Application of Host Cell Reactivation in Evaluating the Effects of Anticancer Drugs and Environmental Toxicants on Cellular DNA Repair Activity in Head and Neck Cancer

DNA repair pathways are targets of numerous anticancer drugs including natural and chemical compounds, which direct cancer cells toward apoptosis. However, different types of cancer cells consist of various alterations in DNA repair genes that make cancer cells become drug-resistant and lead to treatment failure and disease recurrence. On the contrary, cancer cells may also possess defects in certain DNA repair pathway that make them are susceptible to certain compounds, which inhibit another DNA repair pathway inside the cancer cells. As a result, these compounds selectively kill the cancer cells and are less harmful to the normal ones. Understanding the effects of anticancer drugs on DNA repair as well as the DNA repair activity of cancer cells themselves are important for improvement of anticancer treatment. Similarly, this information is helpful for elucidation of the carcinogenicity of environmental toxicants. This chapter introduces the crosstalk between anticancer drugs, environmental toxicants and DNA repair pathways in head and neck cancer. In addition, the application of an easy, fast and measurable in vivo functional assay for nucleotide excision repair (NER) and DNA repair via homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways is shown to examine the cellular DNA repair activity responding to anticancer drugs or environmental toxicants. By which the functional roles of DNA repair genes in response to anticancer treatments and genotoxic substances could be evolved in head and neck cancer cells.


Introduction
DNA repair pathways are targets of numerous anticancer drugs including natural and chemical compounds, which direct cancer cells toward apoptosis. However, different types of cancer cells consist of various alterations in DNA repair genes that make cancer cells become drug-resistant and lead to treatment failure and disease recurrence. On the contrary, cancer cells may also possess defects in certain DNA repair pathway that make them are susceptible to certain compounds, which inhibit another DNA repair pathway inside the cancer cells. As a result, these compounds selectively kill the cancer cells and are less harmful to the normal ones. Understanding the effects of anticancer drugs on DNA repair as well as the DNA repair activity of cancer cells themselves are important for improvement of anticancer treatment. Similarly, this information is helpful for elucidation of the carcinogenicity of environmental toxicants. This chapter introduces the crosstalk between anticancer drugs, environmental toxicants and DNA repair pathways in head and neck cancer. In addition, the application of an easy, fast and measurable in vivo functional assay for nucleotide excision repair (NER) and DNA repair via homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways is shown to examine the cellular DNA repair activity responding to anticancer drugs or environmental toxicants. By which the functional roles of DNA repair genes in response to anticancer treatments and genotoxic substances could be evolved in head and neck cancer cells. 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.

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.

Head and neck cancer
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.

Some HNSCC risk factors are able to inhibit DNA repair
Epidemiological evidences have demonstrated that alcohol drinking, betel quid (BQ) chewing (especially in South Asia and South-West Pacific area including Taiwan), cigarette smoking, and infection of human papillomavirus are risk factors for HNSCC development (Haddad & Shin, 2008;IARC, 2004). The carcinogenicity of betel nut has been approved by the International Agency for Research on Cancer (IARC), a WHO organization, in 2004(IARC, 2004, although the molecular mechanism underlying its carcinogenicity is not fully elucidated. In this regard, we have explored the possible effect of arecoline, a major alkaloid in betel nut, on DNA repair activity using HCR. We found that arecoline could inhibit the repair of UV-induced DNA damages, at least partly, through inactivating p53's expression and transactivation activity (Tsai et al., 2008). Besides, we also showed that arecoline could affect mitotic spindles and deregulated mitotic checkpoint, another key guardian of genome integrity (Wang et al., 2010). These results provide molecular explanation for BQ-associated carcinogenicity that has been shown previously by an increase of mitosis errors and micronucleus (MN) in mammalian cells (Lin, 2010). Micronucleus is a typical sign of GIN and is derived from either DNA strand breaks (clastogenic effect) or whole chromosome lagging during mitosis (aneugenic effect) (Norppa & Falck, 2003). Epidemiological studies also show that the probability of HNSCC development is synergistically increased by simultaneous exposure of BQ, cigarette, and alcohols (Ko et al., 1995;Lee et al., 2005). Regarding the carcinogenic role of cigarette on the aspect of DNA repair, we also found that benzo(a)pyrene (BaP), an important carcinogen in cigarette (IARC, 2010), exhibited negative effects on DNA repair (Lin et al., 2011 manuscript in preparation). The mechanistic study regarding the synergistic effect of arecoline and BaP on regulating DNA repair, especially via p53-and aryl hydrocarbon receptor-dependent pathway, is worthy to be investigated further.

Alterations of DNA repair genes/activity in HNSCC and the relationship with HNSCC development, treatment, as well as patient's outcome
GIN is a hallmark of most human malignancies including HNSCC that elevated microsatellite instability, aneuploidy and various genomic alterations have been found by genome-wide analyses (Bockmuhl et al., 1996;Brieger et al., 2003;Friedlander, 2001;Partridge et al., 1999;Sparano et al., 2006), suggesting that GIN may be involved in the development of HNSCC. Some studies also show that DNA repair activity is reduced in the peripheral blood cells of HNSCC patients when compared with normal individuals (Cheng et al., 1998;Paz-Elizur et al., 2006), implying that altered DNA repair genes and/or activity may play a critical role in the development of HNSCC. Studies using comparative genomic hybridization (CGH) have shown that gene copy numbers at chromosome 11q22-23 (ATM locus) are frequently lost in HNSCC (Bockmuhl et al., 1996;Brieger et al., 2003;van den Broek et al., 2007). Lazar et al. also showed loss of heterozygosity (LOH) at 11q23 in 25% (13/52) of primary HNSCC (Lazar et al., 1998). In addition, we have reported that ATM mRNA is down-regulated in 81.3% (65/80) of laryngeal and pharyngeal cancers, and further show that lower ATM expression (tumor/normal < 0.3) was an independent risk factor for patient's survival (Lee et al., 2011). This is the first study showing that ATM expression is a valuable prognostic marker for HNSCC. One study also shows an absent or reduced ATM protein expression in 31.25% (10/32) of oral cancer (He et al., 2008). These results suggest that alteration of ATM, either in gene sequence or in expression level may be associated with HNSCC.

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Previous investigations showed that LOH of chromosome 17q (BRCA1 locus) were found in 35% to 56% of laryngeal cancer (Kiaris et al., 1995;Rizos et al., 1998). In contrast, studies using CGH found an overrepresentation of 17q in 9% to 47% of HNSCC (Bockmuhl et al., 1996;Brieger et al., 2003), and one array-CGH reported the gain of 17q21 in 33% (7/21) of oral cancer (Sparano et al., 2006). These controversial results by genome-wide analyses may be due to the physically close localization of ERBB2 (HER-2/neu) oncogene and the results need to be clarified by specifically looking at the BRCA1 gene locus. Regarding the expression of BRCA1 in HNSCC, one study showed that BRCA1 immunostaining positivity was lost in 34% (26/77) of tongue cancers, which might be correlated with early-stage tumor progression (Vora et al., 2003). The results of genome-wide studies also suggest that genetic alterations at RAD51 (15q15.1) and XPC (3p25) loci may be present in HNSCC (Bockmuhl et al., 1996;Brieger et al., 2003;Partridge et al., 1999;Sparano et al., 2006;van den Broek et al., 2007). Altered RAD51 protein expression has been reported by one pilot study with twelve head and neck cancer patients (Connell et al., 2006). The patients with high RAD51 protein levels in their pre-treatment tumor biopsies demonstrate poorer cancer-specific survival rates than those with lower RAD51 levels (33.3% vs. 88.9% at 2 years; P = 0.025). These results suggest that RAD51 expression may influence the outcome of with head and neck cancer patients who receive chemotherapy and radiotherapy (Connell et al., 2006). Other reports regarding altered expression of DNA repair genes in HNSCC include Ku80 , NBN and ERCC1 (Hsu et al., 2010;Yang et al., 2006). It has been shown that ERCC1 expression is associated with cisplatin resistance (Handra-Luca et al., 2007;Hsu et al., 2010) and NBN is correlated with outcome of advanced HNSCC patients (Yang et al., 2006). Inactivation of the BRCA/FA pathway via promoter methylation has also been described in HNSCC, and may be related to tobacco and alcohol exposure and survival of these patients (Marsit et al., 2004).

Treatment of HNSCC
Since HNSCC and its treatment can affect important physiological functions, such as speaking, breathing, and swallowing, it is important for choosing the appropriate treatment that not only cures but also benefits to the preservation of organs, physiological functions, and quality of life. The standard treatment for resectable HNSCC is surgical resection with or without postoperative concurrent chemotherapy (cisplatin plus 5-fluorouracil) and radiotherapy (CCRT). Around two-thirds of HNSCC are in advanced stage at time of diagnosis (Specenier & Vermorken, 2009). The majority of these patients with advanced stage tumors finally relapse locoregionally or at distant sites. These patients are usually qualified for palliative treatment only. Recent advances in using cetuximab (anti-EGFR) to prolong patient's survival time in locally advanced HNSCC is a big, but still not a fully satisfied progress (Vermorken et al., 2008). The use of docetaxel (a spindle poison and mitotic catastrophe inducer) can enhance the efficacy of chemotherapy using cisplatin/fluorouracil and improve slightly the overall survival rates of HNSCC patients (Hitt et al., 2005;Posner et al., 2007;Vermorken et al., 2007). These results suggest that a combination regimen exploiting different cell-killing mechanisms may be superior to monotherapy. However, an ideal combination regimen with lower adverse and side effects for efficient treatment of HNSCC is still under looking for.

Understanding the status of DNA repair genes in HNSCC is important for design of an effective therapeutic strategy for this malignancy
Since DNA repair genes/activity play a key role in cancer development and treatment, understanding their expression and genomic/functional alterations may facilitate the www.intechopen.com identification of new predictive or prognostic markers and new therapeutic targets for treatment of HNSCC. For example, recent studies using the strategy of synthetic lethal interaction (SLI) to improve efficacy of cancer treatment have become an attractive strategy (Helleday et al., 2008). Cancer cells that can survive from innumerable genetic alterations are largely dependent on the activities of multiple DNA repair pathways. However, cancer cells may also be defective in certain DNA repair pathway that is inherent or arises during tumorigenesis. Therefore, inhibition of one DNA repair pathway may increase selectively killing of cancer cells that already have another defective DNA repair pathway. For examples, some clinical trials have shown the efficient killing of BRCA1-or BRCA2-defective cancer cells (with defective HR repair) by using PARP1 inhibitors, which block BER pathway (Annunziata & O'Shaughnessy, 2010;Bryant et al., 2005;Farmer et al., 2005;Underhill et al., 2010). Notably, such kind of treatment is less toxic than conventional radiotherapy and chemotherapy. This may benefit to organ preservation of HNSCC patients if one can identify SLI targets (DNA repair genes are good candidates) and develop corresponding regimens for treatment. For this reason, some clinical trials are ongoing to examine the efficacy of anticancer treatments by modulating DNA repair activities that are involved in different DNA repair pathways (Bolderson et al., 2009;Helleday, 2010;Helleday et al., 2008).

Host cell reactivation (HCR) assay
As mentioned above, DNA repair activity plays a critical role in maintaining genome integrity. Regardless the alterations of DNA repair genes at the levels of gene expression or DNA sequence, measurement of DNA repair activity can reflect the overall biological effects that are as consequences of these molecular changes and/or anticancer drug responses. Here we describe an easy and fast functional assay (HCR) to evaluate cellular DNA repair activity in vivo. This method uses a plasmid that can produce luciferase in mammalian cells as a reporter. We choose luciferase as a reporter since its characteristics of high sensitivity and wide dynamic linear range for quantification. Of course, other commonly used reporters, such as chloramphenicol acetyltransferase (CAT), secreted alkaline phosphatase (SEAP) or green fluorescent protein (GFP) can also be used. The reporter is damaged in vitro first and is transfected into host cells. If the damaged reporter plasmid can be repaired in the host cells, the luciferase will be re-expressed. Otherwise, the luciferase activity will be much lower than that transfected with undamaged control plasmid. By this way, one can determine the DNA repair capacity by simply measuring luciferase activity. The reporter plasmid can be damaged using various methods such as UV, chemicals or restriction enzymes and serve as substrates for different DNA repair pathways. In this chapter, we will demonstrate the use of HCR in evaluating DNA repair capacities via NER, HR and NHEJ pathways.

HCR for NER
NER is responsible for the repair of bulky DNA lesions induced by UV and a lot of anticancer drugs. Here we use UV as a method to damage a luciferase reporter plasmid. Other chemicals (such as cisplatin) that cause bulky DNA adducts can also be used.

Materials
1. The reporter plasmid: pCMV-Luc . The firefly luciferase is driven by the cytomegalovirus (CMV) immediate early (IE) gene promoter.

Substrate preparation for NER
1. Amplify the pCMV-Luc and pRL-CMV plasmids in E. coli (Fig. 1A). 3. The supernatants (50 l, adjustable) are transferred into a 96-well plate and 20 l of Dual-Glo™ Luciferase Reagent (Promega) are added to each well. 4. Ten minutes later, the firefly luminescence is measured by a microplate luminometer (Centro LB 960, Berthold, Bad Wildbad, Germany). 5. Add 20 l of Dual-Glo™ Stop & Glo ® Reagent (Promega) to each well and wait for 10 minutes, then the Renilla luminescence is read. 6. The transfection efficiency-adjusted firefly luciferase activity is obtained by dividing the Renilla luciferase activity.

Representation of NER activity by HCR assay
Since the pCMV-Luc is damaged by UV, the DNA repair activity (responsible to UV) can be represented as the Renilla-calibrated firefly luciferase activity derived from UV-damaged pCMV-Luc verse to those from undamaged pCMV-Luc. By this way, one can compare the effects of various environmental toxicants on cellular DNA repair capacity. For example, an inhibitory effect of arecoline on the repair of UV-damaged pCMV-Luc can be found by using HCR assay (Fig. 1C).

HCR for HR repair
HR is a reliable mechanism to accurately repair DNA double strand breaks. Here we use PCR to generate two overlapping DNA fragments that contain i) CMV IE promoter and 5'part of Renilla luciferase gene, ii) 3'-part of Renilla luciferase gene and poly-A tail sequence from pRL-CMV ( Fig. 2A) and serve as substrates for HR (Fig. 2B). The two overlapping DNA fragments can also be produced by restriction enzyme digestion and gel elution.  2. Incubate the PCR reaction mixtures at 94°C for 2 min, then run for 30 cycles of amplification (94°C, 45 sec; 55°C, 1 min; 72°C, 1 min) and additional extension step at 72°C for 5 min. 3. Purify the PCR products of HR13 fragments (1730 bp, containing CMV IE promoter and 5'-part of Renilla luciferase gene) and HR24 fregments (1023 bp, containing 3'-part of Renilla luciferase gene and poly-A tail) from 0.8% agarose gels (Fig. 2C) using the Gel-M TM Gel Extraction System kit (Viogene). 4. Determine DNA concentration and purity by measuring the absorbance at 260 nm and 280 nm with a UV spectrophotometer. Dilute the HR13-PCR products to 17 ng/l and HR24-PCR products to 10 ng/l with distilled H 2 O to make the molar ratio of HR13:HR24 = 1:1, by which the same volume of the two DNA fragments can be used for transfection. Store the purified DNA in aliquots at -20°C. Alternative: the two DNA fragments used for HR can also be generated by using a combination of restriction endonucleases BglII/NheI and PstI/BamHI for pRL-CMV (Progema). The use of former restriction enzymes will produce a DNA fragment containing www.intechopen.com CMV IE promoter and 5'-part of Renilla luciferase gene, the later ones result in 3'-part of Renilla luciferase gene and the poly-A signal. These two DNA fragments contain a 222-bp overlapping region for recombination (Fig. 2D). (D) An alternative way to produce DNA fragments for HR by using restriction enzymes.  (Fig. 3).

HCR for NHEJ repair
NHEJ is another DNA repair mechanism responsible to DSB. Unlike HR repair using sisterchromatids as templates, NHEJ directly joins the broken DNA ends by trimming a few nucleotides on the ends. Therefore, it is thought as an error-prone repair system. In this regard, we prepare two kinds of reporter DNA substrates that are suitable for analyzing the precise and overall NHEJ repair activities, respectively. For overall NHEJ repair, pRL-CMV is linearized with HindIII that cuts the flanking sequence between CMV promoter and the Renilla luciferase coding sequence. The luciferase will express after re-ligation regardless the loss of some nucleotides. For examining precise www.intechopen.com NHEJ, Afl III that digests the coding region of Renilla luciferase gene is used and the luciferase can only be expressed after exact repair (Fig. 4A). The linearized reporter DNA fragments are purified, transfected into host cells and examined for luciferase activity as described above. Below is an example of evaluating the effect of areca nut extracts on precise and overall NHEJ repair (Fig. 4B). Fig. 4. HCR assay for non-homologous end-joining (NHEJ) repair. (A) The Afl III-digested pRL-CMV is used as a substrate for analyzing precise NHEJ repair activity because of the need of exact joining of the Renilla luciferase coding sequence. For overall NHEJ, Hind III that cuts the flanking sequence between CMV promoter and the Renilla luciferase gene is used. The expression of luciferase is not affected by loss of a few nucleotides in this region during the end-joining process. (B) The effect of areca nut extracts (ANE, 800 mg/ml for 24 h) on precise (left panel) and overall (right panel) NHEJ repair.

Conclusion
DNA repair genes play a pivotal role in the maintenance of genome integrity. Alterations of various DNA repair genes, either in gene sequence/structure or in gene expression, are frequently found in most of human malignancies. Since DNA repair activity is able to www.intechopen.com modulate cellular response to DNA-damaging anticancer drugs, alterations of DNA repair genes may be involved in the development of resistance to chemotherapy and radiotherapy. In addition, DNA repair activity plays an important role in preventing the mutagenicity and cytotoxicity induced by numerous environmental carcinogens and toxicants. Cells with reduced DNA repair activity may thus be prone to pathological transformation. Therefore, examining the DNA repair activity of a cell can help us to understand the probability of cellular tumorigenicity associated with exposure of environmental carcinogens and is able to assess the responses of various regimens of anticancer treatment. HCR assay is an easy and fast functional assay that can be applied to investigate several DNA repair pathways and is one of the most useful methods for evaluating cellular DNA repair activity in vivo.