Tumor promotion by hydrogen peroxide in rat liver epithelial cells

Ruo-Pan Huang1, Ao Peng, Mohammad Z. Hossain, Yan Fan, Ajit Jagdale and Alton L. Boynton

Molecular Medicine, Northwest Hospital, 120 Northgate Plaza Suite 230, Seattle, WA 98125, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reactive oxygen species, including H2O2, play an important role in the tumor promotion process. Using an in vitro model of tumor promotion involving the rat liver epithelial oval cell line T51B, the tumor promoting activity of H2O2 in N-methyl-N'-nitro-N-nitrosoguanidine-initiated cells was studied. In this assay system, the promoting effect of H2O2 is evidenced by the formation of colonies in soft agar, appearance of foci in monolayer culture, disruption of gap junction communication (GJC) in foci areas and growth at higher saturation densities. H2O2 preferentially induced the expression of c-fos, c-jun, c-myc and egr-1, while JunB and JunD levels remained almost unchanged. H2O2 also induced hyperphosphorylation of Cx43 and disruption of GJC. The effects of H2O2 on tumor promotion, induction of immediate early (IE) genes and disruption of GJC are blocked by antioxidants. These results suggest that H2O2 acts as a tumor promoter in rat liver non-neoplastic epithelial cells and that the induction of IE genes and disruption of GJC are two possible targets of H2O2 during the tumor promotion process.

Abbreviations: DMF, dimethyl formamide; EGF, epidermal growth factor; GJC, gap junction communication; IE, immediate early; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; NAC, N-acetylcysteine; OA, okadaic acid; ROS, reactive oxygen species; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TPA, 12-O-tetradecanoylphorbol-13-acetate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite the prevalence of many types of cancer, including hepatoma, the carcinogenic process is still poorly understood. The carcinogenic process in animal models is generally believed to involve initiation, in which supposedly irreversible genetic alterations take place, and promotion, in which clonal populations of initiated cells are expanded and ultimately progress to malignancy. Several tumor promoters are known, including 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid (OA), thapsigargin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and H2O2, however, their precise mechanism of action is evasive.

The involvement of reactive oxygen species (ROS), particularly H2O2, in the tumor promotion process is supported by both in vivo and in vitro studies. H2O2 is capable of promoting neoplastic transformation in several two-stage transformation systems, including rat urothelial cells (1), murine myeloid progenitor cells (2), mouse epidermal cells (3) and mouse embryo fibroblasts (4). In vivo studies also suggest that H2O2 is a mouse skin tumor promoter (5). The difference in sensitivity to multistage carcinogenesis between sensitive (SENCAR) and resistant (C57BL/6J) mice is related to the formation of H2O2 by TPA (6). Indirect evidence supporting a role for ROS, including H2O2, in tumor promotion, is that inhibition of ROS generated by tumor promoters such as TPA (2,610), TCDD (11) and chrysarobin (12) profoundly reduces transformation. Treatment of SENCAR mice with chemopreventive agents such as epigallocatechingallate or tamoxifen prior to application of TPA diminished formation of H2O2 (8). trans-tamoxifen and its derivatives are capable of inhibiting H2O2 formation by TPA-treated human neutrophils (9). Sarcophytol, a non-toxic and natural compound which inhibits the in vivo effect of tumor promotors, significantly decreases TPA-induced H2O2 formation in the epidermis of SENCAR mice (10). An antioxidant fraction of Chinese green tea can inhibit the production of H2O2 and TPA-induced tumor promotion in mouse skin (2) and in cultured lung cells it can inhibit oxidant-induced DNA strand breaks (13). Furthermore, the production of ROS and H2O2 is a common feature of tumor promoters such as TPA (14), TCDD (15), UV (16,17), OA (18), peroxisome proliferators (19), steroidal estrogens (20), phenobarbital (21), chlordane (22) and aroclor (23). Therefore, ROS, including H2O2, may play a critical role in the tumor promotion process.

In spite of long speculation on the involvement of H2O2 in the tumor promotion process, current knowledge concerning the mechanism(s) by which H2O2 promotes tumor formation is still scarce. In this report, we have established an in vitro model system for the study of biochemical events associated with tumor promotion by H2O2 at the cellular and molecular level and for characterizing factors that potentiate or inhibit its promoting activity. In this study, we present data to show that H2O2 serves as a tumor promoter in rat liver epithelial T51B cells and that tumor promotion by H2O2 may involve the interruption of gap junction communication (GJC) and the induction of immediate early (IE) genes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
T51B is a rat non-neoplastic epithelial cell line used by us for a variety of studies involving tumor promotion and mechanisms of action of tumor promoters (24,25). The cells were cultured in Eagle's basal medium containing 10% bovine calf serum (Colorado Serum Co., Denver, CO). H2O2, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-acetylcysteine (NAC) and {alpha}-tocopherol were purchased from Sigma Chemical Co. (St Louis, MO).

Tumor promotion protocol
T51B cells (between passages 8 and 10) at a density of 1.375x105 per 60 mm dish were plated on day 0 in complete medium. After 24 h, the cultures were treated for 24 h with the initiating agent, MNNG (0.25(g/ml) or with solvent control, dimethyl formamide (DMF). Twenty-four hours later, the cells were washed twice with serum-free medium and then the cells exposed to H2O2 (50, 100 and 200 µM) with an exchange of the H2O2-containing complete medium every week or twice a week or every other day. Once a week, cells were harvested from individual dishes and passaged at a density of 1.375x105 per 60 mm dish. This process was repeated up to five times. At passage numbers 1, 3 and 5, cells derived from each plate were subjected to the soft agar assay as described previously (26). Briefly, cells (5x104) in 0.26% agar medium were layered onto 0.65% agar-coated 60 mm plates. The plates were cultured for 14–20 days and the viable colonies were stained with p-iodonitrotetrazolium violet (Sigma) overnight. The number of colonies was determined under a microscope and colonies containing >100 cells were scored as positive. Cells treated with H2O2 more than twice a week died during the experiment. For antioxidant treatment, NAC (2.5 mM) or {alpha}-tocopherol (3x10–8 M) was added 30 min prior to H2O2 treatment. The experiments were repeated twice and similar results were obtained.

Northern blot
Total RNA was isolated from cultured cells by the RNAzol method according to the manufacturer's instructions (Cinnc/Biotecx Laboraories, Houston, TX). Northern blotting was performed as described (27). Briefly, 30 µg of total RNA was denatured with formaldehyde and fractionated on a 1.0% agarose gel containing 1.0 M formaldehyde. The RNA was transferred to a nitrocellulose filter by blotting. After UV crosslinking, the filter was hybridized with different [32P]dCTP-labeled (NEN, Boston, MA) probes at 42°C overnight, washed three times for 10 min with an excess amount of 2x SSC, 0.1% SDS at 50°C and twice for 30 min with 0.1% SSC, 0.1% SDS at 50°C. egr-1 cDNA was originally obtained from Dr Vikas (Harvard University) and c-jun, c-fos, junB and junD were a gift from Dr Michel Karin (University of California San Diego, CA). c-myc was obtained from the American Type Culture Collection (Rockville, MD). The intensity of signals was quantitated by densitometry.

Western blot
Western blot assays were performed as previously described (16). Briefly, equal amounts of protein were separated by 7.5% SDS–PAGE for Egr-1 or 10% SDS–PAGE for Cx43 and the proteins transferred to PDVF membranes (Millipore, Bedford, MA). Egr-1 and Cx43 were detected with anti-Egr-1 (28) or anti-Cx43 (29) antiserum coupled with ECL detection (Amersham, Aylesbury, UK).

Measurement of GJC
GJC was assessed by the transfer of the fluorescent dye Lucifer yellow after single cell microinjection as previously described (25). The cells were observed under a fluorescence inverted microscope 10 min after microinjection and the number of neighboring cells labeled with fluorescent dye was recorded. At least 20 injections were performed in each experiment.

Cell viability assay
Cells were seeded in 96-well plates at 1x103 per well and incubated at 37°C overnight. Cells were exposed to different concentrations of H2O2, MNNG or H2O2 plus MNNG. After 48 h, cell viability was assayed with a cell titer 96 non-radiactive cell proliferation assay kit (MTT assay) according to the manufacturer's instructions (Promega, Madison, WI). This assay is based on the conversion of a tetrazolium salt (MTT) into a blue formazan product by viable cells. The values of absorbance at 570 mm were expressed as relative viable cell number. The experiments were repeated twice.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
H2O2 is a tumor promoter in T51B
First, we performed cytotoxicity assays of H2O2 on T51B cells to select the suitable concentration for tumor promotion experiments. T51B cells (1x103 cells/well) were plated in a 96-well plate in complete medium and, 24 h later, H2O2 was added at six different concentrations. Forty-eight hours later, viable cells were determined by the MTT assay. The number of viable cells decreased at concentrations as low as 25 µM. Concentrations above 1000 µM caused a dramatic decrease in viable cell number (Figure 1AGo). Since the tumor promotion protocol also used MNNG, we determined the cytotoxic effect of MNNG and MNNG plus H2O2 by MTT assay. As shown in Figure 1B and C, GoH2O2 plus MNNG showed additive cytotoxicity to T51B cells. Thus, concentrations of 50, 100 and 200 µM for H2O2 and 0.5 µg/ml for MNNG were used in subsequent experiments.





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Fig. 1. Cytotoxicity of H2O2 and MNNG to T51B cells. T51B cells were treated with H2O2 (A), MNNG (B) and H2O2 plus MNNG (C) at the indicated concentrations for 48 h. Cell viability was determined by the MTT assay and expressed as a percentage of untreated cells.

 
To investigate whether H2O2 serves as a tumor promoter in T51B rat liver epithelial cells, we treated MNNG-initiated T51B cells with different concentrations of H2O2. The consequence of H2O2 treatment was transformation, as evidenced by colony formation in soft agar. As shown in Table IGo, only cells initiated with MNNG and treated with H2O2 developed the transformed phenotype of colony formation in soft agar. Transformation was detected as early as passage number 5 and at concentrations of 100 and 200 µM H2O2.


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Table I. Effect of H2O2 on tumor promotion of MNNG-treated T51B rat liver cells
 
The morphology of cells treated with both MNNG and H2O2 was distinct from that of DMF treatment, MNNG treatment or H2O2 treatment. Cells initiated with MNNG and promoted with H2O2 grew as foci in monolayer culture as shown in Figure 2Go. Focus formation is a phenotype associated with transformation.



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Fig. 2. Morphology of T51B cells treated with MNNG and H2O2. T51B cells were treated with DMF, MNNG (0.25 µg/ml), H2O2 (100 µM) or H2O2 (100 µM) plus MNNG (0.25 µg/ml). At passage 5 of treatment, cell morphology was examined under a phase contrast microscope. Cells treated with MNNG and H2O2 were highly transformed in appearance with the formation of foci.

 
Disruption of functional gap junctions is evident in tumor promotion and carcinogenesis. To see whether GJC is altered in H2O2-induced transformed cells, we measured GJC in cells with different treatments. As shown in Table IGo, GJC was significantly reduced in the focus areas of cells treated with MNNG and H2O2 compared with control treatments.

Transformed cells usually proliferate to higher cell densities. As shown in Table IGo, cells initiated with MNNG and promoted with H2O2 grew to higher densities than control cells. This difference was statistically significant (P < 0.01) between MNNG/H2O2-treated cells and control cells, but no significant difference (P < 0.05) was found when cells were treated with DMF, MNNG and H2O2 only.

Taken together, we conclude that H2O2 is a tumor promoter in MNNG-initiated T51B rat liver epithelial cells, as evidenced by the formation of colonies in soft agar, formation of foci in monolayer culture, disruption of GJC in the focus area and growth to high densities.

H2O2 induces expression of early growth response genes
Earlier works indicated that H2O2 can induce the expression of IE genes. To analyze which IE genes may be involved in the process of H2O2-promoted transformation, we examined the expression pattern of several early growth response genes by northern blot analysis. T51B cells were treated with H2O2 (200 µM) and total RNA was analyzed by northern blot at different time points. As shown in Figure 3Go, c-fos, c-jun, c-myc and egr-1 expression was rapidly and transiently induced in response to H2O2. The induction began as early as 30 min and reached peak levels at ~60 min after treament. In contrast, the expression of junD and junB remains almost unchanged. L32, a rRNA, was used as a loading control. Therefore, H2O2 preferentially induces expression of c-fos, c-jun, c-myc and egr-1.




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Fig. 3. Northern blotting analysis of IE gene expression in response to H2O2. (A) Confluent T51B cells were treated with H2O2 (200 µM) and at the times shown were harvested for total RNA preparation. Aliquots of 30 µg of total RNA were used for northern blotting analysis with different cDNA probes as indicated. H2O2 preferentially induces c-fos, c-jun, c-myc and egr-1 expression. (B) The hybridization signals were quantitated using densitometry. The level of IE mRNA plotted is the mean from two experiments at each time point.

 
H2O2 induces phosphorylation of Cx43 and inhibition of GJC
Since inhibition of GJC is a common feature of many tumor promoting agents, we examined the effect of H2O2 on GJC. Confluent T51B cells predominantly express the P1 and P2 phosphorylated forms of Cx43 which are generally believed to be responsible for GJC (30,31). When cells were treated with H2O2, another phosphorylated form of Cx43 (P3) was detected at 30 and 60 min, as shown in Figure 4Go. We have demonstrated that this form of Cx43 (P3) is involved in the disruption of GJC (24). Indeed, addition of H2O2 to T51B cells led to a reduction in GJC from 66 communicating cells in the control to 14 (at 30 min), two (at 1 h) and one (at 2 h), respectively. GJC eventually recovered 3 h after treatment and reached pre-treatment basal levels at 8 h (Figure 4Go). This result clearly demonstrates that H2O2 treatment, especially at a concentration of 200 µM, rapidly and transiently disrupts GJC in T51B cells.



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Fig. 4. H2O2 induces hyperphosphorylation of Cx43 and disruption of GJC. Confluent T51B cells were treated with H2O2 (200 µM) and harvested at the indicated times. Equal amounts of total cell lysate were then subjected to western blot with anti-Cx43 antiserum. Parallel cultures were taken for GJC assays. The numbers in the GJC row represent the mean number of communicating cells in the indicated treatment group.

 
The effect of H2O2 on tumor promotion is inhibited by antioxidants
Since it is known that antioxidants block the tumor promotion process in some in vitro systems and putatively in a clinical setting, we applied antioxidants to test their effects on tumor promotion in our system. T51B cells were preincubated with the antioxidant NAC or {alpha}-tocopherol before treatment with H2O2. As shown in Table IGo, both NAC and {alpha}-tocopherol effectively abrogated the effect of H2O2 on tumor promotion.

Next we examined the effect of NAC on the interruption of GJC by H2O2. As shown in Figure 5Go, both phosphorylation of Cx43 and interruption of GJC by H2O2 (200 µM) were blocked by pretreatment with NAC.



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Fig. 5. Antioxidant NAC blocks the hyperphosphorylation of Cx43 and the disruption of GJC by H2O2. Confluent T51B cells were incubated with NAC (5 mM) for 30 min and then treated with H2O2 (200 µM) for 1 h. Cells were then harvested for Cx43 analysis by western blot. The numbers in the GJC row represent the mean number of communicating cells in the indicated treatment group.

 
We also examined the effect of antioxidants on the induction of IE genes by H2O2. egr-1 was used as a representative IE gene. Consistent with our northern blot data and our previous observation, H2O2 induces expression of Egr-1 protein which peaked at ~2 h. This induction was blocked by antioxidants, as shown in Figure 6Go. To exclude the possibility that antioxidant treatment non-specifically affects global changes inside the cells, we examined the effect of NAC on the induction of Egr-1 by EGF. As shown in Figure 6BGo, NAC did not block the induction of Egr-1 by EGF, suggesting a specific effect of NAC on H2O2.



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Fig. 6. Effect of the antioxidant NAC on the induction of Egr-1 protein expression by H2O2 or EGF. (A) Confluent T51B cells were treated with H2O2 (200 µM) or EGF (20 ng/ml) and harvested at the indicated times. Equal amounts of cell lysates were analyzed for Egr-1 levels with anti-Egr-1 antibody. (B) Confluent T51B were incubated with NAC (5 mM) for 30 min and then treated with H2O2 (200 µM) or EGF (20 ng/ml). Two hours later, cells were harvested and assayed for Egr-1 level by western blot. This experiment was repeated once with similar result.

 
Collectively, these data indicate that the actions of H2O2 on tumor promotion, IE gene expression and GJC interruption are dependent on oxidative stress and antioxidants may be useful in chemoprevention.

The dose effect of H2O2 on the induction of IE genes
Since both 100 and 200 µM H2O2 promoted transformation at similar efficiency in our system, we further determined dose effects of H2O2 on the induction of IE genes. As a representative of IE genes, the induction of Egr-1 protein was examined in a western blot assay. Egr-1 was induced by H2O2 in a dose-dependent manner (Figure 7Go). The dose giving maximal induction was ~200 µM. H2O2 at 100 µM also significantly induced egr-1 expression, while 50 µM H2O2 barely induced egr-1 expression. This result is consistent with the transformation data, suggesting that both concentrations of H2O2 promote transformation via a similar mechanism.



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Fig. 7. Dose-dependent stimulation of Egr-1 protein expression by H2O2. T51B cells were treated with different concentrations of H2O2 for 2 h. Cells were then harvested and equal amounts of protein were subjected to western blot with anti-Egr-1 antiserum. The same blot was reprobed with an antibody to ß-actin. Similar result was obtained from another separate experiment.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chronic infection/inflammation is implicated in the pathogenesis of several forms of cancer, including hepatoma. It is well known that during inflammation, cells release reactive oxygen species, particularly H2O2. However, the role of H2O2 in the development of cancer and the underlying mechanisms are not well defined at this moment. It was our intention to test the hypothesis that H2O2 is involved in the enhancement of liver carcinogenesis and to investigate the possible mechanisms responsible for this process. To achieve this goal, we used T51B rat liver epithelial oval cells as a model system. T51B is a non-neoplastic epithelial cell line derived from rat liver (32). The epithelial nature of T51B cells has been confirmed by the presence of cytokeratin and its cell type is an oval cell, as demonstrated by its reactivity to specific monoclonal antibodies to oval cells developed by Dr Normard Marceau (Laval University) (33). Oval cells are putative stem cells in the liver and the precursor cells from which hepatocellular carcinoma is derived (34,35). In this system, H2O2 profoundly enhanced transformation initiated with MNNG, as shown by colony formation in soft agar, foci formation in monolayer culture, interruption of GJC in the foci area and growth to high cell densities. Although the hepatocarcinogenicity of peroxisome proliferators in rodents has been linked to oxidative DNA damage resulting from excessive leakage of peroxisomal H2O2 and the role of oxidative stress in rat liver tumor promotion has been suggested, to the best of our knowledge, this is the first direct evidence that H2O2 acts as tumor promoter in rat liver epithelial cells.

The mechanisms responsible for tumor promotion by H2O2 still remain unclear. H2O2 may act as a `genotoxicant' or `epigenetic' agent in its role as a promoting agent. Although H2O2 can cause DNA damage, it is, at best, a very weak mutagen in mammalian cells (41). In contrast, our studies, along with other studies, demonstrate that transcriptional modification and post-transcriptional protein modification is the result of exposure to ROS-inducing chemicals. Thus, tumor promotion by H2O2 is likely operating via `epigenetic' rather than `genotoxic' mechanisms in our system.

One aspect of tumor promotion is an effect on cell cycle progression resulting in aberrant proliferative behavior. Proliferation is required to fix mutations in initiated cells and to expand this mutated population of cells. Abnormal cell cycle progression may result from the alteration of expression of IE genes. The role of IE genes in tumor promotion is strongly suggested by the following lines of evidence. Firstly, many tumor promoters induce expression of IE genes. TPA induces AP-1 activity, which mainly consists of c-Fos and c-Jun (10). Subsequent studies demonstrate that other tumor promoters, including TCDD (42), UV (16,43), H2O2 (43), peroxisome proliferators (44), trivalent arsenic (45) and butylated hydroxytoluence hydroperoxide (46), induce the expression of IE genes. Nodularin, a new tumor promoting agent in rat liver, induces expression of the c-jun, c-fos and junB IE genes in hepatocytes (47). Secondly, the carcinogenic process often accompanies the induction of IE genes. High levels of c-Myc have been linked to chemically transformed C3H10T1/2 mouse embryo fibroblasts and to an increased susceptibility to spontaneous transformation of rat fibroblasts (48,49). Induction of c-fos and c-jun is associated with the transformation process in tracheal epithelial cells (50) and a mouse embryonic fibroblast system (51). Thirdly, IE genes play an important role in promoting cell cycle progression, particularly in cell cycle traverse from G0 to G1 and from G1 to S phase (52). Fourthly, inhibition of IE gene expression is correlated with antitumor promoting activity. Curcumin inhibits TPA-induced tumor promotion in mouse skin through the modulation of expression of c-fos, c-jun and c-myc (53). Phenolic antioxidants exert their antitumor promoting activities by inhibition of AP-1 transcriptional activity (54). Inhibition of AP-1 activity by retinoic acid may account for the effects of retinoic acid on the suppression of tumor cell growth (55,56). Furthermore, results from van den Berg's group suggest that overexpression of c-fos in NIH 3T3 cells increases the spontaneous level of chromosomal aberrations and gene mutations (57,58). In T51B cells, H2O2 preferentially induces expression of c-fos, c-jun, c-myc and egr-1. It is our intention to define the specific role of these IE genes in tumor promotion and further identify their target genes in our system.

Cell to cell communication via gap junctions is essential for the maintenance of the homeostatic balance in multicellular organisms (59). Accumulated evidence suggests an important role of GJC in tumor promotion (60). This stems from several findings, including: (i) many tumor promoting agents inhibit GJC (24,59); (ii) GJC is usually down-regulated in tumors (61,62); (iii) on the other hand, up-regulation of GJC is associated with prevention of carcinogenessis (18,24). Results from our laboratory further support this idea. Previously we demonstrated that tumor promoters such as TPA and OA and growth factor such as epidermal growth factor (EGF) and platelet-derived growth factor induce rapid and transient hyperphosphorylation of Cx43 and disruption of GJC in T51B cells (24,25). Recently we observed a profound decrease in GJC as well as in expression of cx43 in prostate cancer cells compared with normal prostate cells (63). Expression of cx43 is profoundly decreased in human brain tumor cells (64,65). Importantly, introduction of Cx43 into these tumor cells reverses the neoplastic phenotype, supporting the notion that cx43 functions as a tumor suppressor gene (65). The expression of Cx43 lengthens the G1 phase time, suggesting a regulatory role of cx43 in cell cycle progression (29,65). Therefore, it is very likely that H2O2-induced disruption of GJC is one of the mechanisms responsible for the clonal expression of initiated cells.

The model system established in this study will allow us to further dissect the molecular changes during tumor promotion and to test inhibitory agents of tumor promotion. Two agents tested in our system were NAC and {alpha}-tocopherol, both of which efficiently inhibit the tumor promoting activity of H2O2. Clinical data suggest that vitamin E supplementation reduces the risk of colon cancer (66) and prostate cancer (67), supporting the application of antioxidants in chemoprevention.

The effects of H2O2 on tumor promotion, induction of IE genes and disruption of GJC are dependent on oxidative stress. Application of antioxidants blocks H2O2-induced tumor promotion, induction of IE genes and disruption of GJC. Our data also indicate that the antioxidant NAC blocks the effect of H2O2 on the induction of hyperphosphorylation and disruption of GJC. Recently Trosko's group reported that H2O2 inhibits GJC in WB-F344 rat liver epithelial cells with an I50 value of 200 µM, but it seems that this effect does not involve oxidative stress because the antioxidants they used did not block the effect of H2O2 on the disruption of GJC (68). The discrepancy between these studies is not clear at this moment. It may be due to several factors, such as different antioxidants, cell lines and concentrations of H2O2.

It is well recognized that the reduction–oxidation (redox) state is important for cell activity. Changes in the redox state are known to influence the DNA-binding activity of several transcription factors, such as Oxy R (69), AP-1 (70), NF-{kappa}B (71), Egr-1 (72), Ets (73), Myb (74) and v-Rel. A redox mechanism is also known to contribute to cell cycle progression (76), hormone receptor interactions (77), bacteriophage DNA replication (78), light signal transduction (78,79), regulation of iron metabolism (80), protein–RNA and protein–DNA interactions and RNA transcription (8183) and protein translation regulation (78,84). It is also known that ROS and H2O2 can induce single-strand breaks in cellular DNA, oxidation of DNA bases, chromosomal aberrations and DNA–protein crosslinks (13,85,86). Important here is the fact that the redox state is involved in tumor promotion (87) and is tightly linked to ROS production. Taken together, the evidence strongly supports the notion that ROS are important mediators for many cellular activities. Although the tumor promotion process in vivo is certainly more complicated, a further characterization of the in vitro model system established in this study should shed light on the mechanism of tumor promotion.


    Acknowledgments
 
This work was supported by NIH grants CA39745 (ALB) and CA57064 (ALB).


    Notes
 
1 To whom correspondence should be addressed Email: rphuang{at}nwlink.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 8, 1998; revised October 27, 1998; accepted .