Scavenging of Extracellular H2O2 by Catalase Inhibits the Proliferation of HER-2/Neu-transformed Rat-1 Fibroblasts through the Induction of a Stress Response

High levels of reactive oxygen species (ROS) are associated with cytotoxicity. Alternatively, nontoxic levels of ROS like hydrogen peroxide (H2O2) can mediate the transmission of many intracellular signals, including those involved in growth and transformation. To identify pathways downstream of endogenous cellular H2O2 production, the response of Rat-1 fibroblasts exhibiting differential HER-2/Neu receptor tyrosine kinase activity to removal of physiological H2O2 concentrations was investigated. The proliferation of all cells was abolished by addition of the H2O2 scavenger catalase to the culture medium. HER-2/Neu activity was not significantly affected by catalase treatment, suggesting that the target(s) of the H2O2 signal lie downstream of the receptor in our model. ERK1/2 phosphorylation was blocked by catalase in fibroblasts expressing wild type Neu, however such a response did not occur in cells possessing activated mutant Neu. This indicates that the ERK1/2 response contributes little to the growth inhibition observed. By contrast, JNK1 activity increased following the addition of catalase or H2O2, regardless of Neu activity or level of cell transformation. Phosphorylation of p38 MAPK was induced by H2O2 but not by catalase. These observations suggest that scavenging of H2O2 from the cellular environment blocks Rat-1 proliferation primarily through the activation of stress pathways. High levels of reactive oxygen species (ROS) are associated with cytotoxicity. Alternatively, nontoxic levels of ROS like hydrogen peroxide (H2O2) can mediate the transmission of many intracellular signals, including those involved in growth and transformation. To identify pathways downstream of endogenous cellular H2O2 production, the response of Rat-1 fibroblasts exhibiting differential HER-2/Neu receptor tyrosine kinase activity to removal of physiological H2O2 concentrations was investigated. The proliferation of all cells was abolished by addition of the H2O2 scavenger catalase to the culture medium. HER-2/Neu activity was not significantly affected by catalase treatment, suggesting that the target(s) of the H2O2 signal lie downstream of the receptor in our model. ERK1/2 phosphorylation was blocked by catalase in fibroblasts expressing wild type Neu, however such a response did not occur in cells possessing activated mutant Neu. This indicates that the ERK1/2 response contributes little to the growth inhibition observed. By contrast, JNK1 activity increased following the addition of catalase or H2O2, regardless of Neu activity or level of cell transformation. Phosphorylation of p38 MAPK was induced by H2O2 but not by catalase. These observations suggest that scavenging of H2O2 from the cellular environment blocks Rat-1 proliferation primarily through the activation of stress pathways. reactive oxygen species stress-activated protein kinase nuclear factor kappa B c-Jun N-terminal kinase mitogen-activated protein kinase extracellular signal-regulated kinase 1 and 2 amino acid(s) heat-inactivated MAPK/ERK kinase 1 5-bromo-2′-deoxyuridine phosphate-buffered saline glutathione S-transferase phosphoinositide 3-OH kinase Oxidative stress is known to induce the cellular apoptotic program by the activation of defined ROS1-responsive signals: the stress-activated protein kinase (SAPK) and nuclear factor kappa B (NF-κB) pathways ((1), for review see Ref. 2Tibbles L.A. Woodgett J.R. Cell. Mol. Life Sci. 1999; 55: 1230-1254Crossref PubMed Scopus (557) Google Scholar). Activation of c-Jun/AP-1 transcription factors by the c-Jun N-terminal kinase (JNK) members of the SAPKs has been shown to induce apoptosis in several cell types (3Leppä S. Bohmann D. Oncogene. 1999; 18: 6158-6162Crossref PubMed Scopus (453) Google Scholar). This signal is in contrast to the other characterized roles of c-Jun/AP-1 activity in survival and growth. For example, Jun proteins associate with Nrf 1 and 2 transcription factors to up-regulate the antioxidant/electrophile response element-mediated gene expression as a defense against oxidative stress (4Venugopal R. Jaiswal A.K. Oncogene. 1998; 17: 3145-3156Crossref PubMed Scopus (493) Google Scholar). Similarly, NF-κB was originally identified as a mediator of tumor necrosis factor α- and ROS-induced apoptosis but more recently has also been characterized as part of a survival response by stimulating antioxidant response element-regulated and anti-apoptotic protein expression (5Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2947) Google Scholar, 6Mercurio F. Manning A.M. Oncogene. 1999; 18: 6163-6171Crossref PubMed Scopus (371) Google Scholar). Indeed, ROS, like H2O2, are associated with the regulation of multiple cellular processes via interaction with proteins and lipid species (7Burdon R.H. Free Radic. Biol. Med. 1995; 18: 775-794Crossref PubMed Scopus (1075) Google Scholar). These observations have led to the idea that ROS are required “cofactors” in the regulation of many intracellular signal transduction cascades. Addition of micromolar concentrations of H2O2 to cells in vitro can activate plasma membrane receptor tyrosine kinases, including the epidermal growth factor receptor/ErbB-1 (8Gamou S. Shimizu N. FEBS Lett. 1995; 357: 161-164Crossref PubMed Scopus (169) Google Scholar), platelet-derived growth factor receptor (9Sundaresan M., Yu, Z.-X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2342) Google Scholar), and insulin receptor (10Schmid E. Hotz-Wagenblatt A. Hack V. Dröge W. FASEB J. 1999; 13: 1491-1500Crossref PubMed Scopus (74) Google Scholar). The p21 Ras-MAPK (mitogen-activated protein kinase) pathway, a key growth signal linking membrane receptors to the nucleus, is also stimulated by oxidative stimuli such as H2O2 (11Guyton K.Z. Liu Y. Gorospe M. Xu Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1148) Google Scholar, 12Zhang J. Jin N. Liu Y. Rhoades R.A. J. Respir. Cell. Mol. Biol. 1998; 19: 324-332Crossref PubMed Scopus (87) Google Scholar). Rapid induction of cellular H2O2 generation has been observed to follow the addition of a variety of peptide growth factors to cells, suggesting an autocrine and/or paracrine role for ROS in growth signaling (13Bae Y.S. Kang S.W. Seo M.S. Baines I.C. Tekle E. Chock P.B. Rhee S.G. J. Biol. Chem. 1997; 272: 217-221Abstract Full Text Full Text PDF PubMed Scopus (1109) Google Scholar, 14Sattler M. Winkler T. Verma S. Byrne C.H. Shrikhande G. Salgia R. Griffin J.D. Blood. 1999; 93: 2928-2935Crossref PubMed Google Scholar, 15Patterson C. Ruef J. Madamanchi N.R. Barry-Lane P. Hu Z. Horaist C. Ballinger C.A. Brasier A.R. Bode C. Runge M.S. J. Biol. Chem. 1999; 274: 19814-19822Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). The specific molecular targets that are critical for H2O2-mediated mitogenesis have yet to be determined; the importance of a given pathway is also dependent on factors such as cell type, environment, and level of differentiation. It follows that the deregulation of ROS levels may be a key factor in neoplasia. Elevations in O⨪2 and H2O2are seen in some human tumor cell lines (16Szatrowski T.P. Nathan C.F. Cancer Res. 1991; 51: 794-798PubMed Google Scholar), and their production from mitochondrial and plasma membrane sources appears to be a requirement of tumor viability and growth (17Oberley T.D. Oberley L.W. Histol. Histopathol. 1997; 12: 525-535PubMed Google Scholar, 18Del Bello B. Paolicchi A. Comporti M. Pompella A. Maellaro E. FASEB J. 1999; 13: 69-79Crossref PubMed Scopus (145) Google Scholar). Mitochondria-localized manganese-containing superoxide dismutase is proposed to act as a both an inhibitor (19Li J.-J. Oberley L.W. St. Clair D.K. Ridnour L.A. Oberley T.D. Oncogene. 1995; 10: 1989-2000PubMed Google Scholar) and activator (20Wenk J. Brenneisen P. Wlaschek M. Poswig A. Briviba K. Oberley T.D. Scharffetter-Kochanek K. J. Biol. Chem. 1999; 274: 25869-25876Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) of growth and transformation. A higher oxidative capacity of cancer cells may also accompany an increase in resistance to ROS stress, which could in turn affect drug resistance. For instance, resistance to H2O2stress by the acquired up-regulation of catalase activity and glutathione levels was shown to be crucial for maintenance of cell viability as well as resistance to cisplatin treatment (21Spitz D.R. Phillips J.W. Adams D.T. Sherman C.M. Deen D.F. Li G.C. J. Cell. Physiol. 1993; 156: 72-79Crossref PubMed Scopus (76) Google Scholar). In theory, the oxidative stress response may be shifted from growth arrest and death to proliferation and transformation for cells exhibiting defects in growth pathway regulation and the apoptotic program (22Ishikawa Y. Kitamura M. Biochem. Biophys. Res. Commun. 1999; 264: 696-701Crossref PubMed Scopus (73) Google Scholar). If the H2O2 signal is involved in these processes, they should be inhibited by its removal from the cellular environment. In the present study the requirement of low levels of H2O2 for Rat-1 fibroblast proliferation was investigated. The effects on defined growth- and stress-associated signaling pathways, produced by the scavenging of physiological H2O2 levels from the extracellular environment, were assessed. Rat-1 clones exhibiting differential growth and transformation properties resulting from specific alterations of the HER-2/Neu receptor were utilized as a model for the responses to removal of the H2O2 stimulus (23Siegel P.M. Muller W.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8878-8883Crossref PubMed Scopus (92) Google Scholar, 24Dankort D.L. Wang Z. Blackmore V. Moran M.F. Muller W.J. Mol. Cell. Biol. 1997; 17: 5410-5425Crossref PubMed Scopus (124) Google Scholar). Multiple signals downstream of Neu are implicated in oncogenesis, and Neu overexpression and/or activating mutation is observed in 25–30% of human breast and ovarian cancers (for review see Ref. 25Reese D.M. Slamon D.J. Stem Cells. 1997; 15: 1-8Crossref PubMed Scopus (204) Google Scholar). The activities of the Neu receptor, extracellular signal-regulated kinase 1 and 2 (ERK1/2) MAPK and JNK1 effector pathways were studied after the addition of the H2O2 scavenger catalase. All Rat-1 clones displayed comparable sensitivities to growth inhibition by this treatment regardless of the level of Neu activity. It was found that scavenging of H2O2 caused stable inhibition of the ERK1/2 signal, transient induction of the JNK1 pathway, and no effect on p38 MAPK. Constitutive Neu activation could rescue the catalase-mediated block of ERK1/2 activity while only slightly increasing resistance to growth inhibition. Therefore, these results indicate that removal of extracellular H2O2 can both down-regulate growth signals and activate stress-associated signals, however, the stress response appears to play a larger role in the anti-proliferative effects observed. All Rat-1 fibroblast clonal cell lines were kindly provided by Dr. W. J. Muller and have been previously characterized (23Siegel P.M. Muller W.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8878-8883Crossref PubMed Scopus (92) Google Scholar, 24Dankort D.L. Wang Z. Blackmore V. Moran M.F. Muller W.J. Mol. Cell. Biol. 1997; 17: 5410-5425Crossref PubMed Scopus (124) Google Scholar). Rat-1NeuN (17Oberley T.D. Oberley L.W. Histol. Histopathol. 1997; 12: 525-535PubMed Google Scholar) clone expresses wild type Neu. The highly transformed Rat-1Neu8142(10) clone expresses Neu having a 12-amino acid (aa 641–652) deletion in the extracellular region of the receptor proximal to the transmembrane domain, resulting in its constitutive dimerization. Rat-1NeuNT (3Leppä S. Bohmann D. Oncogene. 1999; 18: 6158-6162Crossref PubMed Scopus (453) Google Scholar) is another highly transformed clone expressing Neu with an activating point mutation (V664E) in the transmembrane domain (26Bargmann C.I. Hung M.-C. Weinberg R.A. Cell. 1986; 45: 649-657Abstract Full Text PDF PubMed Scopus (901) Google Scholar). Rat-1NeuNT-NYPD (6Mercurio F. Manning A.M. Oncogene. 1999; 18: 6163-6171Crossref PubMed Scopus (371) Google Scholar) is a transformation-impaired clone expressing the NeuNT receptor with C-terminal autophosphorylation sites (Tyr-1028, Tyr-1144, Tyr-1201, Tyr-1226/7, Tyr-1253) replaced with phenylalanine residues. Cells were in culture in a of Rat-1 lines were in with and all were with heat-inactivated in Cells were by Life were with to extracellular H2O2 levels and affect cellular or catalase heat-inactivated catalase for A. H2O2 and the inhibitor The cell growth was by of the cell after of were a and for cells were with and cellular was by the addition of of in 1 in was a The cell was to for cell type by with a from the of known cell of cellular was on cells by the addition of 5-bromo-2′-deoxyuridine for Cells were and with phosphate-buffered of cell was to 2 of with and on for 2 of was and were for an were and cell were in 1 of in were in in of with of for and in 1 of with for The of cells in was by as by was by cells in the cell with 1 for Cells were and to and the were with and in for of a Cells were for protein in 2 1 of protein was in for and in a was as previously were and the were blocked with and with kinase or MAPK were to protein A or and was To ERK1/2 and p38 phosphorylation levels with protein were of and with kinase or MAPK were in and in of cellular protein was to of protein and of or or and were for were in in kinase 2 2 and in a kinase of of kinase 2 of or p38 MAPK protein of c-Jun activation domain or protein of activation domain and of Life for was addition of and of for from was by and to a and of the c-Jun was To c-Jun and N-terminal phosphorylation levels with JNK1 and p38 MAPK the membrane was with or and membrane was Addition of catalase to the culture has been shown by our to block the proliferation of a variety of cell types in a and J. W. J. and G. catalase have also been utilized to the effects of H2O2 in cell culture Y. A.K. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) and overexpression of catalase can and cell growth V. 1998; 19: PubMed Scopus Google Scholar). To catalase activity could a response in our Rat-1 cells were with of catalase or heat-inactivated and the cell after culture was observed The growth of all Rat-1 lines was blocked after catalase removal of this treatment or of the this treatment caused growth arrest induction of the apoptotic as observed by cell and The cell of fibroblasts increasing of treatment was by of and A block of the cell the or was not however, an of cell in and a and inhibition of of cells with an in addition to catalase also the proliferation block 1 that catalase-mediated effects are a of in H2O2 with activities was to cells, suggesting that a defined of extracellular H2O2 is required for cell survival and growth. with the observations of cell and a and was to by catalase as observed by or 2 cellular the addition of H2O2 increased in all lines in with its cell types have been shown to H2O2 the culture C. Ruef J. Madamanchi N.R. Barry-Lane P. Hu Z. Horaist C. Ballinger C.A. Brasier A.R. Bode C. Runge M.S. J. Biol. Chem. 1999; 274: 19814-19822Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, T.P. Nathan C.F. Cancer Res. 1991; 51: 794-798PubMed Google Scholar). This generation is to cell growth of cellular H2O2 production results in to both the of extracellular H2O2 and to levels of proliferation and transformation (19Li J.-J. Oberley L.W. St. Clair D.K. Ridnour L.A. Oberley T.D. Oncogene. 1995; 10: 1989-2000PubMed Google Scholar, J. Brenneisen P. Wlaschek M. Poswig A. Briviba K. Oberley T.D. Scharffetter-Kochanek K. J. Biol. Chem. 1999; 274: 25869-25876Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The endogenous generation of H2O2 by Rat-1 cells was with the of H2O2 produced by clone to the of growth in Addition of A. catalase to the extracellular cells a or higher increase in H2O2 production with the Rat-1NeuN of the differential in HER-2/Neu extracellular and level of transformation by these they were in on molecular responses to catalase The Ras-MAPK signaling has also been shown to be activated by H2O2 (11Guyton K.Z. Liu Y. Gorospe M. Xu Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1148) Google Scholar, Oncogene. 1996; 13: Google Scholar). to the removal of H2O2 ERK1/2 MAPK as for the of this and 2 were of were with ERK1/2 protein Rat-1NeuN cells with H2O2 displayed an increase in MAPK the addition of catalase for to abolished MAPK activity catalase did not such an In contrast, this in ERK1/2 phosphorylation was not observed after the treatment of cells The inhibitor D.T. A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) was to cells for 1 as a for ERK1/2 These observations that the of Neu receptor activity can the response of the MAPK pathway to a in H2O2 the growth response of cells to catalase little from that of Rat-1NeuN cells, this proliferation block appears of the MAPK The response of Rat-1 clones to a of catalase activities was cells displayed a slightly higher resistance to the effects of catalase This may from in MAPK pathway activity or in the of endogenous H2O2 production observed the To for the cells ovarian were also for resistance to catalase These cells extracellular H2O2 a level higher the This increased H2O2 did not to increase in the growth of cells was to the scavenging of cells to removal of H2O2 in the of the inhibitor was also This treatment blocked the growth of Rat-1NeuN cells to a catalase treatment this effect was in the which increased MAPK increased MAPK activity did not rescue cell growth to a appears the H2O2 signal can ERK1/2 are also other intracellular signals that are for the effects of H2O2 scavenging could not be to the of MAPK signals, the response of a the JNK1 The N-terminal phosphorylation of c-Jun by JNK1 is a cell stress response B. M. T. B. T. M. Cell. Full Text PDF PubMed Scopus Google Scholar, R. C. J. 1999; 18: PubMed Scopus Google Scholar). JNK1 activity was by an in vitro kinase All induced a transient increase in JNK1 kinase activity as by level of In Rat-1NeuN cells a level of H2O2 activated JNK1 to with activity 1 after levels of H2O2 could not induce JNK1 activity. catalase induced the JNK1 signal with activity after catalase could also activate JNK1 to a did not in in cell viability and growth as The response of cells was that 2 increased phosphorylation of the c-Jun in an as catalase treatment These observations that of H2O2 the addition of concentrations of H2O2 or removal of endogenous H2O2 levels by can the JNK1 pathway in a the activation of this by catalase is by differential Neu receptor suggesting that may be a critical of the growth inhibition observed with all Rat-1 fibroblasts These indicate that the up-regulation of JNK1 is a response to removal of extracellular H2O2 in our Rat-1 fibroblast model. In a to that observed with JNK1 signal the p38 MAPK was addition of all cells this response to oxidative stress was and no p38 activation was observed after treatment of cells with catalase or heat-inactivated catalase of induction of p38 phosphorylation in both Rat-1NeuN and lines by 2 H2O2 was comparable to that observed for The phosphorylation of p38 MAPK the transcription factor was also the vitro kinase in phosphorylation of peptide by p38 were observed after catalase or H2O2 It is this is a of a of p38 in the cells or the of the utilized to to the p38 the of p38 activation by catalase levels observed to affect ERK1/2 and JNK1 that the block in Rat-1 fibroblast proliferation is of this This is the the effects of the removal of produced H2O2 specific and signaling pathways. To H2O2 from the cellular environment, a catalase was to the culture This was that the molecular signaling responses observed could be to in H2O2 the plasma with this is that the effects observed could from To this the effects of catalase to heat-inactivated catalase in all with the heat-inactivated did not affect cell viability and did affect Neu receptor or JNK1 activity did increase the addition of heat-inactivated but a to level that observed after treatment with catalase. A comparable A. catalase extracellular H2O2 of this treatment blocked the In growth inhibition was of cells with The levels of activity required for this rescue were that the are H2O2 These results that the responses to the catalase its activity. It is that in the of extracellular H2O2 in turn affect intracellular H2O2 can cell Therefore, the effects of the catalase treatment are as a response to the of the environment, which is both and of the plasma the that Neu receptor activity was not by the addition of catalase to cell MAPK signaling indicates that of intracellular signals by this type of treatment such affected signal was the ERK1/2 The inhibition of ERK1/2 phosphorylation observed with catalase addition up-regulation observed with to Rat-1NeuN cells with the that the of the MAPK to H2O2 (11Guyton K.Z. Liu Y. Gorospe M. Xu Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1148) Google J. Jin N. Liu Y. Rhoades R.A. J. Respir. Cell. Mol. Biol. 1998; 19: 324-332Crossref PubMed Scopus (87) Google Scholar). Constitutive activation of the Neu receptor in Rat-1 cells induced ERK1/2 activity and from inhibition. Neu with several signaling including the pathway, in ERK1/2 regulation D.M. Slamon D.J. Stem Cells. 1997; 15: 1-8Crossref PubMed Scopus (204) Google Scholar). The rescue of phosphorylation was by only a increase in resistance of fibroblasts to growth inhibition by catalase. This indicates to the cellular responses ERK1/2 signal is not critical for the the stress-activated JNK1 signal was and induced by that both increased and H2O2 in Rat-1NeuN and JNK1 is known to be to oxidative stress L.A. Woodgett J.R. Cell. Mol. Life Sci. 1999; 55: 1230-1254Crossref PubMed Scopus (557) Google Scholar, M. J. J. Q. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), but our suggest an role for this signal in response to an and members of the of involved in regulation and cell have been shown to activate the through interaction with kinase M., Yu, P. Xu N. T. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, Yu, J. J. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). In is a of the plasma that the extracellular generation of O⨪2 and its dismutase H2O2 T. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). the of this appears to be not only for ROS generation but also for cell survival and growth C. Ruef J. Madamanchi N.R. Barry-Lane P. Hu Z. Horaist C. Ballinger C.A. Brasier A.R. Bode C. Runge M.S. J. Biol. Chem. 1999; 274: 19814-19822Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, Kang Kang FEBS Lett. 1999; PubMed Scopus Google Scholar). It follows that may the pathway through kinase as well as H2O2 our MAPK inhibition and activation after catalase treatment, could be that of the H2O2 stimulus blocks the pathway while other signals like phosphoinositide 3-OH kinase and that can play the kinase kinase was shown to and its activity T. J. Kang J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of our Rat-1 fibroblast lines with a of the inhibitor J. Biol. Chem. Full Text PDF PubMed Google Scholar) inhibited cell proliferation to a comparable regardless of HER-2/Neu activity levels to the signal to the effects JNK1 however, this to be The role of in cell is not c-Jun contributes to a of processes as part of the (3Leppä S. Bohmann D. Oncogene. 1999; 18: 6158-6162Crossref PubMed Scopus (453) Google Scholar). The of activity with other signals such as the could which is In our inhibition and activation are observed with cell growth by R. C. J. 1999; 18: PubMed Scopus Google Scholar) that phosphorylation of c-Jun is specific for stress responses c-Jun in cell is by other indicates that the signal can the cell and in a K. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar), however, the this effect have yet to be Indeed, observed an increase of the of cells in and after with catalase not cells in the of the cell apoptosis may be by a In this that of MAPK to for the growth of Rat-1 ERK1/2 activities are induced the addition of H2O2 and inhibited by its of activated Neu receptor this inhibition but little effect on the block of the stress-activated JNK1 pathway was induced by both the addition and removal of the H2O2 signal, regardless of Neu stress signal, the p38 MAPK pathway, was activated by levels of H2O2 but not by its that the JNK1 response be A of activation may act as a cell of oxidative and to a response of growth arrest and A. P. N. J. M. R. and W. for their and and

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