Oxidant Carcinogenesis and Antioxidant Defenseu PETER CERU?TI,6 GIRISH SHAH, ALEXANDER PESKIN, AND PAUL AMSTAD Department of Carcinogenesis Swiss Institute for fiperitnental Cancer Research I066 Epalinges1Lama nne, Switzerland ROLE OF THE CELLULAR ANTIOXIDANT DEFENSE IN OXIDANT CARCINOGENESIS
Oxidants are ubiquitous in our aerobic environment and formed in situ in tissues and cells by normal metabolism and the metabolism of certain xenobiotics. They are always toxic and produce macromolecular damage. At the same time, oxidants can serve as (patho)physiological signals in growth and differentiation.'-' The sensitivity of cells to oxidants is attenuated by low molecular weight antioxidants and antioxidant enzymes. The biochemistry of the most important enzymes (i.e., superoxide dismutases [SOD], catalase [CAT], GSH peroxidases [GPx], GSH reductase, and GSH-S transferase) has been studied in detaiL4 However, the physiological role of the antioxidant enzymes in situ in the cell is only poorly understood because of complex interactions and interrelationships between the individual components.
Increased Constitutive Antioxidant Defense in Promocable Mouse Epidermal Cells A first indication that the cellular antioxidant defense affects the capacity of oxidants to stimulate the growth of epithelial cells was obtained in a study comparing promotable and nonpromotable epidermal cells, JB6.5 When we measured the specific activities of Cu,Zn-SOD, CAT, and GPx in monolayer cultures of JB6 cells, we discovered that the promotable clone 41 contained approximately twice the activity of SOD and CAT relative to the nonpromotable clone 30, whereas the activities of GPx were comparable. We took advantage of the cross-activity of a rabbit antibody against human CAT to assess CAT protein concentrations by Western blot analysis. Constitutive amounts of CAT were two- to threefold higher in clone 41 than in clone 30, in agreement with the enzyme activity. Northern blots indicated that the higher amounts of CAT and SOD in clone 41 were due to increased stationary concentrations of mRNAs for these genes. (Southern analysis showed that both clones contained the same gene complements.) We conclude that the antioxidant defense of JB6 clone 41 is superior to that of clone 30. The difference between the two clones is particularly remarkable, because the two antioxidant enzymes SOD and CAT are increased coordinately in clone 41. Because the product of the action of S O D is Hz02,an increase in its activity is only beneficial to the cell if 'This work was supported by the Swiss National Science Foundation and the Swiss Association of Cigarette Manufacturers and the Association for International Cancer Research. bTo whom correspondence should be addressed. 158
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it is counterbalanced by a sufficient capacity for the destruction of H202. This is apparently accomplished in clone 41 by an increase in CAT.5 It should be mentioned that SOD and CAT may mutually protect each other from inactivation by active oxygen. Stable Transfectantsof Mouse Epidermal Cells with Increased Complements of Cu,Zn-Superoxide Dismutase and Catalase
We are attempting to gain further insight into the role of the cellular antioxidant defense in oxidant carcinogenesis with the help of stable transfectants of mouse epidermal cells with cDNAs for human Cu,Zn-SOD, Mn-SOD, CAT, and GPx. As it
FIGURE 1. Southern blot analysis of Cu,Zn-SOD and CAT transfectants of mouse epidermal cells JB6. Cellular DNA was restricted with BamHl and after electrophoretic separation transferred to nitrocellulose as described by Southern. Filters were hybridized with a 450-bp AluI to TaqI human Cu,Zn-SOD cDNA probe6 or a 1250 bp Hind111 to PvuII fragment of human CAT cDNA,’ respectively. SOD-transfected clones SOD15 and SOCAT3 show a band at 0.5 kb originating from the transfected SOD gene and a double band at 10 kb from the endogenous mouse gene. CAT-Southern blots in the right-side portion of the figure show a 1.8-kb band for transfected human CAT cDNA.
appears likely that the balance of several antioxidant enzymes will determine oxidant sensitivity, we are in the process of preparing multiple transfectants. It is our experimental strategy to use expression vectors with different selectable markers for each antioxidant enzyme cDNA. This allows sequential selection and the comparison of the properties of related generations of multiple transfectants. Until now we have prepared transfectants with moderately increased complements of Cu,Zn-SOD, CAT, or both. We have fully characterized several clones with increased enzyme activities on the molecular level (i.e., Northern blots, RNAse protection for the expression of the transfected relative to the endogenous genes; Southern blots for the presence and copy number of the transfected genes; immunoblots for the presence of the “transfected” human proteins). The Southern blots shown in
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FIGURE1 document the presence of human Cu,Zn-SOD cDNA in the SOD transfectant SOD15 and of human CAT cDNA in the CAT transfectant CAT4. The blot for the double transfectant SOCAT3 shows bands indicating the presence of both cDNAs. We are characterizing the biological properties of several of the aforementioned stable transfectants and report initial results below. In all experiments so far, an extracellular burst of 0; plus H202 was produced by xanthine/ xanthine-oxidase (X/XO) to test oxidant sensitivity. Growth properties: (a) Cu,Zn-SOD transfectants with two- to threefold increased SOD activity are hypersensitive to growth inhibition by 0; plus H202, (b) CAT transfectants with two- to fourfold increased CAT activity are hyposensitive to 0; plus H202, and (c) transfection of Cu,Zn-SOD clones with CAT corrects their hypersensitivity. The data in FIGURE 2 document these conclusions for an experi-
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2. DNA synthesis capacity of parent JB6 clone 41 and its Cu,Zn-SOD and CAT
transfectants following exposure to an extracellular oxidative burst. Monolayer cultures were grown under standard conditions and exposed to an extracellular burst of active oxygen produced by 15 p.g/ml xanthine and 1.5 pg/ml xanthine oxidase. Incorporation of (3H)thymidine into acid-insoluble material within a I-hour pulse was determined after the indicated lengths of posttreatment incubation. Data are expressed as a percentage of analogousvalues for untreated control cultures for each cell strain (A. Peskin and P. Cerutti, unpublished data).
ment measuring the effect of oxidant treatment on the DNA synthesis capacity of the parent clone 41, SOD 15 with 2.5-fold increased Cu,Zn-SOD activity, CAT4 with 3-fold increased CAT activity, and SOCAT3 with 3-fold higher CAT activity and 1.7-fold higher Cu,Zn-SOD activity than the parent strain. (SOCAT3 was derived from CAT4 by transfection with Cu,Zn-SOD cDNA.) Incorporation of (3H)thymidine within a 1-hour pulse into acid-insoluble material was determined. The data indicate the following order of relative resistance of the four cell strains: CAT4 > SOCAT3 > parent clone 41 > SOD15 (A. Peskin and P. Cerutti, unpublished data). Our data suggest that the balance of Cu,Zn-SOD and CAT (and probably GPx) determines the cellular sensitivity to an extracellular burst of active oxygen. We
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noted that the sensitivity of the transfectants was attenuated in high density cultures, suggesting a protective effect of neighboring cells. Sensitivity to DNA strand breakage: DNA strand breakage was measured by the alkaline elution method. The relative sensitivities of 05 plus H 2 0 2 induced DNA breakage were in decreasing order: Cu,Zn-SOD transfectant > parent > CAT transfectant = Cu,Zn-SOD/CAT double transfectant. These results demonstrate that CAT protects the nuclear DNA from damage by an extracellular burst of active oxygen, while Cu,Zn-SOD augments sensitivity. The fact that a Cu,Zn-SOD/CAT double transfectant was slightly more resistant than the parent clone suggests that a balanced increase in both enzymes results in protection.8 It appears that H 2 0 2 represents the precursor species for DNA strand breakage in mammalian cells exposed to an extracellular burst of active oxygen, but they do not imply that DNA is the immediate target for attack by H202or its radical derivatives9 Inducibility of c-fos: The induction of growth competence-related genes is a necessary prerequisite for growth stimulation. The immediate early gene c-fos is a prototype in this regard, as it was shown previously that its transcription is induced in mouse epidermal cells and fibroblasts by oxidants.I0J1Therefore, we compared the increase in stationary concentration of c-fos mRNA upon active oxygen treatment of the antioxidant gene transfectants. The following order of decreasing inducibility of c-fos was observed: parent > CAT transfectant (CAT4) > Cu,Zn-SOD/CAT double transfectant (SOCAT3) > Cu,Zn-SOD transfectant (SOD15). It is evident that despite the opposite effects of additional CAT and Cu,Zn-SOD on cytotoxicity, the inducibility of c-fos by oxidants is reduced in both types of transfectants. However, the reasons for the decrease in c-fos induction are probably different for CAT and Cu,Zn-SOD transfectants. The former are well protected from excessive H202 toxicity, but at the same time the signal that results in c-fos induction is attenuated. In contrast, increases in Cu,Zn-SOD levels alone augment the intracellular formation of H202, and toxic effects on components of the signal transduction pathways may predominate.6 It should be noted that in the X/XO system used in our work a large amount of 0; plus H202 is generated close to the cell surface. Superoxide may penetrate the membrane sufficiently for dismutation by cytosolic Cu,Zn-SOD. Therefore, enhanced oxidative damage in the SOD transfectants may occur preferentially at the plasma membrane.
MECHANISM OF INDUCTION OF THE PROTOONCOGENE C-FOS BY OXIDANTS Growth promotion by oxidants is observed with cultured human and mouse fibroblasts as well as epidermal It is expected to play a role in inflammation, fibrosis, and t u m o r i g e n e s i ~ Indeed, .~~~~~ oxidants trigger (patho)physiological reactions that resemble those induced by growth and differentiation factors. For example, active oxygen activates the phosphorylation of the ribosomal subunit S6I6 and causes the membrane translocation of protein kinase C as well as its activation.'' In contrast to polypeptide growth factors and hormones, oxidants induce DNA damage, in particular DNA breaks, and as a consequence, they activate ADPR transferase.I8J9As this enzyme is located in the nucleus and accomplishes the posttranslational polyADP-ribosylation of chromosomal proteins, it may represent an important link between DNA damage and oxidant-induced modulation of gene expression.'J5 That small doses of oxidants can stimulate rather than inhibit cell growth implies that they are capable of reprogramming gene expression. Therefore, it was intriguing to find that they induce the immediate early genes c-fos and c-myc. 10~13The transcrip-
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tional induction of these genes by oxidants was suppressed by inhibitors of protein kinases A and C and of ADPR transferase according to Northern blots and nuclear run-onexperiments (P. Amstad et al., unpublished data). These results indicate that the posttranslational modification of proteins participates in the induction mechanism. In contrast, de now protein synthesis was not required. To identify 5'-upstream regulatory sequences that are responsible for the transcriptional induction of c-fos by oxidants, we prepared vectors in which potential 30 min
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FIGURE 3. Induction of factor binding to collagenase TRE-oligonucleotide by oxidants. Gel retardation of (TRE)s pentamer oligonucleotide by nuclear extracts prepared from mouse epidermal cells JB6 clone 30 that had been treated with an extracellular burst of active oxygen produced by X/XO for 30 minutes. When indicated, the cells had been preincubated for 30 minutes with the ADPRtransferase inhibitor benzamide or the protein kinase inhibitors H7 and staurosporine 12, respectively.
1
5' A G C T T G - ( A T G A G T C A G X 5 -C C G G A T C
fos-enhancer motifs are linked to a tk-cat reporter gene (chloramphenicol-acetyltransferase, cat). Transient transfection experiments indicate that the joint DSEAP1 element (dyad symmetry element [DSE]) suffices for oxidant induction. Oxidants also stimulate the binding of protein factors to the fos-AP1 and (TRE)s (TPA responsive element) oligonucleotide motifs. The TRE motif, S'ATGAGTCAG, represents an AP1-like element contained in the S'-upstream regulatory sequences of the collagenase gene which plays a crucial role in its transactivation by the FOS protein.ms21FIGURE 3 shows a gel retardation experiment with the 5-mer
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(TRE)5 and nuclear extracts from X/XO-treated mouse epidermal cells JB6 clone 30. It is evident that factor binding to this element is increased by oxidant treatment for 30 and 60 minutes. Both benzamide (an inhibitor of ADPR transferase) and the protein kinase inhibitors H7 and staurosporine I2 suppress the reaction. As already mentioned, the same drugs suppressed the transcriptional induction of c-fos. Separate experiments indicate that the protein synthesis inhibitor cycloheximide diminished factor binding. From these results we conclude that the posttranslational phosphorylation and polyADP-ribosylation of as yet unidentified proteins are required for the de novo synthesis of regulatory factors that bind to TRE in response to oxidants (P. Amstad and P. Cerutti, unpublished data). Ultraviolet cross-linking experiments and immunocompetition with anti-FOS and anti-JUN antibody indicate that FOS and JUN are among the proteins that bind to TRE. Occupation of TRE with preexisting, possibly modified, FOS and JUN appears to be required for the induction of the collagenase gene by phorbol ester. On the other hand, it is not clear whether the binding of de novo synthesized FOS and JUN plays a role in the activation process. It should be remembered that phorbol ester induction of cat expression was inhibited by cycloheximide in transient transfection experiments with contructs containing a large portion of the collagenase promoter but not with minimal constructs containing only TRE linked to the reporter gene.2w22 Analogous results were obtained in gel retardation experiments with the fos-AP1oligonucleotide motif and nuclear extracts from X/XO-treated cells. As discussed for TRE, it follows that the posttranslational phosphorylation and polyADPribosylation of unidentified proteins is required for the de novo synthesis of protein factors, including FOS, which later bind to fos-AP1. The increase in factor binding to fos-AP1, which is observed 30 minutes following oxidant treatment, does not appear to be involved in the transcriptional induction of c-fos. As the addition of cycloheximide results in transcriptional superinduction of c-fos rather than s ~ p p r e s s i o nit, ~ ~ appears more likely that the de novo synthesized binding proteins participate in the down-regulation of c-fos.22,24 However, down-regulation of c-fos may involve the interaction of de novo synthesized FOS/JUN and other factors with the dyad symmetry element (DSE) of the c-fos promoter rather than with fos-AP1. This is indicated by transient transfection experiments. Constructs containing DSE rather than fos-AP1 linked to a cat reporter gene were down-regulated by FOS overproduction.25 The question arises if the posttranslational modification of the FOS-protein itself affects its binding to fos AP1 and participates in c-fos regulation. Affinity chromatography of polyADP-ribosylated nuclear proteins followed by Western blotting with anti-FOS antibody indicates that FOS protein is only weakly polyADP-ribosylated in response to oxidant treatment. Besides FOS, other chromosomal proteins are polyADP-ribosylated in response to oxidant treat men^^^,^^ PolyADP-ribosylation of histonesZ7in regions containing immediate early genes might participate in their transcriptional induction, because it introduces local relaxation of chromatin conformati0n.~8-3~ Prevention of the conformational change by ADPR-transferase inhibitors may suppress the transcriptional induction of a class of genes that includes c-fos. l5 Instead of participating in transcription initiation, polyADP-ribosylation of chromosomal proteins may be required for efficient message elongation. Oxidantinduced DNA strand breaks may retard message elongation either directly, because they represent blocks to the movement of the transcriptional machinery, or more likely indirectly, because they distort chromatin conformation. As polyADPribosylation is required for the efficient repair of DNA breaks,29its inhibition would be expected to potentiate the inhibitory effect of strand breaks. This model is supported by the observation reported above that the ADPR-transferase inhibitor
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benzamide suppressed transcriptional elongation of c-fos message in oxidant-treated cells according to nuclear run-on experiment^.^' In addition to inducing the ADP-ribosylation of various nuclear proteins, active oxygen may influence the nucleic acid binding properties of transcription factors directly. It is thought that the redox state of two cystein residues located in the basic regions of FOS and JUN is important for their binding to the human metallothionein IIA AP-1 recognition sequence in vitro.Autooxidized FOS and JUN have decreased binding capacities and can be reactivated by the reducing system thioredoxinthioredoxin reductase-NADPH or by high concentrations of d i t h i ~ t h r e i t o l . Fur~~,~~ ther examples in which the redox state of proteins may regulate their activity include ironthe oxy-R encoded protein in bacteria,34 heat shock factor in Dro~ophilu,~~ responsive element binding protein in mammalian cells,36 and PKC (mentioned above)."
SUMMARY
Growth promotion by oxidants is observed with cultured human and mouse fibroblasts as well as epidermal cells. It is expected to play a role in inflammation, fibrosis, and tumorigenesis. Indeed, oxidants trigger (patho)physiological reactions that resemble those induced by growth and differentiation factors. For example, active oxygen activates protein kinases, causes DNA breakage, and induces the growth competence-related protooncogenes c-jos and c-myc. The cellular antioxidant defenses affect the consequences of oxidant exposure. Transfectants of mouse epidermal cells that overproduce Cu,Zn-superoxide dismutase (SOD) were sensitized to the toxic effects of an extracellular burst of 0; plus H202, whereas overproducers of catalase (CAT) were protected. Transfection of SOD overproducers with CAT corrected their hypersensitivity. Inducibility of the protooncogene c-fos by oxidants was diminished in SOD and CAT overproducers, albeit probably for different reasons. It is concluded that a fine balance of the multiple components of the antioxidant defense determines the growth response of cells to oxidative stress. In studies of the mechanism of the transcriptional induction of c-fos by oxidants, we identified the joint DSE-AP1 elements (dyad symmetry element, DSE) as major enhancer motifs in the 5'-upstream regulatory sequences of c-jos. Oxidants also increased the de novo synthesis of protein factors that bind to the fos-AP1 enhancer motif. Protein kinase and ADPR transferase inhibitors suppressed the transcriptional induction of c-fos as well as the increase in factor binding to fos-AP1. We conclude that protein phosphorylation and protein polyADP-ribosylation are required for the transcriptional induction of c-jos and the synthesis of protein factors that bind to fos-AP1. It is likely that the FOS and JUN proteins are among these factors and that they participate in the regulation of c-jos expression by oxidants.
REFERENCES
1. CERUITI,P. 1985. Prooxidant states and tumor promotion. Science 227: 375-381. 2. KOZUMBO,W., M. TRUSH & T. KENSLER. 1985. Are free radicals involved in tumor promotion ? Chem. Biol. Inter. 5 4 199-207. 3. C E R U ~P.,, R. LARSSON & G. KRUPITZA. 1990.I n Genetic Mechanisms in Carcinogenesis and Tumor Progression, C. C. Harris & L. Liotta, eds.: 69-82. Wiley-Liss Inc. New
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& J. MCCORD.(Eds.) 1988. Oxy-radicals in Molecular Biology 4. CERUTTI,P., I. FRIDOVICH and Pathology. Alan R. Liss. New York. D., P. AMSTAD, D. YINFoo & P. CERUTTI.1989. Constitutive and phorbol5. CRAWFORD, myristate-acetate regulated antioxidant defense of mouse epidermal JB6 cells. Molec. Carcinogenesis 2: 136-143. & Y.GRONER.1983. Nucleotide sequence L., N. DAFNI,J. LIEMAN-HURWITZ 6. SHERMAN, and expression of human chromosome 21-encoded superoxide dismutase mRNA. Proc. Natl. Acad. Sci. USA 80: 5465-5469. R., F. QUAN,W. LEWIS,K. GUISE,H. WILLARD, M. HOLMES& R. GRAVEL. 7. KORNELUK, 1984. Isolation of human fibroblast catalase cDNA clones. J. Biol. Chem. 2 5 9 1381913823. P., A. PESKIN, G. SHAH,M. E. MIRAULT, R. MORET,I. ZBINDEN & P. CERUITI. 8. AMSTAD, 1991. The balance between Cu,Zn-superoxide dismutase and catalase affects the sensitivity of mouse epidermal cells to oxidative stress. Biochemistry 3 0 9305-9313. O.,P. SESTILI,F. CATTABENI, G. BELLOMO, S. Pou, M. COHEN& P. CERUTTI. 9. CANTONI, 1989. Calcium chelator Quin 2 prevents hydrogen-peroxide-induced DNA breakage and cytotoxicity. Eur. J. Biochem. 182: 209-212. P. AMSTAD& P. CERUTTI. 1988. Oxidant stress induces the D., 1. ZBINDEN, 10. CRAWFORD, proto-oncogenes c-fos and c-myc in mouse epidermal cells. Oncogene 3: 27-32. 1984. Active oxygen acts as a promotor of transformation R. & P. CERUTTI. 11. ZIMMERMAN, Proc. Natl. Acad. Sci. USA 81: 2085in mouse embryo fibroblast C3H/lOT1/2/C18. 2087. Y.,T. GINDHART, D. WINTERSTEIN, I. TOMITA, J. SEED& N. COLBURN. 1988. 12. NAKAMURA, Early superoxide dismutase-sensitive event promotes neoplastic transformation in mouse epidermal JB6 cells. Carcinogenesis 9 203-207. M., T. KUROKI& K. NOSE. 1988. Induction of DNA replication and 13. SHIBANUMA, expression of protooncogenes c-myc and c-fos in quiescent Balbi3T3 cells by xanthine/ xanthine oxidase. Oncogene 3: 17-21. & L. BROMLEY. 1990. Modulation of fibroblast proliferation by G., M. FRANCIS 14. MURRELL, oxygen free radicals. Biochem. J. 265: 659465. 1991. Inflammation and oxidative stress in carcinogenesis. 15. CERUITI,P. & B. TRUMP. Cancer Cells 3: 1-7. R.& P. CERUITI.1988. Oxidants induce phosphorylation of ribosomal protein 16. LARSSON, S6. J. Biol. Chem. 263: 17452-17458. 1989. Translocation and enhancement of phosphotransferase R. & P. CERUTTI. 17. LARSSON, activity of protein kinase C following exposure of mouse epidermal cells to oxidants. Cancer Res. 4 9 5627-5632. & P. CERUITI.1988. Active oxygen induced DNA strand 18. MUEHLEMAITER, D., R. LARSSON breakage and poly ADP-ribosylation in promotable and non-promotable JB6 mouse epidermal cells. Carcinogenesis 9 239-245. 19. OCHI,T. & P. CERUTTI.1989. Effects of tert-butyl hydroperoxide on promotable and non-promotable JB6 mouse epidermal cells. Chem. Biol. Inter. 71: 339-352. B. STEIN,H. DELIUS,H. RAHMSDORF & P. HERRLICH. 1987. 20. ANGEL,P., I. BAUMANN, 12-0-Tetradecanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5’4anking region. Mol. Cell. Biol. 7: 2256-2266. & H. PONTA.1988. Requirement for fos A., P. HERRLICH, H. RAHMSDORF 21. SCHONTHAL, gene expression in the transcriptional activation of collagenase by other oncogenes and phorbolester. Cell 5 4 325-334. 22. HERRLICH, V. P., H. PONTA,B. STEIN,C. JONAT,S. GOBEL,H. KONIG,R. WILLIAMS, IVANOV & H. RAHMSDORF. 1990. Transcription factors in normal and malignant cells. In Molecular Biology of Cancer Genes. M. Sluyser, ed.: 150-158. Ellis Howard Series in Biomedicine. Amsterdam. W., J. COOPER,T. HUNTER& 1. VERMA.1984. Platelet-derived growth factor 23. KRUIJER, induces rapid but transient expression of the c-fos gene and protein. Nature 3 1 2 711716. P., J. SISSON & I. VERMA. 1988. Serum-induction of fos is under negative 24. SASSONE-CORSI,
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ANNALS NEW YORK ACADEMY OF SCIENCES feedback regulation by the fos protein. A nucleoprotein complex containing AP-1 plus FOS binds to fos promoter region. Nature 334 314-319. F ,BUSCHER,A. SCHONTHAL, H. RAHMSDORF & P. KONIG,H., H. PONTA,U. ~ H M S D O RM. HERRLICH. 1989. Auto-regulation of fos: Dyad symmetry element is the major target of repression. EMBO J. 8 2559-2566. KRUPITZA,G. & P. CERUTTI.1989. ADP-ribosylation of ADPR-transferase and topoisomerase I in intact mouse epidermal cells JB6. Biochemistry 2 8 2034-2040. KRUPITZA,G. & P. CERUTTI.1989. Poly ADP-ribosylation of histones in intact human keratinocytes. Biochemistry 2 8 4054-4060. POIRIER,G., G. DE MURICA,J. JONGSTRA-BILEN, C. NIDERGANG & P. MANDEL.1982. Poly(ADP-ribosy1)ation of poly nucleosomes causes relaxation of chromatin structure. Proc. Natl. Acad. Sci. USA 7 9 3423-3427. (Eds.) 1989. ADP-ribose transfer reactions. Springer JACOBSON, M. & E. JACOBSON Verlag. New York. FENG,J. & B. VILLEPONTEAU. 1990. Serum stimulation of the c-fos enhancer induces reversible changes in c-fos chromatin structure. Mol. Cell. Biol. 10 1126-1133. & P. CERUWI.1992. Mechanism of c-fos induction by active AMSTAD,P., G. KRUPITZA oxygen. Cancer Res. 52: In press. ABATE, C., L. PATEL,F. RAUSCHER & T. CURRAN.1990. Redox regulation of FOS and JUN DNA binding activity in vitro. Science 249 1157-1 161. A. CHUDLEIGH, A. PINTZAS, J. LANG& D. GILLESPIE. FRAME,M., N. WILKIE,A. DARLING, 1991. Regulation of AP-1/DNA complex formation in vitro. Oncogene 6: 205-209. & B. AMEs. 1990. Transcriptional regulator of oxidative STORZ,G., L. TARTAGLIA stress-inducible genes: Direct activation by oxidation. Science 248 189-194. & M. BEST-BELPOMME. 1990. Hydrogen BECKER,J., V. MEZGER,A. M. COURGEON peroxide activates immediate binding of a Drosophila factor to DNA heat-shock regulatory element in vivo and in vitro. Eur. J. Biochem. 1 8 9 553-558. U U S N E R R. , & J. HARFORD.1990. Cis-trans models for post-transcriptional gene regulation. Science 246 870-872.
Oxidants are ubiquitous in our aerobic environment and formed in situ in tissues and cells by normal metabolism and the metabolism of certain xenobiotics. They are always toxic and produce macromolecular damage. At the same time, oxidants can serve as (patho)physiological signals in growth and differentiation.'-' The sensitivity of cells to oxidants is attenuated by low molecular weight antioxidants and antioxidant enzymes. The biochemistry of the most important enzymes (i.e., superoxide dismutases [SOD], catalase [CAT], GSH peroxidases [GPx], GSH reductase, and GSH-S transferase) has been studied in detaiL4 However, the physiological role of the antioxidant enzymes in situ in the cell is only poorly understood because of complex interactions and interrelationships between the individual components.
Increased Constitutive Antioxidant Defense in Promocable Mouse Epidermal Cells A first indication that the cellular antioxidant defense affects the capacity of oxidants to stimulate the growth of epithelial cells was obtained in a study comparing promotable and nonpromotable epidermal cells, JB6.5 When we measured the specific activities of Cu,Zn-SOD, CAT, and GPx in monolayer cultures of JB6 cells, we discovered that the promotable clone 41 contained approximately twice the activity of SOD and CAT relative to the nonpromotable clone 30, whereas the activities of GPx were comparable. We took advantage of the cross-activity of a rabbit antibody against human CAT to assess CAT protein concentrations by Western blot analysis. Constitutive amounts of CAT were two- to threefold higher in clone 41 than in clone 30, in agreement with the enzyme activity. Northern blots indicated that the higher amounts of CAT and SOD in clone 41 were due to increased stationary concentrations of mRNAs for these genes. (Southern analysis showed that both clones contained the same gene complements.) We conclude that the antioxidant defense of JB6 clone 41 is superior to that of clone 30. The difference between the two clones is particularly remarkable, because the two antioxidant enzymes SOD and CAT are increased coordinately in clone 41. Because the product of the action of S O D is Hz02,an increase in its activity is only beneficial to the cell if 'This work was supported by the Swiss National Science Foundation and the Swiss Association of Cigarette Manufacturers and the Association for International Cancer Research. bTo whom correspondence should be addressed. 158
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it is counterbalanced by a sufficient capacity for the destruction of H202. This is apparently accomplished in clone 41 by an increase in CAT.5 It should be mentioned that SOD and CAT may mutually protect each other from inactivation by active oxygen. Stable Transfectantsof Mouse Epidermal Cells with Increased Complements of Cu,Zn-Superoxide Dismutase and Catalase
We are attempting to gain further insight into the role of the cellular antioxidant defense in oxidant carcinogenesis with the help of stable transfectants of mouse epidermal cells with cDNAs for human Cu,Zn-SOD, Mn-SOD, CAT, and GPx. As it
FIGURE 1. Southern blot analysis of Cu,Zn-SOD and CAT transfectants of mouse epidermal cells JB6. Cellular DNA was restricted with BamHl and after electrophoretic separation transferred to nitrocellulose as described by Southern. Filters were hybridized with a 450-bp AluI to TaqI human Cu,Zn-SOD cDNA probe6 or a 1250 bp Hind111 to PvuII fragment of human CAT cDNA,’ respectively. SOD-transfected clones SOD15 and SOCAT3 show a band at 0.5 kb originating from the transfected SOD gene and a double band at 10 kb from the endogenous mouse gene. CAT-Southern blots in the right-side portion of the figure show a 1.8-kb band for transfected human CAT cDNA.
appears likely that the balance of several antioxidant enzymes will determine oxidant sensitivity, we are in the process of preparing multiple transfectants. It is our experimental strategy to use expression vectors with different selectable markers for each antioxidant enzyme cDNA. This allows sequential selection and the comparison of the properties of related generations of multiple transfectants. Until now we have prepared transfectants with moderately increased complements of Cu,Zn-SOD, CAT, or both. We have fully characterized several clones with increased enzyme activities on the molecular level (i.e., Northern blots, RNAse protection for the expression of the transfected relative to the endogenous genes; Southern blots for the presence and copy number of the transfected genes; immunoblots for the presence of the “transfected” human proteins). The Southern blots shown in
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FIGURE1 document the presence of human Cu,Zn-SOD cDNA in the SOD transfectant SOD15 and of human CAT cDNA in the CAT transfectant CAT4. The blot for the double transfectant SOCAT3 shows bands indicating the presence of both cDNAs. We are characterizing the biological properties of several of the aforementioned stable transfectants and report initial results below. In all experiments so far, an extracellular burst of 0; plus H202 was produced by xanthine/ xanthine-oxidase (X/XO) to test oxidant sensitivity. Growth properties: (a) Cu,Zn-SOD transfectants with two- to threefold increased SOD activity are hypersensitive to growth inhibition by 0; plus H202, (b) CAT transfectants with two- to fourfold increased CAT activity are hyposensitive to 0; plus H202, and (c) transfection of Cu,Zn-SOD clones with CAT corrects their hypersensitivity. The data in FIGURE 2 document these conclusions for an experi-
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2. DNA synthesis capacity of parent JB6 clone 41 and its Cu,Zn-SOD and CAT
transfectants following exposure to an extracellular oxidative burst. Monolayer cultures were grown under standard conditions and exposed to an extracellular burst of active oxygen produced by 15 p.g/ml xanthine and 1.5 pg/ml xanthine oxidase. Incorporation of (3H)thymidine into acid-insoluble material within a I-hour pulse was determined after the indicated lengths of posttreatment incubation. Data are expressed as a percentage of analogousvalues for untreated control cultures for each cell strain (A. Peskin and P. Cerutti, unpublished data).
ment measuring the effect of oxidant treatment on the DNA synthesis capacity of the parent clone 41, SOD 15 with 2.5-fold increased Cu,Zn-SOD activity, CAT4 with 3-fold increased CAT activity, and SOCAT3 with 3-fold higher CAT activity and 1.7-fold higher Cu,Zn-SOD activity than the parent strain. (SOCAT3 was derived from CAT4 by transfection with Cu,Zn-SOD cDNA.) Incorporation of (3H)thymidine within a 1-hour pulse into acid-insoluble material was determined. The data indicate the following order of relative resistance of the four cell strains: CAT4 > SOCAT3 > parent clone 41 > SOD15 (A. Peskin and P. Cerutti, unpublished data). Our data suggest that the balance of Cu,Zn-SOD and CAT (and probably GPx) determines the cellular sensitivity to an extracellular burst of active oxygen. We
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noted that the sensitivity of the transfectants was attenuated in high density cultures, suggesting a protective effect of neighboring cells. Sensitivity to DNA strand breakage: DNA strand breakage was measured by the alkaline elution method. The relative sensitivities of 05 plus H 2 0 2 induced DNA breakage were in decreasing order: Cu,Zn-SOD transfectant > parent > CAT transfectant = Cu,Zn-SOD/CAT double transfectant. These results demonstrate that CAT protects the nuclear DNA from damage by an extracellular burst of active oxygen, while Cu,Zn-SOD augments sensitivity. The fact that a Cu,Zn-SOD/CAT double transfectant was slightly more resistant than the parent clone suggests that a balanced increase in both enzymes results in protection.8 It appears that H 2 0 2 represents the precursor species for DNA strand breakage in mammalian cells exposed to an extracellular burst of active oxygen, but they do not imply that DNA is the immediate target for attack by H202or its radical derivatives9 Inducibility of c-fos: The induction of growth competence-related genes is a necessary prerequisite for growth stimulation. The immediate early gene c-fos is a prototype in this regard, as it was shown previously that its transcription is induced in mouse epidermal cells and fibroblasts by oxidants.I0J1Therefore, we compared the increase in stationary concentration of c-fos mRNA upon active oxygen treatment of the antioxidant gene transfectants. The following order of decreasing inducibility of c-fos was observed: parent > CAT transfectant (CAT4) > Cu,Zn-SOD/CAT double transfectant (SOCAT3) > Cu,Zn-SOD transfectant (SOD15). It is evident that despite the opposite effects of additional CAT and Cu,Zn-SOD on cytotoxicity, the inducibility of c-fos by oxidants is reduced in both types of transfectants. However, the reasons for the decrease in c-fos induction are probably different for CAT and Cu,Zn-SOD transfectants. The former are well protected from excessive H202 toxicity, but at the same time the signal that results in c-fos induction is attenuated. In contrast, increases in Cu,Zn-SOD levels alone augment the intracellular formation of H202, and toxic effects on components of the signal transduction pathways may predominate.6 It should be noted that in the X/XO system used in our work a large amount of 0; plus H202 is generated close to the cell surface. Superoxide may penetrate the membrane sufficiently for dismutation by cytosolic Cu,Zn-SOD. Therefore, enhanced oxidative damage in the SOD transfectants may occur preferentially at the plasma membrane.
MECHANISM OF INDUCTION OF THE PROTOONCOGENE C-FOS BY OXIDANTS Growth promotion by oxidants is observed with cultured human and mouse fibroblasts as well as epidermal It is expected to play a role in inflammation, fibrosis, and t u m o r i g e n e s i ~ Indeed, .~~~~~ oxidants trigger (patho)physiological reactions that resemble those induced by growth and differentiation factors. For example, active oxygen activates the phosphorylation of the ribosomal subunit S6I6 and causes the membrane translocation of protein kinase C as well as its activation.'' In contrast to polypeptide growth factors and hormones, oxidants induce DNA damage, in particular DNA breaks, and as a consequence, they activate ADPR transferase.I8J9As this enzyme is located in the nucleus and accomplishes the posttranslational polyADP-ribosylation of chromosomal proteins, it may represent an important link between DNA damage and oxidant-induced modulation of gene expression.'J5 That small doses of oxidants can stimulate rather than inhibit cell growth implies that they are capable of reprogramming gene expression. Therefore, it was intriguing to find that they induce the immediate early genes c-fos and c-myc. 10~13The transcrip-
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tional induction of these genes by oxidants was suppressed by inhibitors of protein kinases A and C and of ADPR transferase according to Northern blots and nuclear run-onexperiments (P. Amstad et al., unpublished data). These results indicate that the posttranslational modification of proteins participates in the induction mechanism. In contrast, de now protein synthesis was not required. To identify 5'-upstream regulatory sequences that are responsible for the transcriptional induction of c-fos by oxidants, we prepared vectors in which potential 30 min
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2
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u!
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FIGURE 3. Induction of factor binding to collagenase TRE-oligonucleotide by oxidants. Gel retardation of (TRE)s pentamer oligonucleotide by nuclear extracts prepared from mouse epidermal cells JB6 clone 30 that had been treated with an extracellular burst of active oxygen produced by X/XO for 30 minutes. When indicated, the cells had been preincubated for 30 minutes with the ADPRtransferase inhibitor benzamide or the protein kinase inhibitors H7 and staurosporine 12, respectively.
1
5' A G C T T G - ( A T G A G T C A G X 5 -C C G G A T C
fos-enhancer motifs are linked to a tk-cat reporter gene (chloramphenicol-acetyltransferase, cat). Transient transfection experiments indicate that the joint DSEAP1 element (dyad symmetry element [DSE]) suffices for oxidant induction. Oxidants also stimulate the binding of protein factors to the fos-AP1 and (TRE)s (TPA responsive element) oligonucleotide motifs. The TRE motif, S'ATGAGTCAG, represents an AP1-like element contained in the S'-upstream regulatory sequences of the collagenase gene which plays a crucial role in its transactivation by the FOS protein.ms21FIGURE 3 shows a gel retardation experiment with the 5-mer
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(TRE)5 and nuclear extracts from X/XO-treated mouse epidermal cells JB6 clone 30. It is evident that factor binding to this element is increased by oxidant treatment for 30 and 60 minutes. Both benzamide (an inhibitor of ADPR transferase) and the protein kinase inhibitors H7 and staurosporine I2 suppress the reaction. As already mentioned, the same drugs suppressed the transcriptional induction of c-fos. Separate experiments indicate that the protein synthesis inhibitor cycloheximide diminished factor binding. From these results we conclude that the posttranslational phosphorylation and polyADP-ribosylation of as yet unidentified proteins are required for the de novo synthesis of regulatory factors that bind to TRE in response to oxidants (P. Amstad and P. Cerutti, unpublished data). Ultraviolet cross-linking experiments and immunocompetition with anti-FOS and anti-JUN antibody indicate that FOS and JUN are among the proteins that bind to TRE. Occupation of TRE with preexisting, possibly modified, FOS and JUN appears to be required for the induction of the collagenase gene by phorbol ester. On the other hand, it is not clear whether the binding of de novo synthesized FOS and JUN plays a role in the activation process. It should be remembered that phorbol ester induction of cat expression was inhibited by cycloheximide in transient transfection experiments with contructs containing a large portion of the collagenase promoter but not with minimal constructs containing only TRE linked to the reporter gene.2w22 Analogous results were obtained in gel retardation experiments with the fos-AP1oligonucleotide motif and nuclear extracts from X/XO-treated cells. As discussed for TRE, it follows that the posttranslational phosphorylation and polyADPribosylation of unidentified proteins is required for the de novo synthesis of protein factors, including FOS, which later bind to fos-AP1. The increase in factor binding to fos-AP1, which is observed 30 minutes following oxidant treatment, does not appear to be involved in the transcriptional induction of c-fos. As the addition of cycloheximide results in transcriptional superinduction of c-fos rather than s ~ p p r e s s i o nit, ~ ~ appears more likely that the de novo synthesized binding proteins participate in the down-regulation of c-fos.22,24 However, down-regulation of c-fos may involve the interaction of de novo synthesized FOS/JUN and other factors with the dyad symmetry element (DSE) of the c-fos promoter rather than with fos-AP1. This is indicated by transient transfection experiments. Constructs containing DSE rather than fos-AP1 linked to a cat reporter gene were down-regulated by FOS overproduction.25 The question arises if the posttranslational modification of the FOS-protein itself affects its binding to fos AP1 and participates in c-fos regulation. Affinity chromatography of polyADP-ribosylated nuclear proteins followed by Western blotting with anti-FOS antibody indicates that FOS protein is only weakly polyADP-ribosylated in response to oxidant treatment. Besides FOS, other chromosomal proteins are polyADP-ribosylated in response to oxidant treat men^^^,^^ PolyADP-ribosylation of histonesZ7in regions containing immediate early genes might participate in their transcriptional induction, because it introduces local relaxation of chromatin conformati0n.~8-3~ Prevention of the conformational change by ADPR-transferase inhibitors may suppress the transcriptional induction of a class of genes that includes c-fos. l5 Instead of participating in transcription initiation, polyADP-ribosylation of chromosomal proteins may be required for efficient message elongation. Oxidantinduced DNA strand breaks may retard message elongation either directly, because they represent blocks to the movement of the transcriptional machinery, or more likely indirectly, because they distort chromatin conformation. As polyADPribosylation is required for the efficient repair of DNA breaks,29its inhibition would be expected to potentiate the inhibitory effect of strand breaks. This model is supported by the observation reported above that the ADPR-transferase inhibitor
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benzamide suppressed transcriptional elongation of c-fos message in oxidant-treated cells according to nuclear run-on experiment^.^' In addition to inducing the ADP-ribosylation of various nuclear proteins, active oxygen may influence the nucleic acid binding properties of transcription factors directly. It is thought that the redox state of two cystein residues located in the basic regions of FOS and JUN is important for their binding to the human metallothionein IIA AP-1 recognition sequence in vitro.Autooxidized FOS and JUN have decreased binding capacities and can be reactivated by the reducing system thioredoxinthioredoxin reductase-NADPH or by high concentrations of d i t h i ~ t h r e i t o l . Fur~~,~~ ther examples in which the redox state of proteins may regulate their activity include ironthe oxy-R encoded protein in bacteria,34 heat shock factor in Dro~ophilu,~~ responsive element binding protein in mammalian cells,36 and PKC (mentioned above)."
SUMMARY
Growth promotion by oxidants is observed with cultured human and mouse fibroblasts as well as epidermal cells. It is expected to play a role in inflammation, fibrosis, and tumorigenesis. Indeed, oxidants trigger (patho)physiological reactions that resemble those induced by growth and differentiation factors. For example, active oxygen activates protein kinases, causes DNA breakage, and induces the growth competence-related protooncogenes c-jos and c-myc. The cellular antioxidant defenses affect the consequences of oxidant exposure. Transfectants of mouse epidermal cells that overproduce Cu,Zn-superoxide dismutase (SOD) were sensitized to the toxic effects of an extracellular burst of 0; plus H202, whereas overproducers of catalase (CAT) were protected. Transfection of SOD overproducers with CAT corrected their hypersensitivity. Inducibility of the protooncogene c-fos by oxidants was diminished in SOD and CAT overproducers, albeit probably for different reasons. It is concluded that a fine balance of the multiple components of the antioxidant defense determines the growth response of cells to oxidative stress. In studies of the mechanism of the transcriptional induction of c-fos by oxidants, we identified the joint DSE-AP1 elements (dyad symmetry element, DSE) as major enhancer motifs in the 5'-upstream regulatory sequences of c-jos. Oxidants also increased the de novo synthesis of protein factors that bind to the fos-AP1 enhancer motif. Protein kinase and ADPR transferase inhibitors suppressed the transcriptional induction of c-fos as well as the increase in factor binding to fos-AP1. We conclude that protein phosphorylation and protein polyADP-ribosylation are required for the transcriptional induction of c-jos and the synthesis of protein factors that bind to fos-AP1. It is likely that the FOS and JUN proteins are among these factors and that they participate in the regulation of c-jos expression by oxidants.
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