Elaine Chu

Writer’s comment: Nitric oxide is best known as a toxic pollutant in the atmosphere, but this gas is also a significant physiological agent with such roles as blood vessel relaxation and defense against invading pathogens. This free radical’s toxicity is mainly due to its reaction with superoxide to produce the potent oxidizing agent peroxynitrite, which can react with DNA and proteins. As part of the body’s defense system, certain cells of the immune system utilize nitric oxide’s biological toxicity as part of normal inflammatory response.
         Under certain conditions, endogenous nitric oxide can also have deleterious physiological effects. The dramatic drop in blood pressure that occurs during septic shock is a well-documented effect of excess endogenous nitric oxide production. What is less well known is that excessive physiological levels of nitric oxide can have cancer-promoting effects on an organism’s cells such as inhibition of apoptosis and facilitation of tumor progression. This paper reviews the body of knowledge accumulated from various studies on one facet of nitric oxide carcinogenicity: prevention of normal DNA repair pathways.
—Elaine Chu

Instructor’s comment: Elaine wrote this fine article in response to a standard assignment in English 104E (Science Writing): the scientific review article. This is a challenging assignment that requires students to find a cutting-edge area of research in their field, research that topic in the scientific literature, and then synthesize that material for an audience of interested professionals. Elaine succeeds superbly on all counts. Her subject is the carcinogenic properties of the free radical, nitric oxide—more specifically, the indirect damage that nitric oxide can cause by inhibiting the ability of DNA to repair itself. The article goes on to explain what current research shows us about exactly how this inhibition of DNA repair works, and concludes by discussing how this research will help scientists in the future to battle cancer.
—Pamela Demory, English Department



I. Introduction
         Nitric oxide (NO), an endogenously produced free radical with diverse physiological roles, has recently gained recognition as a potential carcinogen. Within the past decade, biomedical studies have suggested that NO displays carcinogenic properties under conditions that enhance its presence beyond normal physiological levels. One of the various ways by which NO promotes cancer development is by acting as a mutagen: through DNA damage, NO can introduce mutations in proto-oncogenes and tumor supressors. Scientists have identified both direct and indirect modes of NO-mediated DNA damage. In all cases, the actual damage is caused by oxidized derivatives of NO such as peroxynitrite and nitrate. Some well-documented modes of direct DNA damage by NO include base deamination, single-strand breaks, and adduct formation [1]. In the indirect mode of DNA damage, NO does not cause DNA mutation itself but interferes with the cell’s ability to repair cancer-promoting mutations. Thus, accumulation of DNA damage represents the net result of both direct damage and ineffective repair. Because DNA repair is vital to the integrity of a cell’s genome, the potency of NO’s carcinogenicity may ultimately rely more on its ability to alter this function than on its ability to directly react with DNA. The following report will review recent studies that have contributed important insights into the ways that NO disrupts DNA repair.

II. Targeting of DNA Repair Enzymes: Thiol Nitrosylation and the Zinc Finger Motif
         In general, enzymes that contain thiol groups in their active sites are particularly susceptible to reaction with peroxynitrite that can disrupt their catalytic activity [1]. Unfortunately, DNA repair enzymes—part of the rich and complex cellular machinery that maintains the integrity of the genome—fit this category. By 1998, scientists had established through in vitro studies that peroxynitrite directly nitrosylates thiol groups in the DNA-binding regions of several key DNA repair enzymes including formamidopyrimidine-DNA glycosylase (Fpg), O6-methylguanine DNA-methyltransferase, and xeroderma pigmentosumA (Xpa). The active sites of these nucleotide excision repair enzymes contain a zinc finger motif consisting of a zinc ion coordinated to thiol residues. Disruption of this moiety and irreversible inhibition of enzyme activity has been observed with NO-mediated nitrosylation of the thiol residues [1].
         Recently, Jaiswal et al. have identified another glycosylase, human 8-oxoguanine glycosylase (hOgg1), whose active site zinc finger is irreversibly damaged by NO [2]. This glycosylase removes one of the most abundant oxidative DNA lesions, 8-oxoguanine, which causes GC to AT transitions. After isolating hOgg1 from transfected cholangiocarcinoma cells and treating the protein with NO, Jaiswal et al. assayed the system for hOgg1 activity, nitrosylation of hOgg1, and zinc release. From the data, they concluded that hOgg1 is inhibited by both NO and peroxynitrite through nitrosylation of active site cysteine residues with concurrent loss of zinc. These results, along with those from studies on Fpg and Xpa [1], seem to suggest that base excision repair enzymes are particularly vulnerable to irreversible NO-mediated inactivation due to the critical zinc finger motif within their active sites.

III. Chronic Inflammation Enhances Mutagenicity of NO
         The physiological context in which NO-mediated DNA repair inhibition occurs has helped explain a pattern of cancer development. Normally, endogenous NO production is triggered as part of the immune response when inducible nitric oxide synthase (iNOS) in macrophages is induced by cytokines. However, prolonged iNOS expression resulting from sustained cytokine exposure is a condition currently suspected of predisposing infected tissue to malignant transformation. Carcinogenesis of gastrointestinal tissue, in particular, has repeatedly been linked to chronic infection and inflammation, during which high levels of NO are produced in response to cytokines that accumulate at the infected site [3,4]. This observation, along with the theory that NO can nitrosylate DNA repair proteins, has led to the hypothesis that high levels of cytokine-induced NO is responsible for inhibiting DNA repair and initiating cancer in chronically inflamed tissue. For example, a recent study by Jaiswal et al. shows that inflammatory cytokines in human cholangiocarcinoma cells stimulate the synthesis of NO at sufficient levels to damage DNA [3]. Cholangiocarcinoma is a liver bile duct cancer that occurs with high frequency following chronic inflammation in this area. To demonstrate the presence of catalytically active iNOS in cholangiocarcinoma, Jaiswal et al., used immunohistochemistry to probe for iNOS and 3-nitrotyrosine (which results from the reaction between peroxynitrite and susceptible proteins) in tissue specimens from patients. With three cell lines, Western blot analysis was used to observe the effects of cytokines on nitric oxide synthase expression, a comet assay was used to detect DNA damage via changes in nucleoid structure, and incorporation of radioactive dGMP was used to evaluate the efficiency of DNA repair. The data for all cell lines showed that cytokines inhibited DNA repair activity in a NO-dependent manner. Numerous studies demonstrating similar results have lent substantial evidence to the central hypothesis that NO-induced DNA damage explains how chronic inflammation predisposes tissue to malignant transformation [4].

IV. Alternative Modes of DNA Repair Inhibition
         The preceding findings come from a fairly established area of study that focuses on well-known enzymes that directly regulate DNA repair processes. The studies have also consistently linked damage of these enzymes to NO generated in inflamed tissue. However, studies that suggest alternative routes that NO may take to impede DNA repair and promote carcinogenesis are beginning to emerge and deserve some consideration.

Role of NO in Arsenite-Mediated Inhibition of DNA Repair. Nitric oxide’s mutagenicity has most often been viewed and studied as a link between inflammation and carcinogenesis. However, several studies suggest that NO plays a role in the carcinogenic process of another mutagen, arsenite. Earlier studies have shown that arsenite exposure to cells damages DNA and inhibits DNA repair, suggesting that arsenite directly damages DNA or inhibits repair enzymes [5]. More recent work has demonstrated that DNA enzymes are not affected by arsenite and that, furthermore, NO is involved in arsenite-induced mutagenicity. Liu and Jan demonstrated that arsenite treatment of bovine aortic endothelial cells increased production of NO as well as DNA strand breaks. In addition, they determined that the DNA strand breaks resulted from excision of oxidized bases, an observation linking arsenite exposure to direct NO mutagenicity. From their data, they concluded that the DNA damage that occurs with chronic exposure to arsenite is, in fact, due to nitric oxide [5].
         With NO’s mutagenic role in arsenite-induced carcinogenesis established, D.T. Bau et al. decided to address the question of whether or not arsenite-induced levels of NO are also responsible for inhibiting DNA repair. They transfected Chinese hamster ovary cells with the reporter plasmid pGL2 and irradiated them with UV light to induce formation of pyrimidine dimers. Pyrimidine dimers are targets for nucleotide excision repair enzymes such as Xpa that are known to be nitrosylated and inhibited by NO. To demonstrate NO’s involvement in repair inhibition upon arsenite exposure, they observed the effects of arsenite on the expression of pGL2 in the presence of various reactive oxygen species scavengers. Damage to the pGL2 gene was measured by the loss of its enzyme activity. They noted that nitric oxide synthase inhibitors, but not other reactive oxygen species scavengers, suppressed arsenite inhibition of pGL2 expression. In addition, they noted that treatment of UV-irradiated cells with a nitric oxide donor, in the absence of arsenite, inhibited pGL2 expression by a comparable degree to arsenite. From these observations, they concluded that NO is involved in arsenite inhibition of pyrimidine dimer repair [6].

NO and p53: New Views Concerning Their Interaction. Studies have consistently shown that DNA damage caused by NO activates p53, an important transcription factor that can initiate cell cycle arrest and DNA repair in response to mutagens. In their review on NO and p53 interaction, Ambs et al. report that nitric oxide synthesis is increased in 25-40% of cancer cases in which p53 is mutated and inactivated, indicating that, in normal cells, p53 also prevents NO from further damaging DNA by inhibiting NO synthesis. They suggest that increased nitric oxide levels may contribute to cancer progression by directly introducing mutations into the p53 locus or further damaging the DNA in cells with mutated dysfunctional p53 [7].
         Chazotte-Aubert et al. recently presented a novel insight that may expand the current knowledge of NO’s role in cancerous cells by suggesting that NO can directly interfere with wild-type p53 regulation. They reported that high levels of NO directly modify the structure of wild-type p53 so that it can no longer bind to DNA in vitro [8]. Using immunoprecipitation and Western blotting with anti-NTYR, Chazotte-Aubert et al. showed for the first time that tyrosine residues on p53 are nitrosylated in MCF-7 breast cancer cells that constitutively express nitric oxide synthase [8]. Although Chazotte-Aubert et al. have not yet established whether or not tyrosine nitrosylation of p53 actually results in loss of function, their study has generated a new hypothesis that NO can modify not only the enzymes directly involved in DNA repair but also critical regulators of DNA repair and the cell cycle. To investigate this hypothesis further, Hofseth et al. demonstrated in a similar experiment that specific sites of p53 are phosphorylated and acetylated in MCF-7 cells in response to NO donors [9]. In their case, G1 cell cycle arrest accompanied the p53 modifications, but DNA damage still occurred with increasing exposure to NO, suggesting tentatively that NO interferes with p53’s ability to initiate the repair pathway.

V. Conclusions
         The current literature suggests that NO, a reactive biomolecule, can attack the cell’s DNA repair machinery—and thus promote carcinogenesis—from several vantage points. The most fully elucidated strategy is nitrosylation of repair enzymes, which can lead to activity inhibition and, consequently, accumulation of DNA damage. This mode of attack is a strong candidate for a link between inflammation and carcinogenesis since both inflamed tissue and cancer resulting from inflammation express high levels of NO. On the other hand, NO-mediated repair inhibition—in addition to NO-mediated DNA damage—has recently been suggested to be responsible for the mutagenic effects of arsenite. Further studies will probably be required to confirm this conclusion and elucidate the step-wise mechanism that may link arsenite to NO production. A few recent studies suggest another vantage point upstream of repair enzymes where NO can inhibit DNA repair—the p53 protein. Though research on this topic has not yet demonstrated that NO can actually alter the function of this critical cellular regulator, there is a consensus among reports that NO modifies this protein by reacting with specific residues such as tyrosine. The significance of NO-induced p53 modification may become clearer as further studies are undertaken.
         DNA repair interference is by no means nitric oxide’s only carcinogenic property, but it has received less attention in the current literature than less well-understood properties such as promotion of tumor progression and apoptosis inhibition in certain cells. However, the arsenite and p53 studies suggest that research on NO repair interference may be revived as more carcinogenic processes are linked to NO mutagenicity. After determining the mechanisms by which NO can react with and impair the repair machinery, scientists will probably move their focus towards therapeutic or chemopreventive measures to prevent NO from promoting malignancies.


VI. References

1. Felley-Bosco E. (1998). Role of nitric oxide in genotoxicity. Cancer and Metastasis Reviews 17: 25-37.

2. Jaiswal M., LaRusso N. F., Nishioka N., Nakabeppu Y., & Gores G. J. (2001). Human Ogg1, a protein involved in the repair of 8-oxoguanine, is inhibited by nitric oxide. Cancer Research 61: 6388-6393.

3. Jaiswal M., LaRusso N. F., Burgart L. J. & Gores G. J. (2000). Inflammatory cytokines induce DNA damage and inhibit DNA repair in cholagiocarcinoma cells by a nitric oxide-dependent mechanism. Cancer Research 60: 184-190.

4. Jaiswal M., LaRusso N. F. & Gores G. F. (2001). Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis. American Journal of Physiology Gastrointestinal Liver Physiology 281: G626-G634.

5. Liu F. & Jan K. Y. (2000). DNA damage in arsenite- and cadmium-treated bovine aortic endothelial cells. Free Radical Biology and Medicine 28: 55-63.

6. Bau D.T., Gurr J. R. & Jan K. Y. (2001). Nitric oxide is involved in arsenite inhibition of pyrimidine dimer excision. Carcinogenesis 22: 709-716.

7. Ambs S., Hussain S. P. & Harris C. C. (1997). Interactive effects of nitric oxide and the p53 tumor supressor gene in carcinogenesis and tumor progression. The FASEB Journal 11: 443-446.

8. Chazotte-Aubert L., Hainaut P. & Ohshima H. (2000). Nitric oxide nitrates tyrosine residues of tumor-supressor p53 protein in MCB-7 cells. Biochemical and Biophysical Research Communications 267: 609-613, 2000.

9. Hofseth L.J., Saito S., Espey M. G., Miranda K. M., Wink D. A., Hussain P., Pauls M. K., Zurer I., Rotter V., Appella E. & Harris C. C. (2001). Post-translational modifications to p53 following exposure to nitric oxide. Carcinogenesis 42: 16.