Role of Chronic Inflammation and Resulting DNA Damage in Cervical Carcinogenesis Induced by Human Papillomavirus

Cervical cancer is the second most common cancer among women in the worldwide. Especially, in many regions of developing countries, cervical cancer is the most common among women. Infection with human papillomavirus (HPV) is a necessary event preceding the development of premalignant lesions in the cervical epithelium, which can partially progress to cancer. HPV DNA can be identified in almost all specimens of patients with invasive cervical cancer (Chen & Hunter, 2005; Munoz et al., 2003; Tindle, 2002). Molecular epidemiological studies have demonstrated that specific subtypes of HPV are closely associated with cervical cancer, although the risk of cervical cancer varies with HPV types (Bosch et al., 2002; Chen & Hunter, 2005; International Agency for Research on Cancer [IARC] Working Group, 2007; 2011; Munoz et al., 2003; Sisk & Robertson, 2002; Tindle, 2002). IARC has classified several types of high-risk HPV, including HPV-16 and HPV-18, to be carcinogenic to humans (group 1) (IARC Working Group, 2007; 2011).

associated factor (Huh et al., 2005). E7 also inactivates the cyclin-dependent kinase (CDK) inhibitors, p21 and p27 (Duensing & Munger, 2004). E7-induced RB degradation leads to the release of the transcription factor E2F from the RB/E2F transcriptional repressor complex (von Knebel Doeberitz, 2002). Activation of E2F mediates gene transcription with increased expression of cyclin E and cyclin A and aberrant CDK2 activity (Duensing & Munger, 2004). In addition, these oncoproteins cause genomic instability (Duensing & Munger, 2004). E6 and E7 cooperatively induce numerical centrosome aberrations and eventual aneuploidy in cells overexpressing these oncoproteins (Duensing et al., 2000). E7 protein of high-risk type HPV induced chromosome overduplication associated with aberrant multipolar spindle pole formation, while E6 had no immediate effects on centrosome numbers but potentiated mitotic disturbance (Duensing et al., 2000). On the basis of these numerous studies, E6 and E7 oncoproteins are considered to participate in cervical carcinogenesis by inducing cell immortalization, dysregulation of cell proliferation and chromosomal instability.
However, it has been reported that these oncoproteins are insufficient to transform human cells, and additional cellular events are required for cervical carcinogenesis (Duensing & Munger, 2004). The activation of Ha-ras in HPV16-immortalized human cervical cells resulted in malignancy, while transfection of HPV-16 DNA alone into cervical cells did not (DiPaolo et al., 1989). Human protooncogenes, including the c-Ha-ras gene, can be activated via oxygen radical-induced DNA damage (Du et al., 1994). HPV oncoprotein-expressing cells have an impaired ability to respond to DNA damage (Kessis et al., 1993;Song et al., 1998). These findings raise the possibility that additional factors other than HPV infection mediate DNA damage and participate in carcinogenesis. Recent epidemiological and experimental studies have demonstrated that chronic inflammation contributes to cervical carcinogenesis as described in the following section. In this review, the role of inflammation and resulting DNA damage in cervical carcinogenesis and the molecular mechanisms will be discussed.

Involvement of chronic inflammation in cervical carcinogenesis
In 19th century, Rudolf Virchow noted leucocytes in neoplastic tissues and suggested that the "lymphoreticular infiltrate" reflected the origin of cancer at sites of chronic inflammation (Balkwill & Mantovani, 2001). Since then, there has been a growing research interest in the link between chronic inflammation and carcinogenesis. Actually, many malignancies arise from areas of infection and inflammation (Balkwill & Mantovani, 2001;Coussens & Werb, 2002). Epidemiological and experimental studies have provided evidence indicating that chronic infection and inflammatory conditions contribute to a substantial part of environmental carcinogenesis (Coussens & Werb, 2002;IARC, 2003). A recent review has estimated that chronic inflammation accounts for approximately 25 % of human cancers (S. P. Hussain & Harris, 2007). Infection with bacteria, viruses and parasites contributes to a substantial part of chronic inflammation. IARC has estimated that infectious diseases account for approximately 18 % of cancer cases worldwide, which are largely attributed to infection with oncogenic viruses, including HPV (IARC, 2003) (

Epidemiological studies on chronic inflammation and cervical carcinogenesis
Recent epidemiological and experimental studies have revealed that chronic inflammation is associated with HPV-induced cervical carcinogenesis, although it is still unclear whether HPV infection alone or co-infection with HPV and other pathogens induces inflammatory conditions. An epidemiological study in Costa Rica revealed that there was a positive trend of increasing cervical inflammation associated with high-grade lesions in oncogenic HPVinfected women, and proposed a possibility that cervical inflammation is a cofactor for HPV-induced carcinogenesis (Castle & Giuliano, 2003;Castle et al., 2001). Several epidemiological studies have suggested that other pathogens act in conjunction with HPV infection to increase the risk of cervical cancer. Chlamydia trachomatis infection increased the risk of squamous cervical cancer among HPV-positive women in Brazil and the Philippines (Smith et al., 2002b). Among the HPV DNA-positive women, seropositivity of herpes simplex virus-2 was associated with increased risks of squamous-cell carcinoma and adenoor adenosquamous-cell carcinoma (Smith et al., 2002a). In a study using cervical smears, the relative risk of high grade squamous intraepithelial lesions was significantly higher in patients infected with Gardinerella vaginalis and Chramydia and with dysbacteriosis and nonspecific inflammatory changes (leucocytosis) compared with normal subjects (Roeters et al., 2010). In addition, there is a study showing that biopsy specimens of women infected with carcinogenic HPV type had greater inflammation in the epithelium compared with those of women positive for noncarcinogenic HPV type and negative for HPV, although cervical inflammation varies with HPV cofactors, type of HPV infection, and risk of persistence and www.intechopen.com progression (Kovacic et al., 2008). Recently, meta-analysis showed that bacterial vaginosis is significantly and positively associated with cervical HPV infection (Gillet, et al., 2011), supporting the hypothesis that cervical inflammation is involved in the pathogenesis of HPV-induced cancer.

Molecular epidemiological studies on cervical carcinogenesis
The expression of inflammation-related molecules in cervical tissues and the association with HPV-induced carcinogenesis have been extensively investigated. Activation of the transcription factor nuclear factor (NF)-κB has been observed in cervical tissues of patients with squamous intraepithelial lesions. In normal cervical tissue and low-grade squamous intraepithelial lesions, NF-κB subunits, p50 and p65, and inhibitor IκBα (IκBα) were mainly localized in the cytosol, whereas in high-grade lesions and squamous cell carcinomas, the p50-p65 heterodimer was translocated into the nucleus and the expression of IκBα protein was concurrently decreased (A. Nair et al., 2003). NF-κB is a key player in inflammation and regulates the expression of various genes involved in controlling the inflammatory response, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-1β and tumor necrosis factor (TNF)-α (Karin, 2006;Kundu & Surh, 2008). NF-κB also participates in the promotion and progression of inflammation-related cancer (Karin, 2006;Pikarsky et al., 2004). NF-κB mediates the expression of matrix metalloprotease (MMP) 9 and the angiogenic factor, vascular endothelial growth factor (VEGF) (Karin, 2006), which are considered to participate in tumor progression and metastasis.
COX-2 mediates cancer development via various pathogenic events, including inflammatory responses, apoptosis inhibition and angiogenesis (Chun & Surh, 2004;Warner & Mitchell, 2004;Williams et al., 2000). Molecular epidemiological studies have shown the overexpression of COX-2 in cervical cancer (Kim et al., 2004;Kulkarni et al., 2001). Patients positive for both COX-2 and epidermal growth factor receptor (EGFR) had a higher likelihood of locoregional recurrence and worse prognosis than those negative for one or both proteins (Kim et al., 2004).
In biopsy specimens of cervical intraepithelial neoplasia (CIN) 3 patients, activation of signal transducer and activator of transcription (STAT) 3 and coexpression of MMP9 have been detected in perivascular inflammatory cells. STAT3 induced the expression of the chemokine CCL2, followed by MMP9 expression in tumor-instructed monocytes (Schroer et al., 2011). Recently, it has been reported that the plasma levels of cytokines, especially IL-6, IL-8, TNF-α and macrophage inflammatory protein-1α (MIP-1α), were significantly increased in HPV-positive women relative to HPV-negative controls (Kemp et al., 2010). Therefore, the evidence for the participation of inflammatory responses in cervical carcinogenesis has been accumulating, although further studies are required to clarify the precise molecular mechanism.

DNA damage mediated by reactive species under inflammatory conditions
DNA damage is a key molecular event causing genetic instability involved in induction of human carcinogenesis. Under inflammatory conditions, reactive oxygen species (ROS) and reactive nitrogen species (RNS), including nitric oxide (NO), are generated from inflammatory and epithelial cells. These species are highly reactive and capable of causing oxidative and nitrative DNA damage, which may contribute to carcinogenesis (S. P. Hussain www.intechopen.com J. Nair et al., 2006;Ohshima et al., 2003). ROS can induce the formation of various oxidative DNA lesions, including mutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) (Kawanishi et al., 2001;Tazawa et al., 2003;Wiseman & Halliwell, 1996). If 8-oxodG is not correctly repaired, adenine is preferentially incorporated opposite 8-oxodG during DNA synthesis, leading to G → T transversion (David et al., 2007;Shibutani et al., 1991). Accumulation of 8-oxodG in human body has been demonstrated in patients with cancer and cancer-prone diseases. A significant increase in urinary 8-oxodG levels has been observed in patients with various types of cancer compared with those in control subjects (Erhola et al., 1997;Tagesson et al., 1995;Thanan et al., 2008). The content of 8-oxodG in cervical cells significantly increased with the grade of squamous intraepithelial lesion (Romano et al., 2000), raising the possibility that oxidative DNA damage participates in cervical carcinogenesis.
Whereas ROS are generated from multiple sources, including not only inflammatory cells but also carcinogenic chemicals and electron transport chain in mitochondria, RNS are primarily generated under inflammatory conditions . Therefore, RNS-mediated DNA lesions may play an important role in inflammation-related carcinogenesis, and are expected as potential biomarkers to evaluate the cancer risk. 8-Nitroguanine is a nitrative DNA lesion, formed under inflammatory conditions. NO and superoxide (O 2 • -) are generated from inflammatory and epithelial cells, and react with each other to form peroxynitrite (ONOO -), a highly reactive species causing nitrative and oxidative DNA damage. In vitro experiments revealed that the interaction of guanine with ONOOled to the formation of 8-nitroguanine (Yermilov et al., 1995a), in addition to 8-oxodG (Inoue & Kawanishi, 1995) (Figure 1). In an in vivo experiment, 8-nitroguanine was formed via inflammation in the lung tissues of mice with viral pneumonia (Akaike et al., 2003).
8-Nitroguanine formed in DNA is chemically unstable, and can be spontaneously released, resulting in the formation of an apurinic site (Yermilov et al., 1995b). Adenine is preferentially incorporated opposite an apurinic site during DNA synthesis, leading to G → T transversion (Loeb & Preston, 1986). Translesion DNA synthesis is the process in which error-prone DNA polymerases bypass unrepaired DNA lesions or insert nucleotides opposite the lesions. Cells deficient in subunits of DNA polymerase ζ, were hypersensitive to NO, and translesion DNA synthesis past apurinic site mediated by this polymerase might contribute to extensive point mutations (Wu et al., 2006). It has also been reported that adenine is preferentially incorporated opposite 8-nitroguanine during DNA synthesis mediated by DNA polymerase η and κ, leading to G → T transversion (Suzuki et al., 2005). Therefore, 8-nitroguanine is a potentially mutagenic DNA lesion, which may contribute to inflammation-related carcinogenesis. In the ONOO --treated supF shuttle vector plasmid, which was then replicated in Escherichia coli, the majority of mutations occurred at G:C base pairs, predominantly involving G → T transversions (Juedes & Wogan, 1996; M.Y. . Indeed, this type of mutation occurred in vivo in the ras gene (Bos, 1988) and the p53 tumor suppressor gene in lung and liver cancer (Hsu et al., 1991;Takahashi et al., 1989). G → T transversions were most prominently detected in the omentum of asbestos-exposed rats (Unfried et al., 2002). These findings imply that DNA damage mediated by inflammatory reactions may participate in carcinogenesis via activation of protooncogenes and inactivation of tumor suppressor genes. www.intechopen.com

DNA damage in cervical tissues of CIN patients
To clarify the role of inflammation-mediated DNA damage in cervical carcinogenesis, we performed immunofluorescence staining to examine the formation of 8-nitroguanine and 8-oxodG in biopsy specimens obtained from CIN patients. To detect 8-nitroguanine, we produced a specific antibody and used for experiments (Hiraku & Kawanishi, 2009;Pinlaor et al., 2004a). We compared the fluorescent intensity of these DNA lesions in cervical tissues o f p a t i e n t s w i t h d i f f e r e n t s t a g e s o f C I N c a u s e d b y h i g h -r i s k H P V a n d c o n d y l o m a acuminatum, benign cervical warts caused by low-risk HPV. In biopsy specimens of CIN patients, 8-nitroguanine formation was observed in the nuclei of atypical cells (Figure 2), and 8-oxodG showed a similar staining pattern to 8-nitroguanine. Their staining intensity tended to increase with CIN grades. Statistical analysis revealed that the immunoreactivity of 8-nitroguanine in cervical epithelium was significantly increased in the order of condyloma acuminatum < CIN1 < CIN2-3, while there was no significant difference in 8-oxodG formation (Table 2). iNOS expression was also observed in the cytoplasm of both cervical epithelial cells and infiltrating stromal inflammatory cells in CIN patients. 8-Nitroguanine formation was observed in the majority of iNOS-positive epithelial cells. On the other hand, no or weak 8-nitroguanine formation occurred in cervical tissues from patients with condyloma acuminatum. These results raise the possibility that 8-nitroguanine can be used a potential biomarker to evaluate the risk of cervical carcinogenesis.  The formation of nitrative DNA lesion in relation to cervical carcinogenesis has been supported by a recent study. NO exposure induced DNA damage and increased mutation rates in HPV-positive human cervical epithelial cell lines established from CIN patients (Wei et al., 2009). In addition, NO exposure increased the expression of E6 and E7 genes, resulting in decreased p53 and RB protein levels in these cells (Wei et al., 2009), although precise molecular mechanism has not been understood. These findings raise the possibility that NO serves as a molecular cofactor with HPV infection, and resulting DNA damage and the expression of viral oncoproteins cooperatively contribute to HPV-mediated cervical carcinogenesis. Therefore, modification of local NO concentration in cervical tissues and related molecular events may constitute a strategy to prevent HPV-related cancer. www.intechopen.com

Comparison of 8-nitroguanine formation and p16 expression in CIN patients
There are several reports showing that the cyclin-dependent kinase inhibitor p16 is overexpressed in cervical neoplasia (Gupta et al., 2010;Klaes et al., 2001;Sano et al., 1998;von Knebel Doeberitz, 2002; J. L. Wang et al., 2004), and proposed as a potential biomarker of cervical carcinogenesis. The expression of p16 was significantly higher in CIN and s q u a m o u s c e l l c a n c e r t h a n i n n o r m a l o r i n flammation of the cervix, and p16-positive patients had significantly shorter interval for disease progression from initial biopsy to CIN 3 or invasive cancer than p16-negative patients (J. L. Wang et al., 2004). E7 oncoprotein binds to RB, leading to the release of the transcription factor E2F from the RB/E2F complex (von Knebel Doeberitz, 2002). E2F accumulation leads to the expression of p16-related transcript, although the molecular mechanism remains to be clarified (Khleif et al., 1996). Paraffin sections were incubated with the primary antibodies (rabbit polyclonal anti-8nitroguanine and mouse monoclonal anti-p16 antibodies), and then with the secondary antibodies (Alexa 594-labeled goat anti-rabbit IgG and Alexa 488-labeled goat anti-mouse IgG antibodies). Strong p16 expression (green) was observed in cervical epithelial cells of CIN2 patients and showed a similar pattern to 8-nitroguanine formation (red). In patients with condyloma acuminatum and cervicitis (HPV-16-positive), p16 expression was observed in the basal layer but no or weak 8-nitroguanine formation was detected. Ep, epithelium. St, stroma. Scale bar = 50 µm.

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We performed immunohistochemistry to compare 8-nitroguanine formation and p16 expression in cervical biopsy specimens of HPV-infected patients. Strong p16 expression was observed in cervical epithelial cells of CIN patients and showed a similar pattern to 8nitroguanine ( Figure 3). However, in cervical tissues of patients with condyloma acuminatum and HPV-positive cervicitis, p16 expression was observed in the basal layer, whereas no or weak 8-nitroguanine formation was detected. Statistical analysis revealed that no significant difference in p16 expression was observed among condyloma and CIN groups, whereas there was significant difference in 8-nitroguanine formation as described above (Table 2). These results suggest that p16 is simply a marker to reflect HPV infection, and that 8-nitroguanine is more suitable marker to discriminate high-risk and low-risk cervical lesions.

8-Nitroguanine formation in inflammation-related carcinogenesis
In addition to cervical cancer, we have investigated 8-nitroguanine formation in tissues of patients and animal models of a wide variety of inflammation-related cancer and cancerprone diseases by immunohistochemical analysis. We first demonstrated that this DNA lesion was formed at the site of carcinogenesis induced by bacterial, viral, and parasitic infections (Hiraku, 2010;. The studies on 8-nitroguanine formation in relation to inflammation-related carcinogenesis are summarized in Table 3.  Table 3. 8-Nitroguanine formation in humans and animals in relation to inflammationrelated carcinogenesis

Infectious diseases
We found that 8-nitroguanine was formed in epithelial cells of intrahepatic bile duct using an animal model infected with the liver fluke Opisthorchis viverrini, endemic in northeastern Thailand (Pinlaor et al., 2004a;Pinlaor et al., 2004b;Pinlaor et al., 2003). Administration of the antiparasitic drug praziquantel and the antioxidant curcumin significantly reduced oxidative and nitrative DNA damage Prakobwong et al., 2011). We have also demonstrated that 8-nitroguanine was formed in the gastric gland epithelial cells from gastritis patients with Helicobacter pylori infection (Ma et al., 2004), in the hepatocytes of patients with chronic hepatitis C (Horiike et al., 2005) and in the nasopharyngeal epithelial cells and tumor tissues of patients infected with Epstein-Barr virus (EBV) (Ma et al., 2008). These results suggest that 8-nitroguanine is a promising biomarker providing an assessment of the risk of inflammation-mediated carcinogenesis at the precancerous stage. Moreover, in EBV-infected patients, the staining intensity of 8-nirtroguanine was significantly stronger in nasopharyngeal cancer cells than in epithelial cells of chronic nasopharyngitis patients, suggesting that 8-nitroguanine accumulates during the development of chronic inflammation to cancer (Ma et al., 2008). In addition, 8-nitroguanine formation was observed under inflammatory conditions independent of infection. This DNA lesion was seen in colon epithelial cells of a mouse model of inflammatory bowel disease (Ding et al., 2005), in oral epithelial cells of patients with oral premalignant lesions (Chaiyarit et al., 2005;Ma et al., 2006) and in bronchial epithelial cells of asbestos-exposed mice .
Several studies have raised a possibility that DNA damage is involved in tumor progression. In patients with inhrahepatic cholongiocarcinoma, oxidative and nitrative DNA damage in tumor and adjacent tissues was associated with tumor invasion  8-Nitroguanine staining was observed in tumor tissues of patients with malignant fibrous histiocytoma, a soft tissue tumor, which is proposed to be accompanied with inflammatory responses. The statistical analysis using the Kaplan-Meier method revealed that strong 8-nitroguanine formation was significantly associated with a poor prognosis of the patients (Hoki et al., 2007a). These findings indicate that this DNA lesion contributes to not only tumor initiation but also tumor progression and poor prognosis of cancer patients.

Mechanism of inflammation-related carcinogenesis and the role of DNA damage
On the basis of our previous studies, possible mechanism of inflammation-related carcinogenesis and tumor development via DNA damage is shown in Figure 4. Various infectious agents, including bacteria, viruses and parasites, inflammatory diseases and environmental factors can induce inflammatory responses and the production of ROS and RNS from inflammatory and epithelial cells. A wide variety of inflammatory cytokines mediate the activation of transcription factors, including NF-κB and STATs. NF-κB regulates the expression of a wide variety of inflammation-related molecules including iNOS (Karin, 2006;Kundu & Surh, 2008) and participates in multiple steps of carcinogenesis (Karin, 2006;Pikarsky et al., 2004). RNS can induce the activation of NF-κB under certain circumstances (Janssen-Heininger et al., 2000). Therefore, reciprocal and positive regulation between RNS and NF-κB may lead to persistent inflammatory reactions and nitrative DNA damage, contributing to carcinogenesis.
www.intechopen.com STAT1 and STAT3 are known to mediate the expression of iNOS (Lo et al., 2005;Tedeschi et al., 2003). STAT3 interacts with EGFR in the nucleus to form a complex, which mediates transcriptional activation of iNOS (Lo et al., 2005). Actually, phosphorylated STAT3 (active form) and EGFR were strongly expressed and colocalized in the nucleus of cancer cells of nasopharyngeal cancer patients (Ma et al., 2008). A recent study has demonstrated that excess ONOOmediates the activation of JAK/STAT signalling pathway in experimental animals (H. Wang et al., 2009). These findings imply that a positive loop between STATs and iNOS may exist.
Hypoxia-inducible factor (HIF)-1α is an oxygen-sensing transcription factor, which is upregulated in a hypoxic environment during tumor growth. HIF-1α mediates the transcription of various genes, including iNOS and VEGF (Harris, 2002). An increase in www.intechopen.com iNOS-catalyzed NO production induces the accumulation and activation of HIF-1α (Mateo et al., 2003;Thomas et al., 2004). HIF-1α and 8-nitroguanine were colocalized and associated with poor prognosis of cancer patients (Hoki et al., 2007b). Therefore, reciprocal activation of HIF-1α and iNOS may lead to persistent DNA damage in tumor tissues, contributing to poor prognosis of cancer patients. A recent study has demonstrated that IκB kinase (IKK)-β, involved in NF-κB activation, is required for HIF-1α protein accumulation under hypoxia in cultured cells and animals (Rius et al., 2008), whereas NF-κB bas been reported to be regulated under hypoxia in an HIF-1α-dependent manner (Walmsley et al., 2005). Thus, HIF-1α and NF-κB may positively regulate each other and participate in tumor progression. Collectively, molecular events mediated by various pathogens converge to nitrative stress, and resulting DNA damage contributes to the accumulation of genetic alterations in tissues throughout the carcinogenic process. In particular, 8-nitroguanine formation may participate in inflammation-related carcinogenesis as a common mechanism, regardless of etiology. Therefore, 8-nitroguanine could be used as a potential biomarker to evaluate the cancer risk and predict the prognosis of cancer patients. In addition, certain pathogens possess unique molecules mediating abnormal cell proliferation and survival, such as E6 and E7 proteins of HPV as described below. Therefore, 8-nitroguanine formation may contribute to carcinogenesis in cooperation with pathogen-specific molecular events.
A recent study has demonstrated that 8-nitro-cGMP, formed by the reaction of RNS with cGMP, regulates the redox-sensor signaling proteins, via S-guanylation of cysteine sulphydrils, and mediates an adaptive response against oxidative and nitrative stress (Sawa www.intechopen.com et al., 2007). S-Nitrosylated proteins, formed via reaction of thiols with NO or its reactive metabolites, possess cytoprotective properties (Ishima et al., 2007). In inflammation-related carcinogenesis, nitrated or nitrosylated molecules may participate in protection of initiated cells bearing genetic alterations due to oxidative and nitrative DNA damage, and contribute to tumor development.

Mechanism of HPV-mediated cervical carcinogenesis via inflammatory reactions
A possible mechanism of HPV-induced cervical carcinogenesis involving inflammatory reactions is shown in Figure 5. We have demonstrated that inflammation-mediated DNA lesions were formed in cervical tissues of CIN patients (Hiraku et al., 2007). To date, no evidence suggesting that HPV infection alone induces inflammatory states has been provided. Therefore, it is speculated that inflammatory reactions are derived from HPV infection and co-infection with other pathogens, although the precise mechanism remains to be clarified. DNA damage mediated by inflammatory reactions would play a substantial role in tumor initiation and also the following steps of carcinogenesis. In addition, HPV oncoproteins E6 and E7 mediate cell immortalization, dysregulation of cell proliferation and chromosomal abnormalities by interacting with numerous target proteins. The molecular events mediated by these oncoproteins may promote the proliferation and transformation of cells initiated by oxidative and nitrative DNA damage, and contribute to tumor promotion and progression.
In addition, several studies have demonstrated that HPV oncoproteins mediate inflammatory responses. The expression of E6 and E7 oncoproteins derived from highly carcinogenic HPV-16 enhanced the release of IL-1α from cultures of normal cervical keratinocytes, and E7 proteins that strongly bound to RB protein (high risk types HPV-16 and HPV-18) induced more IL-1α release than those that bound poorly (low-risk type HPV-6) (Iglesias et al., 1998). A recent study has shown that E7 expression increased the promotor activity of COX-2 and the downstream molecule IL-32 in HPV-positive cervical cancer cell lines (Lee et al., 2011). IL-32 induces the expression of various inflammatory cytokines, including IL-1β, IL-6, TNF-α and chemokines (S.H. . IL-32 expression was detected in cervical tissues of patients with squamous cell carcinoma and increased with the tumor stage (Lee et al., 2011). Therefore, these HPV oncoproteins may participate in inflammatory responses in cervical tissues and contribute to carcinogenesis. In addition, a recent study has shown that NO treatment mediates the expression of E6 and E7 in cultured cells (Wei et al., 2009). A COX-2-selective inhibitor suppressed the expression of E7 in cultured cervical cancer cells (Lee et al., 2011). Inflammatory reactions may reciprocally mediate the expression of HPV oncoproteins, resulting in the persistence of inflammation-related DNA damage and dysregulated cell proliferation, contributing to tumor progression.

Conclusion and future perspective
Chronic infection and inflammation are known to contribute to a substantial part of environmental carcinogenesis. Cervical cancer is the second most common cancer among women, and HPV infection is involved in almost all cases. Recent epidemiological studies revealed that chronic inflammation participates in cervical carcinogenesis. Under www.intechopen.com inflammatory conditions, reactive oxygen and nitrogen species are generated, and resulting DNA damage may play an important role in carcinogenesis.
We performed immunohistochemical analysis and demonstrated that 8-nitroguanine, a mutagenic DNA lesion formed during inflammation, was formed in cervical epithelial cells in CIN patients, and its immunoreactivity was significantly increased with CIN grades. The comparison of 8-nitroguanine formation and p16 expression revealed that 8-nitroguanine is more suitable to detect cervical lesions mediated by high-risk types of HPV. Taken together with this and previous studies, it is concluded that 8-nitroguanine can be used as a potential biomarker to evaluate the risk of inflammation-related carcinogenesis, including cervical carcinogenesis. In addition, HPV oncoproteins E6 and E7 are known to mediate cell immortalization, dysregulated cell proliferation and chromosomal instability. Several studies have suggested that these oncoproteins crosstalk with signalling pathways related to inflammatory responses. Therefore, oxidative and nitrative DNA damage mediated by inflammatory reactions and oncoprotein-mediated molecular events may cooperatively contribute to cervical carcinogenesis.
Establishment of methods for quantitative analysis of 8-nitroguanine in biological samples, such as blood and urine, would be useful for evaluation of the risk of inflammation-related carcinogenesis. 8-Nitroguanine formed in DNA is chemically unstable, and is likely to be released from DNA. Thus, this property may hamper the quantitative analysis. An attempt has been made to utilize free 8-nitroguanine in urine for quantitative analysis using highperformance liquid chromatography coupled with electrochemical detection and immunoaffinity purification (Sawa et al., 2006). 8-Nitroguanine has also been measured by liquid chromatography with mass spectrometry and glyoxal derivatization (Ishii et al., 2007).
Moreover, the development of therapeutics targeting inflammation-related molecules may contribute to prevention of cervical carcinogenesis and improvement of prognosis of cancer patients. Animal experiments demonstrated that iNOS inhibitors suppress inflammatory responses and effectively reduce inflammation-related carcinogenesis, although evidence has not yet been provided for their inhibitory effect on cervical carcinogenesis. ONO-1714, an iNOS-specific inhibitor, significantly decreased the degree of cholangitis and reduced the incidence of intrahepatic biliary tumors in bilioenterostomized hamsters (Mishima et al., 2009). Another iNOS inhibitor, 1400W, reduced tumorigenesis in the mammary glands of γirradiated mice treated with diethylstilbestrol (Inano & Onoda, 2005).
Moreover, administration of the antioxidant curcumin significantly reduced the incidence of liver fluke-induced carcinogenesis via suppression of oxidative and nitrative DNA damage (Prakobwong et al., 2011). To develop a strategy for prevention of HPV-mediated cervical cancer, further studies are needed to clarify the precise molecular mechanisms and the role of chronic inflammation.

Acknowledgement
The author is grateful to Dr. Tsutomu Tabata (Mie University Graduate School of Medicine, Japan) and Professor Ning Ma (Suzuka University of Medical Sciences, Japan) for clinical and technical advices, respectively. The author is also grateful to Professor Shosuke Kawanishi (Suzuka University of Medical Sciences), Professor Mariko Murata (Mie www.intechopen.com University Graduate School of Medicine) and all collaborators for the encouragement throughout this work. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labour and Welfare of Japan.