H2AX a promising biomarker for lung cancer: a review.

Cancer Investigation, 31:582–599, 2013 ISSN: 0735-7907 print / 1532-4192 online C 2013 Informa Healthcare USA, Inc. Copyright DOI: 10.3109/07357907.2013.849721

ORIGINAL ARTICLE

H2AX a Promising Biomarker for Lung Cancer: A Review D. Matthaios,1 P. Hountis,2 P. Karakitsos,3 D. Bouros,4 andS. Kakolyris1

Cancer Invest Downloaded from informahealthcare.com by Ondokuz Mayis Univ. on 05/07/14 For personal use only.

Department of Oncology, Democritus University of Thrace, Alexandroupolis, Greece,1 Department of Thoracic Surgery, Athens Naval Hospital, Athens, Greece,2 Department of Cytopathology, University of Athens Medical School, “Attikon” University Hospital, Athens, Greece,3 Department of Pneumonology, Democritus University of Thrace, Alexandroupolis, Greece,4 in higher eukaryotes, are not transcriptionally coupled to Sphase like core histone H2A genes, and comprise between 2 and 25% of the total histone H2A complement. The H2AX proteins have an extended C-terminal tail with an SQ motif at S139 that is phosphorylated in response to DNA damage [5]. Phoshorylation of H2AX can be detected using specific antibodies and therefore enumerate the number of DSBs in a cell, and as a result give valuable information of the DDR status of the cell. This review will focus on the possible applications of H2AX as a key regulator of DDR, as a biomarker of sensitivity to chemo and radiotherapy of lung tumors, as a marker of lung tumor’s sensitivity, as a marker of environmental genotoxicity, as a prognostic factor for survival in lung cancer patients, and as a therapeutic target in lung cancer (Table 1).

Histone’s H2A variant (H2AX) phosphorylation is an early indicator of DNA double-strand breaks formation and DNA damage response. Thus, it may act as a novel biomarker to monitor genotoxic events that can drive cancer development and tumor progression. This review will focus on the possible applications of H2AX as a key regulator of DNA damage response in lung cancer and as a biomarker of: sensitivity of lung tumors to chemotherapy and radiotherapy, treatment with PARP inhibitors, bystander effect, multistep lung carcinogenesis, environmental smoking, and chemical genotoxicity, chemoprevention, prognosis, and also as therapeutic targets in lung cancers. Keywords: H2AX, Lung cancer, DNA damage response, PARP inhibitors, Carcinogenesis

METHODS INTRODUCTION

Literature search A literature search using Medline was conducted from 1980 onwards, searching for articles with relevant key words such as γ -2, DDR, histones, and lung cancer. Appropriate additional references were found from the bibliographies of identified papers of interest. Any relevant scientific proceedings or medical texts were checked when necessary.

Lung cancer is the most common cause of cancer mortality worldwide for both men and women, causing approximately 1.2 million deaths per year [1]. In the United States, there will be an estimated 221,000 new cases of lung cancer and 157,000 deaths in 2011 [2]. The traditional evaluation of prognosis in nonsmall cell lung carcinoma (NSCLC) has relied, as in most other malignant tumors, on the stage of disease at presentation. Other factors commonly considered include performance status, weight loss, and presence or absence of symptoms at diagnosis [3], as well as time-honored pathologic parameters, e.g. tumor size, tumor differentiation, and histologic subtype. However, advances in molecular biology have provided important insights into other potentially significant prognostic biomarkers during the last decade [4]. Phosphorylation of the H2AX histone is an early indicator of DNA double-strand breaks (DSB) and of the resulting response (DNA damage response, DDR). Histones are the basic units utilized by cells to compact their genome into the nucleus. Two copies of each of four core histones—H2A, H2B, H3 and H4—form an octamer around which approximately 150 bp of DNA is wrapped to form the nucleosome core particle. The histone H2AX variants exist as single-copy genes

H2AX a principal player in DNA damage response H2A functions as a core component of the histone octamer that forms the nucleosome. The isoform H2AX represents 2 to 25% of the total histone H2A expressed by cells and has been established to play a crucial role in the maintenance of genomic stability [6]. H2AX is implicated in the genomic response to DNA damage, and phosphorylated H2AX (γ -H2AX) accumulates at DNA DSBs, where it restructure chromatin and assist in the recruitment of DNA repair and signaling factors [6]. DNA damage-induced phosphorylation of H2AX at Ser139 is mediated by members of the PIKK group of protein kinases, including ATM, ATR, and DNAPK [6]. Studies of knockout cells demonstrate that there is a primary role for ATR in DNA replication stress and for ATM in response to low doses of gamma-radiation [6]. ATM

Correspondence to: Dimitrios Matthaios Department of Oncology, Democritus University of Thrace, Ainou 12, Alexandroupolis, Greece, PO 68100. email: [email protected]

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H2AX and Lung Cancer  Table 1. Potential Applications of γ -H2AX in Lung Cancer


Potential applications of γ -H2AX in lung cancer Evaluation of cisplatin resistance and of compounds that enhance cisplatin cytotoxicity Evaluation of chemotherapy cytotoxicity Evaluation of cytotoxicity of new molecules Monitoring of the process of lung carcinogenesis Biomarker in lung cancer chemoprevention studies Measurement of the toxicity of cigarettes and smoking Measurement of the DNA damage caused by environmental factors causing lung cancer (RN)a IRb biodosimeter Predictor of radiosensitivity Evaluation of radiosensitizing agents Measurement of the efficacy of combined chemo- and radiotherapy protocols Monitoring of radiation-induced bystander effect Efficacy of PARPc -inhibition in clinical trials that use PARP-inhibitors H2AX and γ -H2AX peptides and antibodies as chemotherapeutic agents Prognostic marker a

Radonium. Irradiation. c Poly (ADP-ribose) polymerase. b

is activated by its autophosphorylation at Ser1981 position, which leads to dissociation of the inactive ATM dimers into single protein molecules with increased kinase activity [7]. A tri-protein complex, the MRN complex (MRE11RAD50-NBS1) recognizes DNA damage, recruits ATM to the site of damage, and targets ATM in order to start phosphorylation of the respective substrates [8]. ATM activation needs prior ATM acetylation, mediated by Tip60 histone acetyltransferase [9]. Apart from H2AX, the target substrates phosphorylated by ATM are BRCA1, 53BP1, and MDC1 as well as checkpoint proteins, Chk1 and Chk2. ATR phosphorylates H2AX in response to single-stranded DNA breaks and during replication stress, such as replication fork arrest [10] (Figure 1, Figure 2). DNA-PK mediates phosphorylation of H2AX in cells under hypertonic conditions and during apoptotic DNA fragmentation [11]. However, DNA damage caused by ionizing radiation (IR) leads to phosphorylation of H2AX that is mediated by all PIKK kinases, ATM, ATR, and DNA-PK [12]. It was reported that some other events occur before H2AX phosphorylation in mammalian cells. Ayoub et al. [13] observed that DNA breaks mobilize heterochromatin protein 1β (HP1-β), a chromatin factor bound to histone H3 methylated on lysine 9 (H3K9me). In response to DNA damage, HP1-β was rapidly phosphorylated at threonine 51 (Thr51) by casein kinase 2 (CK2). This phosphorylation results to the release of HP1-β from chromatin which leads to mobilization from chromatin. Although it is still not known what signals CK2 to phosphorylate HP1, the phosphorylation and mobilization of HP1 seems to be important for H2AX phosphorylation [13]. Unrepaired DSBs induce genome instability and promote tumorigenesis. Two domains, which are frequently found in proteins involved in DDR, have been described to specifically recognize phosphorylated amino acid residues. The forkhead-associated (FHA) C 2013 Informa Healthcare USA, Inc. Copyright

domain recognizes phosphorylated threonine residues in a specific aminoacid sequence context [14]. In addition, two consecutive BRCT domains (BRCA1 C-terminal domain) can create a structural element with phosphopeptide binding capacity [15,16]. It was shown that the BRCT repeats of mediator of DNA damage checkpoint protein 1 (MDC1) build the predominant recognition module of γ -H2AX [17]. Interaction between MDC1 and γ -H2AX can be recognized as the first step in which the site of the DSB is prepared for DNA damage signaling and repair. This is because there is experimental evidence that MDC1 also directly interacts in a highly dynamic manner with NBS1 [17], which together with other proteins of the MRN complex is required for the activation of ATM [18]. CK2 phosphorylates MDC1 and this promotes phosphorylation-dependent interactions with NBS1, through its closely opposed FHA domain and two BRCA repeats [19]. As a result, a positive feed-back loop is constructed that extends H2AX phosphorylation to large DNA regions. The recruitment of ATM to DSBs is dependent on NBS1 and NBS1 forms the complex with both ATM and γ -H2AX. Thus, both γ -H2AX and NBS1 contribute to the recruitment of ATM to DSB sites and its activation of cell-cycle checkpoints. Kobayashi et al. have investigated the mechanism involved in ATM recruitment to DSBs [20]. Kobayashi et al. showed that ATM forms a complex with γ -H2AX via NBS1 or MDC1 and this might be crucial for the recruitment of ATM to DSB sites and induction of ATM-dependent checkpoints [20]. Several protein phosphatases, PP2A, PP4, PP6, and Wip1, have been shown to dephosphorylate γ -H2AX and negatively regulate H2AX functions [21]. The phosphorylation of six SDTDXD/E repeats near the N terminus of MDC1 serves to recruit NBS1 (Nijmegen Breakage Syndrome 1) and to regulate the intra-S-phase checkpoint in response to DNA damage [22]. In addition, MDC1 also recruits E3 ubiquitin ligase RNF8 in a phosphorylation-dependent manner, with the latter being responsible for tethering 53BP1 (p53-binding protein 1), BRCA1 (breast and ovarian cancer susceptibility protein 1), and RAP80 (receptor-associated protein 80)containing complexes at damage sites [23]. Furthermore, a signal amplification loop that includes the factors H2AX, MDC1, and probably NBS1 activates and/or retains ATM at DSB sites and leads to the spreading of H2AX phosphorylation up to megabase regions surrounding DSBs [24]. This long-range γ -H2AX/MDC1 localization adjacent to DSBs serves as a landing site for the accumulation of other DDR proteins. Besides MDC1, MCPH1 (also known as BRIT1) is also recruited to DSB sites via its direct interaction with γ -H2AX, as is mediated by the C-terminal BRCT domains of MCPH1 [25]. The shortening or uncapping of telomeres is a crucial event in the context of DDR system. It will reveal unprotected double-stranded ends and trigger a DDR, in which damage response proteins, such as γ -H2AX, will accumulate at the dysfunctional telomeres [26]. In mTR−/− cells (cells lacking the RNA component of telomerase), γ -H2AX has been shown to localize to the telomere in late generation, thereby suggesting that critically shortened telomeres are recognized directly as DNA breaks [27]. It


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Figure 1. H2AX phosporylation and its role in DDR in lung carcinogenesis. A variety of factors that are known to have a causal relationship with lung carcinogenesis are also implicated in the creation of DSBs that in turn lead to the phoshorylation of H2AX, a key molecule of the DNAdamage repair (DDR) system. A series of the main molecules that are implicated (a) in the amplification and stabilization of H2AX phosphorylation (feedback loop) (b) in the accumulation at DSBs sites, like cohesins and complexes for chromatin remodeling and (c) in checkpoint control are depicted.

has also been reported that subtelomeric regions in budding yeast are constitutively modified by γ -H2AX, suggesting that telomeres can be recognized as a form of DSB damage [28]. H2AX is neither necessary for the normal mitotic telomere maintenance, nor required for the chromosome fusions caused by dysfunctional telomeres. As far as lung cancer is concerned, telomere shortening is an early event in bronchial carcinogenesis, preceding P53/Rb pathway inactivation and telomerase reactivation, and leading to DDRs. Telomere shortening occurs at the earliest stage of lung carcinogenesis as an initiating event. In contrast, dismiss suppression of DDR, through γ -H2AX and p-CHK2 downregulation, is a late event associated with SCC and ADC progression [29]. Raynaud et al. found that there are clear molecular differences at the telomeric and DDR level between primary lung NSCLC tumors and their corresponding metastases [30]. In addition to being ubiquitylated, histones are also acetylated following DNA damage. For example, histone

acetyltransferase TIP60 participates in DDR [31], partially by promoting histone H4 acetylation and the accumulation of repair proteins, including RAD51, at DSB sites [32]. Tip60 also acetylates and activates ATM at DSB sites [9]. As far as H2AX is concerned, TIP60 has been shown to catalyze H2AX acetylation, independently of its phosphorylation status, in response to ionizing irradiation [33]. Furthermore, acetylation of H2AX by TIP60 is proposed to be prerequisite for H2AX ubiquitylation [33]. This acetylation-dependent H2AX ubiquitylation by TIP60–UBC13 complex may result in the release of H2AX from damaged chromatin, thereby enhancing chromatin dynamics and allowing the access of repair proteins to DSB sites. Such complex procedures involving phosphorylation, acetylation, ubiquitylation, and SUMOylation of H2AX and other DDR proteins constitute a complex network that is activated in response to DNA damage. The INO80 complex is a multisubunit, ATP-dependent chromatin remodeling complex, whose ability to regulate Cancer Investigation


H2AX and Lung Cancer 

Figure 2. The central role of H2AX in DDR system. Abbreviations: DNA-damage repair (DDR), Double-strand break (DSB), ataxia telangiectasia mutated (ATM), ataxia telangiectasia and rad3-related (ATR), DNA-dependent protein kinase (DNA-PK), nibrin (NBS1), mediator of DNA damage checkpoint protein 1 (MDC1), Protein phosphatases (PP2A and PP4c), p53-binding protein 1 (53BP1), activation-induced cytidine deaminase (AID), recombination-activating protein (RAG v(D)J), reactive oxygen species (ROS), topoisomerase (TOP), homologous recombination (HR), O6-methylguanine (06MeG), mismatch repair (MMR), nucleotide excision repair (NER), non-homologous end joining (NHEJ), direct repair (DR), checkpoint kinase 1 and 2 (CHK1 and CHK2), translesion DNA synthesis (TLS), base excision repair (BER). There are many factors that directly or indirectly can cause DSBs like: ROS, smoking, radiation, drugs, and telomere shortening, during v(D)J recombination, class switch recombination (CSR) and meiosis, mutation in DDR proteins, FHIT loss, and infections. HR and NHEJ are involved in the repair of DSBs. ATM, ATR, and DNA-PK are key molecules participating in the initial H2AX phosphorylation. A signal amplification loop including H2AX, NBS1, and MDC1 stimulates ATM and increases H2AX phosphorylation. PP2A and PP4c dephosphorylate γ -H2AX. NBS1 and MDC1 bind to γ -H2AX and after that, a series of DDR proteins accumulate like: MRE11–RAD50–NBS1 complex, rNF8, BRCA1,p53-binding protein 1 (53BP1). TIP60 and UBC13 regulate H2AX acetylation which is essential for the ubiquitylation (Ub) of H2AX and its release from chromatin. C 2013 Informa Healthcare USA, Inc. Copyright


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transcriptional processes is well established [34]. Several studies suggested that γ -H2AX might have some impact on NHEJ (nonhomologous end joining) [35, 36]. Since, as mentioned above, γ -H2AX is involved in the accumulation of DNA repair proteins, such as 53BP1 and MRN complex at DSB sites, and these proteins are known to play a role in some specialized NHEJ processes, γ -H2AX can contribute to certain types of NHEJ. Although H2AX is likely dispensable for classic NHEJ repair, it plays an additional role in specific NHEJ processes that require the stable accumulation of several DNA damage repair proteins, including MRN, 53BP1, and BRCA1. Similar to its role in NHEJ, H2AX only modulates HR repair efficiency [37, 38]. In the absence of H2AX, MRN complex can recognize DSB sites and initiate DNA end resection and HR repair. H2AX may perform an accessory function in HR repair via its ability to stabilize and accumulate DNA damage repair proteins at or near DSB sites. The moderate role of the H2AX-MDC1 axis in HR repair is also supported by mouse genetic studies. Depletion of components directly or indirectly involved in HR pathway such as, ATR [39], MRN complex [40], BRCA1 [41], BRCA2 [42], CtIP [43], and RAD51 [44], in mice all resulted in embryonic lethality, implying that the intact HR pathway is crucial during embryogenesis. In fact, mice lacking other factors involved in the H2AX-MDC1 axis, such as ATM [45], and MDC1 [46], all display increased genomic instability and cancer susceptibility; however, these mice are viable. Again, such observations indicate that an HR-mediated DSB repair pathway can occur, however, at lower efficiency, in cells or animals deficient in any of the components involved in the H2AX-mediated DNA damage-signaling cascade. Ebi et al. found that knockdown of RB-induced γ -H2AX foci formation in NSCLC cells with wild-type RB, in association with growth inhibition and reactive oxygen species (ROS) generation, which was canceled by overexpression of miR-17–92. Conversely, induction of γ -H2AX was observed in a miR-17–92-overexpressing SCLC cell line with miR-20a antisense oligonucleotides. These findings suggest that miR-17–92 overexpression may serve as a fine-tuning influence to counterbalance the generation of DNA damage in RB-inactivated SCLC cells, thus reducing excessive DNA damage to a tolerable level and consequently leading to genetic instability [47]. Huang et al. examined hypothesized contributions of the replicative DNA polymerase δ (pol δ) subunit POLD4 to the generation of genomic instability in lung cancer. The lung cancer cell line ACC-LC-48 was found to have low POLD4 expression, with higher histone H3K9 methylation and lower acetylation in the POLD4 promoter, as compared with the A549 cell line with high POLD4 expression. POLD4 overexpression reduced intrinsically high induction of γ -H2AX [48]. Polo-like kinase 2 gene (Plk2/Snk) is a direct target for transcriptional regulation by p53 and that silencing Plk2 sensitizes cancer cells to taxol-induced apoptosis. Matthew et al. found that following knockdown of Plk2 in wild-type p53-expressing H460 human NSCLC cells, there was a significant increase in cell death observed in aphidicolin-treated cells and a further increase after release from aphidicolin-block. The highest

levels of cell death were observed when Plk2-deficient cells were released from both aphidicolin and etoposide treatment. They also observed higher levels of Serine 139 H2AX phosphorylation in Plk2-deficient as compared to control cells before and after aphidicolin treatment indicating that there is more DNA damage when Plk2 is depleted [49]. Methods of γ -H2AX detection γ -H2AX detection methods are nowadays easily applicable and therefore γ -H2AX adoption as a promising biomarker in lung cancer is on the road. The quantitative measurement of DSBs was based on methods, such as pulse-field gel electrophoresis (PFGE), DNA elution tests, or the “comet assay” (single-cell gel electrophoresis—SCGE). Comet assay is a versatile, sensitive, and widely used one. In this method, individual cells with damaged DNA embedded in agarose gels are subjected to an electric field and generate a characteristic pattern of DNA distribution forming a tail that, after staining with fluorescence dye, can be analyzed by fluorescence microscopy. The extent and length of the comet’s tail correlates with the severity of DNA damage [50]. The sensitivity of the comet assay, however, depends on proper calibration and its specificity is not excellent [51]. Development of an antibody, that is specific to γ -H2AX, enabled the detection of H2AX phosphorylation and thus detection of DNA damage and repair in situ in individual cells. The presence of γ H2AX in chromatin can be detected shortly after induction of DSBs in the form of discrete nuclear foci. Since each focus represents a single DSB, their frequency reports the incidence of DSBs [52]. In comparison to other methods of DNA damage assessment as mentioned above, the immunocytochemical approach is easier and offers much greater sensitivity [52, 53]. The presence of γ -H2AX-containing nuclear foci can be measured by microscopy, flow cytometry, and possibly Western blotting of cell/tissue lysates, with normalization for the total H2AX levels. Measurement of γ -H2AX with the use of multiparameter flow or laser scanning cytometry seems to be particularly advantageous as H2AX phosphorylation can be determined in individual cells with high sensitivity and accuracy and γ -H2AX expression in cell populations can be correlated with DNA content or induction of apoptosis [52, 53]. In the everyday clinical context, patient blood samples can be conveniently collected and used for determination of γ -H2AX foci [54–56]. The tracking of γ H2AX foci in PBL is common in the literature in comparison to any other normal tissues in assessing DNA damage. DSB induction and repair in PBL has been studied using samples derived from in vivo or ex vivo irradiation and repair over 24 hrs ex vivo. Patient blood samples have been collected and DNA damage and repair were assayed using the Comet assay or γ -H2AX foci to determine interindividual differences in the rate of γ -H2AX resolution [55, 56]. One ELISA-based method using an electrochemiluminescent detection system (an assay derived from the Meso Scale Discovery Technology or MSD assay) to measure γ -H2AX in tumors biopsies after irradiation was recently reported [57]. A high-throughput screening system, called the RABIT (Rapid Automated Biodosimetry Tool), using a γ -H2AX IF assay to Cancer Investigation


H2AX and Lung Cancer  directly measure DSBs level, was developed, which would allow the screening of 6,500 samples a day [58]. Circulating tumor cells (CTCs) in peripheral blood of patients reflects a fraction of solid tumor cells available for more frequent pharmacodynamic assessment of chemotherapy activity than using tumor biopsy. However, currently available CTC assays are limited to cell membrane antigens. Wang et al. described an assay that directly examines changes in levels of γ -H2AX in individual CTCs of patients treated with chemotherapeutic agents [59]. Currently available CTC assay systems are limited to cell membrane antigens, such as HER2, MUC1, and EGFR. In addition, currently used methods for detection of DNA damage in patient samples, including γ -H2AX immunofluorescence, alkaline comet assay, immunohistochemistry, and FACS analysis can be labor- and time-intensive and have limited applicability in the clinic to monitor tumor response to chemotherapeutic agents. For instance, whereas FACS analysis of γ -H2AX is more sensitive than the alkaline comet assay [55], it does not distinguish CTCs from normal blood cells, and the analytical sensitivity is too low to detect CTCs, even if it could distinguish them; therefore, γ H2AX response to drug treatment could be established only in nontumor cells (e.g., peripheral blood mononuclear cells). Compared with other approaches, the combination of CTC enrichment with nuclear γ -H2AX detection is not only a unique and advanced technique, but also feasible for incorporation into clinical trials of molecular-targeted oncology drugs [60]. Finally, Qian et al. developed a computer-aided cytologic diagnosis (CACD) system to recognize expression of the cancer biomarkers histone H2AX in lung cancer cells and then tested the accuracy of this system to distinguish resected lung cancer from preneoplastic and normal tissues [60]. γ -H2AX and lung tumor sensitivity. The example of cisplatin resistance Platinum-based combination chemotherapy remains until now the main treatment option for lung cancer. Cisplatin is adopted widely and used effectively in the first-line chemotherapy. Unfortunately, development of cisplatin resistance is a major obstacle to the success of lung cancer chemotherapy. Cisplatin is a cell-cycle-non-specific cytotoxic drugs and its main target is DNA. Thus, defective DNA damage repair is one of the main mechanisms of cisplatin resistance. To investigate the kinetics of cisplatin adduct formation and DNA damage signaling at a cellular level, Liedert et al. analyzed cisplatin-treated tumors for the presence of PtDNA adducts using a Pt-1,2-d(GpG) intrastrand cross-linkspecific monoclonal antibody [61]. This antibody recognizes the most frequently occurring adduct formed by cisplatin, which is associated with its cytotoxicity and anticancer activity [61, 62]. Pt-DNA adducts were detected in the lung as early as 3 hrs after a single dose of cisplatin. Platinum adduct formation can cause stalling of replication forks, which leads to collapse and the generation of DNA DSBs. This leads to activation of checkpoint kinases ATM and ATR, and their downstream substrates, Chk2 and Chk1, which recruit other repair proteins to sites of damaged DNA [63]. In cisplatinC 2013 Informa Healthcare USA, Inc. Copyright

treated KrasG12D/+ tumors, Oliver et al. detected γ -H2AX 4 hrs (the earliest time point examined) after cisplatin treatment, with maximal staining 12 to 24 hrs following. Basal phosphorylation of the checkpoint kinase Chk2 (Thr68) was detected in untreated tumors, and increased phosphorylation of both Chk1 (Ser345) and Chk2 (Thr68) was clearly evident after cisplatin treatment. These data demonstrate that tumors sense DNA damage in response to cisplatin within 4 hrs, and respond by cell-cycle arrest and cell death associated with activation of both Chk1 and Chk2. In KrasG12D/+;p53fl/fl lung tumors analyzed 4to 24 hrs after a single dose of cisplatin, Oliver et al. did not detect obvious differences in DNA damage signaling compared with p53 wild-type tumors .The authors also observed very few tumors with patterns of BrdU or γ -H2AX staining that deviated significantly from the mean at the indicated time points, suggesting that most tumors initially respond to cisplatin-induced DNA damage in this model [64]. Increased DNA repair has been proposed as a mechanism of platinum resistance [65]. Oliver et al. observed a clear difference in the dynamics of phosphorylation of these two DNA damage signaling proteins. Chk1 phosphorylation occurred in response to cisplatin in tumors from both na¨ıve and long-term treated mice (G1 vs. G2, and G3 vs. G4) demonstrating that resistant tumors activate the DDR, and, thus, that cisplatin is entering these cells [64]. In contrast, Chk2 phosphorylation was induced after an initial dose of cisplatin (G1 vs. G2), and then remained high even in tumors that had not been given cisplatin for several weeks [65]. This finding suggests that these two signaling pathways may be responding to distinct DNA damage signals as a result of cisplatin treatment—one that is transient (Chk1), and another that is persistent (Chk2). Furthermore, it suggests there is a fundamental difference in the DDR mechanism in na¨ıve and long-term cisplatin-treated lung tumors. As a result, it seems that increased DNA damage repair is the predominant mechanism of cisplatin resistance in vivo in this model. In long-term cisplatin-treated tumors, carboplatin produces fewer adducts and reduced DNA damage signaling, evident by γ -H2AX staining compared with na¨ıve tumors [65]. These data indicate that, just as encountered in clinical resistance [66], cisplatin-resistant tumors in this model are cross-resistant to other platinum analogs. Since many human cancers have premalignant stages of tumor progression, it will be important to investigate whether treating low-grade tumors with DNA-damaging agents can facilitate tumor progression. This knowledge will be validated as in the next years oncologists will detect more frequently earlier-stages of lung cancer disease. Rabik et al. reported that O6-Benzylguanine (BG) enhances cisplatin [cisdiammine dichloroplatinum (II)]-induced cytotoxicity and apoptosis in head and neck cancer cell lines by an unknown mechanism. However, in A549 NSCLC cell line did not enhance the cisplatin cytotoxicity [67]. Bulky cisplatin lesions are repaired primarily by nucleotide excision repair (NER), in which the structure-specific endonuclease XPF–ERCC1 is a critical component. It has been suggested that expression of ERCC1 correlates with cisplatin resistance in NSCLC. Arora et al., using NSCLC, ovarian, and breast cancer cells,


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showed that the XPF–ERCC1 complex is a valid target to increase cisplatin cytotoxicity and efficacy [68]. They assessed the repair of cisplatin-ICL-induced DSBs by monitoring γ H2AX focus formation. Interestingly, XPF protein levels were significantly reduced following ERCC1 downregulation, but the converse was not observed. The repair of both types of cisplatin-DNA lesions was decreased with downregulation of XPF, ERCC1, or both XPF–ERCC1. The ICL-induced DSBs persist in the absence of XPF–ERCC1. The suppression of the XPF–ERCC1 complex significantly decreases the cellular viability which correlates well with the decrease in DNA repair capacity [68]. Insulin-like growth factor-1 (IGF-1) eliminates the effect of cisplatin that induces DNA damage through the formation of platinum–DNA adducts. Jeon et al. investigated the effects of IGF-1 on the DNA DSBs repair system induced by cisplatin. NCI-H1299 and H460 NSCLC cells treated with IGF-1 recovered from cisplatin-derived inhibited proliferation and apoptosis [69]. Decreased tail length in comet assay and suppressed phosphorylation of histone H2AX at Ser139 with IGF-1 cotreatment indicates that IGF-1 attenuates cisplatin-induced DNA damage. Suppression of the IGF system with AG1024 or siRNA of insulin receptor substrate1 (IRS-1), a major adaptor molecule of the IGF system, augmented cisplatin-induced γ -H2AX, Ser1981-pATM, and Ser428-pATR generation. ATM strongly binds with IRS-1 under the influence of cisplatin, and the interaction was partially inhibited by IGF-1. Immunocytochemistry revealed that cisplatin induces nuclear translocation of IRS-1 with Ser1981-pATM, which is suppressed by cotreatment with IGF-1 [69]. Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. Iwasa et al. investigated the effects of the combination of YM155, a novel small-molecule inhibitor of survivin expression, and platinum compounds (cisplatin and carboplatin) on human NSCLC cell lines. Immunofluorescence analysis of histone γ -H2AX showed that YM155 delayed the repair of DSBs induced in nuclear DNA by platinum compounds. The authors suggest that YM155 sensitises tumor cells to platinum compounds both in vitro and in vivo, and that this effect is likely attributable to the inhibition of DNA repair and consequent enhancement of apoptosis [70]. Pyriplatin, cis-diammine(pyridine)chloroplatinum(II), a platinum-based antitumor drug candidate, is a cationic compound with anticancer properties in mice and is a substrate for organic cation transporters that facilitate oxaliplatin uptake. Unlike cisplatin and oxaliplatin, which form DNA cross-links, pyriplatin binds DNA in a monofunctional manner. On a platinum-per-nucleotide basis, pyriplatin-DNA adducts are less cytotoxic than those of cisplatin and oxaliplatin [71]. Mutations of NPRL2 in yeast cells are associated with resistance to cisplatin-mediated cell killing. Restoration of NPRL2 in NPRL2-negative and cisplatin-resistant cells resensitize lung cancer cells to cisplatin treatment both in vitro and in vivo. Jayachandran et al. showed that sensitization of NSCLC cells to cisplatin by NPRL2 is accomplished through the regulation of key components in the DNA-damage checkpoint pathway [72]. NPRL2 can phosphorylate ataxia telangiectasia-mutated (ATM) kinase acti-

vated by cisplatin and promote downstream of γ -H2AX formation in vitro and in vivo, which occurs during apoptosis concurrently with the initial appearance of high-molecular weight DNA fragments. Moreover, this combination treatment results in higher Chk1 and Chk2 kinase activity than does treatment with cisplatin alone and can activate Chk2 in pleural metastases tumor xenograft in mice. Ectopic expression of NPRL2 activates the DNA damage checkpoint pathway in cisplatin-resistant and NPRL2-negative cells; hence, the combination of NPRL2 and cisplatin can resensitize cisplatin nonresponders to cisplatin treatment through the activation of the DNA damage checkpoint pathway, leading to cell arrest in the G2 –M phase and induction of apoptosis. The direct implication of this study is that combination treatment with NPRL2 and cisplatin may overcome cisplatin resistance and enhance therapeutic efficacy [72]. Wang et al. identified two small-molecule compounds, obtusilactone A (OA) and (−)-sesamin from C. kotoense, as potent Lon protease inhibitors. The compounds are able to cause DNA DSBs and activate checkpoints. Treatment with OA and (−)-sesamin induced p53-independent DDRs in NSCLC cells, including G1 –S checkpoint activation and apoptosis, as evidenced by phosphorylation of checkpoint proteins (H2AX, Nbs1, and Chk2), caspase-3 cleavage, and sub-G1 accumulation. [73]. Sulindac is known to enhance the cellular responsiveness of tumors toward chemotherapeutic drugs. Combination treatment with As2 O3 and sulindac induced oxidative DNA damage in a time-dependent fashion, which was evaluated by H2AX phosphorylation along with HO-1 induction [74]. γ -H2AX and chemoprevention in lung cancer The approach of chemoprevention in lung cancer offers the possibility to interfere with the process of cancerogenesis by the use of natural or synthetic chemical compounds and either to prevent DNA damage or to step the proliferation of premalignant lung cells. With regard to bronchial carcinoma, chemoprevention in the first place implies cessation of smoking but the currently used procedures are not extremely efficient and many ex-smokers experience an increased risk of acquiring a lung tumor over a prolonged period of time (secondary prevention). In the past 20 years, many details of the process of tumorigenesis have been revealed and this knowledge has promoted the targeted use of chemoprotective compounds. In addition to the already known such as vitamin A, beta-carotene, vitamins E, C, and B12 as well as selenium, the last few years have seen the development of new compounds, such as retinoids, dithiols, cyclooxygenase inhibitors, epidermal growth factors and others, the mechanisms of action of which provide interesting new approaches. In addition, the introduction of biomarkers such as γ -H2AX allows to monitor the process of tumorigenesis in its different stages, from early to late, and thereby offers the perspective to perform studies on chemoprevention more rapidly and effectively as in previous years. Tocopherols, which exist in α, β, γ , and δ forms, are antioxidative nutrients also known as vitamin E. Although α-tocopherol (α-T) is the major form of vitamin E found in the blood and tissues, γ - and δ-T have been suggested to have stronger antiinflammatory activities. Cancer Investigation


H2AX and Lung Cancer  Yang et al. using a tocopherol mixture that is rich in γ -T (γ -TmT, which contains 57% γ -T) demonstrated the inhibition of inflammation as well as of cancer formation and growth in the lung and colon in animal models. γ -TmT also decreased the levels of 8-hydroxydeoxyguanosine, γ -H2AX, and nitrotyrosine in tumors [75]. Lu et al. have demonstrated the inhibitory activity of a mixed tocopherol preparation (cTmT) against the formation and growth of lung cancer. In both the carcinogenesis and the tumor growth models, the inhibitory action of g-TmT was associated with enhanced apoptosis and lowered levels of 8-hydroxydeoxyguanine, γ H2AX, and nitrotyrosine in the tumors of the g-TmT–treated mice [76]. Moreover, (2)-epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to inhibit tumorigenesis and cancer cell growth in animal models. Li et al. characterized the inhibitory actions of EGCG against human lung cancer H1299 cells in culture and in xenograft tumors. Tumor cell apoptosis and oxidative DNA damage, assessed by the formation of 8-hydroxy-2#-deoxyguanosine (8-OHdG) and γ -H2AX, were dose dependently increased by EGCG treatment. However, the levels of 8-OHdG and γ H2AX were not changed by the EGCG treatment in host organs [77]. Resveratrol decreases cancer risk and improves health of laboratory animals. However, it can also promote genomic instability. Rusin et al. examined how resveratrol influenced the growth of human cancer cell lines of different origin: osteosarcoma (U-2 OS) and lung adenocarcinoma (A549) and how it modulated the expression as well as the localization of key proteins, involved in DNA repair and cell cycle regulation. Resveratrol-induced growth arrest was associated with signs of stress-induced senescence. Moreover, resveratrol-induced telomeric instability in U-2 OS cells and the activation of DNA damage signaling in both cell lines manifested as the phosphorylation of H2AX at serine 139 and of p53 at serines 15 and 37 [78]. γ -H2AX and the multistep lung carcinogenesis model Carcinogenesis is thought to be a multistep process consisting of the accumulation of gene mutations. Preneoplastic lesions may be morphological phenotypes of the different steps in this progression from normal to malignant tissue. The World Health Organization’s tumor classification system defines preneoplastic lesions of the bronchial epithelium: squamous dysplasia and carcinoma in situ, referred to here as low-grade and high-grade squamous dysplasia (DL and DH), which may be precursors to squamous cell carcinoma; atypical adenomatous hyperplasia (AAH), which may be the progenitor lesion for adenocarcinoma. Preneoplastic conditions include squamous metaplasia (progressing to DL and DH) and adenomatous hyperplasia (progressing to AAH). Gorgoulis et al. [79] and Barkova et al. [80] have performed analyses of precancerous and cancer lesions of lung and other cancers to determine when DDR checkpoint is activated. Activation of the DDR was detected by monitoring expression of γ -H2AX, phosphorylated Chk2 (pChk2), and other proteins and by immunohistochemical staining of fixed tissues. In these studies, the hyperplasias showed activation of DNA damage checkpoint pathway. Dysplasias C 2013 Informa Healthcare USA, Inc. Copyright

and cancers exhibited also signs of DDR activation, shown by 53BP1 localization and γ -H2AX and pChk2 expression. The results suggested that a DNA-damage checkpoint is activated in preneoplastic lesions. The conclusions of these studies were that in precancerous lesions, replication stress, leading to formation of DNA DSBs, activates a DNA-damage checkpoint, which in turn induces cell-cycle arrest or apoptosis. Authors proposed that tumor suppressor loci would eventually be targeted, releasing the cells from the suppressive effects of the DNA-damage checkpoint and leading to tumor progression [79, 80]. To illustrate that the cells within the lesions had undergone replication stress that could have led to DNA DSBs, investigators showed that there was allelic loss specifically at the fragile FHIT/FRA3B locus in the preneoplastic lesions [79, 80]. Chromosome fragile regions are known to be highly susceptible to formation of small deletions due to replication stress. Since the FHIT/FRA3B locus was damaged in the lesions examined, damage to this fragile locus may itself cause activation of the checkpoint [81] and subsequent loss or reduction of expression of the haploinsufficient Fhit tumor suppressor may contribute to alteration or inactivation of checkpoint responses [82], allowing lesion progression. Nuciforo et al. found activated DDR markers in morphologically normal tissues, also in association with inflammation. 53BP1 loss occurs early at the transition from normal to dysplastic change, whereas the activated forms of ATM and CHK2, but not γ -H2AX, initially accumulate in preinvasive lesions and are then lost during tumor progression. In individual lung tumors, the activation of ATM, CHK2, and the presence of 53BP1 were consistently correlated, whereas γ -H2AX did not correlate with activated ATM. Finally, the study of associations between critical clinicopathological parameters and activated DDR factors showed a significant correlation between reduced local tumor extension and the phosphorylation of ATM, CHK2, and the presence of 53BP1[83]. Blanco et al. used a silica-induced multistep lung carcinogenesis model driven by chronic inflammation to study the evolution of molecular markers and genetic alterations [84]. Blanco et al. found that DDR pathway is activated in preneoplastic lesions, in association with inducible nitric oxide synthase and p53 induction. p16 was also induced in early tumorigenic progression and was inactivated in bronchiolar dysplasias and tumors. Lack of mutations of Ras and epidermal growth factor receptor, and a very low frequency of Tp53 mutations suggest that they are not required for tumorigenesis in this model. In contrast, epigenetic alterations in p16 (CDKN2A), CDH13, and APC, but not in Rassf1 and Nore1A, were clearly observed [84]. Progressive DNA damage in live cells by oxidants is responsible for cell aging and leads to tumor transformation. The strategies to slow aging or prevent cancer rely on protection of DNA from the damage. Since cells reside within intercellular matrix, it is crucial to understand whether matrix elements possess properties of modulating oxidative DNA damage. Zhao et al. explored the effect of hyaluronate (HA), the ubiquitous component of the matrix, on extent of DNA damage induced by exogenous and endogenously generated oxidants. The data indicate that HA

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protected DNA from damage caused by the exogenous oxidant H2 O2 . The authors postulate that expression of CD44 in some cell types such as stem cells may provide the means to internalize HA by endocytosis and one of the functions of the internalized HA may be protection of DNA from oxidants [85]. Finally, as we have mentioned above, Lantuejoul et al. found that telomere length critically shortened at bronchial metaplasia stage to increase gradually from dysplasia to invasive SCC (squamous cell carcinoma), in bronchioloalveolar lesions, telomere length decreased from normal to AIS (adenocarcinoma in situ) and increased from stage I to II to stage III to IV ADC (invasive adenocarcinoma) [29]. γ -H2AX as a biomarker of environmental genotoxicity, smoking, and chemical compounds. Induction of γ -H2AX is found following exposure of cells to suspected DNA-damaging compounds such as cigarette smoke, polycyclic aromatic compounds, dinitrobenzo[e] pyrene, norethindrone, chromium, crude oil, electromagnetic fields, microwaves from mobile phones, and extreme heat, all demonstrating a potential role for γ -H2AX detection in determining potential genotoxics. The primary effects induced by all these genotoxic agents are ssDNA lesions such as pyrimidine dimers, 6–4 (T-C) photoproducts, 29 or 8-oxo-7,8-dihydro-2 -deoxyguanosine (oxo8dG), and base ring fragmentation. Such lesions are induced regardless of the phase of the cell-cycle [86–89]. In addition, outside of Earth’s atmosphere, the biological effects of high charge and energy ions during space exploration are a major concern for astronaut health. As a consequence, γ -H2AX may be useful in elucidating the effects of space travel-induced DNA damage [90]. Finally, γ -H2AX could be used as a biodosimeter of irradiation caused by bioterrorism. Toyooka et al. showed that cigarette sidestream smoke (CSS) generated γ -H2AX, in a human pulmonary epithelial cell model, A549. Treatment with CSS drastically induced discrete foci of γ -H2AX within the nucleus in a dose-dependent manner. CSS increased intracellular oxidation, and N-acetylcysteine (NAC), an antioxidant, significantly attenuated the formation of γ -H2AX, suggesting that ROS produced from CSS partially contributed to the phosphorylation. CSS in fact induced DSBs, which was also inhibited by NAC. DSBs are the worst type of DNA damage, related to genomic instability and carcinogenesis [91]. Attempts have been made to construct reduced toxicity cigarettes, presumed to have diminished genotoxic potential. One such product on the market is the tobacco and nicotine free (T&N-free) cigarette type made from lettuce and herbal extracts. Jorgensen et al. have developed a sensitive assay of the genotoxicity of CS based on cytometric analysis of induction of the DDR in normal human pulmonary endothelial or A549 pulmonary adenocarcinoma cells. The authors observed that exposure of A549 cells to CS from T&N-free cigarettes induced a smoke–dose-dependent DDR as evidenced by phosphorylation (activation) of the ATM protein kinase and of the γ -H2AX. The extent of DDR induced by T&N-free smoke was distinctly greater than that induced by comparable doses of CS from reference cigarettes (2R4F) containing tobacco and nicotine. The

pattern of DDR induced by T&N-free smoke was similar to that of 2R4F cigarettes in terms of the cell-cycle phase specificity and involvement of ROS. The data also imply that similar to 2R4F exposure of cells to T&N-free smoke leads to formation of double-strand DNA breaks (DSBs) resulting from collapse of replication forks upon collision with the primary ssDNA lesions induced by smoke. Since DSBs are potentially carcinogenic, Jorgensen et al. conclude that smoking tobacco and nicotine-free cigarettes is at least as hazardous as smoking cigarettes containing tobacco and nicotine [92]. To obtain a more complete view of the DDR, Zhao et al. explored the correlation between ATM activation, H2AX phosphorylation, and activation of Chk2 through its phosphorylation on Thr68, and phosphorylation of p53 on Ser15 in NHBE and A549 cell exposed to CS (Cigarette smoke). ATM and Chk2 were phosphorylated 1 hr prior to phosphorylation of H2AX and p53. The dephosphorylation of ATM, Chk2, and H2AX was seen after 2 hrs following CS exposure. The dose-dependence and kinetics of DDR were essentially similar in both cell types, which provide justification for the use of A549 cells in the assessment of genotoxicity of CS in lieu of normal bronchial epithelial cells. The observation that DDR was more pronounced in S-phase cells is consistent with the mechanism of induction of DSBs occurring as a result of collision of replication forks with primary lesions such as DNA adducts that can be caused by S-generated oxidants [93]. Msiska et al. compared the induction of DNA DSBs in normal (small airway epithelial) cells and cancer cells (A549) after exposure to asbestos (crocidolite), a proven carcinogen, silica, a suspected carcinogen, and titanium dioxide (TiO2 ), an inert particle recently reported to be carcinogenic in animals. The results indicate that crocidolite induced greater DNA DSBs than silica andTiO2 , regardless of cell type. DNA DSBs caused by crocidolite were higher in normal cells than in cancer cells. Silica and TiO2 induced higher DNA DSBs in cancer cells than in normal cells [94]. In light of the knowledge that CS increases oxidative stress and induces cell proliferation in the lungs of smokers, the high propensity of S-phase cells to develop DSBs upon exposure to CS has to be considered as a crucial event in smoke-induced tumor development. Albino et al. also reported the cell-cycle phase specificity in both the induction of DSBs by CS and their prevention by free radical scavengers. The detection of γ -H2AX to assess the induction of CS-induced DSBs and their relationship to cell-cycle phase provides a convenient tool to explore approaches to protect cells from this type of genotoxic damage [95]. Radon (222Rn) gas produces decay progeny that emits high energy alpha-particles. In this study, Bio-plex multiplex technology was employed to investigate modulations of 27 proinflammatory cytokines following exposure of human monocytic cells to 1.5 Gy of α-particle radiation. Concurrently, DNA damage was assessed by examining the formation of phosphorylated γ -H2AX sites. The authors concluded that α-particle radiation causes dysregulation in the production of a number of proinflammatory cytokines and results in significant DNA damage [96]. As we have mentioned, hypoxia-inducible factor 1 (HIF-1) pathway is Cancer Investigation


H2AX and Lung Cancer  induced in many tumors included lung cancer and associated with poorer outcome. The hypoxia-responsive transcription factor HIF-1alpha dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT), which is also an important binding partner for the aryl hydrocarbon receptor (AhR). AhR is an important mediator in the metabolic activation and detoxification of carcinogens, such as the environmental pollutant benzo[a]pyrene (BaP). Schults et al. demonstrated that the BaP-induced hypoxanthine-guanine phosphoribosyltransferase mutation frequency and γ -H2AX foci were markedly amplified when the HIF-1 pathway was induced. BaPDNA adducts were only marginally increased, and transient strand breaks were diminished by HIF-1 induction, indicating changes in DNA repair. These data indicate that concurrent exposure of tumor cells to hypoxia and exogenous genotoxins can enhance genetic instability [97]. Glutathione S-transferases (GSTs) are enzymes that are significant in carcinogen detoxification. GST-Mu class is showing the high activity toward most polycyclic aromatic hydrocarbon (PAH) epoxide. Tang et al. demonstrated that the expression of GST-M2 in tumor tissues is significantly lower than in paired adjacent nonmalignant tissues. Protection against B[a]P-induced DNA damage by GST-M2 in lung cancer cells was detected by Comet assay and γ -H2AX [98]. Similarly, halting DNA replication using the DNA polymerase α inhibitor aphidicolin prevents activation of ATM and H2AX phosphorylation induced by UV or by oxidants [99]. γ -H2AX as a biomarker of chemo- and radio-therapy in lung cancer treatment strategies Exposure to sources of IR, including X-rays, γ -radiation, α-particles, and heavy ions leads to the direct induction of DSBs in cellular DNA [100, 101]. In addition, treatment of cells with cytotoxic agents, including but not limited to DNA synthesis inhibitors, DNA alkylating agents, topoisomerases I and II inhibitors, bleomycin, and hydrogen peroxide, also lead to the formation of DSBs which induce γ -H2AX formation [102, 103]. This DNA damage occurs during the repair or attempted repair of other non-DSB DNA lesions, many of which occur because of interference with replication and transcription complex progression. Thus, the central position of γ -H2AX in DNA DSB detection/repair may give it a significant role in new lung cancer drug development and treatment optimization through clinical trials. Persistence of γ H2AX foci after the initial induction of DNA damage indicates that some of the damage remains unrepaired, making γ -H2AX an attractive candidate for the rapid assessment of radiation sensitivity in individuals and cell lines [104] leading to the identification of cell lines and human subjects with defective DNA repair. Therefore, γ -H2AX may be useful as a biodosimeter [105] for exposure to IR and as a predictor of radiosensitivity [106] making γ -H2AX a potentially useful tool to enhance the clinical efficacy of radiation treatment. Chemotherapy strategies Roscovitine, a cyclin-dependent kinases (CDKs) inhibitor, has been reported to have antitumor effects in some canC 2013 Informa Healthcare USA, Inc. Copyright

cer cell lines by inducing apoptosis. However, the exact underlying mechanisms are not fully understood. Zhang et al. reported that roscovitine induces expression and cleavage of the universal CDK inhibitor p21Waf1/Cip1 in NSCLC A549 cells in a dose-dependent manner. They also showed that roscovitine induced an enhanced expression of γ -H2AX, which was blocked by caspase-3 inhibition, suggesting that p21Waf1/Cip1 cleavage may interfere with DNA repair, leading to increased frequency of DSBs and enhanced apoptosis [107]. The DNA topoisomerase I (topo1) inhibitor topotecan (TPT) and topo2 inhibitors doxorubicin, etoposide, and mitoxantrone (MXT) are widely used antitumor drugs. Zhao et al. explored a relationship between H2AX phosphorylation and activation of checkpoint kinase 2 (Chk2) in human lung carcinoma A549 cells treated with TPT or with MXT. In the untreated cells, activated Chk2 was present predominantly in centrosomes. Upon treatment with TPT or MTX, the activated Chk2 presented itself in form of either minute or large IF foci in the cell’s nucleoplasm. H2AX phosphorylation whether induced by TPT or MXT was rapid, with the maximal rate occurring during the initial 2 hrs and peaking at 2 hrs of treatment. While TPT-induced H2AX phosphorylation and Chk2 activation were maximal in S phase cells, MTX-induced H2AX phosphorylation was maximal in G1 cells while Chk2 activation was maximal in G2M and minimal in G1 cells [108]. REV3 is the catalytic subunit of DNA translesion synthesis polymerase ζ . Inhibition of REV3 expression can increase sensitivity to cisplatin-based cancer therapy but also can be used for susceptible cancers as potential monotherapy [109]. Inhibition of REV3 expression in of lung (A549, Calu-3) cancer cells leads to an accumulation of persistent DNA damage as indicated by an increase in phospho-ATM, 53BP1, and γ -H2AX foci formation, subsequently leading to the activation of the ATM-dependent DDR cascade. Knobel et al. conclude that their findings indicate that depletion of REV3 not only can amend cisplatin-based cancer therapy but also can be applied for susceptible cancers as a potential monotherapy [109]. Kropotov et al. studied etoposide induced γ -H2AX in human lung cancer cells in which Peroxiredoxin (PrxV) activity was downregulated (knockdown, KD-clones) or compromised by overexpression of redox-negative (RD) protein. In KD clones, but not in RD-clones, formation of etoposideinduced γ -H2AX was increased, indicating that PrxV inhibits conversion of topoisomerase II cleavage complexes into double-strand DNA breaks but this inhibition is not caused by its antioxidant activity [110]. Docetaxel is a member of the taxane antimicrotubule class of chemotherapeutic agents, which are currently widely used in lung cancer therapy. Zhang et al. found that docetaxel induces replication-dependent γ -H2AX formation which plays a crucial role in docetaxel-triggered apoptosis. The DNA polymerase inhibitor aphidicolin dose-dependently prevents docetaxel-induced γ -H2AX formation, as well as apoptosis. In addition, Wortmannin pretreatment caused elevated γ -H2AX level, which was accompanied with increased


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apoptosis. This effect was due to the inhibition of DNA repair process by Wortmannin, as downregulation of p21Waf1/Cip1 and DNA repair proteins such as Ku70, Ku80, DNA-PKcs and Rad50, were detected [111]. The same authors in another paper showed that Wortmannin downregulated the expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) which is involved in DNA double stand break (DSB) repair, as a result, leading to the inhibition of DSBs rejoining, as indicated by increased level of γ -H2AX at 24 hrs after IR [112]. Deng et al. found that Gefitinib, at the cellular level, radiosensitizes EGFR with NSCLC H358 by blocking EGFR nuclear translocation as one of its mechanisms. Immunostaining for confocal microscopy suggested that more nuclear γ -H2AX foci are present in the gefitinib-interfering group than in the X-ray group. The nuclear γ -H2AX foci also stayed longer in the gefitinib-interfering group [113]. Similar results were found by Tanaka et al. that concluded in their study with the use of γ -H2AX that gefitinib enhances the radioresponse of NSCLC cells by suppressing cellular DNA repair capacity, thereby prolonging the presence of radiation-induced DSBs [114]. Hubaux et al. evaluated the anticancer efficacy of a HDAC inhibitor (valproate: VPA) on SCLC cells in combination with the standard chemotherapeutic first-line regimen (cisplatin + etoposide). The authors showed that VPA induces apoptosis of small cell lung cancer cell lines and improves efficacy of cisplatin combined with etoposide. VPA hyperacetylates histone H3. The mechanism of VPA proapoptotic activity involves induction of p21, inhibition of Bcl-xL, cleavage of Bid, and phosphorylation of Erk and H2AX [115]. Securin and γ H2AX have been shown to regulate cell survival and genomic stability. Jiang et al. conclude that the opposing effects of baicalein, a natural flavonoid extracted from the Scutellaria baicalensis root, on securin and γ -H2AX levels may be involved in the regulation of cell viability and genomic stability by this compound [116]. Takahashi et al. suggested that the number of γ -H2AX foci is correlated with the temperature dependence of cell killing. During periods when cells were exposed to heat, the cell-cycle–dependent pattern of cell killing was the same as the cell-cycle pattern of γ -H2AX foci formation. The authors also found that thermotolerance was due to a depression in the number of γ -H2AX foci formed after heating when the cells were pre-treated by heat [117]. Krynetskaia et al. dissected the roles of HMGB1associated proteins in DDR in lung cancer A549 cell line. The authors concluded that phosphorylation of p53 and phosphorylation of H2AX occur in two distinct branches of the DDR. These findings identify new molecular components of the DNA damage signaling cascade and provide novel promising targets for chemotherapeutic intervention [118]. Kamal et al. evaluated a series of new 4bacrylamidopodophyllotoxin derivatives. The compounds (13j, 13k, and 13l) showed G2 –M cell-cycle arrest and the apoptotic event was found to be due to both the single-strand DNA breaks as observed by comet assay as well as DSBs as observed by the large accumulation of γ -H2AX foci [119].

Owonikoko et al. found that in A549 and 128–88T cell lines, vorinostat potentiated carboplatin induction of γ H2AX and increased α-tubulin acetylation (a marker for stabilized microtubules) [120]. Galluzi et al. used microarray technology to identify miRNAs that were upregulated by NSCLC A549 cells in response to cisplatin (CDDP). In addition, pre-miR-630 blocked early manifestations of the DDR, including the phosphorylation of the ATM kinase and of two ATM substrates, histone H2AX and p53 [121]. Radiotherapy strategies Ghosh et al. found that oxygen beam was three times more cytotoxic than γ - radiation in A549 lung adenocarcinoma cells, as determined by γ -H2AX counting [122]. Carbon beams (5.16 MeV/u, LET = 290 keV/ m) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness (RBE) than low LET radiation. The same team of Ghosh et al. found that Carbon beam was three times more cytotoxic than γ -rays, despite the fact that the numbers of γ -H2AX foci were same [123]. Roig et al. examined that the DNA DSB damage response induced by high energy charged particles on lung fibroblast cells embedded in a 3-dimensional (3-D) collagen tissue equivalents was investigated using antibodies to γ -H2AX and phosphorylated DNA-PKcs (p-DNA-PKcs). Patterns of discrete DNA damage streaks across nuclei or saturated nuclear damage were observed, with saturated nuclear damage being more predominant as samples were positioned closer to the physical Bragg peak [124]. In lung cancer tumors, tumor cell inactivation which is not apoptosis-related, such as mitotic catastrophe, seems to be more frequent event after exposure to DNAdamaging agents. However, there are still many unexplained questions for the clarification of the mechanisms of mitotic catastrophe. Kodym et al. found that a large fraction of HCC2279 NSCLC cells underwent mitotic catastrophe after irradiation. Cells were arrested in metaphase with chromosomal damage indicated by DNA fragments displaced from the metaphase plate and considerable numbers of residual γ -H2AX foci [125]. Sak et al., studied the role of Rad51dependent homologous recombination in the radiation response of NSCLC cell lines. There was no correlation between the clonogenic survival at 2Gy and the percentage of initial Rad51 or γ -H2AX foci after IR. The most reliable predictive factor for radiosensitivity of NSCLC cell lines was the relative fraction of Rad51 foci remaining at 24 hrs after IR. Although most of the Rad51 foci are co-localized with γ H2AX foci, no correlation of the relative fraction of persisting γ -H2AX foci and SF2 is evident [126]. Pappas et al. examined the ability of adenoviral-mediated PTEN(Ad-PTEN) to modulate the repair of radiation-induced DNA DSBs in NSCLC cell line H1299 using the detection of repair foci positive for γ -H2AX. Compared with controls, the repair of radiationinduced DSBs was retarded in H1299 cells pretreated with Ad-PTEN, consistent with the radiosensitizing effect of the vector [127]. Franken et al. indicated that RBE values for IRinduced foci (IRIF) (DNA-DSB) induction provide little valid information on other biologically relevant end points in cells

Cancer Investigation

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exposed to high-LET radiations in lung cancer cell line SW1573 with the use of γ -H2AX [128]. Combined chemo-radiotherapy strategies Fukushima et al. demonstrated that gimeracil enhanced the efficacy of X-ray irradiation against lung as well as head and neck cancer xenografts in a dose-dependent manner. Furthermore, they observed decreased expression of γ -H2AX, in LC-11 tumors treated with X-ray irradiation and gimeracil compared to that observed in tumors treated with X-ray irradiation alone, suggesting that gimeracil may inhibit rapid repair of X-ray-induced DNA damage in tumors [129]. Huang et al. examined a novel camptothecin derivative (TLC388) with higher efficacy and reduced toxicity. The formation of γ -H2AX foci was observed after TLC388 or radiation exposure and when the cells were treated with 30 nM TLC388 plus radiation at a dose of 2 Gy, the percentage of cells containing γ -H2AX foci increased significantly [130]. Patel et al. found that the marketed anti-parasitic drug albendazole possesses DNA damaging and microtubule disrupting capabilities, as examined with the use of γ -H2AX. At clinically achievable concentrations, albendazole in combination with irradiation causes enhanced apoptotic induction in melanoma and small cell lung cancer SCLC, which have an increased propensity to metastasize to the brain [131]. Xiong et al. delivered of a single-chain antibody variable region fragment (ScFv 18–2) to the cell nucleus. ScFv 18–2 binds to a regulatory region of the DNA-dependent protein kinase (DNA-PK). ScFv 18–2 showed significant radiosensitization. The authors concluded that this molecule inhibits repair of radiation-induced DSBs, as evidenced by the persistence of γ -H2AX-stained foci and by inhibition of staining with anti-DNA-PKcs phosphoserine 2056 [132]. Geng et al. studied the effect of LBH589 a novel inhibitor of class I and II HDACs (Histone deacetylases). The findings that LBH589 confines HDAC4 to the cytoplasm and increases the duration of gamma-H2AX foci in irradiated cell lines suggest that HDAC4 participates in DNA damage signaling following IR [133]. To evaluate the interaction between histone deacetylase inhibitors and irradiation in NSCLC, Cuneo et al. studied NVP-LAQ824 in mouse models of human lung cancer. Immunostaining for γ -H2AX nuclear foci was performed to determine the effect of LAQ824 on radiation-induced DNA DSBs. Combined modality treatment delayed the resolution of γ -H2AX foci with over 30% of cells staining positive 6 hrs after treatment versus approximately 5 and 3% in cells treated with LAQ824 or radiation alone [134]. Zhang et al. examined whether the HDAC inhibitors m-carboxycinnamic acid bishydroxamide (CBHA) and depsipeptide FK228 affect H2AX phosphorylation (γ -H2AX). CBHA and FK228, but not 5-fluorouracil, enhanced IR-induced γ -H2AX in A549 and other cancer cell lines. Overexpression of p300 similarly augmented IR-induced γ -H2AX [135]. Using a highthroughput, unbiased screening approach, Lally et al. have identified 4 -bromo-3 -nitropropiophenone (NS-123) as a radiosensitizer of human glioma cells in vitro and in vivo. A549 lung adenocarcinoma cells were also radiosensitized C 2013 Informa Healthcare USA, Inc. Copyright

by NS-123 in vitro [136]. Iwasa et al. have investigated the effect of CP-751,871, a fully human monoclonal antibody specific for IGF-IR, on the sensitivity of human NSCLC cell lines to radiation. Radiation induced damage was evaluated by immunofluorescence analysis of the histone γ -H2AX and Rad51. These results show that CP-751,871 sensitizes NSCLC cells to radiation both in vitro and in vivo, and that this effect of CP-751,871 is likely attributable to the inhibition of DNA repair and enhancement of apoptosis that result from attenuation of IGF-IR signaling [137]. Moreover, Gupta et al. found that Soy isoflavones increased A549 cell killing induced by radiation. Multiple γ -H2AX foci were detectable at 1 hr after radiation but decreased at 24 hrs after radiation. Soy isoflavones augment radiation effect by inhibiting APE1/Ref1 DNA repair activity in NSCLC [138]. Hudson et al. demonstrated that tissues of younger animals are much more susceptible to IR-induced DNA damage. Younger animals exhibited higher levels of γ -H2AX formation which partially correlated with cellular proliferation and expression of DNA repair proteins. The lowest focal induction was seen in lung and brain of young animals. The mechanisms of cell and tissue specificity of in vivo IR responses need to be further elucidated. This study provides a roadmap for the future analyses of DNA damage and repair induction in young individuals [139]. Guo et al. in a study investigated the effects of a novel phage display single-chain variable fragment (scFv) antibody targeting Prx I on human lung carcinoma cell line A549 radiosensitivity. They found that protein expression of radiosensitivity-related proteins, RAD51 and γ -H2AX were modulated [140]. Iwasa et al. investigated the effect of YM155, a small-molecule inhibitor of survivin expression, on the sensitivity of human NSCLC cell lines to γ -radiation. Radiation-induced DNA damage was evaluated on the basis of histone H2AX phosphorylation and foci formation. Immunofluorescence analysis of γ -H2AX showed that YM155 delayed the repair of radiation-induced DSBs in nuclear DNA [141]. Owonikoko et al. found that in A549 and 128–88T cell lines, vorinostat potentiated carboplatin induction of γ H2AX and increased α-tubulin acetylation (a marker for stabilized microtubules) [121]. γ -H2AX and bystander effect Ionizing radiation confers a large amount of biological effects in unirradiated cells. This so-called radiation-induced bystander effect (RIBE) manifests in various ways including changes in gene expression, genetic and epigenetic alterations, as well as increases in cell transformation and cell death. The radiation induced bystander effect, which can be monitored through DSB induction, can also be measured using γ -H2AX formation [142]. In bystander cells, γ -H2AX foci are specifically detected in late S–G2 phase and are associated with Rad51 foci that signify the function of homologous recombination repair, possibly on DNA replication–mediated DSBs. The results point to enhanced NHEJ as a mechanism of AR and suggest that AR may be

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transmitted to bystander cells through factors generating replication-mediated DSBs [143]. γ -H2AX in the era of PARP-inhibitors Cells that are homozygous for defect in homologous recombination are exquisitely sensitive to PARP inhibition even without exposure to a DNA-damaging agent [144]. In this situation, the background endogenous base damage discussed above, which is normally repaired by BER, will lead to collapsed replication forks as there is no effective DSB repair to compensate [145]. Aguilar-Quesada et al. have used targeted kinase inhibition to inactivate BRCA1, handicap the homologous recombination DNA repair machin-ery and selectively sensitize transformed cells to PARP inhibition. This approach avoids the use of toxic DNA-damaging chemotherapeutic drugs, thereby providing the potential to extend welltolerated PARP inhibition to treatment for BRCA-proficient cancers. PARP inhibition also activates ATM, and induces γ H2AX foci in an ATM-dependent manner [146]. Albert et al. investigated the combination of PARP-1 inhibition with radiation in lung cancer models. ABT-888, a novel potent PARP-1 inhibitor, was used to explore the effects of PARP-1 inhibition on irradiated tumors and tumor vasculature. ABT-888 reduced clonogenic survival in H460 lung cancer cells, and inhibited DNA repair as shown by enhanced expression of γ -H2AX [147]. Assays to measure HR proficiency and PARP activity in vivo will be vital to the primary or acquired resistance to PARP inhibitors in the clinical studies. Pharmacodynamic biomarker assays to measure levels of PAR, γ -H2AX foci, RAD51 foci in vivo were recently developed [148, 149] and applied in several clinical studies [150–152]. An immunoassay ELISA method or IHC can measure PAR levels either in patient’s tumor biopsies or in blood cells, and afterwards measurement of γ -H2AX and RAD51 foci can monitor the DNA repair status. Further clinical studies are needed to evaluate if low levels of RAD51, γ -H2AX, or other DNA repair proteins constitute biomarkers of PARP inhibitors efficacy. The adoption of PAR, γ -H2AX, and RAD51 measurement in tumor biopsies of patient’s blood prior to, during, and after chemotherapy may add valuable information for the discrimination of the subgroup of patients that respond to therapies with PARP inhibitors. γ -H2AX as a therapeutic target in lung cancer While H2AX and the PI3 kinases that phosphorylate H2AX have both been proposed as potential therapeutic targets, no drugs directed against these targets are known to be currently in clinical use or development. However, PI3 kinase inhibitors have been developed for research purposes and are available through AstraZeneca/KuDos [153, 154]. Because H2AX is ubiquitous to all cells, serves a structural role in the integrity of chromatin, and has a relatively long half-life in the cell, the H2AX protein itself may be a problematic drug target. Many commercial H2AX and γ -H2AX peptides and antibodies are available from a variety of companies. Inhibition of the phosphorylation of H2AX is probably a more practical therapeutic strategy than alteration of H2AX levels.

Peptide inhibitors of H2AX phosphorylation may be useful as chemotherapeutic agents [155, 156]. The effect of H2AX peptides on IR sensitivity was examined using human squamous cell carcinoma cell lines that were either radiosensitive (SCC-61) or radioresistant (SQ-20B). The peptide mimics were found to inhibit γ -H2AX focal formation in both cell lines in response to 3 Gy IR and to decrease cell survival following irradiation [156]. These results indicate that H2AX could potentially be targeted to enhance the efficiency of radiation therapy. In addition, inhibition of H2AX phosphorylation through interference with upstream kinase activities may be an attractive target for drug development. Caffeine and wortmannin which inhibit H2AX phosphorylation are also radiosensitizers [12]. However, though many tumor cell lines exhibit higher spontaneous levels of γ -H2AX, inhibition of H2AX phosphorylation may be expected to deleteriously affect all cells, not just cancer cells [157]. γ -H2AX as a prognostic factor of survival in lung cancer patients Our research team assessed the expression of γ -H2AX in a cohort of 96 patients with NSCLC and evaluated its role as a prognostic indicator in resectable NSCLC patients. Moreover, we correlated γ -H2AX levels with other clinical and pathological parameters. Low γ -H2AX expression was associated with a significantly better survival as compared with those having high γ -H2AX expression (23.2 months for high γ -H2AX expression vs. 35.3 months for low γ -H2AX expression, p = .009; HR = 1.95, 95% CI = 1.15–3.30). Further investigation with multivariate Cox proportional hazards regression analysis revealed that high expression of γ -H2AX remained independent prognostic factor of worse overall survival (HR = 2.15, 95% CI = 1.22–3.79, p = .026). Finally, we found that patients with tumors that had either p53 high/γ H2AX low or p53 low/γ -H2AX high expression levels were 2.54 (95% CI = 1.36–4.73, p = .003) times and patients with tumors that had p53 and γ -H2AX high expression levels were 3.55 (95% CI = 1.66–7.33, p = .001) times more likely to die of cancer than patients with tumors that had both p53 and γ -H2AX low expression levels. In conclusion, our study demonstrated that overexpression of γ -H2AX is an independent prognostic indicator of reduced overall survival in patients with NSCLC [158]. CONCLUSIONS γ -H2AX is a major biomarker of DDR. Its wide spectrum of clinical implications in the diagnosis, prognosis, and management of lung cancer would render it in the future a valuable molecule in the everyday clinical practice of lung cancer management. Since lung cancer is still a neoplasm with dismal prognosis, new molecules and pathways are needed to understand the mechanisms of lung cancer carcinogenesis. γ -H2AX is a promising candidate for this purpose [159]. As Euripides, an ancient Greek playwright said: “Time will explain it all. He is a talker, and needs no questioning before he speaks” [160] and this will also happen with the role of DDR and γ -H2AX in lung cancer. Cancer Investigation

H2AX and Lung Cancer  ACKNOWLEDGMENTS DM had the initial idea of the review writing, had developed the tables and figures, and wrote the manuscript. PH contributed to the writing of the manuscript, PK, DB, and SK corrected and supervised the review.


DECLARATION OF INTEREST

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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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ABBREVIATIONS

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NSCLC DSB DDR SCC AIS ADC NHEJ SCLC ROS HR PFGE SCGE CTCs CACD FACS RABIT BG NER EGCG CSS OA NPR2 PRXV

non-small cell lung cancer double strand breaks DNA damage response squamous cell carcinoma adenocarcinoma in situ invasive adenocarcinoma Non-Homologous End Joining Small Cell lung carcinoma reactive oxygen species homologous recombination pulse-field gel electrophoresis single cell gel electrophoresis Circulating tumor cells computer-aided cytologic diagnosis Fluorescence Activated Cell Sorting Rapid Automated Biodosimetry Tool O6-Benzylguanine nucleotide excision repair Epigallocatechin-3-gallate cigarette sidestream smoke obtusilactone A NPR2 nitrogen permease regulator 2 Peroxiredoxin

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