**4.1.2 Substrate preparation for NER**

470 Selected Topics in DNA Repair

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

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

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

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.

1. The reporter plasmid: pCMV-Luc (Liu et al., 2004). The firefly luciferase is driven by the

Other chemicals (such as cisplatin) that cause bulky DNA adducts can also be used.

cytomegalovirus (CMV) immediate early (IE) gene promoter.

(Bolderson et al., 2009; Helleday, 2010; Helleday et al., 2008).

(SEAP) or green fluorescent protein (GFP) can also be used.

repair capacities via NER, HR and NHEJ pathways.

**4.1 HCR for NER** 

**4.1.1 Materials** 

**4. Host cell reactivation (HCR) assay** 

