**7.** *BRCA1* **and its encoded product as potentially molecular targets for cisplatin for cancer therapy**

In recent years, there has been significant progress made in evaluating what happens when BRCA1 is inactivated so it cannot respond to DNA damage in cancer cells, in other words, taking advantage of the inherent weakness of the BRCA1 dysfunction in cancer cells. These cells have increased sensitivity to DNA-damaging agents that eventually result in major genomic instability and cell death (Amir et al., 2010; Ashworth, 2008; Helleday et al., 2008; Lieberman, 2008; Powell & Bindra, 2009; Quinn et al., 2009; Tassone et al., 2009; Zhu et al., 2009). Cancerous cells with inactivated BRCA1 had defects in DNA repair of double strand breaks (DSBs) (Farmer et al., 2005; Kennedy et al., 2004; Litman et al., 2008). Moreover, extensive investigations have revealed the relevance of the BRCA1-mediated ubiquitination to DNA repair functions. Mutations in the BRCA1 RING domain resulted in the loss of the E3 ubiquitin ligase activity, and conferred hypersensitivity of the cancerous cells to DNAdamaged chemotherapy and γ-irradiation (Ransburgh et al., 2010; Ruffer et al., 2001; Wei et al., 2008).

It was initially reported that overexpression of BRCA1 in the human breast cancer MCF7 cell line resulted in an increased resistance to cisplatin (Husain et al., 1998). Furthermore, antisense or siRNA-based inhibition of endogenous BRCA1 expression promoted the increased sensitivity to cisplatin that was associated with the decreased DNA repair by NER and an increased apoptosis (Lafarge et al., 2001; Quinn et al., 2003). This indicates that the reduced BRCA1 expression observed in sporadic cancers may also be exploited for DNA damage-based chemotherapy (James et al., 2007; Quinn et al., 2009). In a similar situation, BRCA1-deficient mouse embryonic stem cells displayed defective DNA repair and a 100-fold increased sensitivity to the alkylating agent mitomycin C and cisplatin than those containing wild-type BRCA1 (Bhattacharyya et al., 2000; Moynahan et al., 2001). This sensitivity was reversed upon correction of the BRCA1 mutation in mouse embryonic fibroblast cells with a disrupted BRCA1 (Fedier et al., 2003). Reconstitution of BRCA1 in the cells via transfection meant that BRCA1 functions were regained, and resulted in a reduced level of cancer cell death, following treatment with cisplatin or other DNA damaging agents (Quinn et al., 2003). Moreover, more recent evidence has revealed the implication of BRCA1 in cisplatin-resistant breast and ovarian cancer cell lines. These cells that acquired resistance to DNA-damaging agents was mediated by a secondary mutation in BRCA1. This mutation restored the BRCA1 protein expression and function for DNA repair, causing the cancer cells to become more tolerant to cisplatin (Swisher et al., 2008; Tassone et al., 2003; Wang & Figg, 2008). Recently, a number of clinical studies have examined the utilization of this BRCA1 dysfunction in response to the DNA-damaging drug cisplatin. A pathological complete response (pCR) with excellent compliance was observed in cancer patients with BRCA1 mutations (Byrski et al., 2009; Font et al., 2010; Quinn et al., 2007; Silver et al., 2010; Taron et al., 2004). This indicates that patients with BRCA1 dysfunction gain more benefit from treatments that exert their effects by causing DNA damage.

Therefore, it is important to continue elucidating *BRCA1*/BRCA1-dependent pathways to design molecular–targeted therapy for the platinum treatment of cancer cells by taking advantage of their impairment of the *BRCA1*/BRCA1 repair capacity and BRCA1 dependent ubiquitination inactivated by cisplatin.

A DNA Repair Protein BRCA1 as a Potentially

Molecular Target for the Anticancer Platinum Drug Cisplatin 215

based drugs (Chang et al., 2005). Recombination pathways can also be involved as repair systems responsible for DNA damage induced by the anticancer drug cisplatin. Recombination-deficient *E. coli* mutants were sensitive to cisplatin and exhibited a decreased survival by four orders of magnitude in comparison with the parental strain at a cisplatin concentration of 75-80 µM (Zdraveski et al., 2000). Many recombination-deficient strains showed a sensitivity to the drug equal to that of the NER-deficient strains. Double mutations in recombination and NER proteins were approximately 4-fold more sensitive to cisplatin than the corresponding single mutants. This indicates that recombination and NER pathways play roles that are independent of each other in protecting cells from cisplatininduced damage. Impaired recombination DNA repair in yeast and prostate cancer cell lines

The one hybrid GAL4 transcription assay is used to study the effect of cisplatin on transcriptional transactivation. The level of transcriptional transactivation is inversely proportional to the amount of platinum-*BRCA1* adducts. The results are most likely due to inhibition of transcription of the reporter plasmid that resulted from interstrand crosslinks (Ratanaphan et al., 2009). The transcriptional transactivation activity of *BRCA1* has previously been reported by fusing the C-terminal domain of *BRCA1* to a heterogenous DNA-binding domain (Chapman and Verma, 1996). The BRCT domain (amino acids 1380- 1863) of human *BRCA1* scores positively in transcriptional activation trap experiments using various forms of so-called "one hybrid assay".The *BRCA1*-fused DNA-binding domain activates transcription in a cell-free system to a similar extent as a dose of the powerful activator, VP16 (Scully et al., 1997). A *GAL4:BRCA1* has also been introduced in yeast- and mammalian-based transcription assays to characterize the deleterious mutations in the 3/ terminal region of the *BRCA1* (Vallon-Christersson et al., 2001). The transcriptional activity

In order to investigate whether the drug-damaged *BRCA1* is able to transactivate the expression of a firefly luciferase gene, DNA repair-proficient MCF-7 cells were transiently transfected with the cisplatin-damaged pBIND-BRCT along with the reporter plasmid pG5Luc. The firefly luciferase activity was significantly decreased at a cisplatin

It has been hypothesized that the BRCT domain could transactivate the expression of another reporter gene. The reporter gene pSV-β-galactosidase was used for this purpose. It was of interest, that the level of transactivation was significantly higher when co-transfected with the pBIND-BRCT than with the parental pBIND (Fig. 5). This indicated that the GAL4-BRCT domain may stimulate the pSV-β-galactosidase. However, the expression of β-galactosidase was decreased to the level of β-galactosidase alone when co-transfected with the platinated pBIND-BRCT. It was again of interest that, β-galactosidase expression was dramatically diminished when both the pSV-β-galactosidase and the pBIND-BRCT were platinated (Fig. 6). Expression of β-galactosidase from the pSV-β-galactosidase can be transactivated both by the GAL4 domain of the pBIND and pBIND-BRCT. Acting upon the GAL4 DNA sequence similarity, the GAL4 protein alone can stimulate the expression of β-galactosidase. However, the degree of transactivation was slightly higher by the pBIND-BRCT. This indicates that the BRCT domain on the fusion protein is able to transactivate the β-galactosidase gene-bearing pSV-β-galactosidase. When platinated pSV-β-galactosidase is co-transfected with the pBIND

also showed an increased sensitivity to cisplatin (Wang et al., 2005).

**7.2 Inhibition of** *BRCA1* **transcriptional transactivation** 

reflects a tumor-suppressing function of the BRCA1 protein.

concentration of 12.5 μM (Fig. 4).

#### **7.1 Cellular repair of cisplatin-damaged** *BRCA1*

Preliminary results from our laboratory have indicated that the cisplatin-modified *BRCA1*  gene sequence was resistant to restriction endonuclease cleavage, and indicated that cisplatin preferentially formed 1,2-intrastrand d(GpG) cross-links (Ratanaphan et al., 2009). The drug inhibited *BRCA1* amplification in a dose-dependent manner. It has been found that cisplatin-treated, *BRCA1* exon 11, of adenocarcinoma MCF-7 cells exhibited a time dependent recovery after drug exposure to the cells at 37°C for 6 h, with an initial low level of lesion removal during the first 4 h (Fig. 3). A more complete lesion removal was observed with over 90% of 50 μM cisplatin after 18 h of repair time. However, only 30% of the lesion repair was observed at a higher cisplatin concentration of 200 μM (Ratanaphan et al., 2009). From a host cell reactivation assay, the result indicated that a reduction in cellular reactivation of the drug-damaged reporter gene encoding plasmid was a consequence of an increase in platination levels within the transcribed reporter gene. This indicated that the cellular response to cisplatin reflected its intrinsically low capacity for removal of cisplatin-*BRCA1* adducts. Following cisplatin-induced *BRCA1* adducts, a number of cellular repair proteins, excluding BRCA1, are responsible for recognizing and processing the removal of DNA damage. NER is a major process for removing platinum-damaged DNA. This process requires an ATP-dependent multiple protein complex that recognizes the bending induced on DNA by cisplatin. The NER complex has a dual role that can unwind the DNA strands (helicase), and excise the damage strand (endonuclease) of about 24-32 nucleotides in length, containing a platinum lesion. DNA resynthesis factors are recruited at the site of the incised DNA, and employ the opposite strand as template to fill in the gap in concert with DNA ligases. Two distinct sub-pathways of NER that may be involved, are transcription-coupled repair (TCR) and/or global genomic repair (GGR). TCR preferentially repairs transcribed strands of the RNA polymerase II-transcribed active gene, while GGR repairs throughout

the genome (Shuck et al., 2008). Recently, the suppression of ERCC1 expression in a HeLa S3 cell line by small interfering RNA (siRNA) led to a decrease in the repair activity of cisplatin-induced DNA damage along with a decrease in cell viability against platinum-

Fig. 3. Cellular repair of cisplatin-damaged 3,426-bp *BRCA1* exon 11. MCF-7 cells were incubated with medium plus cisplatin at various concentrations (50–200 μM) for 6 h. The cells were washed twice with PBS and fresh medium was added. The genomic DNA was then extracted at 2, 4, 8 and 18 h and used as the template for the QPCR assay (Ratanaphan et al., 2005, 2009).

Preliminary results from our laboratory have indicated that the cisplatin-modified *BRCA1*  gene sequence was resistant to restriction endonuclease cleavage, and indicated that cisplatin preferentially formed 1,2-intrastrand d(GpG) cross-links (Ratanaphan et al., 2009). The drug inhibited *BRCA1* amplification in a dose-dependent manner. It has been found that cisplatin-treated, *BRCA1* exon 11, of adenocarcinoma MCF-7 cells exhibited a time dependent recovery after drug exposure to the cells at 37°C for 6 h, with an initial low level of lesion removal during the first 4 h (Fig. 3). A more complete lesion removal was observed with over 90% of 50 μM cisplatin after 18 h of repair time. However, only 30% of the lesion repair was observed at a higher cisplatin concentration of 200 μM (Ratanaphan et al., 2009). From a host cell reactivation assay, the result indicated that a reduction in cellular reactivation of the drug-damaged reporter gene encoding plasmid was a consequence of an increase in platination levels within the transcribed reporter gene. This indicated that the cellular response to cisplatin reflected its intrinsically low capacity for removal of cisplatin-*BRCA1* adducts. Following cisplatin-induced *BRCA1* adducts, a number of cellular repair proteins, excluding BRCA1, are responsible for recognizing and processing the removal of DNA damage. NER is a major process for removing platinum-damaged DNA. This process requires an ATP-dependent multiple protein complex that recognizes the bending induced on DNA by cisplatin. The NER complex has a dual role that can unwind the DNA strands (helicase), and excise the damage strand (endonuclease) of about 24-32 nucleotides in length, containing a platinum lesion. DNA resynthesis factors are recruited at the site of the incised DNA, and employ the opposite strand as template to fill in the gap in concert with DNA ligases. Two distinct sub-pathways of NER that may be involved, are transcription-coupled repair (TCR) and/or global genomic repair (GGR). TCR preferentially repairs transcribed strands of the RNA polymerase II-transcribed active gene, while GGR repairs throughout the genome (Shuck et al., 2008). Recently, the suppression of ERCC1 expression in a HeLa S3 cell line by small interfering RNA (siRNA) led to a decrease in the repair activity of cisplatin-induced DNA damage along with a decrease in cell viability against platinum-

**7.1 Cellular repair of cisplatin-damaged** *BRCA1*

**D N A** 

et al., 2005, 2009).

**amplification (%)**

0 5 10 15 20 **Repair time (h)**

Fig. 3. Cellular repair of cisplatin-damaged 3,426-bp *BRCA1* exon 11. MCF-7 cells were incubated with medium plus cisplatin at various concentrations (50–200 μM) for 6 h. The cells were washed twice with PBS and fresh medium was added. The genomic DNA was then extracted at 2, 4, 8 and 18 h and used as the template for the QPCR assay (Ratanaphan

**50 µM 100 µM 200 µM** based drugs (Chang et al., 2005). Recombination pathways can also be involved as repair systems responsible for DNA damage induced by the anticancer drug cisplatin. Recombination-deficient *E. coli* mutants were sensitive to cisplatin and exhibited a decreased survival by four orders of magnitude in comparison with the parental strain at a cisplatin concentration of 75-80 µM (Zdraveski et al., 2000). Many recombination-deficient strains showed a sensitivity to the drug equal to that of the NER-deficient strains. Double mutations in recombination and NER proteins were approximately 4-fold more sensitive to cisplatin than the corresponding single mutants. This indicates that recombination and NER pathways play roles that are independent of each other in protecting cells from cisplatininduced damage. Impaired recombination DNA repair in yeast and prostate cancer cell lines also showed an increased sensitivity to cisplatin (Wang et al., 2005).
