**5. BRCA1 and protein ubiquitination**

The BRCA1 protein displays an E3 ubiquitin ligase activity through its RING domain, and this activity is enhanced when it exists as a heterodimer with the BARD1 RING domain (Xia et al., 2003). In vitro and in vivo studies have indicated that the BRCA1-BARD1 complex was capable of autoubiquitination that paradoxically stabilized the protein complex, and that also activated its in vitro E3 ligase activity with other proteins (Chen et al., 2002; Wu-Baer et al., 2010). However, the substrate specificity of the BRCA1 E3 ligase activity and its biological relevance to tumor suppression function are still unknown. Putative substrates for ubiquitination by the BRCA1-BARD1 RING complexes have recently emerged from in vitro and in vivo studies such as the nucleosomal histones H2A and its variant H2AX, RNA polymerase II, γ-tubulin, nucleophosmin/B23, and estrogen receptor α (ERα) (Eakin et al., 2007; Horwitz et al., 2007; Parvin, 2009; Sato et al., 2004; Starita et al., 2005; Thakar et al., 2010). BRCA1 can form a RING heterodimer E3 ligase activity with BARD1, and this is required for the recruitment of BRCA2 and RAD51 to damaged sites for HR repair (Ransburgh et al., 2010). Many cancer-predisposing mutations in the BRCA1 RING domain, that inhibited the E3 ligase activity and its ability to accumulate at damaged sites, were defective in homologous recombination that is critical for tumor suppression (Morris et al.,

A DNA Repair Protein BRCA1 as a Potentially

that reacts with cellular targets.

**6.1 Cisplatin-DNA adducts** 

Molecular Target for the Anticancer Platinum Drug Cisplatin 211

cervical and lung cancers (Kelland, 2007). Although widely used as a well established anticancer drug in cancer chemotherapy, cisplatin displays major toxic side effects, such as nephrotoxicity, nausea and vomiting and neurotoxicity. In addition to its toxic side effects, a major limitation of cisplatin chemotherapy is the development of genetic mechanisms of resistance. The effectiveness of cisplatin depends on the drug uptake, and the actual amount

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

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

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

diaqua form (2.38x10-5 s-1 compared to 1.4x10-5 s-1) (Cubo et al., 2009).

been postulated to potentiate the anticancer activity of the drug.

**6.2 Cisplatin-protein adducts** 

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 process after DSBs.

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.

#### **6. Cisplatin**

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, cervical and lung cancers (Kelland, 2007). Although widely used as a well established anticancer drug in cancer chemotherapy, cisplatin displays major toxic side effects, such as nephrotoxicity, nausea and vomiting and neurotoxicity. In addition to its toxic side effects, a major limitation of cisplatin chemotherapy is the development of genetic mechanisms of resistance. The effectiveness of cisplatin depends on the drug uptake, and the actual amount that reacts with cellular targets.
