antioxidants inhibit the enhancement of malignant cell

8/2/2019 Antioxidants Inhibit the Enhancement of Malignant Cell

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Carcinogenesis vol.17 no.6 pp . 1273-1278, 1996

Antioxidants inhibit the enhancement of malignant celltransformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Detlef Wolfle and Hans MarquardtDepartment of Toxicology, University of Hamburg Medical School, andDepartment of Toxicology and Environmental Medicine of the FraunhoferSociety, Grindelallee 117, D-20146 Hamburg, GermanyThe mechan isms of the tumor promoting activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were studied using asin vitro model the enhancement ('promotion') of malignanttransformation of C3H/M2 mouse fibroblasts induced byA f-methyl-./V'-nitro-./V-nitrosoguanidine or 3-methylchol-anthrene. In this assay, the promoting effect of TCDD wasmaximal at a very low concentration of 1.5 pM and wascomparable to the effect of the reference tumor promoter,12-0-tetradecanoylphorbol-13-acetate (TPA, 0.25 |ig/ml).The role of reactive oxygen species in the promotingaction was investigated: mannitol, a scavenger of hydroxylradicals, or antioxidants, i.e. ascorbic acid plus a-tocoph-erol, abolished the in vitro promoting effects of TPA andTCDD. Furthermore, the involvement of protein kinase C(PKC) activation was studied: the protein kinase inhibitorH-7 markedly reduced the in vitro promoting activity ofTPA but did not affect the promotion by TCDD. In accordwith these results, TPA, but not TCDD, enhanced the PKCactivity in C3H/M2 fibroblasts. Since the TPA-mediatedactivation of PKC was not affected by ascorb ate p lus 99%pure. The following compounds were purchased from the indicated companies:

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D.Wolfle and H.MarquardtMNNG, TPA and H-7 (Sigma, Munich, Germany); MCA (Eastman OrganicChemicals, Rochester, NY); bisindolylmaleimide (Boehringer Mannheim,Mannheim, Germany). Basal Eagle's medium and fetal calf serum wereobtained from Gibco (Eggenstein, Germany) and [y- 32P]ATP from AmershamBuchler (Braunschweig, Germany).Malignant transformation of C3H/M2 mouse fibroblasts in vitroThis assay w as carried out as described previously (6) and adjusted to m easuretumor promotion according to procedures described (36). Briefly, cellsharvested from logarithmically growing stock cultures (between passages 5and 20) were plated on day 0 in basal Eagle's medium supplemented with10% fetal calf serum into 60-mm dishes to determine their plating efficiency(100 cells/dish) and the transformation rate (1000 cells/dish). After 24 h, thecultures were treated for 24 h with initiating agents, i.e. MNNG (0.1 ^g/ml),MCA (1 Hg/ml), or solvent control, dimethylsulfoxide (0.5%). Thereafter, themedium was renewed and the cells were allowed to grow in fresh medium.Beginning on day 5 until the end of the experiment, with each of the (twice-weekly) medium renewals TCDD, the positive control, i.e. TPA (0.25 u.g/ml),or solvent were added. The media were further supplemented with mannitol(10 mg/ml), the antioxidants ascorbate (10 nM) plus a-tocopherol (100 U.M),H-7 (1 ng/ml) or bisindolylmaleimide (1 u.g/ml), as indicated. The cells werefixed and stained after 2 weeks (to determine their plating efficiency) or after8 weeks (to determine the transformation rate).Protein kinase C assaySoluble and paniculate PKC were assayed based on published procedures(37,38). C3H/M2 fibroblasts from two dishes were combined for eachdetermination. Dishes were washed with Hank's buffer and cells wereharvested in 3 ml of the homogenization buffer containing 20 mM Tris-HCl(pH 7.5), 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 2 mM PMSF, 10 u.g/mlleupeptin and 0.25 M sucrose and then sonicated. The homogenates werecentrifuged at 100 000 g for 1 h to obtain the cytosolic fraction. M embranepellets were resuspended in the same buffer containing, in addition. 1%Nonidet P-40.PKC fractions were partially purified by DE 52 chromatography and PKCactivity was measured with histone INS and 25 mM ATP ([y-"P]ATP; 5X10 5cpm) as described previously (31). PKC activity was determined as thedifference in activity between samp les incubated in the presence of phosp hatid-ylserine, 1,2-dioleoyl-sn-glycerol and calcium and those incubated with0.5 mM EGTA instead.StatisticsStatistical significance was calculated with Student's Mest for unpairedobservations. A value of P < 0.05 was considered significant.ResultsPromotion of C3H/M2 malignant transformation by TCDDThe effect of TCDD on the induction of transformation by theinitiating agents MNNG or MCA was studied in C3H/M2fibroblasts (Table I). Although the transformation rate wasvariable, the promoting effect of TCDD was seen in allexperiments; the average enhancement by TCDD was 3.5-fold and 3.8-fold in MCA- and MNNG-pretreated cultures,respectively. The phorbol ester, TPA, was used as a referencetumor p romo ter . TPA alone did not induce cell transformation(Tables II, m and IV). However, in MNN G- or MC A-initiatedcultures the number of transformed foci was significantlyenhanced by subsequent exposure to TPA. In accordance withpreviously reported findings (3), TCDD was also ineffectiveat initiating transformation, even at concentrations up to thenanomolar range (data not shown). However, exposure ofMN NG - or MCA -pretreated cultures to very low concentrationsof TCDD (0.5-1.5 pM) resulted in a maximal enhancementof the induction of transformed foci, similar to that observedwith TPA (Table II). The decrea sing prom oting effect at higherconcentrations of TCDD (25 pM) was not associated with areduction of the plating efficiency. These data on TCDD arein accord with those previously reported (3).Inhibition of the TPA- or TCD D-m ediated promotion bymannitol or antioxidantsMannitol, a potent scavenger of the hydroxyl radical, or acombination of the antioxidants a-tocopherol and ascorbate1274

Table I. Enhancement of C3H/M2 transformation by TCDDDMSO(0.5%) MNNG(0.1 ng/ml) MCA(I.Oug/ml)

DM SO (0 . 5%)TCDD (1 . 5 pM )

0/149 a(0.00) b0/163(0.00)

12/115(0.10)48/123(0.38)

15/150(0.10)58/168(0.35)

aNumber of transformed foci per dishes treated.bNumerical value, of the ratio.

were used in non-toxic concentrations to examine the role ofreactive oxygen species in the process of malignant transforma-tion. While the mechanisms of both antioxidants, i.e. a-tocopherol as a major chain-breaking agent in the lipoproteinfraction and ascorbate as an electron acceptor in the aqueousmedium, are widely accepted, there has been considerablespeculation on the role of these antioxidants as pro-oxidants(39,40). However, it has been reported that ascorbate preventsthe oc-tocopherol-mediated peroxidation of lipids (40). Thus,the protection against radical formation by a single antioxidant(especially at high doses) is not unequivocal; therefore, amixture of a-tocopherol and ascorbate was used to studytheir antioxidant effect on tumor promotion. Mannitol or theantioxidants had no effect on the induction of transformationby suboptimal doses of the initiating agents, MNNG or MCA,but abolished the promoting effects of TPA and TCDD onthe malignant transformation of MNNG- or MCA-pretreatedfibroblasts (Table III).Effect of the PKC inhibitor H-7 on the promotion by TPAor TCDDThe well documented inhibition of the promoting activity ofTPA by H-7 and other PKC inhibitors (41,42) was also foundusing the C3H/M2 fibroblast transformation assay. Treatmentof M2 fibroblasts with the PKC inhibitor H-7 in a non-toxicconcentration had no significant effect on the transformationby MNNG or MCA, but markedly inhibited the promotingaction of TPA (Table IV). In contrast, the promotion by TCD Dwas not affected by cotreatment with H-7. Preliminary datawith the more specific PKC inhibitor bisindolylmaleimide (1(ig/ml) confirmed the findings with H-7 that the promotingaction of TPA, but not that of TCDD is inhibited (datanot shown).PKC activity after treatment with TPA or TCDDC3H/M2 fibroblast cultures were treated with promoting con-centrations of TPA or TCDD and the calcium- and phospho-lipid-dependent PKC activities were determined in the cytosolicand membrane fractions of the cells. In accord with the timecourse of PKC activation by TPA reported by others (37),short-term treatment (10 min) with TPA increased membrane-associated PK C activity (Figure 1) and concomitantly decreasedsoluble enzyme activity (data not shown). This TPA-inducedtranslocation of PKC activity to the membrane fraction wasnot inhibited by a concomitant treatment of the fibroblastswith a-tocopherol plus ascorbate. After long-term treatment(2 days) with TPA, PKC activity was depleted in both, thecytosolic and the membrane fraction of the cells. However, noeffect of TCDD (1 pM) on PKC activity was observed atdifferent time points from 3 h up to 2 weeks. The latter resultis contrary to our findings in primary rat hepatocytes (31), in
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Antioxidants inhibit the enhancement of malignant cell transformation

Table II.Promotor

DMSOTPATCDD

Dose-dependent enhancementConcentration

(0.5%)0.25 ug/ml0.1 pM0.5 pM1.5 pM6.0 pM25.0 pM

of C3H/M2 transformationDMSOPE a26272737292829

by TCDD

Tx a0/130/160/70/60/140/140/13

MNNGPE28313131262525

Tx1/15 (0.07)b3/14(0.21)0/7 (0.00)2/5 (0.40)4/14 (0.29)4/15 (0.27)(1)/14 (0.07)

MC APE30313139272528

Tx4/15 (0.27)26/16(1.63)2/8 (0.25)3/7 (0.43)

24/14(1.71)9/12 (0.75)9/12 (0.75)Cultures were pretreated for 24 h with DMSO or the initiating agents, MNNG (0.1 ug/ml) or MCA (1.0 ug/ml).Results are a summary of two separate experiments.aPE, plating efficiency (%); Tx, number of transformed foci per dishes treated.bNumbers in parentheses, numerical value of the ratio.

Table III. InhibitionTreatment

DMSO (0.5%)DMSO + MannitolDMSO + Toc/AscTPA (0.25 ug/ml)TPA + MannitolTPA + Toc/AscTCDD (1.5 pM)TCDD + MannitolTCDD + Toc/Asc

of TCDD-induced enhancementDMSOPE a

202726212523212929

of C3H/M2

Tx a

0/210/90/200/150/190/90/200/170/11

transformation by mannitolMNNGPE

182215202617162026

or antioxidants

Tx

3/22 (0.14)b

3/20(0.15)2/19(0.11)7/13 (0.54)3/16(0.19)1/12 (0.08)14/18 (0.78)2/18 (0.11)3/10 (0.30)

MC APE

232419232421222522

Tx

2/21 (0.10)3/20(0.15)2/23 (0.09)7/15 (0.47)2/15 (0.13)2/13 (0.15)16/22 (0.73)1/16 (0.06)2/15 (0.13)Cultures were pretreated for 24 h with DMSO or the initiating agents, MNNG (0.1 |ig/ml) or MCA (1.0 ug/ml). Thereafter, cultures were treated for 6 weekswith mannitol (10 mg/ml) or tx-tocopherol (10"4 M) plus ascorbic acid (10 5 M) (Toc/Asc) in the presence or absence of TPA and TCDD, respectively.Results are a summary of two separate experiments.aPE, p lating efficiency (%); Tx, number of transformed foci per dishes treated.bNumbers in parentheses, numerical value of the ratio.

Table IV. Effects of the protein kinase inhibitor H-7 on the enhancement of C3H/M2 transformation by TPA and TCDDTreatment DMSO MNNG MCA

DMSO (0.5%)DMSO + H-7 (1 ng/ml)TPA (0.25 u.g/ml)TPA + H-7 (1 ug/ml)TCDD (1.5 pM)TCDD + H-7 (1 ug/ml)

PE a273122232823

Tx a0/260/270/240/220/270/28

PE272521272525

Tx4/26 (0.15)b3/30(0.10)10/21 (0.48)3/20(0.15)11/24 (0.46)11/29 (0.38)

PE273223252825

Tx5/27(0.19)3/29 (0.10)9/21 (0.43)4/22(0.18)15/29 (0.52)14/31 (0.45)

Cultures were pretreated for 24 h with DMSO or the initiating agents, MNNG (0.1 Ug/ml) or MCA (1.0 ug/ml).Results are a summary of three separate experiments.aPE, plating efficiency (%); Tx, number of transformed foci per dishes treated.bNumbers in parentheses, numerical value of the ratio.

which TCDD (1 pM) significantly activated PKC activity inthe membrane fraction.DiscussionA major unresolved problem in tumor promotion is to definethe critical cellular targets which lead to the clonal expansionof initiated cells. The present study compares the effects oftwo chemically unrelated tumor promoters, i.e. TPA andTCDD, on malignant transformation in vitro. Using C3H/M2mouse fibroblasts initiated with MN NG or M CA, the maximal

promoting effect of TCDDwhich was equivalent to that ofTPA (0.4 mM)was found at a concentration of 1.5 pM, i.e.at a concentration which was five orders of magnitude lowerthan that of TPA. This difference between TCDD and TPApotency in vitro was also reported by others (3), but was notseen to the same extent in vivo (43). This discrepancy betweenin vitro and in vivo effects might be due to differences in thetoxicokinetics and the interactions of TPA and TCDD withdifferent cell types in vivo. However, there is a good correlationof the in vitro/in vivo (rat liver) potencies when chemicals ofa related structure, i.e. dioxins (3) and/or polychlorinated1275
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CON T P A10min 2d T C D D3h 2d 14dFig. 1. PKC activity in the paniculate fractions of TPA- or TCDD-treatedC3H /M2 fibroblasts. Fibroblasts were treated with 1 |iM TPA (dark bars) or1 pM TCDD (hatched bars) for the indicated times (d, day). Paniculatefractions were prepared and assayed for PKC as described. Data representthe mean SD (duplicate cultures from two representative experiments).biphenyls (44) are investigated. In the promotion/ transforma-tion assay with C3H10T1/2 cells initiated with MNNG, themaximal effect of TCDD had been observed at 40 pM (3) andin a transformation assay with rat tracheal cells the promotingeffect of TCDD was maximal at 300 pM (4). Neoplastictransformation of immortal human keratinocytes was achievedwith 10 nM or higher concentrations of TCDD (5). Moreover,in primary rat hepatocytes TCDD at 1 pM maximally stimulatedreplicative DNA synthesis and mitosis (45,46). Thus, mousefibroblasts and rat hepatocytes appear to be very sensitive withrespect to TCDD-mediated effects associated with tumorpromotion.

Furthermore, the results of this study suggest that theproduction of ROS may be a common mechanism in thepromotion induced by TPA and TCDD. It is known fromin vivo studies that antioxidants inhibit tumor promotion byTPA (23,24) and other promoters (25). In in vitro studies, theaddition of the hydroxyl radical scavenger mannitol resultedin a dose-dependent inhibition of the promoting effect of TPAon JB6 mouse epidermal cell transformation (47) and on thetransformation of Balb 3T3 cells initiated with MCA (48).The results of our study with mannitol (at the maximallyeffective concentration reported by Saito et al.\ 48) in thetransformation assay with MCA- and MNNG-initiated C3H/M2 fibroblasts are in accord with these observations and show,in addition, that the in vitro promoting effect of TPA is alsoabolished by the antioxidants ascorbate plus a-tocopherol.Thus, our findings are consistent with the hypothesis thattumor promotion by TPA is mediated by the productionof reactive oxygen species. For phorbol ester-type tumorpromoters, a correlation has been found between their in vivopromoting potencies and the formation of H 2O 2 and oxidizedDNA bases by these agents (14). TPA is known to decrease1276

the activities of superoxide dismutase, catalase and glutathioneperoxidase in vivo and the ratio of reduced glutathione/oxidizedglutathione in mouse skin (49,50). Treatment in vitro ofepidermal cells with tumor promoters rapidly increases thesteady state levels of hydroperoxides by stimulation of theprooxidant activities of various endogenous enzymic and non-enzymic sources of ROS (51). ROS are able to induce DNAstrand-breaks, chromosomal damages and enhanced expressionof the protooncogenes c-myc and c-fos (52). Highly persistent.c-myc levels correlate w ith anchorage-independent growth andfocus formation in chemically transformed C3H 10T1/2 mouseembryo fibroblasts and with an increased susceptibility tospontaneous transformation of rat fibroblasts (53,54). Further-more, TPA activates the transcription factors AP-1 and NF-K B ; the latter is known as an oxidative stress-responsivetranscription factor which is strongly activated by H 2O2- Theactivation of N F - K B by phorbol esters is potentiated by H 2O2and suppressed by antioxidants (55). Thus, tumor promoter-mediated formation of ROS results in an altered regulation oftranscription factors which may be associated with hyperplasiaand cell transformation. The production of free radicals andROS is not restricted to phorbol ester-type tumor promoters,but is a common feature of tumor promoters, e.g. peroxisomeproliferators (56), endogenous and synthetic steroidal estrogens(57), phenobarbital (17), chlordane (58) and Aroclor (59).Treatment with high doses of TCDD has also been reportedto stimulate RO S production in vivo (18). In the present study,we demonstrate that mannitol or antioxidants inhibit thepromoting effect of very low TCDD concentrations (1.5 pM)on the malignant transformation of C3H/M2 cells induced byMNNG or MCA. Since the synergism between ascorbate anda-tocopherol in the inhibition of lipid peroxidation is wellestablished (39,40), we tentatively conclude from our resultswith these antioxidants and mannitol that tumor promotion byTPA and TCDD involves lipid peroxidation possibly via theformation of hydroxyl radicals.

Many biochemical effects of TCDD, especially TCDD-induced early events, e.g. the activation of PKC and theelevation of AP-1 transcription factor activity (30), resemblethe TPA-induced effects. Recently we have reported that inprimary rat hepatocytes after treatment (348 h) with 1 pMTCDD the particulate PKC activity is significantly enhanced(31). In the present study, therefore, the involvement of PKCin the promoting effect of TPA and TCDD was investigated.The inhibition of the promoting activity of TPA by variousinhibitors of PKC in vitro (41) and in vivo (42) is wellestablished and our in vitro data on the inhibiton of the TPA-mediated enhancement of transformation by H-7 are in accordwith these observations. In contrast to TPA-treated M2-fibroblasts, the enhanced transformation rate of TCDD-treatedcells was not affected by H-7 or the more specific PKCinhibitor bisindolylmaleimide (preliminary data, not shown).In addition, TCDD failed to enhance PKC activity in M2mouse fibroblasts. Our previous observation in primary rathepatocytes that antioxidants inhibit TCDD-enhanced PKCactivity (unpublished results) suggest that this TCDD effect israther an indirect than a direct activation of PKC. In contrast,the PKC activation by short-term treatment of M2-fibroblastswith TPA was not affected by antioxidants. In conclusion, itappears that PKC activation by TPA is an agent-specificpathway which is not involved in the promoting activity ofTCDD. There is further evidence that PKC activation is notgenerally associated with enhanced cell transformation: (i) the
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Antioxidants inhibit the enhancement of malignant cell transformation

PKC inhibitors staurospo rine (60 ), H-7 and HA 1004 (61) werereported to be tumor promoters; (ii) triphenylmethane, achemopreventive agent, stimulates PKC activity in C3H10T1/2 cells (62); (iii) the high incidence of cell transformation ina cell variant of Balb/c3T3 was not associated with PKCactivation (63); (vi) a C3H10T1/2 cell line stably overexpress-ing PKCpi was found to have an untransformed phenotypeand to be dependent on TPA for focus formation suggestingthat other TPA-mediated events in addition to PKCpi activationmay be necessary (64); (v) no differences between normal andSV40-transformed rat embryo fibroblasts were found withrespect to quantity or distribution of total PKC or PKC a (65);(vi) PKC activities in the epidermis of a TPA promotion-sensitive mouse strain (SENECAR) and that of a TPA-resistantstrain (C57BL/6J) were roughly the same, whereas the activityof 8-lipoxygenase and the levels of oxidants were higher inepidermal cells of the sensitive mouse strain as compared tothe resistant strain (13,15). Thus, sensitivity to TPA-mediatedpromotion may not be exclusively determined by PKC activa-tion but also by additional mechanisms. Many investigationson PKC are now focused on the role of PKC isoenzymes andtheir subcellular location (65): PKC isoenzymes exert manybiological effects and are differentially regulated during carci-nogenesis (66,67) and in cell transformation (68), probablydepending also on the cell type under investigation.

In summary, different classes of tumor promoters, e.g.phorbol esters and polychlorinated hydrocarbons, share anumber of common biological and pathological responses,including stimulation of cellular growth (43,44,67), malignantcell transformation and tumor promotion. Inhibitors of thecellular metabolism, including antioxidants, are valuable toolsto elucidate essential mechanisms involved in tumor promotion,possibly the formation of ROS. A further characterization ofthe modulation of ROS pathways by different promoting agentsshould provide deeper insight into the mechanisms of tumorpromotion.AcknowledgementsThe authors are grateful to E.Becker, A.Piasecki and A.Ruge for excellenttechnical assistance. The work was funded by grants of the Bundesministeriumfur Bildung und Forschung (07 DIXI2).References

1. Harris.C.C. (1991) Chem ical and physical carcinogenesis: advances andperspectives for the 1990s. Cancer Res., 51, 5023-50442. Pitot,H.C, Goldsworthy,T., Campbell.H.A. and Poland,A. (1980)Quantitative evaluation of the promotion by 2,3,7,8-tetrachlorodibenzo-p-dioxin of hepatocarcinogenesis from diethylnitrosamine. Cancer Res., 40 ,3616-3620.3. Abernethy.D.J. and B oreiko.C.J. (1987) Promotion of C3H/1OT1/2 morpho-logical transformation by polychlori-nated dibenzo-p-dioxin isomers.Carcinogenesis (Land.), 8, 1485-1490.4.Tanaka,N., Nettesheim.P., Gray,T., Nelson.K. and BarrettJ.C. (1989) 2,3,7,8-Tetrachlorodibenzo-p-dioxin enhancement of A/-methyl-/v"-nitro-A'-nitroso-guanidine-induced transformation of rat tracheal epithelial cells in culture.Cancer Res., 49, 2703-2708.5. Yang,J.-H., Thraves.R, Dritschilo,A. and RhimJ.S. (1992) Neoplastictransformation of immortalized human keratinocytes by 2,3,7,8-tetra-chlorodibenzo-p-dioxin. Cancer Res., 52, 3478-3482.6.Marquardt,H., Philips.F.S. and Sternberg.S.S. (l976)Tumo rigenicity in vivoand induction of malignant transformation and mutagenicity in cell culturesby adriamycin and daunomycin. Cancer Res., 36, 2065-2069.7.Trush,M.A. and Kensler.T.W. (1991) An overview of the relationshipbetween oxidative stress and chemical carcinogenesis. Free Rad. Biol.Med., 10, 201-209.8.Cerutti,P.A. (1991) Oxidant stress and carcinogenesis. Eur. Clin. Invest.,

2 1 , 1-5.

9.Troll,W. and Wiesner.R. (1985) The role of oxygen radicals as a possiblemechanism of tumor promotion. Ann. Rev. Pharmacol. Toxicol., 25 ,509-528.10. Marnett.L.J. (1987) Peroxyl free radicals: potential mediators of tumorinitiation and promotion. Carcinogenesis, 8, 1365-1373.11. Weitzman.S.A., Weitberg.A.B., Clark,E.P. and Stossel.T.P. (1985) Phago-cytes as carcinogens: Malignant transformation produced by humanneutrophils. Science, 227, 1231-1233.12.Clemens,M.R. (1991) Free radicals in chemical carcionogenesis. Klin.

Wochenschr., 69 , 1123-1134.13.Fischer,S.M., BaldwinJ.K., Jasheway :D.W., Patrick,K.E. andCameron.G.S. (1988) Phorbol ester induction of 8-lipoxygenase in inbredSENCAR (SSIN) but not C57B L/6J mice correlated with hyperplasia,edema, and oxidant generation but not ornithine decarboxylase induction.Cancer Res., 48, 658-664.

14.Wei,H. and Frenkel,K. (1993) Relationship of oxidative events and DNAoxidation in SENCAR mice to in vivo promoting activity of phorbol ester-type tumor promoters. Carcinogenesis, 14, 1195-1201.15.Wei,L., Wei.H. and Frenkel.K. (1993) Sensitivity to tumor promotion ofSENCAR and C57BL/6J mice correlates with oxidative events and DNAdamage. Carcinogenesis, 14, 841847.16.0gawa,T., Higashi.S., Kawarada.Y. and Mizumoto,R. (1995) Role ofreactive oxygen in synthetic estrogen induction of hepatocellularcarcinomas in rats and preventive effect of vitamins. Carcinogenesis, 16,831-836.17.Scholz,W., Schiitze.K., Kunz,W. and Schwarz,M. (1990) Phenobarbitalenhances the formation of reactive oxygen in neoplastic rat liver nodules.Cancer Res., 50, 7015-7022.18.Stohs,S.J., Shara,M.A., Alsharif.N.Z., Wahba.Z.Z. and Al-Bayati,Z.A.F.(1990) 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced oxidative stress infemale rats. Toxicol. Appl. Pharmacol., 106, 126-135.19.Howe,G.R. etal. (1990) Dietary factors and risk of breast cancer: com binedanalysis of 12 case-control studies. J. Natl Cancer Inst., 82, 561-569.2O.Mergens,W.J. and Bhagavan.H.N. (1989) Alpha-tocopherols (vitamin E).In Moon.T.E. (ed.), Nutrition and Cancer Prevention: Investigation of theRole of Micronutrients. Marcel Dekker, New York, pp. 305340.21.Block,G. (1992) Vitamin C status and cancer: epidemiologic evidence ofreduced risk. Ann. NY Acad. Sci, 669, 280-290.22.Bostick,R.M, PotterJ.D., McKenzie.D.R., Sellers.T.A., Kushi.L.H.,Steinmetz.K.A. and Folsom.A.R. (1993) Reduced risk of colon cancerwith high intake of vitamin E: the Iowa women's health study. CancerRes., 53, 4230-4237.23.Perchellet,J.-P., Owen,M.D., Posey.T.D., Orten,D.K. and Schneider,B.A.(1985) Inhibitory effects of glutathione level-raising agents and D-a-tocopherol on ornithine decarboxylase induction and mouse skin tumor

promotion by 1 2-0-tetradecano ylphorbo l-l 3-acetate. Carcinogenesis, 6,567-573.24. Sm art.R .C, Huang,M.-T., Han.Z.T., Kap lan.M .C, Focella.A. andConney.A.H. (1987) Inhibition of 12-O-tetradecanoylphorbol-13-acetateinduction of ornithine decarboxylase activity, DNA synthesis, and tumorpromotion in mouse skin by ascorbic acid and ascorbyl palmitate. CancerRes., 47, 6633-6638.25 . Battalora,M.S.J., Kruszewski.F.H. and DiGiovanni,J. (1993) Inhibition ofchrysarobin skin tumor promotion in SENCAR mice by antioxidants.Carcinogenesis, 14, 2507-2512.26 . Imamoto.A ., Beltran.L.M. and DiGio vann iJ. (1990) Differentialmechanism for the inhibition of epidermal growth factor binding toits receptor on mouse keratinoytes by anthrones and phorbol esters.Carcinogenesis, 11, 1543-1549.27.Slaga,T.J., Solanki.V. and Logani,M. (1983) Studies on the mechanism ofantitumor promoting agents: suggestive evidence for the involvement offree radicals in promotion. In Nygaard.O.F. and Simic.M.G. (eds.),Radioprotectors and Anticarcinogens. Academic Press, New York, pp.

28 . Wei.H., Wei,L., Fenkel.K., Bowen.R. and Barnes.S. (1993) Inhibition oftumor promoter-induced hydrogen peroxide formation in vitro and in vivoby genistein. Nutr. Cancer, 20, 1-12.29 . Kadrihassani.N., Leger.C.L. and Descomps.B. (1995) The fatty acidbimodal action on superoxide anion production by human adherentmonocytes under phorbol 12-myristate 13-acetate or diacylglycerolactivation can be explained by the modulation of protein kinase C andp47 (phox) translocation. J. Biol. Chem., 270, 15111-15118.30.Puga,A., Nebert.D.W. and Carrier.F. (1992) Dioxin induces expression ofc-fos and c-jun proto-oncogenes and a large increase in transcription factorAP-1. DNA Cell Biol., 11, 269-281.31.Wolfle,D., Schmutte.C. and Marquardt.H. (1993) Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on protein kinase C and inositol phosphate1277
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D.Wolfle and H.Marquardtmetabolism in primary cultures of rat hepatocytes. Carcinogenesis, 14 ,2283-2287.32.Moser,G.J. and Smart,R.C. (1989) Hepatic tumor-promoting chlorinatedhydrocarbons stimulate protein kinase C activity. Carcinogenesis, 10 ,851-856.33.Hahnel,R. and Gschwendt.M. (1995) The interaction between proteinkinase C (PKC) and estrogens. Int. J. Oncol., 7, 11-16.34.Guyton,K.Z., Dolan.P.M. and Kensler,T.W. (1994) Quinone methidemediates in vitro induction of ornithine decarboxylase by the tumorpromoter butylated hydroxy-toluene hydroperoxide. Carcinogenesis, 15 ,817-821 .

35 . Larsson,R. and Cerutti.P. (1989) Translocation and enhancement ofphosphotransferase activity of protein kinase C following exposure inmouse epidermal cells to oxidants. Cancer Res., 49, 5627-5632.36.Sanchez,J.H., Abernethy.D.L. and Boreiko.C.J. ( 1986) Reversibleexpression of morphological transformation in C3H 10T1/2 mouse embryocultures exposed to 12-0-tetra deca-n oylpho rbol-l 3-acetate.Carcinogenesis, 7, 1793-1796.37 . Pittner.A.R. and FainJ.N. (1990) Vasopressin and norepinephrinestimulation of inositol phosphate accumulation in rat hepatocytes aremodified differently by protein kinase C and protein kinase A. Biochim.Biophys. Acta, 1043, 211-217.38 . Kikkawa,U., Minakuchi.R., Takai.Y. and Nishizuka,Y. (1983) Calcium-activated, phospholipid-dependent protein kinase (protein kinase C) fromrat brain. Methods Enzymol, 99, 288-298.39.Wayner,D.D.M, Burton.G.W. and Ingold,K.U. (1986) The antioxidantefficiency of vitamin C is concentration-dependent. Biochim. Biophys.Ada, 884, 119-123.40 . Bowry.V.W. and Stocker,R. (1993) Tocopherol-mediated peroxidation. Theprooxidant effect of vitamin E on the radical-initiated oxidation of humanlow-density lipoprotein. J. Am. Chem. Soc, 115, 6029-6044.41 . Semba.M. and Inui.N. (1990) Effects of activators and inhibitors of proteinkinase C on two-stage transformation in BALB/3T3 cells. Toxicol. Lett.,

51 , 7-12.42.Yamada,S., Hirota.K., Chida.K. and Kuroki,T. (1988) Inhibition of phorbolester-caused induction of ornithine decarboxylase and tumor promotion inmouse skin by staurosporine, a potent inhibitor of protein kinase C.Biochem. Biophys. Res. Commun., 157, 9-15 .43 . Poland,A., Palen.D. and Glover.E. (1982) Tumor promotion by TCDD inskin of HRS/J hairless mice. Nature, 300, 271-273.44. W61fle,D., K iihler.A. and M arquardt,H. (1995) Assessment of the cytotoxicand tumor promoting potential of 2,3,7,8-TCDD and PCB congenersin vitro: comparison of in vitro and in vivo data. OrganohalogenCompounds, 25,195-198.45 . W61fle,D., Becke r.E. and Schmutte,C. (1993) G rowth stimulation of primaryrat hepatocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cell Biol. Toxicol.,9, 15-31.46.Schrenk,D., Krager.A., Lipp,H.-P. and Bock.K.W. (1992) 2,3,7,8-Tetrachlorodibenzo-p-dioxin and ethinyl-estradiol as co-mitogens incultured rat hepatocytes. Carcinogenesis, 13, 453-456.47.Nakamura,Y., Colbum.N.H. and Gindhart.T.D. (1985) Role of reactiveoxygen in tumor promotion: implication of superoxide anion in promotionof neoplastic transformation in JB-6 cells by TPA. Carcinogenesis, 6,229-235.48.Saito,M, Tanaka,N., Ohkawa.Y. and Inui,N. (1987) Inhibition of 12-O-tetradecanoylphorbol-13-acetate-enhanced transformation in vitro byradical scavengers. Cancer Lett., 35, 167-171.49.Solanki,V., Rana.R.S. and Slaga,T.J. (1981) Diminution of mouse epidermalsuperoxide dismutase and catalase activities by tumor promoters.Carcinogenesis, 2, 1141-1146.5O.Reiners,J.J.,Jr., Thai.G., Pavone.A., Rupp.T. and Kodari.E. (1990)Modulation of catalase activities in murine epidermal cells as a functionof differentiation and exposure to 12-O-tetradecanoylphorbol-13-acetate.Carcinogenesis, 11 , 957-963 .51.PerchelIet,J.P. and Perchellet.E.M. (1988) Phorbol ester tumor promotersand multistage skin carcinogenesis. ISI Atlas of Science: Pharmacol., 2,325-333.52.Amstad,P. and Cerutti,P. (1990) Genetic modulation of the cellularantioxidant defence capacity. Environ. Health Perspect., 88 , 77-82.53.Shuin,T, BiIlings,P., Lillehaug,J., Patiemo.S., Roy-Burman,P. andLandolphJ. (1986) Enhanced expression of c-myc and decreased express-ion of c-fos protooncogenes in chemically and radiation-transformed C3H10T1/2 Cl 8 mouse embryo cell lines. Cancer Res., 46, 5302-5311.54.Mougneau,E., Cemi,C, Tillier.A. and Cuzin.F. (1987) EMBL Conferenceon 'Oncogenes and Growth Control', Abstract 127, Heidelberg.

55 . Meyer.M, Schreck.R. and Baeuerle.P.A. (1993) H 2O 2 and antioxidantshave opposite effects on activation of NF-KB and AP-1 in intact cells:AP-1 as secondary antioxidant-responsive factor. EMBOJ., 12, 2005-2015.56.Reddy,J.K. and Rao.M.S. (1986) Peroxisome proliferators and cancer:mechanisms and implications. TIPS, 11 , 438441.57.Li,Y, Trash,M.A. and Yager,J.D. (1994) DNA damage caused by reactiveoxygen species originating from a copper-dependent oxidation of the 2-hydroxy catechol of estradiol. Carcinogenesis, 15, 1421-1427.58.Hassoun,E., Bagchi.M, Bagchi.D. and Strohs,S.J. (1993) Comparativestudies on lipid peroxidation and DNA-single strand breaks induced bylindane, DDT, chlordane and endrin in rats. Comp. Biochem. Phvsiol.,104C, 427-431.59.Faux,S.P., Francis.J.E., Smith.A.G. and ChipmanJ.K. (1992) Induction of8-hydroxydeoxyguanosine in Ah-responsive mouse liver by iron andAroclor 1254. Carcinogenesis, 13, 247-250.60 . Yoshizawa.S., Fujiki.H., Suguri.H., Suganuma.M, Nakayasu.M,Matsushima.R. and Sugimura.T. (1990) Tumor-promoting activity ofstaurosporine, a protein kinase inhibitor on mouse skin. Cancer Res., 50 ,4974-^1978.61.Kischel,T, Harbers.M, Stabel,S., Borowski.P., Miiller.K. and Hilz.H.(1989) Tumor promotion and depletion of protein kinase C in epidermalJB6 cells. Biochem. Biophys. Res. Commun., 165, 981-987.62.Cooney,R.V, Pung.A., Harwood.P.J., Boynton.A.L., Zhang,L.-X.,Hossain.M.Z. and Bertram,.!.S. (1992) Inhibition of cellular transformationby triphenylmethane: a novel chemopreventive agent. Carcinogenesis, 13 ,1107-1112.63.Tatsuka,M., Owada.M.K. and Mitsui,H. (1992) Expression of platelet-derived growth factor-independent phenotypes in BA LB/c 3T3 cell variantwith high susceptibility to chemically or physically induced neoplasticcell transformation: dissociation from activation of protein kinase C.Cancer Res., 52, 4232^*241.64 . Krauss.R.S., Housey.G.M, Johnson,M.D. and Weinstein.I.B. (1989)Disturbances in growth control and gene expression in a C3H/10T1/2 cellline that stably overproduces protein kinase C. Oncogene, 4, 991-998.65 . Hyatt,S.L., Klauck.T. and Jaken.S. (1990) Protein kinase C is localized infocal contacts of normal but not transformed fibroblasts. Mol.Carcinogenesis, 3 , 45-53 .66 . LaPorta.C.A.M. and Comolli.R. (1994) Membrane and nuclear proteinkinase C activation in the early stages of diethylnitrosamine-induced rathepatocarcinogenesis. Carcinogenesis, 15, 1743-1747.67 . LaPorta.C.A.M. and Comolli.R. (1995) Over-expression of protein kinaseC is associated with a delay in preneoplastic lesion development indiethylnitrosamine-induced rat hepatocarcinogenesis. Carcinogenesis, 16,1233-1238.68 . Yamanishi.D.T., Graham ,M., Bu ckm eierJ.A . and M eyskens,F.L.,Jr. (1991)The differential expression of protein kinase C genes in normal humanneonatal melanocytes and metastatic melanomas. Carcinogenesis, 12,105-109.69 . Sawada,N., Staecker,R.J. and Pitot.H.C. (1987) Effects of tumor-promotingagents 12-O-tetradecanoylphorbol-13-acetate and phenobarbital on DNAsynthesis of rat hepatocytes in primary culture. Cancer Res., 47,5665-5671.

Received on November 27, 1995; revised on January 10, 1996; accepted onFebruary 13, 1996

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antioxidants inhibit the enhancement of malignant cell

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