**3.1.3 Epstein-Barr virus and nasopharyngeal carcinoma**

NPC is a human epithelial tumor with a high prevalence in the southern Chinese population. In southern China, the incidence rate is about 25–50 per 100,000 person-year and 100-fold higher than that in the Western world. In contrast to other head cancer and epithelial malignancy in general, a unique feature of NPC is its strong association with Epstein-Barr virus (EBV) (McDermott et al., 2001). Various transcription factors are known to participate in iNOS expression including signal transducers and activators of transcription (STATs), such as STAT1 and STAT3 (Tedeschi et al., 2003; Lo et al., 2005). Epidermal growth factor receptor (EGFR) physically interacts with STAT3 in the nucleus, leading to transcriptional activation of iNOS (Lo et al., 2005). STAT3 is repeatedly activated through phosphorylation via the expression of latent membrane protein 1 (LMP1) as well as EGFR (Chen et al., 2003; Tao et al., 2005), and interleukin-6 (IL-6) is required for LMP1-mediated STAT3 activation (Chen et al., 2003). In addition, LMP1-mediated iNOS expression was reported in EBV-infected epithelium cell lines, which play a role in colonization independent of anchorage and tumorigenicity in nude mice (Yu et al., 2002). We performed double immunofluorescent staining method to examine the formation of DNA lesions, 8-nitroguanine and 8-oxodG in surgical and biopsy specimens of nasopharyngeal tissues from NPC patients and chronic nasopharyngitis patients. We also examined the expression of iNOS, STAT3, EGFR and IL-6 in these specimens to examine contribution of these molecules to iNOS expression for 8-nitroguanine formation.

Using biopsy and surgical specimens of nasopharyngeal tissues from NPC patients in southern China, we performed double immunofluorescent staining to examine the formation of 8-nitroguanine and 8-oxodG (Ma et al., 2008; Huang et al., 2011). Intensive immunoreactivity of iNOS was detected in the cytoplasm of 8-nitroguanine-positive cancer cells. DNA lesions and iNOS expression were also observed in epithelial cells of EBV-positive patients with chronic nasopharyngitis but weaker than those in NPC patients (Fig. 7.). No or few DNA lesions were observed in EBV-negative subjects. EGFR and phosphorylated STAT3 were strongly expressed in cancer cells of NPC patients, suggesting that the STAT3-dependent mechanism is important to the carcinogenesis (Ma et al., 2008). IL-6 was expressed mainly in inflammatory cells of nasopharyngeal tissues of EBV-infected patients. We also found that serum levels of 8-oxodG were significantly higher in NPC patients than control subjects (Huang et al., 2011). Collectively, these findings indicate that the nuclear accumulation of EGFR and activation of STAT3 by IL-6 play a key role in iNOS expression and resultant DNA damage, leading to EBV-related NPC.

NPC is a human epithelial tumor with a high prevalence in the southern Chinese population. In southern China, the incidence rate is about 25–50 per 100,000 person-year and 100-fold higher than that in the Western world. In contrast to other head cancer and epithelial malignancy in general, a unique feature of NPC is its strong association with Epstein-Barr virus (EBV) (McDermott et al., 2001). Various transcription factors are known to participate in iNOS expression including signal transducers and activators of transcription (STATs), such as STAT1 and STAT3 (Tedeschi et al., 2003; Lo et al., 2005). Epidermal growth factor receptor (EGFR) physically interacts with STAT3 in the nucleus, leading to transcriptional activation of iNOS (Lo et al., 2005). STAT3 is repeatedly activated through phosphorylation via the expression of latent membrane protein 1 (LMP1) as well as EGFR (Chen et al., 2003; Tao et al., 2005), and interleukin-6 (IL-6) is required for LMP1-mediated STAT3 activation (Chen et al., 2003). In addition, LMP1-mediated iNOS expression was reported in EBV-infected epithelium cell lines, which play a role in colonization independent of anchorage and tumorigenicity in nude mice (Yu et al., 2002). We performed double immunofluorescent staining method to examine the formation of DNA lesions, 8-nitroguanine and 8-oxodG in surgical and biopsy specimens of nasopharyngeal tissues from NPC patients and chronic nasopharyngitis patients. We also examined the expression of iNOS, STAT3, EGFR and IL-6 in these specimens to examine contribution of these molecules to iNOS expression for 8-nitroguanine

Using biopsy and surgical specimens of nasopharyngeal tissues from NPC patients in southern China, we performed double immunofluorescent staining to examine the formation of 8-nitroguanine and 8-oxodG (Ma et al., 2008; Huang et al., 2011). Intensive immunoreactivity of iNOS was detected in the cytoplasm of 8-nitroguanine-positive cancer cells. DNA lesions and iNOS expression were also observed in epithelial cells of EBV-positive patients with chronic nasopharyngitis but weaker than those in NPC patients (Fig. 7.). No or few DNA lesions were observed in EBV-negative subjects. EGFR and phosphorylated STAT3 were strongly expressed in cancer cells of NPC patients, suggesting that the STAT3-dependent mechanism is important to the carcinogenesis (Ma et al., 2008). IL-6 was expressed mainly in inflammatory cells of nasopharyngeal tissues of EBV-infected patients. We also found that serum levels of 8-oxodG were significantly higher in NPC patients than control subjects (Huang et al., 2011). Collectively, these findings indicate that the nuclear accumulation of EGFR and activation of STAT3 by IL-6 play a key role in iNOS expression and resultant DNA damage, leading to EBV-related

antibody and anti-PCNA or anti-3-nitrotyrosine or anti-iNOS, p53 antibody using double immunofluorescence technique. 8-Nitroguanine formation is observed mainly in the nucleus of oral epithelial cells. Strong expression of PCNA and p53 was observed in the nucleus of 8 nitroguanine-positive cells in the basal layer. iNOS expression is strongly observed in inflammatory cells and weakly in the cytoplasm of epithelial cells. Moreover, strong 8 nitroguanine and 3-nitrotyrosine immunoreactivities are observed in the nucleus of the

same epithelial cells. Scale bar represents 50 m.

formation.

NPC.

**3.1.3 Epstein-Barr virus and nasopharyngeal carcinoma** 

Fig. 7. Formation of 8-nitroguanine and 8-oxodG and expression of LMP, iNOS, STAT3 and EGFR in the nasopharyngeal tissues of NPC and chronic nasopharyngitis patients.

8-Nitroguanine, a Potential Biomarker to

Evaluate the Risk of Inflammation-Related Carcinogenesis 217

Fig. 8. Formation of 8-nitroguanine, 8-oxodG, iNOS and p53 in the mouse model of

infiltrated cells. Double immunostaining for 8-nitroguanine and p53, significant p53

represents 50 m.

inflammatory bowel disease. 8-Nitroguanine is accumulated in the nuclei and the cytoplasm of epithelial cells. 8-Nitroguanine is also present in infiltrated cells. 8-oxodG formation is observed in the nuclei of epithelial cells and the infiltrated cells in lamina propria, 8-oxodG and 8-nitroguanine co-localized in the nuclei of most epithelial cells and infiltrated cells, iNOS is expressed in the cytoplasm of epithelial cells and infiltrated cells in lamina propria, and iNOS and 8-nitroguanine colocalized in the cytoplasm of many epithelial cells and some

expression is observed in the nuclei of regenerated epithelial cells and some infiltrated cells. P53 is also induced in the cytoplasm of many epithelial cells in lower portions of the gland. P53 is expressed in most of 8-nitroguanine-immunoreactive epithelial cells. Scale bar

8-Nitroguanine and 8-oxodG are co-localized in cancer cells. These DNA lesions are formed mainly in the nucleus and weakly in the cytoplasm. The formation of these DNA lesions was also observed in inflammatory cells in stroma in NPC patients and the epithelium in patients with chronic nasopharyngitis. Immunoreactivity of LMP1 is clearly observed along cell membrane and the cytoplasm of cancer cells. iNOS and 8-nitroguanine are colocalized in cancer cells of NPC patients. Expression of EGFR and STAT3 are colocalized in nasopharyngeal epithelical cells of EBV-positive chronic nasopharyngitis patients. Scale bar represents 50 m.

#### **3.1.4 Inflammatory bowel diseases and colon cancer**

Ulcerative colitis and Crohn's disease are well known as chronic inflammatory diseases in the lower bowel, and share many clinical and pathological characteristics. These diseases are referred to as inflammatory bowel disease (IBD), which leads to long-term impairment of intestinal structure and function (Podolsky, 2002). A large number of immunological abnormalities have been noted in patients with IBD (Bouma & Strober, 2003). It is well established that an increased cancer risk occurs in tissues undergoing chronic inflammation. Epidemiological studies have suggested that the incidence of colorectal cancer in IBD is greater than the expected incidence in the general population (Ekbom et al., 1990; Langholz et al., 1992; Choi & Zelig, 1994; Pikarsky et al., 2004). The histological and molecular signatures suggest an inflammation-driven carcinogenesis process in IBD patients. To evaluate whether nitrative DNA damage plays a role in the carcinogenic process triggered by IBD, we prepared a mouse model of IBD induced by transfer of CD45RBhighCD4+ T cells lacking regulatory T cells to SCID mice (Powrie et al., 1993; Philippe et al., 2003), since mouse models of IBD can result from either excessive effector T cell function or deficient regulatory T cell function (Bouma & Strober, 2003). We performed a double immunofluorescent staining procedure to examine the formation of 8-nitroguanine and 8 oxodG in the colon tissues. We also examined the expression of iNOS by immunohistochemistry. To evaluate the proliferating activity of colonic epithelial cells and their response to DNA damage, we also examined the expression of p53 in the colon tissues.

In theCD45RBhighCD4+Tcell-transferred mouse, significant 8-nitroguanine accumulation was induced in the nuclei and the cytoplasm of epithelial cells, and was also present in infiltrated cells supposed to be inflammatory cells in lamina propria. 8-Nitroguanine was formed in most of 8-oxodG-immunoreactive nuclei of epithelial cells and infiltrated cells (Fig. 8). When the sections were pretreated with RNase, 8-nitroguanine immunoreactivity was more clearly observed in the nuclei of epithelial cells (Ding et al., 2005). This result suggests that 8-nitroguanine was formed in genomic DNA. iNOS was expressed in the cytoplasm of epithelial cells and infiltrated cells in the lamina propria of the IBD mouse model (Fig. 8). iNOS was expressed mainly in 8-nitroguanine immunoreactive epithelial cells (Fig. 8). However, no or little 8-nitroguanine, 8-oxodG and iNOS were observed in nontreated control mice. In the IBD mouse model, significant p53 was accumulated in the nuclei of regenerated epithelial cells. P53 was also expressed in some infiltrated cells. P53 expression was overlapped with 8-nitroguanine (Fig. 8). The mice of IBD model that we used by transfer of CD45RBhighCD4+ T cells showed the similar results of histopathological analysis and immunohistochemical staining. No or little expression of p53 was observed in non-treated control mice.

8-Nitroguanine and 8-oxodG are co-localized in cancer cells. These DNA lesions are formed mainly in the nucleus and weakly in the cytoplasm. The formation of these DNA lesions was also observed in inflammatory cells in stroma in NPC patients and the epithelium in patients with chronic nasopharyngitis. Immunoreactivity of LMP1 is clearly observed along cell membrane and the cytoplasm of cancer cells. iNOS and 8-nitroguanine are colocalized in

nasopharyngeal epithelical cells of EBV-positive chronic nasopharyngitis patients. Scale bar

Ulcerative colitis and Crohn's disease are well known as chronic inflammatory diseases in the lower bowel, and share many clinical and pathological characteristics. These diseases are referred to as inflammatory bowel disease (IBD), which leads to long-term impairment of intestinal structure and function (Podolsky, 2002). A large number of immunological abnormalities have been noted in patients with IBD (Bouma & Strober, 2003). It is well established that an increased cancer risk occurs in tissues undergoing chronic inflammation. Epidemiological studies have suggested that the incidence of colorectal cancer in IBD is greater than the expected incidence in the general population (Ekbom et al., 1990; Langholz et al., 1992; Choi & Zelig, 1994; Pikarsky et al., 2004). The histological and molecular signatures suggest an inflammation-driven carcinogenesis process in IBD patients. To evaluate whether nitrative DNA damage plays a role in the carcinogenic process triggered by IBD, we prepared a mouse model of IBD induced by transfer of CD45RBhighCD4+ T cells lacking regulatory T cells to SCID mice (Powrie et al., 1993; Philippe et al., 2003), since mouse models of IBD can result from either excessive effector T cell function or deficient regulatory T cell function (Bouma & Strober, 2003). We performed a double immunofluorescent staining procedure to examine the formation of 8-nitroguanine and 8 oxodG in the colon tissues. We also examined the expression of iNOS by immunohistochemistry. To evaluate the proliferating activity of colonic epithelial cells and their response to DNA damage, we also examined the expression of p53 in the colon tissues. In theCD45RBhighCD4+Tcell-transferred mouse, significant 8-nitroguanine accumulation was induced in the nuclei and the cytoplasm of epithelial cells, and was also present in infiltrated cells supposed to be inflammatory cells in lamina propria. 8-Nitroguanine was formed in most of 8-oxodG-immunoreactive nuclei of epithelial cells and infiltrated cells (Fig. 8). When the sections were pretreated with RNase, 8-nitroguanine immunoreactivity was more clearly observed in the nuclei of epithelial cells (Ding et al., 2005). This result suggests that 8-nitroguanine was formed in genomic DNA. iNOS was expressed in the cytoplasm of epithelial cells and infiltrated cells in the lamina propria of the IBD mouse model (Fig. 8). iNOS was expressed mainly in 8-nitroguanine immunoreactive epithelial cells (Fig. 8). However, no or little 8-nitroguanine, 8-oxodG and iNOS were observed in nontreated control mice. In the IBD mouse model, significant p53 was accumulated in the nuclei of regenerated epithelial cells. P53 was also expressed in some infiltrated cells. P53 expression was overlapped with 8-nitroguanine (Fig. 8). The mice of IBD model that we used by transfer of CD45RBhighCD4+ T cells showed the similar results of histopathological analysis and immunohistochemical staining. No or little expression of p53 was observed in

cancer cells of NPC patients. Expression of EGFR and STAT3 are colocalized in

**3.1.4 Inflammatory bowel diseases and colon cancer** 

represents 50 m.

non-treated control mice.

Fig. 8. Formation of 8-nitroguanine, 8-oxodG, iNOS and p53 in the mouse model of inflammatory bowel disease. 8-Nitroguanine is accumulated in the nuclei and the cytoplasm of epithelial cells. 8-Nitroguanine is also present in infiltrated cells. 8-oxodG formation is observed in the nuclei of epithelial cells and the infiltrated cells in lamina propria, 8-oxodG and 8-nitroguanine co-localized in the nuclei of most epithelial cells and infiltrated cells, iNOS is expressed in the cytoplasm of epithelial cells and infiltrated cells in lamina propria, and iNOS and 8-nitroguanine colocalized in the cytoplasm of many epithelial cells and some infiltrated cells. Double immunostaining for 8-nitroguanine and p53, significant p53 expression is observed in the nuclei of regenerated epithelial cells and some infiltrated cells. P53 is also induced in the cytoplasm of many epithelial cells in lower portions of the gland. P53 is expressed in most of 8-nitroguanine-immunoreactive epithelial cells. Scale bar represents 50 m.
