. Bermudez, E., Everitt, J., and Walker, C. Expression of growth factor and growthfactor receptor RNA in rat pleural mesothelial cells in culture. Exp. Cell Res., 190:91—98,1990.

15. Walker, C., Bermudez, E., Stewart, W., Bonner, J., Molloy, C. J., and Everitt, J.Characterization of platelet-derived growth factor and platelet-derived growth factorreceptor expression in asbestos-induced rat mesotheioma. Cancer Ret., 52: 301—306,1992.

16. Lee, T. C., Thang, Y., Aston, C., Hints, R., Jagirdar, J., Perle, M. A., Burt, M., and Rom,w. N.Normalhumanmesothelialcellsandmesotheliomacelllinesexpressinsulin-likegrowth factor I and associatedmolecules.Cancer Res., 53: 2858-2864, 1993.

17. Gerwin, B. I., Lechner, J. F., Reddel, R. R., Roberts, A. B., Robbins, K. C.,Gabrielson, E. W., and Harris, C. C. Comparison of production of transforminggrowth factor-beta and platelet-derived growth factor by normal human mesotheialcells and mesothelioma cell lines. Cancer Res., 47: 6180—6184, 1987.

18. Quantin, B., and Breathnach, R. Epidermal growth factor stimulates transcription ofthe c-jun proto-oncogene in rat fibroblasts. Nature (Lond.), 334: 538—539,1988.

19. Schlessinger, J., and Ullrich, A. Growth factor signaling by receptor tyrosine kinases.Neuron, 9: 383—391,1992.

20. Hensey, C. E., Boscoboinik, D., and Azzi, A. Suramin, an anti-cancer drug, inhibitsprotein kinase C and induces differentiation in neuroblastoma cell clone NB2A. FEBSLett.,258:156—158,1989.

21. Levitzki. A., and Gazit, A. Tyrosine kinase inhibition: an approach to drug development. Science (Washington DC), 267: 1782—1788,1995.

22. Mohammadi, M., Honegger, A., Sorokin, A., Ullrich, A., Schlessinger, J., andHurwitz, D. R. Aggregation-induced activation of the epidermal growth factor receptor protein tyrosine kinase. Biochemistry, 32: 8742—8748,1993.

23. Stevenson, M. A., Pollock, S. S., Coleman, C. N., and Calderwood, S. K. X-irradiation, phorbol esters, and H2O2stimulate mitogen-activated protein kinase activity inNIH-3T3 cells through the formation of reactive oxygen intermediates. Cancer Res.,54: 12—15,1994.

24. Guyton, K. Z., Liu, Y., Gorospe, M., Xu, Q., and Holbrook, N. J. Activation ofmitogen-activated protein kinase by H202. J. Biol. Chem., 271: 4138—4142, 1996.

25. Janssen, Y. M., Barchowsky, A., Treadwell, M., Driscoll, K. E., and Mossman, B. T.Asbestos induces nuclear factor kappa B (NF-kappa B) DNA-binding activity andNF-kappa B-dependent gene expression in tracheal epithelial cells. Proc. Natl. Aced.Sci. USA, 92: 8458—8462, 1995.

26. Ames, B. N., and Gold, L. S. Too many rodent carcinogens: mitogenesis increasesmutagenesis. Science (Washington DC), 249: 970—971, 1990.

27. Ito, T., and May, W. S. Drug development train gathering steam: inhibitors of specificsignaling molecules may provide future treatments for acute lymphocytic leukemia.Nat. Med., 2: 403-404, 1996.

A. B.

1 2 @3 4 56

0@ 5―15― 1' 5'It!@!:@!!J 0@ 5―15―2Hrs1@@ I

EGF Croc. EGF Croc.(5ng!mI)(5jiglcm2) (5ngImI)(5@sg!cm2)

Fig. 5. Autophosphorylation kinase activity of the EGF-R after immunoprecipitation.

Because of their large surface area and rigid structure, it is unlikelythat asbestos fibers interact with the EGF-R with the specificityattributed to ligand. However, in receptor binding studies, we have

found that crocidolite asbestos interferes with the binding of EGF toits receptor in membrane preparations.4 This interference appears to

be due to interactions of the fibers with the membranes and/or with theEGF-R itself. In binding studies between fibers and EGF in theabsence of membranes, there was no significant binding of EGF to theasbestos. Therefore, the interference with binding is not due to adsorption of the EGF to the fibers. The ability of crocidolite to blockthe binding of growth factor to receptor makes it unlikely that thetemporal delay between fiber exposure and ERK1/2 response is due tocrocidolite inducing autocrine feedback on cells, because these growthfactors would presumably be blocked as well.

One hypothesis of carcinogenesis, based on microscopic studies invitro, is that asbestos is genotoxic due to direct interaction of fibers

with chromosomes due to their penetration of the discontinuousnuclear membrane during mitosis (4, 5). A number of reports mdicating that asbestos does not cause cell transformation or genotoxicityin a variety of standard bioassays and the inability to demonstrate

asbestos fibers within mesothelial cells in rodent inhalation experi

ments or in tissues from patients with mesothelioma have brought thistheory into question (reviewed in Ref. 1). Recently, chronic inflammation and mitogenesis have been proposed as more plausible mechanisms to explain the carcinogenicity of inhaled fibers (26). Ourstudies here provide a mechanistic framework for a signal transduction pathway that triggers chronic cell proliferation by asbestos afterinteraction of fibers with the cell surface. Moreover, because inhibilion of ERK responses to asbestos could be achieved with use ofAG1478 (a tyrphostin inhibiting EGF-R activation), additional strategies based on perturbation of asbestos-induced signaling for devel

opment of therapeutic agents in the treatment of asbestos-associatedtumors may be plausible (27).

Acknowledgments

We thank J. J. M. Bergeron (McGill University, Montreal, Quebec, Canada)for EGF-R antibodies; A. Levitzki (Hebrew University, Jerusalem, Israel) forAG 1478; the Drug Synthesis & Chemistry Branch, Developmental Therapeu

4 C. L. Zanella, S. M. Shreeve, T. R. Trinon, and B. T. Mossman, unpublished

observations.

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1996;56:5334-5338. Cancer Res   Christine L. Zanella, James Posada, Thomas R. Tritton, et al.   ReceptorCascade after Phosphorylation of the Epidermal Growth FactorSignal-regulated Kinase 1 Mitogen-activated Protein Kinase Asbestos Causes Stimulation of the Extracellular

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