br Materials and methods br Results br Discussion We

Materials and methods
Results
Discussion We found that mRNA expression of PCNA significantly was down-regulated in BCP-ALL glucose transporter following co-treatment with doxorubicin and NU7441 (Fig. 6e). Our result is congruent with Gehen et al. study reporting that down-regulation of PCNA potentiates growth suppression of lung epithelial cells in response to oxidative DNA damage [32]. Therefore, these results indicate that NU7441 might hinder cell growth and proliferation of BCP-ALL cells through alteration of c-Myc-mediated GADD45 and PCNA expression (Fig. 9). The γH2AX serves as a rapid and sensitive marker for the presence of DSBs induced by genotoxic agents [5]. In the present study, NU7441 in concert with doxorubicin could enhance the frequency of DSBs in both cell lines, correlating with a dramatic increase in γ-H2AX foci (Fig. 8). These data imply that enhancement of doxorubicin toxicity in response to DNA-PK inhibition is a result of increased unrepaired DSBs. High-level accumulation of antiapoptotic protein Bcl-2 has been reported in BCP-ALL cells which blocks apoptosis and promotes cancer cell survival [33]. Gaizo MV et al.demonstrated Bcl-2 dependency of ALL cells supporting the potential ability of Bcl-2 antagonists as single agents or combination therapies in ALL [34]. In addition, it is well established that doxorubicin can induce Bcl-2 expression via NF-kB pathway [35], [36]. Intriguingly, treatment of BCP-ALL cells with doxorubicin caused up-regulation of Bcl-2 gene which was significantly suppressed in the presence of NU7441 (Fig. 5). Notably, Basu et al. reported that DNA-PK activates NF-kB pathway following DNA damage in glioblastoma cells [37]. Thus, our findings support this idea that Bcl-2 down-regulation induced by inhibition of DNA-PK may be attributed to suppression of NF-kB pathway in BCP-ALL cells, but needs to be clarified further. In conclusion, these data provide mechanistic evidence for the first time that the combination of DNA-PK inhibitor NU7441 with doxorubicin potentiates the inhibition of cell growth and the induction of apoptosis in BCP-ALL cells. Therefore, targeting DNA-PK might be considered an important though as of yet not fully exploited avenue to enhance therapeutic efficacy of DNA-damaging chemotherapeutic agents in BCP-ALL.
Conflict of interest
Acknowledgment This study was supported by the grant No. 26071 from Iran University of Medical Sciences.
Introduction DNA replication constitutes one of the most important processes within the cell and allows for the accurate duplication of genomic information prior to cell division. While this process occurs rapidly in unperturbed cells, numerous endogenous and exogenous sources may either slow or stall replication fork progression [1]. Such disruption results in the exposure of single-stranded DNA (ssDNA) that must be stabilised to prevent replication fork collapse and double-stranded DNA break formation [2]. This process includes the nucleation of replication protein A (RPA) onto exposed ssDNA, as well as the subsequent accumulation and activation of the ataxia telangiectasia and Rad3-related (ATR) kinase [3]. ATR may then phosphorylate Chk1 to promote cell cycle checkpoint activation [4], as well as other proteins involved in restarting replication [5]. In addition to ATR, numerous additional regulatory enzymes are also activated by replication stress, including the related PI3K-like kinases, ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) [6], [7], [8]. Activation of these enzymes results in the coordinated regulation of numerous DNA repair proteins and is exemplified by the ATM, ATR and DNA-PK-mediated phosphorylation of the RPA 32kDa subunit (RPA32) [8], [9], [10]. Recently our group and others provided evidence that the restart of stalled replication forks is promoted by human single-stranded DNA-binding protein 1 (hSSB1; also known as Nucleic Acid Binding Protein 2 NABP2) [11], [12], a ssDNA-binding protein that also functions in the repair of double-stranded DNA breaks [13] and oxidative nucleotide lesions [14]. Following the disruption of replication, hSSB1 was found to localise with stalled replication forks where it promotes recruitment and subsequent activation of the ATR kinase. The importance of hSSB1 in these processes is highlighted by the inefficiency of hSSB1-depleted cells to activate checkpoint signalling following replication stress, as well as their inability to restart stalled replication forks. These deficiencies are manifested by hypersensitivity to multiple replication inhibitors [11].