**6.1 Cisplatin-DNA adducts**

210 DNA Repair

2006, 2009; Ransburgh et al., 2010). Moreover, BRCA1 accumulation at the sites of DSBs occurred rapidly (within 20 s), and the RING structure was required (residue 1-200 of BRCA1) for the rapid recruitment with Ku80 at damaged sites in response to nonhomologous end joining (Wei et al., 2008). Missense mutations in the BRCA1 RING domain significantly reduced their accumulations at DSBs, and abolished the association with Ku80. Therefore, the loss of the BRCA1 E3 ligase activity rendered cancerous cells hypersensitive to DNA-damaging agents,and clearly demonstrated a significant role for ubiquitnation in the DNA damage response and DNA repair activity (Ransburgh et al., 2010; Ruffner et al., 2001). Thus ubiquitination is involved in key steps that properly conduct the DNA repair

Several reports have shown that the BRCA1 E3 ligase was capable of in vitro monoubiquitination of histones H2A and its variant H2AX (Thakar et al., 2010). This implied a BRCA1 function in regulating chromatin structure in the context of transcriptional regulation and DNA repair. Hyperphosphorylated RNA polymerase II (RNAPII) at its carboxyl terminal domain (CTD), consists of multiple repeats of the heptapeptide (YSPTSPS), involved in a generalized response to UV irradiation. It also served as a substrate for the BRCA1-dependent ubiquitination that was proposed to facilitate BRCA1 function in DNA repair by inhibiting DNA transcription, and then recruiting other DNA repair proteins at a lesion (Starita et al., 2005). Recently, It was found that the BRCA1 mediated ubiquitination of RNAPII prevented a stable association of some transcription factors (TFIIE and TFIIH) in the transcriptional preinitiation complex, and thus blocked the initiation of mRNA synthesis (Horwitz et al., 2007). Ubiquitination of the preinitiation complex was not targeting proteins for degradation by proteasome but rather the ubiquitin moiety itself interfered with the assembly of basal transcription factors at the promoter (Horwitz et al., 2007). Nucleoplasmin B23 and γ-tubulin were found to be the candidate substrates of the BRCA1 E3 ligase activity in vivo (Parvin, 2009; Sato et al., 2004). Both proteins were present in centrosomes, and apparently were not targeted for degradation by BRCA1 mediated modifications. The results indicated that ubiquitination of nucleoplasmin B23 and γtubulin played a vital role in regulating the centrosome number and maintenance of genomic stability by unknown mechanisms. Recently, the BRCA1 protein has been shown to inhibit ERα transcriptional activity, and to induce repression of estrogen response genes and cell proliferation (Xu et al., 2005). A potential explanation for the regulation of estrogen signaling by BRCA1 was the ERα ubiquitination and degradation mediated by the BRCA1 E3 ligase activity (Dizin & Irminger-Finger, 2010; Eakin et al., 2007). Conversely, the BRCA1-associated protein 1 (BAP1) is a deubiquitinating enzyme that can interact with the BRCA1 RING domain (Jensen et al., 1998). It was shown that BAP1 inhibited the BRCA1 autoubiquitination, and the nucleophosmin/B23 ubiquitination mediated by the BRCA1 E3 ligase activity (Nishikawa et al., 2009). Down-regulation of BAP1 in cells also resulted in the retardation of the S phase and ionizing irradiation hypersensitivity, a phenotype similar to BRCA1 deficiency. This again indicated that the BRCA1-BARD1 complex and the BAP1 protein coordinately regulated

ubiquitination during a DNA damage response and the cell cycle.

Cisplatin [*cis*-diamminedichloroplatinum(II)] is the platinum-based anticancer drug and is most effective in the treatment of metastatic testiscular cancers, ovarian, head, neck, bladder,

process after DSBs.

**6. Cisplatin** 

It is generally accepted that DNA is the most important intracellular target of cisplatin. When cisplatin is dissolved in aqueous solution, chloride ions are displaced to allow the formation of aquated species, which are the reactive forms of the compound (Pinto and Lippard, 1985). The concentration of chloride ions influences the reactivity of cisplatin. After intravenous administration it is relatively less reactive in the extracellular space where the physiological chloride concentration is about 100 mM, but on crossing the plasma membrane, it is activated in the intracellular space where the chloride concentration drops to 2-3 mM. Chlorine groups of cisplatin are easily replaced by water molecules to allow the formation of aqauted species in a stepwise manner. Activated cisplatin is a potent electrophile that will react with any nucleophile, including the sulfhydryl groups on proteins and nucleophilic groups on nucleic acids. DNA is attacked by activated cisplatin at guanine residues in position N7, in double stranded DNA from the side of the major groove. The attack is apparently preceeded by an electrostatic attraction between the positively charged platinum (II) complex and the negatively charged phosphodeoxyribose DNA backbone and facilitated by bidirectional diffusion along the backbone. The initial attack of DNA by activated cisplatin is followed by the replacement of the remaining chloro ligand before the adduct forms an intramolecular attack on a second purine residue (either guanine or adenine). The hydration rate constant of the monoaqua form was faster than that of diaqua form (2.38x10-5 s-1 compared to 1.4x10-5 s-1) (Cubo et al., 2009).

The anticancer activity of cisplatin potentially results from the modification of DNA through a covalent cross-link or platinum (Pt)-DNA adduct (Fig. 2). The DNA adducts interfere with DNA replication and transcription, and ultimately lead to cell death by cancer (Ahmad, 2010; Wang & Lippard, 2005). The predominant adducts formed by cisplatin in vitro are 1,2 intrastrand crosslinks. Quantitative studies show that the 1,2-intrastrand d(GpG), and d(ApG) crosslinks account for 65% and 25%, respectively (Fichtinger-Schepman et al., 1985; Eastman, 1986). They alter the DNA structure, block replication and transcription and activate a programmed cell death (apoptosis). X-ray diffraction of the crosslinked dinucleotide *cis*-Pt(NH3)2[d(pGpG)] reveals that the intrastrand cisplatin crosslink produces a severe local distortion in the DNA double helix, leading to unwinding and kinking. These crosslinks bend and unwind the duplex. The altered structure is recognized by high-mobility-group (HMG) proteins and other proteins. The binding of HMG proteins to cisplatin-modified DNA has been postulated to potentiate the anticancer activity of the drug.

#### **6.2 Cisplatin-protein adducts**

The interaction of cisplatin with proteins is of particular significance, and is believed to play an important role in distribution of the drug and the inactivation responsible for determining its efficacy and toxicity (Casini et al., 2008; Sun et al., 2009; Timerbaev et al., 2006). It is intriguing, that protein adducts affect some crucial aspects of protein structure

A DNA Repair Protein BRCA1 as a Potentially

**cisplatin for cancer therapy** 

al., 2008).

DNA damage.

dependent ubiquitination inactivated by cisplatin.

Molecular Target for the Anticancer Platinum Drug Cisplatin 213

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

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

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-

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

Fig. 2. Common cisplatin-DNA adducts

and functions. For instance, the platination of human serum albumin caused partial unfolding of the protein structure at a high drug concentration, and induced intermolecular crosslinks possibly at Cys34 and/or Met298 via bifunctional adducts or via NH3 release (Ivanov et al., 1998; Neault & Tajmir-Riahi, 1998). Myoglobin, a small protein, containing a heme group required for the transport of oxygen in skeletal muscles and myocardial cells, formed intramolecular mono- and bi-functional adducts with cisplatin. Its putative platinum-binding sites were His116 and His119 (Zhao & King, 2010). A number of intramolecular crosslinks also occurred with ubiquitin adducts (Casini et al., 2009). The loss of activity of the C-terminal heat shock protein 90 after protein aggregation was reported to be a consequence of cisplatin binding but it did not exhibit any conformational change (Ishidaa et al., 2008). It is intriguing, that cisplatin can cause a structural perturbation of a synthetic peptide containing a Zn2+ finger domain. The platinum coordinates to Zn2+ binding sites to induce Zn2+ ejection and subsequently the loss of the protein tertiary structure. This implies that cisplatin can inhibit critical biological functions regulated by Zn2+ finger proteins. Such a mechanism has been discussed in the apoptosis process mediated by the interaction of cisplatin and platinum-based compounds with Zn2+ finger transcriptional factors (Bose et al., 2005). Likewise, the nucleocapsid Zn2+ finger NCp7 protein, a protein required for the recognition and packaging of viral RNA, became attached to some platinum compounds, when its ability to bind nucleic acid was changed and prevented viral infectivity (de Paula et al., 2009; Musah, 2004).

and functions. For instance, the platination of human serum albumin caused partial unfolding of the protein structure at a high drug concentration, and induced intermolecular crosslinks possibly at Cys34 and/or Met298 via bifunctional adducts or via NH3 release (Ivanov et al., 1998; Neault & Tajmir-Riahi, 1998). Myoglobin, a small protein, containing a heme group required for the transport of oxygen in skeletal muscles and myocardial cells, formed intramolecular mono- and bi-functional adducts with cisplatin. Its putative platinum-binding sites were His116 and His119 (Zhao & King, 2010). A number of intramolecular crosslinks also occurred with ubiquitin adducts (Casini et al., 2009). The loss of activity of the C-terminal heat shock protein 90 after protein aggregation was reported to be a consequence of cisplatin binding but it did not exhibit any conformational change (Ishidaa et al., 2008). It is intriguing, that cisplatin can cause a structural perturbation of a synthetic peptide containing a Zn2+ finger domain. The platinum coordinates to Zn2+ binding sites to induce Zn2+ ejection and subsequently the loss of the protein tertiary structure. This implies that cisplatin can inhibit critical biological functions regulated by Zn2+ finger proteins. Such a mechanism has been discussed in the apoptosis process mediated by the interaction of cisplatin and platinum-based compounds with Zn2+ finger transcriptional factors (Bose et al., 2005). Likewise, the nucleocapsid Zn2+ finger NCp7 protein, a protein required for the recognition and packaging of viral RNA, became attached to some platinum compounds, when its ability to bind nucleic acid was changed and

Fig. 2. Common cisplatin-DNA adducts

prevented viral infectivity (de Paula et al., 2009; Musah, 2004).
