**2. Roles of DNA repair genes/pathways in cancer development and treatment**

DNA repair genes play a pivotal role in the maintenance of genome integrity. Defects or dysregulation of DNA repair genes can result in genomic instability (GIN), which is a common feature of cancer cells (Hanahan & Weinberg, 2000). To prevent this, human cells evolve several

Application of Host Cell Reactivation in Evaluating the Effects of Anticancer Drugs

repair assay, host cell reactivation (HCR), in cancer research.

**using chemotherapy or radiotherapy** 

the patient's outcome.

**3. Head and neck cancer** 

and Environmental Toxicants on Cellular DNA Repair Activity in Head and Neck Cancer 467

tumor tissues, DNA repair genes, including *ATM, CHEK1/2*, and *TP53*, are found predominantly to be highly activated in the precancerous stage of bladder, colon and lung epithelia when DNA damages are emerging inside these cells (Bartkova et al., 2005; Gorgoulis et al., 2005; Venkitaraman, 2005). Further, DNA repair genes also play a key role in the oncogene-induced senescence and prevent cell transformation (Bartkova et al., 2006; Braig et al., 2005; Di Micco et al., 2006). In other word, cells that are unable to activate DNA repair genes in the early-stage of tumorigenesis are susceptible for malignant transformation. These data demonstrated in somatic cancers strongly indicate that defects or inactivations of DNA repair genes/pathways are prerequisite for tumor development. Besides, several cancer predisposition syndromes are linked to hereditary mutations or deletions of DNA repair genes, such as *ATM* in ataxia telangiectasia, *BRCA1* and *BRCA2* in familial breast and ovarian cancers, *XPC* and *DDB2* in Xeroderma pigmentosum. Hence, people with, either inherited or sporadic, inactivated DNA repair genes/pathways are prone to cancer development. In this chapter, we will use head and neck cancer as an example to illustrate the important role of DNA repair genes/pathways in the development and treatment of this malignancy, and demonstrate the application of a functional DNA

**2.3 DNA repair activity is a critical determinant for efficacy of anticancer treatment** 

The cell-killing mechanisms of radiotherapy and most regimens of chemotherapy are dependent on the induction of severe DNA damages, which result in apoptosis of cancer cells. Therefore, the DNA repair activity of cancer cells can play an important role in modulating patient's response to these anticancer treatments. For example, the platinum-based anticancer chemical, cisplatin is one of the most popular DNA-damaging chemotherapeutic drugs used in clinical management. It causes DNA adducts by interstrand crosslinking, which is repaired by a combination of NER and HR (Helleday et al., 2008; Miyagawa, 2008). Mutations of NER genes, such as *XPF* or *ERCC1*, may increase the sensitivity of cells toward cisplatin (Martin et al., 2008; Saldivar et al., 2007). In contrast, elevated expression of NER genes usually confers resistance to chemotherapy using DNA-damaging regimens. The expression level of *BRCA1*, which plays a primary role in HR and may has a regulatory role in NER (Hartman & Ford, 2002; Takimoto et al., 2002), is also correlated with chemotherapy efficacy. It has been shown that cells with reduced or inactivated *BRCA1* are more sensitive to cisplatin but, in contrast, are resistant to taxanes, the microtubule-interfering drugs (Husain et al., 1998; Lafarge et al., 2001; Mullan et al., 2001). Overexpression of *RAD51*, a member of BRCA/FA complex involved in HR, is also correlated with cisplatin resistance (Bhattacharyya et al., 2000). For *ATM*, an *in vitro* study showed that partial loss of distal 11q (*ATM* locus) was associated with decreased IR sensitivity in head and neck cancer cell lines (Parikh et al., 2007). Therefore, understanding the status of DNA repair genes/activity is thought to be important for the selection of appropriate chemotherapeutic regimens and may have a great impact on the clinical treatment as well as

Head and neck squamous cell carcinoma (HNSCC) is the most popular head and neck cancer and is the sixth most common cancer in the world. They include malignancies

originated from the epithelia of larynx, pharynx, oral and nasal cavities.

DNA repair pathways that may interplay each other to repair various types of DNA damages. These DNA repair mechanisms include pathways of base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), double strand break (DSB) repair through homologous recombination (HR) or non-homologous end-joining (NHEJ) and direct repair of DNA lesions such as O6-methylguanine by O6-methylguanine-DNA methyltransferase (Sancar et al., 2004). Collectively, there are hundreds of DNA repair genes involved in various DNA repair pathways that include processes of sense and recognition of DNA lesions, amplification and transmission of the damage signal, recruitment of repair proteins to the damage sites, and execution of DNA repair (Sancar et al., 2004; Wood et al., 2005).

#### **2.1 DNA repair genes/signaling in HR and NER pathways**

Upon DNA damaged, histone H2AX, a histone H2A variant, is quickly phosphorylated (denoted as -H2AX) in an ataxia telangiectasia mutated (*ATM*)-dependent manner (Uziel et al., 2003). The phosphorylated H2AX serves as an important marker for DNA damages. Some genes are also involved in the recognition of DNA damages. They are members of MRN complex (*Mre11A, RAD50, NBN*) for DSB and damage-specific DNA binding protein 1 and 2 (*DDB1* and *DDB2*), Xeroderma pigmentosum (XP) complementation group C (*XPC*) for UV-induced damages and bulky DNA adducts, which are produced by DNA-damaging chemotherapeutic drugs and can be repaired through NER pathway. In addition to H2AX, ATM also phosphorylates p53, BRCA1, CHEK1/2 and results in activation of various DNA repair pathways as well as induction of cell cycle arrest (Sancar et al., 2004). Generally, the genes involved in HR repair include *BRCA1, BRCA2*, members of *RAD51* and Fanconi anemia (FA) families, as well as the Bloom syndrome, RecQ helicase-like (*BLM*) and Werner syndrome, RecQ helicase-like (*WRN*). The genes in NER pathway consists of XP complementation group A to G (*XPA* to *XPG*), XP complementation group variant (*XPV*), excision repair cross-complementing rodent repair deficiency, complementation group 1 (*ERCC1*), replication protein A (*RPA*), and so on (Sancar et al., 2004; Wood et al., 2005). *ATM* is the key gene for initiating DNA repair signaling. Its downstream targets, both *TP53* and *BRCA1* are capable of regulating multiple DNA repair pathways (Deng, 2006; Helton & Chen, 2007). *BRCA1* encodes a multifunctional protein that maintains genome integrity through regulating gene transcription, cell cycle checkpoints, DNA repair (Deng, 2006; Yoshida & Miki, 2004), and centrosome duplication (Deng, 2002; Xu et al., 1999). In addition to the role in HR, BRCA1 is involved in NER through transactivating the expression of *DDB2* and *XPC* (Hartman & Ford, 2002; Takimoto et al., 2002), both of them can also be transactivated by p53 (Adimoolam & Ford, 2002; Hwang et al., 1999). Thus, both *BRCA1* and *TP53* can regulate NER pathway. RAD51 is the human homolog of bacteria *recA* and forms a complex with BRCA1 and BRCA2. This interaction is important for proper regulation of RAD51 activity inside a cell. Loss of the binding between RAD51 and BRCA complex may be a key event leading to GIN and tumorigenesis (Martin et al., 2007). RAD51 contributes the key step of HR by mediating homologous pairing and strand exchange between two homologous DNA (Richardson, 2005). It has been shown that *RAD51* overexpression is correlated with GIN and that p53 can transcriptionally inhibits *RAD51* expression (Arias-Lopez et al., 2006; Richardson et al., 2004).

#### **2.2 DNA repair activity is important for preventing cancer development**

Activation of DNA repair genes has been shown as a critical anticancer barrier in early human tumorigenesis. By examining various stages from precancerous lesions to late-stage

DNA repair pathways that may interplay each other to repair various types of DNA damages. These DNA repair mechanisms include pathways of base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), double strand break (DSB) repair through homologous recombination (HR) or non-homologous end-joining (NHEJ) and direct repair of DNA lesions such as O6-methylguanine by O6-methylguanine-DNA methyltransferase (Sancar et al., 2004). Collectively, there are hundreds of DNA repair genes involved in various DNA repair pathways that include processes of sense and recognition of DNA lesions, amplification and transmission of the damage signal, recruitment of repair proteins to the damage sites, and

Upon DNA damaged, histone H2AX, a histone H2A variant, is quickly phosphorylated (denoted as -H2AX) in an ataxia telangiectasia mutated (*ATM*)-dependent manner (Uziel et al., 2003). The phosphorylated H2AX serves as an important marker for DNA damages. Some genes are also involved in the recognition of DNA damages. They are members of MRN complex (*Mre11A, RAD50, NBN*) for DSB and damage-specific DNA binding protein 1 and 2 (*DDB1* and *DDB2*), Xeroderma pigmentosum (XP) complementation group C (*XPC*) for UV-induced damages and bulky DNA adducts, which are produced by DNA-damaging chemotherapeutic drugs and can be repaired through NER pathway. In addition to H2AX, ATM also phosphorylates p53, BRCA1, CHEK1/2 and results in activation of various DNA repair pathways as well as induction of cell cycle arrest (Sancar et al., 2004). Generally, the genes involved in HR repair include *BRCA1, BRCA2*, members of *RAD51* and Fanconi anemia (FA) families, as well as the Bloom syndrome, RecQ helicase-like (*BLM*) and Werner syndrome, RecQ helicase-like (*WRN*). The genes in NER pathway consists of XP complementation group A to G (*XPA* to *XPG*), XP complementation group variant (*XPV*), excision repair cross-complementing rodent repair deficiency, complementation group 1 (*ERCC1*), replication protein A (*RPA*), and so on (Sancar et al., 2004; Wood et al., 2005). *ATM* is the key gene for initiating DNA repair signaling. Its downstream targets, both *TP53* and *BRCA1* are capable of regulating multiple DNA repair pathways (Deng, 2006; Helton & Chen, 2007). *BRCA1* encodes a multifunctional protein that maintains genome integrity through regulating gene transcription, cell cycle checkpoints, DNA repair (Deng, 2006; Yoshida & Miki, 2004), and centrosome duplication (Deng, 2002; Xu et al., 1999). In addition to the role in HR, BRCA1 is involved in NER through transactivating the expression of *DDB2* and *XPC* (Hartman & Ford, 2002; Takimoto et al., 2002), both of them can also be transactivated by p53 (Adimoolam & Ford, 2002; Hwang et al., 1999). Thus, both *BRCA1* and *TP53* can regulate NER pathway. RAD51 is the human homolog of bacteria *recA* and forms a complex with BRCA1 and BRCA2. This interaction is important for proper regulation of RAD51 activity inside a cell. Loss of the binding between RAD51 and BRCA complex may be a key event leading to GIN and tumorigenesis (Martin et al., 2007). RAD51 contributes the key step of HR by mediating homologous pairing and strand exchange between two homologous DNA (Richardson, 2005). It has been shown that *RAD51* overexpression is correlated with GIN and that p53 can transcriptionally inhibits *RAD51* expression (Arias-

execution of DNA repair (Sancar et al., 2004; Wood et al., 2005).

**2.1 DNA repair genes/signaling in HR and NER pathways** 

Lopez et al., 2006; Richardson et al., 2004).

**2.2 DNA repair activity is important for preventing cancer development** 

Activation of DNA repair genes has been shown as a critical anticancer barrier in early human tumorigenesis. By examining various stages from precancerous lesions to late-stage tumor tissues, DNA repair genes, including *ATM, CHEK1/2*, and *TP53*, are found predominantly to be highly activated in the precancerous stage of bladder, colon and lung epithelia when DNA damages are emerging inside these cells (Bartkova et al., 2005; Gorgoulis et al., 2005; Venkitaraman, 2005). Further, DNA repair genes also play a key role in the oncogene-induced senescence and prevent cell transformation (Bartkova et al., 2006; Braig et al., 2005; Di Micco et al., 2006). In other word, cells that are unable to activate DNA repair genes in the early-stage of tumorigenesis are susceptible for malignant transformation. These data demonstrated in somatic cancers strongly indicate that defects or inactivations of DNA repair genes/pathways are prerequisite for tumor development. Besides, several cancer predisposition syndromes are linked to hereditary mutations or deletions of DNA repair genes, such as *ATM* in ataxia telangiectasia, *BRCA1* and *BRCA2* in familial breast and ovarian cancers, *XPC* and *DDB2* in Xeroderma pigmentosum. Hence, people with, either inherited or sporadic, inactivated DNA repair genes/pathways are prone to cancer development. In this chapter, we will use head and neck cancer as an example to illustrate the important role of DNA repair genes/pathways in the development and treatment of this malignancy, and demonstrate the application of a functional DNA repair assay, host cell reactivation (HCR), in cancer research.

#### **2.3 DNA repair activity is a critical determinant for efficacy of anticancer treatment using chemotherapy or radiotherapy**

The cell-killing mechanisms of radiotherapy and most regimens of chemotherapy are dependent on the induction of severe DNA damages, which result in apoptosis of cancer cells. Therefore, the DNA repair activity of cancer cells can play an important role in modulating patient's response to these anticancer treatments. For example, the platinum-based anticancer chemical, cisplatin is one of the most popular DNA-damaging chemotherapeutic drugs used in clinical management. It causes DNA adducts by interstrand crosslinking, which is repaired by a combination of NER and HR (Helleday et al., 2008; Miyagawa, 2008). Mutations of NER genes, such as *XPF* or *ERCC1*, may increase the sensitivity of cells toward cisplatin (Martin et al., 2008; Saldivar et al., 2007). In contrast, elevated expression of NER genes usually confers resistance to chemotherapy using DNA-damaging regimens. The expression level of *BRCA1*, which plays a primary role in HR and may has a regulatory role in NER (Hartman & Ford, 2002; Takimoto et al., 2002), is also correlated with chemotherapy efficacy. It has been shown that cells with reduced or inactivated *BRCA1* are more sensitive to cisplatin but, in contrast, are resistant to taxanes, the microtubule-interfering drugs (Husain et al., 1998; Lafarge et al., 2001; Mullan et al., 2001). Overexpression of *RAD51*, a member of BRCA/FA complex involved in HR, is also correlated with cisplatin resistance (Bhattacharyya et al., 2000). For *ATM*, an *in vitro* study showed that partial loss of distal 11q (*ATM* locus) was associated with decreased IR sensitivity in head and neck cancer cell lines (Parikh et al., 2007). Therefore, understanding the status of DNA repair genes/activity is thought to be important for the selection of appropriate chemotherapeutic regimens and may have a great impact on the clinical treatment as well as the patient's outcome.
