The role of nitric oxide in neoplastic transformation of C3H 10T1/2 embryonic fibroblasts

Terrilea Burnett, Ao Pung, John S. Bertram and Robert V. Cooney1

University of Hawaii Cancer Research Center, 1236 Lauhala Street, Honolulu, HI 96813, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nitric oxide synthase inhibitors block the neoplastic transformation of C3H 10T1/2 cells in vitro. Evidence presented herein suggests that they mediate their effects early in the carcinogenic process as brief treatment with the NOS inhibitor aminoguanidine (AG) during log phase cell growth (initiation phase) is sufficient to block foci formation. In contrast, treatment initiated after formation of a confluent monolayer was associated with diminished protection, while treatment commencing late in the promotional phase had no protective effect and appeared to enhance the number and stage of foci observed. These findings suggest that while AG treatment can inhibit transformation during the early promotional phase, it most effectively inhibits transformation during the initiation phase. In general AG enhanced growth of both normal and tumor cells, suggesting that effects on growth were unrelated to its anti-transformation properties, however, these effects could be related to the effect on tumor cell stage noted above. Although induction of inducible nitric oxide synthase (iNOS) by treatment with LI during the last 2 weeks of the assay was associated with enhanced transformation, the efficacy of AG in protecting against transformation was not clearly associated with substantial reductions in NO synthesis. The data suggest that AG inhibits transformation early in the transformation process independently of iNOS inhibition and that AG may have deleterious effects late in the process, possibly through stimulation of tumor cell growth.

Abbreviations: AG, aminoguanidine; BME, Eagle's basal medium; DAN, 2,3-diaminonaphthalene; IFN-{gamma}, {gamma}-interferon; iNOS, inducible nitric oxide synthase; LI, lipopolysaccharide plus {gamma}-interferon; LPS, lipopolysaccharide; MCA, 3-methylcholanthrene; MF, mutation frequency; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; NAT, 2,3-naphthatriazole; NOx, nitrogen oxides; PBS, phosphate-buffered saline; PE, plating efficiency; ROS, reactive oxygen species.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Environmental agents have been implicated in the development of cancer since 1775, when Sir Percival Pott noted the high incidence of scrotal cancer in chimney sweeps (1). Since, hundreds of exogenous factors, including chemical carcinogens, radiant energy and oncogenic viruses, have been reported to induce neoplastic transformation of cells in vitro and to cause malignant tumors in experimental animals. Recent observations that oxidized DNA bases occur in mammalian cells in significant quantities in the apparent absence of exposure to exogenous carcinogens or mutagens suggest that endogenous mutagens may have an etiological role in cancer as well (24). A number of reactive oxygen species (ROS) and free radicals are synthesized in vivo as a result of aerobic metabolism or host defense mechanisms, including superoxide (·O2), hydrogen peroxide (H2O2), hydroxyl radical (·OH) and nitrogen oxides (NOx) (25). Under homeostatic conditions, these highly reactive and potentially toxic species are maintained at low levels and their toxic effects can be moderated by dietary and endogenous antioxidants as well as signal transduction and repair pathways (3,5). However, when antioxidant defenses are inadequate, lipids, proteins, nucleic acids and other biomolecules may become targets for free radical-mediated reactions by oxidative or nitrosative species (25).

Oxidative damage to DNA bases increases the risk for formation of hereditary genomic alterations and may contribute to the process of carcinogenesis if growth regulatory genes are affected (3). While most oxidative lesions can be repaired by specific DNA repair enzymes, if repair is not completed prior to DNA synthesis, significant genotoxicity may occur in proliferating cells (68). The clinical relevance of oxidative damage is suggested by observations that chronic infection and/or inflammation, conditions resulting in increased ROS synthesis and cell proliferation subsequent to cell injury/death, is associated with increased risk for site-specific cancer, e.g. hepatitis B virus with liver cancer, human papilloma virus with cervical cancer and Helicobacter pylori with stomach cancer (2,4,710).

Of special interest among endogenous reactive species is nitric oxide (NO). Produced by a host of mammalian cell types, this relatively stable free radical is the first gas known to function as a cellular messenger. Essential for maintaining normal physiological homeostasis through functions which include neurotransmission, vasodilation, smooth muscle relaxation, angiogenesis, cell signal transduction and regulation of cell proliferation, NO also serves as a mediator of non-specific host defense during infection or inflammation (1118). NO has been shown to be cytotoxic to numerous pathogens via mechanisms involving reaction of NO with cellular and mitochondrial targets (11). Following its oxidative metabolism to NOx, NO has been demonstrated to be mutagenic to mammalian cells (19,20), possibly through nitrosation of primary or secondary amines (2123). Its genotoxic spectrum includes chromosome aberrations, sister chromatid exchanges and single-strand breaks (20,24,25). In some cases, genotoxic damage may be exacerbated by NO-mediated inhibition of DNA repair enzymes (26).

We were interested in examining the potential for cellular toxicity by NO and its relation to carcinogenesis. In an earlier investigation, we reported that NO-mediated mutagenicity in bacteria was enhanced by cellular proliferation, was oxygen dependent and increased as a function of the square of the NO concentration (27). C3H 10T1/2 murine fibroblasts, one of the most widely used cell lines for the study of carcinogenesis, synthesize NO when exposed to bacterial lipopolysaccharide (LPS) and {gamma}-interferon (IFN-{gamma}) and in the process form chemical species capable of nitrosating secondary amines present in the culture medium (28). Inhibitors of the inducible isoform of the enzyme nitric oxide synthase (iNOS) block NO synthesis by activated 10T1/2 cells and suppress neoplastic transformation of carcinogen-initiated cells when treated during the promotional phase of the transformation assay (28). These data suggest that NO may play a role in the process of mammalian neoplastic transformation. In the current investigation, we sought to better characterize the mechanism by which NO synthase inhibitors prevent neoplastic transformation in C3H 10T/2 cells.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
C3H 10T1/2 mouse fibroblasts (ATCC no. CCL 226), passages 8–15, were used for all experiments (29). Unless otherwise stated, cells were seeded in plastic tissue culture dishes at a density of 4000 cells/ml in Eagle's basal medium (BME) supplemented with 5% bovine calf serum (HyClone Laboratories) and gentamicin sulfate (25 µg/ml) and incubated at 37°C in a humidified atmosphere of 5% CO2/95% air. At this seeding density, confluent monolayers are normally formed by day 6 or 7 after plating. The neoplastic MCA cell line was obtained from Dr L.Mordan (University of Hawaii Cancer Research Center). Stock solutions of IFN-{gamma} (murine recombinant; Gibco BRL), LPS (Escherichia coli serotype 0127:B8) and aminoguanidine (AG) were filter sterilized and stored at –70°C until use. All reagents were purchased from Sigma unless otherwise specified.

Nitrite analysis
The concentration of nitrite (NO2) in the culture medium was determined by utilizing a modification of the Saltzman assay (30). Cell culture medium (40 µl) was added to 960 µl of absorbing reagent (0.5% sulfanilic acid, 0.002% N-1-naphthylethylenediamine dihydrochloride, 14% glacial acetic acid) and incubated at room temperature for 15 min. Absorbance was measured at 550 nm using a Shimadzu model UV160U spectrophotometer. Solutions of sodium nitrite added to the absorbing reagent were used to plot a standard curve against which experimental values were compared. The detection of nitrite with this assay was linear over the range 0.4 to at least 10 µM.

N-nitrosation of the primary amine 2,3-diaminonapthalene (DAN)
N-nitrosation of DAN was analyzed using a modification of the technique described by Miles et al. (31). Cultures were treated for 5 days with LPS (10 µg/ml), IFN-{gamma} (30 ng/ml) and/or AG (300 µM). DAN (0.1 mM in acetone), a primary aromatic amine that can be nitrosated to yield the fluorescent 2,3-naphthatriazole (NAT) derivative, or acetone as a control (0.5%) was then added to the culture medium. Some control cultures were treated with sodium nitrite (25 µM) plus DAN. After an incubation period of 24 h, cell culture medium (0.5 ml) was added to 2.5 ml of 10 mM NaOH (used to enhance fluorescence) and mixed. Fluorescence emission spectra, determined by luminescence spectrophotometry (Shimadzu model DR-15) with excitation at 375 nm and emission at 415 nm, were compared with values for control cultures incubated with DAN plus phosphate-buffered saline (PBS) and corrected for background static measurements found in cell-free BME treated with DAN.

Assay for determination of ouabain resistance
The ouabain assay was used to characterize the mutagenic potential of NO (32). Confluent cultures in 100 mm culture dishes (4 dishes/group) were treated for 5 days with LPS (10 µg/ml), IFN-{gamma} (30 ng/ml) and/or AG (300 µM) and, following replenishment with fresh culture medium, re-treated for 2 days. Cells were then typsinized and three 60 mm dishes were seeded with 200 cells/dish in BME for determination of plating efficiency (PE); remaining cells were plated at equal density into three 100 mm culture dishes in ouabain-supplemented culture medium (2 mM; Sigma) for determination of mutation frequency (MF). After 1–3 weeks incubation, cultures were fixed in methanol and stained with Giemsa for quantitation of colony formation.

The protocol was modified for determination of the mutagenic potential of NO gas. Cells (4000 cells/ml) plated into 100 mm dishes (8 dishes/group) were grown to mid log phase (~day 5). Prior to gas exposure, cells were washed and the BME replaced with 2 ml of PBS. Dishes placed onto a rocker contained within a sterile polyethylene glove bag were exposed to varying concentrations of NO gas diluted with N2 or to N2 alone (Scott Specialty Gases, San Bernadino, CA) for 30 min. Positive control cultures were treated with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (1 µg/ml; Sigma) for 30 min prior to gas exposure. Negative control cultures were incubated with PBS prior to N2 exposure. Following gas exposure, cultures were re-incubated in BME for 5 days. Cells were then subdivided into three 60 mm dishes for determination of PE and five 100 mm dishes for MF and incubated as described above.

PE was calculated as the percentage of cells plated in BME that formed microscopically discrete colonies within 7–10 days of plating. MF was determined by dividing the number of ouabain-resistant colonies per dish by the number of surviving cells (number of cells plated/dishxPE). The means ± SEM were determined for each treatment group and normalized to MF/105 cells.

Transformation assay
Cultures were seeded at a density of 1000 cells/60 mm culture dish and treated 24 h later with the carcinogen 3-methylcholanthrene (MCA) (10 µM; Sigma) for 1 day. The cell culture medium was then replaced with fresh medium and the cells re-incubated. At the weekly changes of culture medium cultures were treated with AG (300 µM) as indicated in Results. In some cases cultures were treated with combinations of LPS, IFN-{gamma} or AG without exposure to MCA. Approximately 5 weeks after formation of a confluent monolayer cultures were fixed with methanol, stained and scored for the presence of type II and III foci according to the guidelines established by Reznikoff et al. (33). These types of morphologically transformed foci have been repeatedly demonstrated to be tumorigenic in syngeneic mice.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Potential of endogenous NO to nitrosate primary amines
We earlier demonstrated the potential for nitrosation of secondary amines by lipopolysaccharide plus {gamma}-interferon (LI)-stimulated C3H 10T1/2cells (28). We sought to confirm the ability of activated 10T1/2 cells to catalyze formation of N-nitrosamines from primary amines, to determine if the iNOS inhibitor AG would suppress this process and to rule out a role for nitrite-mediated nitrosation. Stimulation of cultures with LPS and IFN-{gamma} resulted not only in synthesis of NO2 during the exposure period to the primary amine DAN but also in formation of NAT, the highly fluorescent N-nitrosation product of DAN, indicating that NO is converted to a form capable of nitrosation in cell culture (Table IGo). Nitrosation of DAN was increased by ~12.7-fold in LI-treated cultures compared with unstimulated control cultures incubated with DAN, while simultaneous treatment with LI and AG inhibited both NO2 formation and nitrosation of DAN. Addition of exogenous NaNO2 + DAN to the culture medium of unstimulated cultures did not enhance NAT formation relative to background levels, indicating that nitrosation was mediated by a precursor of nitrite, the formation of which was effectively blocked by AG.


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Table I. Nitrosation of DAN and NO2 production by C3H 10T1/2 cells
 
Mutagenic effects of NO on C3H 10T1/2 cells
As illustrated in Table IIGo, exposure of log phase C3H 10T1/2 cells to NO gas enhanced formation of ouabain-resistant colonies in a dose-dependent manner, indicating the mutagenic potential of exogenous NO in this cell line. Exposure to 20 p.p.m., the lowest concentration evaluated, was insufficient to significantly induce colony formation relative to negative control cultures. However, elevating the exposure dosage to 50 or 91 p.p.m. increased the MF by ~11- and 16-fold, respectively, over baseline levels. The combination of MNNG with NO gas at 91 p.p.m. yielded 8.7- and 5.6-fold increases in MF in comparison with cultures exposed to NO (91 p.p.m.) or MNNG independently, indicating a synergistic interaction. Although the decrease in PE for cells treated with both MNNG and NO indicates increased cellular toxicity for this treatment combination, the PE remained within the normally reported range of 12–30% for C3H 10T1/2 cells in the ouabain assay (29,32,34).


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Table II. Mutagenic potential of exogenous NO
 
The ouabain assay was also used to investigate the mutagenic potential of endogenously generated NO in C3H 10T1/2 cells (Table IIIGo). LI-treated cultures synthesized NO2 (~37 µM) during the 48 h period preceding subdivision of cells into ouabain-supplemented medium. In comparison, NO2 in cultures treated with LI + AG or AG alone remained at background levels (data not shown). Ouabain-resistant clones were not observed in cultures treated with LI. However, it should be noted that in contrast to the sparse population of background cells observed in cultures treated with AG or PBS, cultures treated with LI were virtually devoid of viable cells, suggesting that ouabain toxicity is enhanced when combined with LI treatment. Surprisingly, the MF of cultures treated with AG was ~4-fold higher than that of cultures treated concomitantly with LI and AG. The PE for each treatment group was well within the reported range of 12–30% for C3H 10T1/2 cells (29,32,34), indicating that independently neither endogenous nitrogen oxides at the levels generated nor the treatment agents induced cytotoxicity.


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Table III. Mutagenic potential of endogenous NO
 
Differential efficacy of AG to suppress neoplastic transformation during the initiation and promotional phases of the transformation assay
Earlier studies suggested that endogenous NO may play a role during the promotional phase of the transformation assay, as continuous treatment with various inhibitors of NO synthase from day 9 to 44 suppressed neoplastic transformation (28). To better define the mechanism by which iNOS inhibitors act, we sought to define the time period(s) during the ~44 day transformation assay in which AG most effectively blocks transformation. As seen in Table IVGo, positive control cultures treated with the carcinogen MCA formed a total of 19 foci/12 dishes following 6 weeks incubation. In this group there was a predominance, albeit modest, of type II and type III foci. Total foci decreased by ~37% (P = 0.18) in cultures co-treated with MCA plus AG for a 24 h period. Treatment with AG during the initiation phase (days 2–9) inhibited foci formation by ~58% (P = 0.05). Continuous exposure to AG throughout all or most of the transformation assay (days 1 or 2–44) was equivalent in effectiveness to cultures in which treatment with AG was limited to day 1, suggesting that AG blocks early events in the transformation assay. We also examined specific periods within the promotional phase of the transformation assay which were not evaluated during our earlier study. Treatment with AG continuously upon formation of a microscopically confirmed confluent monolayer (days 16–44) was less effective than treatment during the initiation phase, while treatment late in the promotional phase (days 30–44) failed to protect against foci formation. Interestingly, the ratio of type II to type III foci was reversed in cultures in which AG exposure was limited to the promotional phase, resulting in a greater number of type III foci. Medium NO2 remained at baseline levels for all groups throughout the period of observation (data not shown).


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Table IV. Effects of NOS inhibition on formation of transformed foci
 
Potential for neoplastic transformation by endogenous NO
We then sought to determine if induction of iNOS in the absence of initiation with a known carcinogen was sufficient to induce development of neoplastic foci (Table VGo). Cultures treated with LI continuously from day 1 until formation of a confluent monolayer (day 21) generated copious quantities of NO2 (48 µM). Following exposure to fresh untreated culture medium on day 21, these cultures continued to synthesize NO2, resulting in a final peak concentration of ~88 µM after 28 days of the assay. Surprisingly, these high levels of NO were not sufficient to induce the development of neoplastic foci. Cultures treated for 1 week periods during the last 4 weeks of the assay generated similar quantities of NO2 at termination of treatment, but foci were observed only in cultures treated during the last 2 weeks of the assay, suggesting that LI treatment late in the transformation assay may enhance foci development.


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Table V. Effects of iNOS induction on formation of transformed foci
 
Effects of AG on tumor cell proliferation
In an effort to clarify the mechanism by which AG suppresses neoplastic transformation, we also investigated the effects of treatment with AG on tumor cell proliferation. Neoplastic cells derived from a MCA-transformed C3H 10T1/2 cell line were treated with AG and cell growth determined daily (Figure 1Go). At termination of the study on day 8, cell numbers in AG-treated cultures were nearly 2-fold higher (P < 0.0001) than those of untreated controls, indicating that AG does not function by inhibiting neoplastic tumor cell proliferation. Indeed, these results indicate that inhibition of NO synthesis may increase the growth rate of existing tumor cells.



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Fig. 1. Effects of AG on the growth of neoplastic MCA cells. Cells were seeded at a density of 5000 cells in 35 mm culture dishes on day 0 and treated with AG (300 µM) on day 1. Cell counts were determined daily. Values represent the means of three dishes. P values were significant (<0.01) on all days other than day 1.

 

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 Materials and methods
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We used the transformation assay as a model to further examine the role of NO in neoplastic transformation. The assay consists of exposing log phase cells to known or suspected carcinogens and, following an incubation period of ~6 weeks, scoring for morphologically transformed foci (33). Rapid cell proliferation during the initiation phase (days 1–9) is thought to enhance fixation into the genome of carcinogen-mediated lesions, resulting in a population of `initiated' cells. During the promotional phase (days 9–44) episodic growth stimulation of proliferatively quiescent cells by weekly exposure to growth factors may `promote' clonal expansion of initiated cells, resulting in formation of morphologically transformed clones. Although log phase growth is traditionally associated with the initiation phase and the promotional phase is characterized by growth quiescence, the time period required for formation of a confluent monolayer of proliferatively quiescent cells is dependent on the cell culture conditions.

Results from our previous study indicated that treatment with the NOS inhibitors N{omega}-nitro-L-arginine methyl ester, NG-methyl-L-arginine and AG during the post-initiation promotional phase (days 9–44) reduces the number of transformed foci in a dose-dependent manner. Due to the similar efficacy of the three NOS inhibitors, in the present study we examined the ability of AG to protect against neoplastic transformation during specific time periods within the transformation assay. The period of optimal efficacy observed in our study was days 2–9. Total foci in these cultures were reduced by 58% in comparison with positive controls treated with MCA. Protection was lower in cultures treated for 24 h on day 1, however, despite the brevity of treatment, this group was almost equivalent in efficacy to treatment on days 1–44. In contrast, treatment initiated after formation of a confluent monolayer (visually confirmed on day 16) was associated with diminished protection, while treatment commencing late in the promotional phase conferred no protection. These findings suggest that while AG treatment can inhibit transformation during the promotional phase, it most effectively inhibits transformation during the initiation phase.

Suppression of neoplastic foci by chemical inhibitors of iNOS suggests a relation between NO synthesis and the carcinogenic process. Nitrosation of biomolecules is thought to represent one of the mechanisms by which NO/NOx promotes mutagenesis and carcinogenesis (22,23,35,36). In our studies LI-stimulated cultures incubated with the primary amine DAN generated the N-nitrosation product NAT at levels ~13-fold over that observed in control cultures. Addition of AG to the treatment protocol inhibited both nitrosation and nitrite synthesis, indicating that the nitrosating species is derived from the L-arginine->NO biosynthetic pathway. Previously we reported nitrosation of the secondary amine morpholine by 10T1/2 cultures treated with LI (28), indicating that activated C3H 10T1/2 cells can catalyze formation of N-nitrosamines from both primary and secondary amines. N-nitrosation of primary amines in DNA bases results in rapid deamination via N-nitrosamine intermediates (19). In the absence of repair, base substitution mutations or point mutations can occur. Mutations, particularly those in genes regulating cell proliferation, are hypothesized to contribute to the initiation, promotion and progression stages of carcinogenesis (210,37). We observed exogenous NO to be highly mutagenic in a dose-dependent manner to 10T1/2 cells. The mutagenic properties of endogenous NO, as analyzed in the current study, remain inconclusive due to toxicity. The mechanisms underlying the pattern of extensive cell loss in LI-treated cultures exposed to ouabain-supplemented culture medium are unclear. Cytotoxicity associated with ouabain treatment has been previously reported in rat macrophages treated with lipopolysaccharides (38).

Based on earlier reports that nitrite levels increased during the course of the transformation assay (28), we surmised that the mechanism for suppression of neoplastic transformation by iNOS inhibitors was related to protection against NO-induced cellular damage. However, a number of findings in the current study fail to support a significant role for inducible NO in the transformation process. Nitrite levels, measured weekly in the culture medium, remained consistent with background levels in all groups not exposed to LI. Hence, it is doubtful that quantities of NO sufficient to induce significant cellular damage were synthesized during the course of the assay. We also noted decreased foci in cultures treated with AG on day 1 for 24 h, yet inhibition of NOS during this period may be minimal, as the lag period required for commencement of NO synthesis in C3H 10T1/2 cells has been observed to be ~24–48 h in our laboratory (data not shown). Moreover, it has been reported that fibroblasts in early log growth synthesize low or undetectable levels of NO (28,39), yet exposure to AG during this period inhibited transformation by up to 58%. Conversely, the synthesis of significant quantities of nitrite by uninitiated cultures treated with LI continuously for the first 21 days of the assay failed to result in formation of transformed foci, suggesting that NO exposure during the early phase of the assay of itself may be insufficient to induce the process of neoplastic transformation.

In the light of our observations that the efficacy of AG to protect against transformation, under the conditions of the transformation assay, was not clearly associated with substantial reductions in NO synthesis, we searched for alternative explanations. We did observe an apparent association of AG efficacy with growth phase of MCA-treated cells at the time of AG treatment. In both the present study and our earlier study (28) we noted that addition of AG to the culture medium of log phase cells, whether during the initiation or early promotional phase, resulted in decreased formation of transformed foci relative to MCA-initiated controls. In comparison, AG treatment after formation of a microscopically confirmed confluent monolayer (promotional phase) conferred diminished protection against neoplastic transformation. In our study AG significantly enhanced growth of transformed cells. To further investigate this, we specifically targeted the late promotional phase for AG treatment, when nascent neoplastic foci are present. In these cultures we observed increased foci number and an increased ratio of type III to type II foci in comparison with cultures treated with AG early in the assay during log growth. Whether the effects on foci development are secondary to the presence of AG or to decreased levels of NO is not entirely clear. Our observation of foci formation in AG-treated confluent cultures incubated in ouabain-supplemented culture medium suggests that AG may under some circumstances promote mutagenesis, perhaps by altering the balance of oxidative radicals. Alternatively, it is possible that low levels of NO synthesis during the promotional phase of the transformation assay may in a limited manner serve to protect against neoplasia, conceivably by regulating growth or inducing apoptosis in cell populations with genetic or cytotoxic damage. NO has been observed to induce apoptosis (40) and inhibit tumor cell growth (41). Indeed, an inverse relation between NO and transformation is not unprecedented, as recent reports indicate that tamoxifen, while increasing NO synthesis, was found to decrease neoplastic transformation (42).

The significant inhibition of foci by treatment with AG during the initiation phase suggests that this is a critical period within the context of transformation. AG may attenuate the carcinogenicity of MCA, perhaps by blocking one or more of the sequential steps required for MCA activation and/or initiation. If so, treatment of initiated cell populations with AG might be less effective, consistent with our observation of diminished protection when AG treatment is commenced later in the promotional phase. Data from another investigation support the concept that differential efficacy of AG to inhibit transformation may be due to multiple and, as yet, poorly understood mechanisms. In this study high concentrations of fully phosphorothioated antisense oligodeoxynucleotides specific for iNOS mRNA were found to inhibit NO synthesis, but significantly increased formation of neoplastic foci (43). Upon addition of AG to the treatment protocol, however, the number of foci diminished. The results raise the possibility that protection by AG may in some cases be unrelated to its ability to form inhibitory complexes with the iNOS protein, as oligodeoxynucleotides specifically targeted to iNOS were shown to significantly diminish iNOS protein levels and NO production. Yet, AG still effectively decreased focus formation by 37% (similar to its effect seen here). An alternative mode of protection by AG during the initiation phase may serve to unravel the paradox that we observed during our study whereby exposure of log phase cells to substantial levels of endogenously generated NO failed to induce transformed foci, while `protection' against NO by AG treatment during the same time period significantly reduced transformation.

In a recent study using whole animal models treatment with polycyclic aromatic hydrocarbons, including MCA, was shown to enhance H2O2 synthesis and induce formation of oxidative DNA base damage at levels exceeding formation of MCA-mediated polycyclic aromatic hydrocarbon–base adducts by 10-fold (44). The possibility that AG and other NOS inhibitors may interfere with P450 enzyme metabolism should be considered in studies demonstrating effects for these inhibitors. If reactive species of oxygen are endogenously synthesized during the promotional phase by MCA-initiated cells, then inhibition of iNOS may actually enhance oxidative damage by decreasing NO to levels inadequate to quench reactive species and may account for the increase in transformation that we observed in cultures treated late in the assay compared with cultures treated earlier, as well as the transformation enhancing properties of iNOS antisense oligodeoxynucleotides (43). Moreover, Wink and colleagues noted that NO, by abrogating cytotoxicity mediated by superoxide or hydrogen peroxide, protected against cellular damage by these species (45).

In summary, we have demonstrated inhibition of transformation by treatment during the initiation and early promotional phases by a chemical inhibitor of iNOS. In contrast, treatment late in the transformation assay conferred no protection. The available evidence suggests that the actions of AG in preventing neoplastic transformation are unrelated to its function as an iNOS inhibitor, rather that NO may have beneficial effects in preventing neoplasia despite its potential for mutation at higher doses. Clearly, more research is warranted to fully elucidate the role of NO and NO synthesis inhibitors in carcinogenesis.


    Notes
 
1 To whom correspondence should be addressed Email: bob{at}crch.hawaii.ed Back


    Acknowledgments
 
This research was supported by a grant in aid from the American Cancer Society (CN-158).


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 4, 1999; revised April 18, 2000; accepted June 30, 2000.





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