**3.3 Treatment of HNSCC**

468 Selected Topics in 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

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

**3.2 Alterations of DNA repair genes/activity in HNSCC and the relationship with** 

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

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

**3.1 Some HNSCC risk factors are able to inhibit DNA repair** 

lagging during mitosis (aneugenic effect) (Norppa & Falck, 2003).

**HNSCC development, treatment, as well as patient's outcome** 

gene sequence or in expression level may be associated with HNSCC.

may play a critical role in the development of HNSCC.

pathway, is worthy to be investigated further.

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.

#### **3.4 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

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

MEM® I reduced serum medium (Invitrogen, Cat. No. 31985).

1. Amplify the pCMV-Luc and pRL-CMV plasmids in *E. coli* (Fig. 1A).

nm and 280 nm with a UV spectrophotometer.

and *Renilla* luciferase activities.

**4.1.2 Substrate preparation for NER** 

**4.1.3 Transfection** 

**4.1.4 Dual-luciferase assay** 

20°C for one minute.

and Environmental Toxicants on Cellular DNA Repair Activity in Head and Neck Cancer 471

2. The internal control plasmid used for calibrating transfection efficiency: pRL-CMV (Promega, Cat. No. E2261). The *Renilla* luciferase is driven by the CMV IE promoter. 3. Transfection reagents: LipofectamineTM 2000 (Invitrogen, Cat. No. 11668) and Opti-

4. The Dual-GloTM Luciferase assay system (Promega, Cat. No. E2940) for analyzing firefly

2. Harvest bacteria by centrifugation at 13,000 rpm for 15 minutes, discard the supernatant completely and purify plasmids using the Plasmid Midi Kit (Geneaid, Cat. No. PI025). 3. Determine plasmid DNA concentration and purity by measuring the absorbance at 260

4. Prepare UV-damaged luciferase reporter plasmid (pCMV-Luc) with a UV-crosslinker (CL-1000, UVP). The plasmids are placed within inner side of an opened eppendorf lid (Fig. 1B) or 35 mm petri dish without lid. The UV dose for irradiation is dependent on cell types because of differential intrinsic DNA repair capacities of various cells. We use 1000 J/m2 for 293 (human embryonic kidney), Beas-2B (human bronchial epithelium), H1299 (human lung cancer), HEp-2 (human laryngeal cancer), SAS, Ca9-22, (human oral cancer) and 500 J/m2 for KB (human oral cancer) cells. It is important to keep above parameters (the same plasmid amount in a fixed volume for UV irradiation) consistently in each experiment, or prepare an enough quantity of UV-dameged

1. The HEp-2 cells (6104) are seeded in 24-well plates 24 h prior to transfection (the

2. Prepare DNA-LipofectamineTM 2000 (Invitrogen) complexes for each sample as follows: a. Add 0.5 g of UV-damaged or undamaged (serve as a control) pCMV-Luc together with 0.05 g of internal control plasmid (pRL-CMV, Promega) in 50 μl of Opti-

b. Mix 1 l LipofectamineTM 2000 gently in 50 μl of Opti-MEM medium and incubate

c. Combine the diluted DNA with the diluted LipofectamineTM 2000 (total volume is 100 μl). Mix gently and incubate for 20 min at room temperature to allow the

3. Add the 100 μl of DNA-LipofectamineTM 2000 complexes to each well and incubate at 37°C in a CO2 incubator for 6 h. Then the cells can be treated with toxicants (such as arecoline) or anticancer drugs for another 24 h. Note: cells can be treated with toxicants or anticancer drugs prior to transfection that is dependent on experimental design.

1. After 24 h post-transfection, cells are harvested in 100 l (adjustable) lysis buffer (0.1 M HEPES, pH 7.8, 1% Triton X-100, 1 mM CaCl2 and 1 mM MgCl2) with cell scrapers. 2. The cell lysates are transferred to eppendorf tubes and centrifuged at 13,000 rpm at

plasmids that can be stored in aliquots at -80°C once for all experiments.

appropriate cell numbers for seeding are dependent on cell types).

formation of DNA-LipofectamineTM 2000 complexes.

MEM (Invitrogen) medium, mix gently.

for 5 minutes at room temperature.

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).
