**2.4 Cellular genes induction**

46 Carcinogenesis, Diagnosis, and Molecular Targeted Treatment for Nasopharyngeal Carcinoma

Fig. 1. Schematic representation of the functional regions of ZEBRA and its structure. The transactivation (TA) domain is shown in grey and the DNA contact region and the dimerization region are shown in green and blue. The amino acid sequence of the DNA binding and dimerization domain is expanded below. The location of the region with homology to leucine zippers (ZIP) and the additional region required for dimerization

During the lytic phase of the EBV life cycle, the activation of viral DNA synthesis is related to ZEBRA efficient recognition of a large (1 kb) complex intergenic region that serves as the origin of replication. This region, known as oriLyt, consists of essential and auxiliary segments (Hammerschmidt & Sugden 1988).The two essential components of oriLyt, the upstream and downstream elements, together constitute the minimal origin of DNA replication (Rennekamp, Wang & Lieberman 2010). The auxiliary component serves as an enhancer element that augments DNA replication (Cox, Leahy & Hardwick 1990). ZEBRA recognizes the origin of lytic DNA replication (oriLyt) by interacting with seven ZEBRA-binding sites (Schepers, Pich & Hammerschmidt 1993). Mutation of all seven binding motifs in the background of a recombinant virus drastically reduces production of infectious virus particles (Feederle & Delecluse 2004).These ZEBRA binding elements are located in two noncontiguous regions of oriLyt. Four elements are present in the upstream core region of oriLyt and overlap with the promoter of the BHLF1 open reading frame and three additional ZEBRA binding elements located mainly in the enhancer region are dispensable for viral replication (Schepers, Pich & Hammerschmidt

The current model for the role of ZEBRA in lytic DNA replication suggests that the protein serves as a physical link between oriLyt and core components of the replication machinery. The six core replication factors encoded by EBV are the DNA polymerase (BALF5); the polymerase processivity factor (BMRF1); the helicase (BBLF4); the primase (BSLF1); the primase associated factor (BBLF2/3), and the single-stranded DNA binding protein (BALF2) (El-Guindy, Heston & Miller 2010). The function of tethering replication proteins to oriLyt is not limited to ZEBRA; the transactivation domains of Sp1 and ZBP89 interact with BMRF1 and BALF5 and target them to the downstream region of oriLyt (Baumann et al. 1999). Similarly, ZBRK1, a cellular DNA binding zinc finger protein, serves as a contact point for

function (CT) are indicated below the sequence

**2.3 Role of ZEBRA during viral replication** 

BBLF2/3 on oriLyt (Liao et al. 2005).

1996).

The EBV lytic transactivator ZEBRA not only initiates expression cascade of viral lytic genes but also induces some cellular genes involved in immune regulation (Cayrol & Flemington 1995; Chen, C., Li & Guo 2009). ZEBRA can turn on gene expression through binding to and activation of the target promoters. Notably, a previous study shows that ZEBRA induces transcription of human interleukin 10 (IL-10) in B cells (Mahot et al. 2003).

Table 2 lists a number of genes whose expression is perturbed by ZEBRA. In each case, the regulation occurs in the absence of other viral genes. Regulation at the RNA level implies that ZEBRA may act as transcription factors on the cellular promoters by direct binding or via their associations with other transcription factors. However, the mechanisms of regulation of the cellular genes have not been assessed further as yet. Several of these genes have also been shown to be regulated at the protein level, suggesting relevance. Of special note is the identification of a series of cell cycle regulatory genes such as p21CIP1, p53, CDC25A and E2F1.


Table 1. Cell genes regulated by ZEBRA

In NPC, ZEBRA was found to be a potent inducer of IL-8, increasing IL-8 at both protein and RNA levels and activating the IL-8 promoter suggesting that the EBV lytic infection may contribute to the inflammation-like microenvironment of NPC by the upregulation of chemokines (Hsu et al. 2008).

During EBV reactivation in NPC cells, the lytic protein ZEBRA not only induces GM-CSF expression but also upregulates COX-2 that increases production of PGE2 (Dolcetti et al. 2010; Kared et al. 2008). The secreted GM-CSF and PGE2 may cooperatively promote IL-10 production from monocytes (Lee et al. 2011). Thus, through the Zta-induced immunomodulators, EBV lytic infection in NPC cells may drive nearby monocytes into IL-10 producing cells, facilitating local immunosuppression.

Role of the Epstein-Barr Virus ZEBRA Protein

transcription (cis sequence).

carcinogenic (Munoz et al. 2003).

**3.1 HPV and head and neck cancer** 

significance in the field of molecular virology (Levinson, 2008).

and HPV in the Carcinogenesis of Nasopharyngeal Carcinoma 49

forth only when cervical cancer's proximity with HPV was proved and with its increasing

HPVs are small non enveloped virus containing double stranded DNA as their genetic material and are about 55 nm in size. HPV are strictly epitheliotropic viruses infecting skin or mucosal surfaces, and displaying a very high selectivity for the specific epithelium infected [7,8], and are one of the most common viruses which are transmitted sexually and are found in both men and women. Its ratio is much higher in western countries as compared to other regions of the world. The genome is a circular molecule of doublestranded DNA 8000 base pairs or so. Ten open reading frames (POL) are grouped in a region L (early) coding non-structural protein and an L (late) region encoding the capsid proteins. The non-coding region (NCR of 850 bp for HPV 16) is located between the POL L1 and E6/E7. It contains the promoters of early genes, regulatory sequences (original site) and

According to their ability to transform epithelial cells, HPV genotypes are divided into lowrisk and high-risk types. Low-risk types are associated with benign lesions such as warts, while infections with high-risk types progress to malignant lesions (Munoz et al. 2003). More than 100 different HPV genotypes have been described, but only 30 genotypes identified in the female genital tract are associated with epithelial neoplasms ranging from benign common warts to malignant carcinoma of the uterine cervix (McGlennen 2000). It is widely reported that in addition to HPV 16 and 18, which are frequently found in association with cervical cancer (CC), HPVs 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82, while other three as probable high-risk types (types 26, 53, and 66) are also considered as

Approximately 15% of malignant diseases are caused by infectious agents. HPV can be frequently found in oral carcinomas, especially tonsillar cancer. A group of HPV-infected tumours shows clear signs for a virally induced transformation process: high-risk HPVs can be detected in all tumour cells, the viral oncogenes E6 and E7 are constantly expressed and lead to upregulation of cellular p16(INK4a), a cyclin-dependent kinase inhibitor. The patients frequently lack typical risk factors associated with head and neck cancers such as drinking and smoking. An association of herpes viruses with head and neck cancer has been for long time suspected and there is good evidence there, but only for a relationship of EBV nasopharyngeal carcinoma. HPV aetiology is now accepted for up to 20% of head and neck cancer. However, the relationship between carcinogenesis and HPV infection is not as clear as with cervical cancer. HPV is not detectable in many head and neck cancers, and it is frequently detected in normal oral mucosa, which is why HPV infections were in head and neck cancer is often regarded only as an accompanying infection. Initial investigations into a causal role for HPV in the aetiology of head and neck lesions relied upon electron microscopy (EM) or immunohistochemical staining. Human papillomavirus virions could be identified by EM from specimens of papillomas (Frithiof & Wersall 1967), fibromas (Gross et al. 1982), verruca, condyloma acuminatav (Shaffer, Reimann & Gysland 1980; Syrjanen, K. J. & Surjanen 1981), focal epithelial hyperplasia, and oral nodular leukoplakias (Jenson et al. 1982). Immunohistochemical staining has revealed the presence of HPV capsid antigens in HPV-infected cells (Loning et al. 1984). Capsid antigen, however, has rarely been

By initiating or enhancing leukocyte infiltration, the lytic-cycle-induced chemokines may contribute to an inflammation-like microenvironment, where the interaction between immune infiltrates and tumour cells is crucial for NPC development (Sbih-Lammali et al. 1999). The contribution possibly occurs not only in the developed NPC tumours but also at the precancer stage where an inflammation like microenvironment predisposes precancerous cells to tumour formation (Lu, H., Ouyang & Huang 2006), which may account for how EBV reactivation serves as a risk factor before the onset of NPC (Chien et al. 2001).

#### **2.5 Prognostic value of ZEBRA**

Antibodies against ZEBRA are produced during primary EBV infection, and thus, the detection of ZEBRA-specific antibodies may allow an early diagnosis of EBV infections. In 1991, Joab and al were able to detect IgG anti-ZEBRA antibodies (IgG/ZEBRA) in 87% of NPC patients. These antibodies were absent in control sera (Joab et al. 1991). In a more recent study, IgG-ZEBRA antibodies have been shown to represent a sensitive marker for the diagnosis of NPC in children than IgA-VCA and IgA-EA antibodies, which have been recognized as specific markers for this tumour. This study also indicates that Zp125, identified as the most immunogenic epitope of the activation domain of ZEBRA protein, showed a high degree of immunoreactivity with sera from children, young adult and older adult patients with NPC (Dardari et al. 2008). The analysis of antibody patterns in patients with NPC indicates that IgG-ZEBRA had better prognostic value than IgA-EA and IgA-VCA. Stable low IgG-ZEBRA antibody titer, or a striking decline in IgG-ZEBRA antibodies, was observed in children during treatment (Gutierrez et al. 2001; Schaade, Kleines & Hausler 2001). Of note, children showing low IgG-ZEBRA titers were also negative for IgAVCA and IgA-EA; the latter have been identified as being produced following frequent reactivation of latent EBV, repeated EBV infection, or both (Gutierrez et al. 2001; Yip et al. 1994).

Epigenetic dysregulation plays significant role in oncogenesis. Methylation changes in both global and targeted genes have been attributed to EBV+ lymphomas and carcinomas (Niller, Wolf & Minarovits 2009). These studies suggest the contribution of EBV genome in the epigenetic dysregulation of genes involved in tumorigenesis. Systemic analyses of epigenetic alterations under the expression of specific viral gene that may help to specify the contribution of EBV genome. Although results from the Ying-Fan Chen (Chen, Y. F. et al. 2011) suggest that the expression of major viral lytic protein Zta has no effect on changing DNA methylation in the host genome, the comprehensive methods established by him provide a useful platform to investigate genomic methylation changes upon various conditions. These results from these studies will lead to a better understanding of the EBV pathogenesis and may facilitate the development of new therapies.

#### **3. Human Papilloma Virus**

The papilloma viruses and its viral nature were first seen in human warts in 1907, and the first papilloma virus was isolated from a rabbit that was identified by Richard Shoppe in the year 1983. Even it was an early start for the detection of human papilloma viruses; this topic remained closed till 1970's. Studies related with papilloma viruses were allowed to move

By initiating or enhancing leukocyte infiltration, the lytic-cycle-induced chemokines may contribute to an inflammation-like microenvironment, where the interaction between immune infiltrates and tumour cells is crucial for NPC development (Sbih-Lammali et al. 1999). The contribution possibly occurs not only in the developed NPC tumours but also at the precancer stage where an inflammation like microenvironment predisposes precancerous cells to tumour formation (Lu, H., Ouyang & Huang 2006), which may account for how EBV reactivation serves as a risk factor before the onset of NPC (Chien et al.

Antibodies against ZEBRA are produced during primary EBV infection, and thus, the detection of ZEBRA-specific antibodies may allow an early diagnosis of EBV infections. In 1991, Joab and al were able to detect IgG anti-ZEBRA antibodies (IgG/ZEBRA) in 87% of NPC patients. These antibodies were absent in control sera (Joab et al. 1991). In a more recent study, IgG-ZEBRA antibodies have been shown to represent a sensitive marker for the diagnosis of NPC in children than IgA-VCA and IgA-EA antibodies, which have been recognized as specific markers for this tumour. This study also indicates that Zp125, identified as the most immunogenic epitope of the activation domain of ZEBRA protein, showed a high degree of immunoreactivity with sera from children, young adult and older adult patients with NPC (Dardari et al. 2008). The analysis of antibody patterns in patients with NPC indicates that IgG-ZEBRA had better prognostic value than IgA-EA and IgA-VCA. Stable low IgG-ZEBRA antibody titer, or a striking decline in IgG-ZEBRA antibodies, was observed in children during treatment (Gutierrez et al. 2001; Schaade, Kleines & Hausler 2001). Of note, children showing low IgG-ZEBRA titers were also negative for IgAVCA and IgA-EA; the latter have been identified as being produced following frequent reactivation of latent EBV, repeated EBV infection, or both (Gutierrez et al. 2001; Yip et al.

Epigenetic dysregulation plays significant role in oncogenesis. Methylation changes in both global and targeted genes have been attributed to EBV+ lymphomas and carcinomas (Niller, Wolf & Minarovits 2009). These studies suggest the contribution of EBV genome in the epigenetic dysregulation of genes involved in tumorigenesis. Systemic analyses of epigenetic alterations under the expression of specific viral gene that may help to specify the contribution of EBV genome. Although results from the Ying-Fan Chen (Chen, Y. F. et al. 2011) suggest that the expression of major viral lytic protein Zta has no effect on changing DNA methylation in the host genome, the comprehensive methods established by him provide a useful platform to investigate genomic methylation changes upon various conditions. These results from these studies will lead to a better understanding of the EBV

The papilloma viruses and its viral nature were first seen in human warts in 1907, and the first papilloma virus was isolated from a rabbit that was identified by Richard Shoppe in the year 1983. Even it was an early start for the detection of human papilloma viruses; this topic remained closed till 1970's. Studies related with papilloma viruses were allowed to move

pathogenesis and may facilitate the development of new therapies.

**3. Human Papilloma Virus** 

2001).

1994).

**2.5 Prognostic value of ZEBRA** 

forth only when cervical cancer's proximity with HPV was proved and with its increasing significance in the field of molecular virology (Levinson, 2008).

HPVs are small non enveloped virus containing double stranded DNA as their genetic material and are about 55 nm in size. HPV are strictly epitheliotropic viruses infecting skin or mucosal surfaces, and displaying a very high selectivity for the specific epithelium infected [7,8], and are one of the most common viruses which are transmitted sexually and are found in both men and women. Its ratio is much higher in western countries as compared to other regions of the world. The genome is a circular molecule of doublestranded DNA 8000 base pairs or so. Ten open reading frames (POL) are grouped in a region L (early) coding non-structural protein and an L (late) region encoding the capsid proteins. The non-coding region (NCR of 850 bp for HPV 16) is located between the POL L1 and E6/E7. It contains the promoters of early genes, regulatory sequences (original site) and transcription (cis sequence).

According to their ability to transform epithelial cells, HPV genotypes are divided into lowrisk and high-risk types. Low-risk types are associated with benign lesions such as warts, while infections with high-risk types progress to malignant lesions (Munoz et al. 2003). More than 100 different HPV genotypes have been described, but only 30 genotypes identified in the female genital tract are associated with epithelial neoplasms ranging from benign common warts to malignant carcinoma of the uterine cervix (McGlennen 2000). It is widely reported that in addition to HPV 16 and 18, which are frequently found in association with cervical cancer (CC), HPVs 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82, while other three as probable high-risk types (types 26, 53, and 66) are also considered as carcinogenic (Munoz et al. 2003).
