Carcinogenesis vol.17 no.6 pp.

1273-1278, 1996

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

Detlef Wolfle and Hans Marquardt

Department of Toxicology, University of Hamburg Medical School, and Department of Toxicology and Environmental Medicine of the Fraunhofer Society, Grindelallee 117, D-20146 Hamburg, Germany

The mechanisms of the tumor promoting activity of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) were studied using as in vitro model the enhancement ('promotion') of malignant transformation of C3H/M2 mouse fibroblasts induced by Af-methyl-./V'-nitro-./V-nitrosoguanidine or 3-methylcholanthrene. In this assay, the promoting effect of TCDD was maximal at a very low concentration of 1.5 pM and was comparable 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 promoting action was investigated: mannitol, a scavenger of hydroxyl radicals, or antioxidants, i.e. ascorbic acid plus a-tocopherol, abolished the in vitro promoting effects of TPA and TCDD. Furthermore, the involvement of protein kinase C (PKC) activation was studied: the protein kinase inhibitor H-7 markedly reduced the in vitro promoting activity of TPA but did not affect the promotion by TCDD. In accord with these results, TPA, but not TCDD, enhanced the PKC activity in C3H/M2 fibroblasts. Since the TPA-mediated activation of PKC was not affected by ascorbate plus <xtocopherol, it is concluded that the antioxidants interfere with tumor promotion at a step beyond PKC activation. Thus, the results suggest that the enhancement of malignant cell transformation by TPA and TCDD is dependent on a common mechanism, possibly induced by oxygen radicals, and, in addition, on further mechanisms that may involve agent-specific signalling pathways (e.g. PKC activation by TPA).

Introduction The multi-step model of carcinogenesis involves initiation, in which supposedly irreversible genetic alterations take place, and promotion, in which the clonal population of initiated cells is expanded and ultimately progresses to malignancy (1). However, the current knowledge concerning the mechanisms of tumor promotion is still scarce. 2,3,7,8-Tetrachlorodibenzop-dioxin (TCDD*), a prototype of many halogenated aromatic hydrocarbons, is one of the most powerful tumor promoters in rodent bioassays (2). Nevertheless, only a limited number of in vitro models exist for the study of biochemical events associated with tumor promotion by TCDD at a cellular and molecular level and for characterizing factors that potentiate or inhibit its promoting activity. Such in vitro models used for TCDD include two stage transformation-systems, e.g. the
Abbreviations: H-7, l-(5-isoquinolinylsulfonyl)-2-methylpiperazine; MCA, 3-methylcholanthrene; MNNG, A'-methyl-/v"-nitro-/V-nitrosoguanidine; PKC, protein kinase C; ROS, reactive oxygen species; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TPA, 12-0-tetradecanoylphorbol-13-acetate. Oxford University Press

transformation of embryonic fibroblasts such as C3H 10T1/2 (3) and rat tracheal epithelial cells (4) initiated with JV-methyl/V'-nitro-AZ-nitrosoguanidine (MNNG). Furthermore, human epidermal keratinocytes immortalized by adenovirus 12-SV 40 are transformed by TCDD (5). We used the nontumorigenic mouse fibroblast cell line C3H/M2 which can be malignantly transformed to tumorigenic cells by different chemical carcinogens (6). The involvement of reactive oxygen species (ROS) and free radicals in the multi-step process of carcinogenesis, particularly in tumor promotion, has often been postulated with supportive evidence from in vivo and in vitro studies (7-12). The role of free radicals in tumor promotion is strongly suggested by the following observations: (i) free radical generating organic peroxides and H2O2 are known to be mouse skin tumor promoters (12) and (ii) the in vivo and in vitro tumor promoting activity of the mouse skin tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), is associated with oxidative events such as an increased H2O2 production and oxidative DNA damage (13-15). Moreover, non phorbol ester-type tumor promoters (16,17), including TCDD (18), have been reported to enhance the formation of ROS. Indirect evidence supporting a role for free radicals and ROS in carcinogenesis is the inhibitory effect of antioxidants (19,20). Antioxidants, e.g. vitamin C (21) and vitamin E (22), which have been inversely associated with cancer incidence for a variety of sites in humans, have also been shown to inhibit tumor promotion by TPA (23,24) and non-phorbol estertype tumor promoters (16,25,26). Additionally, the phenolic antioxidants, butylated hydroxyanisole and butylated hydroxytoluene, have been shown to inhibit tumor promotion by both, TPA and benzoyl peroxide (27). The anticarcinogenic effects of many agents, e.g. naturally occurring flavones such as genistein, may also be due to their antioxidant properties (28). A possible mechanism of ROS production is the activation of protein kinases (8,29), e.g. protein kinase C (PKC), which is the intracellular receptor for phorbol esters. Interestingly, PKC is also activated by TCDD (30,31) and other tumor promoters, e.g. chlorinated hydrocarbons (32), estrogens (33) and quinones (34). Low concentrations of superoxide have also been implicated in the activation of PKC and this activation may be triggered by an oxidation of critical sulfhydryl groups in the regulatory lipid-binding domain of PKC (35). Thus, it is tempting to speculate that the action of tumor promoters involve similar signal transfer pathways, including the activation of PKC and the formation of ROS. In the present study, the PKC inhibitor H-7 and the antioxidants, ascorbate and a-tocopherol, were used to investigate the role of PKC activation and ROS formation in the promotion of carcinogen-induced transformation of C3H/M2 mouse fibroblasts by TCDD or TPA. Materials and methods
Chemicals TCDD was obtained from Okometric (Bayreuth, Germany) and was >99% pure. The following compounds were purchased from the indicated companies:

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1273

D.Wolfle and H.Marquardt MNNG, TPA and H-7 (Sigma, Munich, Germany); MCA (Eastman Organic Chemicals, Rochester, NY); bisindolylmaleimide (Boehringer Mannheim, Mannheim, Germany). Basal Eagle's medium and fetal calf serum were obtained from Gibco (Eggenstein, Germany) and [y-32P]ATP from Amersham Buchler (Braunschweig, Germany). Malignant transformation of C3H/M2 mouse fibroblasts in vitro This assay was carried out as described previously (6) and adjusted to measure tumor promotion according to procedures described (36). Briefly, cells harvested from logarithmically growing stock cultures (between passages 5 and 20) were plated on day 0 in basal Eagle's medium supplemented with 10% 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, the cultures 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, the medium 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 (twiceweekly) 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 were fixed and stained after 2 weeks (to determine their plating efficiency) or after 8 weeks (to determine the transformation rate). Protein kinase C assay Soluble and paniculate PKC were assayed based on published procedures (37,38). C3H/M2 fibroblasts from two dishes were combined for each determination. Dishes were washed with Hank's buffer and cells were harvested 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/ml leupeptin and 0.25 M sucrose and then sonicated. The homogenates were centrifuged at 100 000 g for 1 h to obtain the cytosolic fraction. Membrane pellets were resuspended in the same buffer containing, in addition. 1% Nonidet P-40. PKC fractions were partially purified by DE 52 chromatography and PKC activity was measured with histone INS and 25 mM ATP ([y-"P]ATP; 5X105 cpm) as described previously (31). PKC activity was determined as the difference in activity between samples incubated in the presence of phosphatidylserine, 1,2-dioleoyl-sn-glycerol and calcium and those incubated with 0.5 mM EGTA instead. Statistics Statistical significance was calculated with Student's Mest for unpaired observations. A value of P < 0.05 was considered significant.

Table I. Enhancement of C3H/M2 transformation by TCDD DMSO (0.5%)

DMSO (0.5%) TCDD (1.5 pM) 0/149a (0.00) b 0/163 (0.00)

MNNG (0.1 ng/ml)

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

MCA (I.Oug/ml)
15/150 (0.10) 58/168 (0.35)

Number of transformed foci per dishes treated. Numerical value, of the ratio.

were used in non-toxic concentrations to examine the role of reactive oxygen species in the process of malignant transformation. While the mechanisms of both antioxidants, i.e. atocopherol as a major chain-breaking agent in the lipoprotein fraction and ascorbate as an electron acceptor in the aqueous medium, are widely accepted, there has been considerable speculation on the role of these antioxidants as pro-oxidants (39,40). However, it has been reported that ascorbate prevents the 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, a mixture of a-tocopherol and ascorbate was used to study their antioxidant effect on tumor promotion. Mannitol or the antioxidants had no effect on the induction of transformation by suboptimal doses of the initiating agents, MNNG or MCA, but abolished the promoting effects of TPA and TCDD on the malignant transformation of MNNG- or MCA-pretreated fibroblasts (Table III). Effect of the PKC inhibitor H-7 on the promotion by TPA or TCDD The well documented inhibition of the promoting activity of TPA by H-7 and other PKC inhibitors (41,42) was also found using the C3H/M2 fibroblast transformation assay. Treatment of M2 fibroblasts with the PKC inhibitor H-7 in a non-toxic concentration had no significant effect on the transformation by MNNG or MCA, but markedly inhibited the promoting action of TPA (Table IV). In contrast, the promotion by TCDD was not affected by cotreatment with H-7. Preliminary data with the more specific PKC inhibitor bisindolylmaleimide (1 (ig/ml) confirmed the findings with H-7 that the promoting action of TPA, but not that of TCDD is inhibited (data not shown). PKC activity after treatment with TPA or TCDD C3H/M2 fibroblast cultures were treated with promoting concentrations of TPA or TCDD and the calcium- and phospholipid-dependent PKC activities were determined in the cytosolic and membrane fractions of the cells. In accord with the time course of PKC activation by TPA reported by others (37), short-term treatment (10 min) with TPA increased membraneassociated PKC activity (Figure 1) and concomitantly decreased soluble enzyme activity (data not shown). This TPA-induced translocation of PKC activity to the membrane fraction was not inhibited by a concomitant treatment of the fibroblasts with a-tocopherol plus ascorbate. After long-term treatment (2 days) with TPA, PKC activity was depleted in both, the cytosolic and the membrane fraction of the cells. However, no effect of TCDD (1 pM) on PKC activity was observed at different time points from 3 h up to 2 weeks. The latter result is contrary to our findings in primary rat hepatocytes (31), in

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Results Promotion of C3H/M2 malignant transformation by TCDD The effect of TCDD on the induction of transformation by the initiating agents MNNG or MCA was studied in C3H/M2 fibroblasts (Table I). Although the transformation rate was variable, the promoting effect of TCDD was seen in all experiments; the average enhancement by TCDD was 3.5fold and 3.8-fold in MCA- and MNNG-pretreated cultures, respectively. The phorbol ester, TPA, was used as a reference tumor promoter . TPA alone did not induce cell transformation (Tables II, m and IV). However, in MNNG- or MCA-initiated cultures the number of transformed foci was significantly enhanced by subsequent exposure to TPA. In accordance with previously reported findings (3), TCDD was also ineffective at initiating transformation, even at concentrations up to the nanomolar range (data not shown). However, exposure of MNNG- or MCA-pretreated cultures to very low concentrations of TCDD (0.5-1.5 pM) resulted in a maximal enhancement of the induction of transformed foci, similar to that observed with TPA (Table II). The decreasing promoting effect at higher concentrations of TCDD (25 pM) was not associated with a reduction of the plating efficiency. These data on TCDD are in accord with those previously reported (3). Inhibition of the TPA- or TCDD-mediated promotion by mannitol or antioxidants Mannitol, a potent scavenger of the hydroxyl radical, or a combination of the antioxidants a-tocopherol and ascorbate 1274

Antioxidants inhibit the enhancement of malignant cell transformation

Table II. Dose-dependent enhancement of C3H/M2 transformation by TCDD Promotor Concentration DMSO PEa DMSO TPA TCDD (0.5%) 0.25 ug/ml 0.1 pM 0.5 pM 1.5 pM 6.0 pM 25.0 pM 26 27 27 37 29 28 29 Txa 0/13 0/16 0/7 0/6 0/14 0/14 0/13 MNNG PE 28 31 31 31 26 25 25 Tx 1/15 (0.07)b 3/14(0.21) 0/7 (0.00) 2/5 (0.40) 4/14 (0.29) 4/15 (0.27) (1)/14 (0.07) MCA PE 30 31 31 39 27 25 28 Tx 4/15 (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. a PE, plating efficiency (%); Tx, number of transformed foci per dishes treated. b Numbers in parentheses, numerical value of the ratio.

Table III. Inhibition of TCDD-induced enhancement of C3H/M2 transformation by mannitol or antioxidants Treatment DMSO PEa DMSO (0.5%) DMSO + Mannitol DMSO + Toc/Asc TPA (0.25 ug/ml) TPA + Mannitol TPA + Toc/Asc TCDD (1.5 pM) TCDD + Mannitol TCDD + Toc/Asc 20 27 26 21 25 23 21 29 29 Txa 0/21 0/9 0/20 0/15 0/19 0/9 0/20 0/17 0/11 MNNG PE 18 22 15 20 26 17 16 20 26 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) MCA Downloaded from http://carcin.oxfordjournals.org/ by guest on March 7, 2012 PE 23 24 19 23 24 21 22 25 22 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 weeks with 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. a PE, plating efficiency (%); Tx, number of transformed foci per dishes treated. b Numbers 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 TCDD Treatment DMSO PE 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) 27 31 22 23 28 23
a

MNNG Tx
a

MCA Tx 4/26 (0.15)b 3/30(0.10) 10/21 (0.48) 3/20(0.15) 11/24 (0.46) 11/29 (0.38) PE 27 32 23 25 28 25 Tx 5/27(0.19) 3/29 (0.10) 9/21 (0.43) 4/22(0.18) 15/29 (0.52) 14/31 (0.45)

PE 27 25 21 27 25 25

0/26 0/27 0/24 0/22 0/27 0/28

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. a PE, plating efficiency (%); Tx, number of transformed foci per dishes treated. b Numbers in parentheses, numerical value of the ratio.

which TCDD (1 pM) significantly activated PKC activity in the membrane fraction. Discussion A major unresolved problem in tumor promotion is to define the critical cellular targets which lead to the clonal expansion of initiated cells. The present study compares the effects of two chemically unrelated tumor promoters, i.e. TPA and TCDD, on malignant transformation in vitro. Using C3H/M2 mouse fibroblasts initiated with MNNG or MCA, the maximal

promoting effect of TCDDwhich was equivalent to that of TPA (0.4 mM)was found at a concentration of 1.5 pM, i.e. at a concentration which was five orders of magnitude lower than that of TPA. This difference between TCDD and TPA potency in vitro was also reported by others (3), but was not seen to the same extent in vivo (43). This discrepancy between in vitro and in vivo effects might be due to differences in the toxicokinetics and the interactions of TPA and TCDD with different cell types in vivo. However, there is a good correlation of the in vitro/in vivo (rat liver) potencies when chemicals of a related structure, i.e. dioxins (3) and/or polychlorinated 1275

D.Wolfle and H.Marquardt

1200-

CON

TPA 10min 2d

3h

TCDD 2d 14d

Fig. 1. PKC activity in the paniculate fractions of TPA- or TCDD-treated C3H/M2 fibroblasts. Fibroblasts were treated with 1 |iM TPA (dark bars) or 1 pM TCDD (hatched bars) for the indicated times (d, day). Paniculate fractions were prepared and assayed for PKC as described. Data represent the mean SD (duplicate cultures from two representative experiments).

biphenyls (44) are investigated. In the promotion/ transformation assay with C3H10T1/2 cells initiated with MNNG, the maximal effect of TCDD had been observed at 40 pM (3) and in a transformation assay with rat tracheal cells the promoting effect of TCDD was maximal at 300 pM (4). Neoplastic transformation of immortal human keratinocytes was achieved with 10 nM or higher concentrations of TCDD (5). Moreover, in primary rat hepatocytes TCDD at 1 pM maximally stimulated replicative DNA synthesis and mitosis (45,46). Thus, mouse fibroblasts and rat hepatocytes appear to be very sensitive with respect to TCDD-mediated effects associated with tumor promotion. Furthermore, the results of this study suggest that the production of ROS may be a common mechanism in the promotion induced by TPA and TCDD. It is known from in vivo studies that antioxidants inhibit tumor promotion by TPA (23,24) and other promoters (25). In in vitro studies, the addition of the hydroxyl radical scavenger mannitol resulted in a dose-dependent inhibition of the promoting effect of TPA on JB6 mouse epidermal cell transformation (47) and on the transformation of Balb 3T3 cells initiated with MCA (48). The results of our study with mannitol (at the maximally effective concentration reported by Saito et al.\ 48) in the transformation 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 also abolished by the antioxidants ascorbate plus a-tocopherol. Thus, our findings are consistent with the hypothesis that tumor promotion by TPA is mediated by the production of reactive oxygen species. For phorbol ester-type tumor promoters, a correlation has been found between their in vivo promoting potencies and the formation of H2O2 and oxidized DNA bases by these agents (14). TPA is known to decrease 1276

the activities of superoxide dismutase, catalase and glutathione peroxidase in vivo and the ratio of reduced glutathione/oxidized glutathione in mouse skin (49,50). Treatment in vitro of epidermal cells with tumor promoters rapidly increases the steady state levels of hydroperoxides by stimulation of the prooxidant activities of various endogenous enzymic and nonenzymic sources of ROS (51). ROS are able to induce DNA strand-breaks, chromosomal damages and enhanced expression of the protooncogenes c-myc and c-fos (52). Highly persistent. c-myc levels correlate with anchorage-independent growth and focus formation in chemically transformed C3H 10T1/2 mouse embryo fibroblasts and with an increased susceptibility to spontaneous transformation of rat fibroblasts (53,54). Furthermore, TPA activates the transcription factors AP-1 and NFKB; the latter is known as an oxidative stress-responsive transcription factor which is strongly activated by H2O2- The activation of NF-KB by phorbol esters is potentiated by H2O2 and suppressed by antioxidants (55). Thus, tumor promotermediated formation of ROS results in an altered regulation of transcription factors which may be associated with hyperplasia and cell transformation. The production of free radicals and ROS is not restricted to phorbol ester-type tumor promoters, but is a common feature of tumor promoters, e.g. peroxisome proliferators (56), endogenous and synthetic steroidal estrogens (57), phenobarbital (17), chlordane (58) and Aroclor (59). Treatment with high doses of TCDD has also been reported to stimulate ROS production in vivo (18). In the present study, we demonstrate that mannitol or antioxidants inhibit the promoting effect of very low TCDD concentrations (1.5 pM) on the malignant transformation of C3H/M2 cells induced by MNNG or MCA. Since the synergism between ascorbate and a-tocopherol in the inhibition of lipid peroxidation is well established (39,40), we tentatively conclude from our results with these antioxidants and mannitol that tumor promotion by TPA and TCDD involves lipid peroxidation possibly via the formation of hydroxyl radicals. Many biochemical effects of TCDD, especially TCDDinduced early events, e.g. the activation of PKC and the elevation of AP-1 transcription factor activity (30), resemble the TPA-induced effects. Recently we have reported that in primary rat hepatocytes after treatment (348 h) with 1 pM TCDD the particulate PKC activity is significantly enhanced (31). In the present study, therefore, the involvement of PKC in the promoting effect of TPA and TCDD was investigated. The inhibition of the promoting activity of TPA by various inhibitors of PKC in vitro (41) and in vivo (42) is well established and our in vitro data on the inhibiton of the TPAmediated enhancement of transformation by H-7 are in accord with these observations. In contrast to TPA-treated M2fibroblasts, the enhanced transformation rate of TCDD-treated cells was not affected by H-7 or the more specific PKC inhibitor bisindolylmaleimide (preliminary data, not shown). In addition, TCDD failed to enhance PKC activity in M2 mouse fibroblasts. Our previous observation in primary rat hepatocytes that antioxidants inhibit TCDD-enhanced PKC activity (unpublished results) suggest that this TCDD effect is rather an indirect than a direct activation of PKC. In contrast, the PKC activation by short-term treatment of M2-fibroblasts with TPA was not affected by antioxidants. In conclusion, it appears that PKC activation by TPA is an agent-specific pathway which is not involved in the promoting activity of TCDD. There is further evidence that PKC activation is not generally associated with enhanced cell transformation: (i) the

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Antioxidants inhibit the enhancement of malignant cell transformation

PKC inhibitors staurosporine (60), H-7 and HA 1004 (61) were reported to be tumor promoters; (ii) triphenylmethane, a chemopreventive agent, stimulates PKC activity in C3H10T1/ 2 cells (62); (iii) the high incidence of cell transformation in a cell variant of Balb/c3T3 was not associated with PKC activation (63); (vi) a C3H10T1/2 cell line stably overexpressing PKCpi was found to have an untransformed phenotype and to be dependent on TPA for focus formation suggesting that other TPA-mediated events in addition to PKCpi activation may be necessary (64); (v) no differences between normal and SV40-transformed rat embryo fibroblasts were found with respect to quantity or distribution of total PKC or PKC a (65); (vi) PKC activities in the epidermis of a TPA promotionsensitive mouse strain (SENECAR) and that of a TPA-resistant strain (C57BL/6J) were roughly the same, whereas the activity of 8-lipoxygenase and the levels of oxidants were higher in epidermal cells of the sensitive mouse strain as compared to the resistant strain (13,15). Thus, sensitivity to TPA-mediated promotion may not be exclusively determined by PKC activation but also by additional mechanisms. Many investigations on PKC are now focused on the role of PKC isoenzymes and their subcellular location (65): PKC isoenzymes exert many biological effects and are differentially regulated during carcinogenesis (66,67) and in cell transformation (68), probably depending also on the cell type under investigation. In summary, different classes of tumor promoters, e.g. phorbol esters and polychlorinated hydrocarbons, share a number of common biological and pathological responses, including stimulation of cellular growth (43,44,67), malignant cell transformation and tumor promotion. Inhibitors of the cellular metabolism, including antioxidants, are valuable tools to elucidate essential mechanisms involved in tumor promotion, possibly the formation of ROS. A further characterization of the modulation of ROS pathways by different promoting agents should provide deeper insight into the mechanisms of tumor promotion. Acknowledgements
The authors are grateful to E.Becker, A.Piasecki and A.Ruge for excellent technical assistance. The work was funded by grants of the Bundesministerium fur Bildung und Forschung (07 DIXI2).

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