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ISSN 1007-9327 CN 14-1219/R  World J Gastroenterol  2008 October 28; 14(40): 6163-6170

VIRAL HEPATITIS

Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection


Fan Zhao, Ning-Bo Hou, Xiao-Li Yang, Xiang He, Yu Liu, Yan-Hong Zhang, Cong-Wen Wei, Ting Song, Li Li, Qing-Jun Ma,

Hui Zhong


Fan Zhao, Ning-Bo Hou, Xiang He, Yan-Hong Zhang, Cong-Wen Wei, Ting Song, Li Li, Qing-Jun Ma, Hui Zhong, Molecular Genetics Department, Beijing Institute of Biotechnology, Beijing 100850, China

Xiao-Li Yang, Yu Liu, Biochemical and Immunology Analyze Center, The General Hospital of Chinese People’s Armed Police Forces, Beijing 100039, China

Author contributions: Zhao F and Hou NB contributed equally to this work; Ma QJ, Li L, Zhang YH and Zhong H designed research; Yang XL, He X, Liu Y, Zhang YH, Wei CW, Song T and Li L performed research; Zhong H wrote the paper.

Supported by National Natural Science Foundation of China, No. 30700413

Correspondence to: Hui Zhong, PhD, Molecular Genetics Department Beijing Institute of Biotechnology, Beijing 100850, China. towall@yahoo.com

Telephone: +86-10-66931809  Fax: +86-10-88272563

Received: April 14, 2008         Revised: June 21, 2008

Accepted: June 28, 2008

Published online: October 28, 2008

 

Abstract

AIM: To explore whether acute cellular DNA damage response is induced upon hepatitis B virus (HBV) infection and the effects of the HBV infection.

METHODS: We incubated HL7702 hepatocytes with HBV-positive serum, mimicking a natural HBV infection process. We used immunoblotting to evaluate protein expression levels in HBV-infected cells or in non-infected cells; immunofluorescence to show ATR foci ands Chk1 phosphorylation foci formation; flow cytometry to analyze the cell cycle and apoptosis; ultraviolet (UV) radiation and ionizing radiation (IR)-treated cells to mimic DNA damage; and Trypan blue staining to count the viable cells.

RESULTS: We found that HBV infection induced an increased steady state of ATR protein and increased phosphorylation of multiple downstream targets including Chk1, p53 and H2AX. In contrast to ATR and its target, the phosphorylated form of ATM at Ser-1981 and its downstream substrate Chk2 phosphorylation at Thr-68 did not visibly increase upon infection. However, the level of Mre11 and p21 were reduced beginning at 0.5 h after HBV-positive serum addition. Also, HBV infection led to transient cell cycle arrest in the S and the G2 phases without accompanying increased apoptosis. Research on cell survival changes upon radiation following HBV infection showed that survival of UV-treated host cells was greatly increased by HBV infection, owing to the reduced apoptosis. Meanwhile, survival of IR-treated host cells was reduced by HBV infection.

CONCLUSION: HBV infection activates ATR DNA damage response to replication stress and abrogates the checkpoint signaling controlled by DNA damage response.

 

© 2008 The WJG Press. All rights reserved.

 

Key words: Hepatitis B virus; DNA damage response; Cell cycle; p21; Mre11

 

Peer reviewer: Dr. Michael Torbenson, Department of Pathology, Johns Hopkins University School of Medicine, 1503 E Jefferson, Room B314, Baltimore 21231, United States

 

Zhao F, Hou NB, Yang XL, He X, Liu Y, Zhang YH, Wei CW, Song T, Li L, Ma QJ, Zhong H. Ataxia telangiectasia-mutated-Rad3-related DNA damage checkpoint signaling pathway triggered by hepatitis B virus infection. World J Gastroenterol 2008; 14(40): 6163-6170  Available from: URL: http://www.wjgnet.com/1007-9327/14/6163.asp  DOI: http://dx.doi.org/10.3748/wjg.14.6163

 

INTRODUCTION

Eukaryotic cells employ multiple strategies of checkpoint signaling and DNA repair mechanisms to monitor and repair damaged DNA[1-5]. There are two branches of the checkpoint response pathway, ataxia telangiectasia-mutated (ATM) pathway and ATM-Rad3-related (ATR) pathway. The major difference between ATM and ATR is the type of DNA damage to which each responds. For example, ATM responds to ionizing radiation (IR) and other agents that cause double-strand breaks (DSBs) in DNA. ATR responds to ultraviolet radiation (UV) radiation and other agents that induce the accumulation of stalled replication forks and subsequent single-stranded breaks (SSBs) in DNA. The DSBs are recognized by the Mre11-Rad50-Nbs1 complex, which recruits and activates ATM kinase[6]. The SSBs are coated by replication protein A, and it recruits a complex of ATR kinases and ATR-interacting protein, which is then activated by the Rad9-Rad1-Hus1 complex and other factors. Accumulating evidence suggests that checkpoint signaling through ATR is intimately linked to the process of DNA replication[7,8].

    A variety of checkpoint and DNA repair proteins have been identified as substrates for ATM and ATR kinases, including the checkpoint kinases Chk1 and Chk2, as well as p53, Smc1 and H2AX. Chk1 was first identified in Schizosaccharomyces pombe because of its role in the checkpoint arrest at G2/M, and it is mainly phosphorylated by ATR in response to UV, hydroxyurea and aphidicolin[9-11]. p21 was initially identified as a component of quaternary complex containing cdk-cyclin kinases and PCNA. Previous study has shown that p21 protein is degraded after low doses of UV; this degradation is essential for optimal DNA repair and is ATR-dependent[12]. The MRN complex consisting of Mre11, Rad50, and Nbs1 is a target of both ATM and ATR and is involved in both pathways[13-15].

    Virus replication presents the host cells with large amounts of exogenous genetic material, including DNA ends and unusual structures. Therefore, infected cells recognize viral replication as a DNA damage stress and elicit DNA damage signal transduction, which ultimately induces apoptosis as part of host immune surveillance. However, recent reports have shown that viruses evolve a variety of mechanisms to manipulate DNA damage signaling for their replication and propagation. For example, Epstein-Barr Virus (EBV)[16] abrogates the p53 checkpoint signaling pathway through the interaction of the BZLF1 protein and p53 to avoid apoptosis. Other viruses such as human immunodeficiency virus type 1 (HIV-1)[17-20], herpes simplex virus type 1 (HSV-1)[21,22] and human cytomegalovirus[23] can activate and exploit a cellular DNA damage response, which aids viral replication. Adenovirus blocks ATM signaling and concatemer formation through targeting the DNA repair complex of MRN for degradation and mislocalization[24,25]. Thus, under some circumstances, viruses have co-opted endogenous checkpoint regulators to ensure their own efficient replication[26,27].

    Hepatitis B virus (HBV) is a partially enveloped double-stranded DNA virus with a genome of 3.2 kb. Upon infection, the viral genome is transported into the cell, where it is converted into a covalently closed circular DNA (cccDNA). The cccDNA serves as a template for transcription by host cell RNA polymerase . The pregenomic RNA is then reverse transcribed into DNA replicative intermediates in the cytoplasm within immature viral core particles, by the virally encoded polymerase. Integration into host chromosome may happen during its replication.

    There is no evidence thus far that the ATM/ATR kinases or their downstream pathways are triggered by HBV infection. The present study was undertaken by culturing normal hepatocyte cell line HL7702 and primary hepatocytes from a healthy liver donor with HBV-positive serum, mimicking natural HBV infection. We showed here, perhaps for the first time, that HBV infection elicited acute cellular DNA damage response dependent on ATR. However, the ATR checkpoint signaling was blocked downstream of p53-dependent and p53-independent pathways to evade apoptosis.

 

MATERIALS AND METHODS

Chemicals

Mimosine and aphidicolin were obtained from Sigma. The stock concentration of mimosine was 100 mmol/L, the stock concentration of aphidicolin was 10 mmol/L; both were dissolved in Dimethyl Sulphoxide (DMSO).

 

Cell culture, synchronization and infection

The human hepatocyte cell line HL7702, which was isolated from a HBV-seronegative individual, was obtained from Shanghai Biochemistry Institute. HL7702 were cultured in RPMI-1640 with 10% heat-inactivated FBS (Gibco). Serum samples from HBV carriers were analyzed. The patient was anti-HBsAg-positive, as detected by ELISA (SIIC Ke-Hua, Shanghai), and HBV DNA in the serum sample was quantified using FQ-PCR (Da-An Gene Corp). The patient had received no antiviral therapy prior to the study and was not infected with HCV or HIV. The number of serum HBV viral particles was 7 × 109 copies/mL, as quantified by FQ-PCR. Normal serum was obtained from healthy non-infected individuals as a control. The sera were stored at -80 until use.

    When synchronized, the HL7702 cells were cultured in RPMI-1640 containing 0.1% FBS for 2 d; the culture medium was then replaced with fresh RPMI-1640 including 10% FBS and 200 mmol/L mimosine for 24 h, in order to arrest the cells at G1-S phase. The arrested cells were then washed twice, and the culture medium was replaced with RPMI-1640 containing normal or HBV-positive serum. The cells were then harvested at different times after mimosine release. All procedures were performed under level P2 biosafety conditions to minimize the possibility of cross-contamination.

 

Primary culture of human hepatocytes

Hepatocytes were prepared from a 35-year-old healthy male liver donor according to previously described procedures[28]. Briefly, the liver tissues were cut with scissors into 0.1-0.5 mm3 pieces and were shattered with a 5-mL syringe into single cells or cell aggregates. Cells were seeded into 12-well culture dishes and incubated with 1 mL of 10% FBS in RPMI-1640 at 37 under 50 mL/L CO2. The medium was changed after the first 48 h with serum-free medium. The serum-free medium was composed of DMEM/F12 (1:1) and 0.01 nmol/L nicotinamide, 0.02 ng/L epidermal growth factor (EGF), 0.02 ng/L basic fibroblast growth factor (bFGF), 0.365 ng/L glutamate, B27 (1:50, Sigma), 0.1 U/L penicillin, 0.1 ng/L streptomycin, and 0.1 ng/L fluconazole. HBV-positive serum was added to the culture medium 2 wk after hepatocyte phenotype cell development, and cells were harvested for immunoblotting assay 3 h later.

 

Immunoblotting assay

Cell extracts were lysed in ice-cold Tris buffer (50 mmol/L, pH 7.5) containing 5 mmol/L EDTA, 300 mmol/L NaCl, 0.1% Igepal, 0.5 mmol/L NaF, 0.5 mmol/L Na3VO4, 0.5 mmol/L PMSF, and antiprotease mixture (Roche Molecular Biochemicals) for 30 min on ice and centrifuged at 13000 g for 10 min. The supernatant protein concentration was determined by the Bradford procedure (BioRad). The proteins were resolved on 15% SDS-PAGE and transferred onto nitrocellulose membranes. Blots were blocked in TBST containing 5% non-fat dried milk (NFDM) and incubated with primary antibodies as follows: antibodies against p21, Mre11, ATR, (Santa Cruz) and tubulin (Sigma) were incubated at room temperature for 1 h. Antibodies against ATM phosphoserine 1981 (ATMp), Chk2 phosphothreonine 68 (Chk2p), Chk1 phosphoserine 345 (Chk1p), p53 phosphoserine 15 (p53p) and H2AX phosphoserine 139 (H2AXp) (Cell Signaling) were incubated at 4 overnight. Secondary antibodies were from Jackson Laboratories. Horseradish-peroxidase-based detection was performed using a chemiluminescence reagent (Amersham Biosciences), according to the manufacturer’s instructions.

 

DNA damage sensitivity assays

For radiation sensitivity assays, cells were irradiated with the indicated doses of g rays from a 137Cs source for indicated times, or 254 nm UV light with complete medium plated in triplicate. HBV-positive serum was added to the cultures before indicated doses of UV or IR radiation. Cells were washed extensively to remove viral inputs 24 h after HBV-positive serum addition, and then treated with different dose of UV or IR radiation. After 48 h of UV and 4 d of IR treatment, cells were collected and surviving cells were counted with Trypan blue staining. The percentage survival was determined by quantization of the relative viable number of treated cells divided by the viable number of untreated cells.

 

Flow cytometry

For propidium iodide staining, cells were harvested by trypsinization, fixed with ice-cold 70% ethanol, and resuspended in a solution containing 50 mg/L propidium iodide, 0.1% Triton X-100, 50 mg/L RNase A, and 5 mmol/L EDTA at room temperature (RT) for 1 h. Cells were then diluted 1:1 in 1% BSA PBS for cytometric analysis.

 

Immunofluorescence assay

HL7702 cells were grown on glass coverslips for 12 to 18 h prior to infection. At 0.5 h post-infection, cells were washed briefly in phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde (PFA) in PBS for 10 min, then permeabilized for 10 min in 1% Triton X-100 in PBS. For the visualization of detergent-resistant or chromatin-associated nuclear proteins, an in situ extraction method that removed the cytoplasm and nucleosolic proteins was used. Cells were pre-extracted for 5 min on ice with 0.5% Triton X-100 in cytoskeletal buffer as described previously[29] then fixed in 4% PFA for 10 min. After washing with PBS and blocking in 2% fetal bovine serum (FBS) in PBS, cells were incubated with primary antibodies diluted in 2% FBS overnight at 4. Anti-Chk1p was used at a concentration of 1:100, while anti-ATR was used at 1:400. After washing with PBS three times, cells were incubated with secondary antibodies diluted 1:200 in 2% FBS for 30 min, then washed again three times with PBS. Next, the nuclei were stained with DAPI diluted in PBS for 10 min. After a final wash in PBS, samples were preserved in glycerol and images were captured by using a Zeiss LSM510 confocal microscope.

 

RESULTS

HBV infection induced a cellular DNA damage response dependent on ATR

To explore whether acute cellular DNA damage response was induced upon HBV infection, we incubated HL7702 hepatocytes with HBV-positive serum to examine the phosphorylation status of DNA damage response proteins. 105 HL7702 monolayer cells in a 6-cm plate were infected with HBV-positive serum containing 106 HBV at 37 under 50 mL/L CO2. Serum from uninfected individuals was used as a non-infected control. Cells were washed eight times to remove excess viral inputs before harvesting. Whole-cell lysates from the HBV-infected and non-infected cultures were examined for the status of the DNA damage response proteins. Figure 1A reveals that HBV infection induced an increase in the steady state levels of the ATR protein and in the phosphorylation levels of its downstream substrates Chk1, p53 and H2AX. An increase in Chk1 phosphorylation at Ser-345 was evident at 3 h from the start of HBV-positive serum addition, with further increase from 6 h of infection (hoi) to 48 hoi. The phosphorylation of p53 Ser-15 was elevated beginning at 24 hoi and increased greatly at 48 hoi. Finally, there was a sharp increase in the amount of phosphorylated H2AX Ser-139 beginning at 24 hoi. To confirm these results, primary hepatocytes from a healthy male liver donor were obtained and incubated with HBV-positive serum for 3 h; obvious Chk1 phosphorylation was detected in HBV-infected cells (Figure 1B). We then used immunofluorescence to examine the localization of these proteins early after infection. Figure 1C shows that ATR foci as well as the Chk1 phosphorylation foci in non-infected cells were very faint, as opposed to the HBV infected cells where the foci were larger, more numerous and much brighter.

    In contrast to ATR and its targets, the phosphorylated form of ATM at Ser-1981 was not visibly increased upon infection, and phosphorylation of its downstream substrate Chk2 at Thr-68 began to decrease starting from 3 hoi (Figure 1A), with slight recovery at later time points, indicating that the p53-independent pathway was blocked. Phosphorylation of p53 at Ser-15 in response to DNA damage usually correlates with the ability of p53 to trans-activate downstream target genes. Therefore, we examined expression levels of p53 transcriptional targets p21cip1/waf1, a cyclin-dependent kinase inhibitory protein. Figure 1A shows that the amount of p21 decreased substantially with time after infection, suggesting that p53-dependent downstream signaling was blocked during HBV infection, despite the appearance of phosphorylated p53. By 0.5 h after HBV-positive serum addition, downregulation of Mre11 began to be detected. This reduction in Mre11 protein occurs shortly after HBV infection, implying that  an incoming virion protein may lead to this degradation. Further investigation is required to explain this phenomenon.

    Since HBV infection activated DNA damage checkpoint pathway that responded to replication stress, we asked if HBV infection would effect cell cycle progression and cell death. Cells were synchronized in G1 phase by mimosine, an inhibitor of DNA synthesis initiation, for 24 h. Arrested cells were then washed twice and media replaced with RPMI-1640 containing HBV serum or normal serum. Cells were harvested at different times after mimosine release. The results of fluorescence-activated cell sorting (FACS) analysis showed that approximately 5% of the cells were in early S phase with 95% in G1 after synchronization (Figure 2A), indicating that the synchronization was fulfilled. Control cells treated with normal serum had approximately 44.4% of cells in S phase after 12 h release from mimosine, whereas cells treated with HBV-positive serum still had 56.6% of cells in S phase (Figure 2A and B). The percentage of total cells in S phase indicates the effect of virus in extending the period of DNA synthesis. Toward the end of the cell cycle, there was an accumulation of cells in G2 phase in HBV-infected cells (5.5% for control cells versus 17.5% for HBV infection cells) without the appearance of fragments with less than 2N DNA (Figure 2A and C). These findings indicate that DNA damage pathway responding to replication stress induced by HBV infection will lead to transient cell cycle arrest without accompanying increased apoptosis.

 

Checkpoint signaling pathway was compromised in HBV-infected cells

Previous results show that HBV infection did not show increased apoptosis even though a cellular DNA damage response dependent on ATR was activated (Figure 2A). To better understand the consequences of the ATR signaling pathway triggered by HBV infection, we examined if HBV infection had an impact on host cell survival after IR and UV radiation. Cells were washed extensively to remove viral inputs 24 h after HBV-positive serum addition and then treated with different doses of UV or IR radiation. After 48 h of UV and 4 d of IR treatment, cells were collected and surviving cells were counted with Trypan blue staining. Figure 3A and B showed that the survival rate of UV-irradiated cells was enhanced by HBV infection, while the survival rate of IR-irradiated cells was reduced by HBV infection. Figure 3C shows that while the cell survival rate after 8 s of UV or 4 Gy of IR was about 30%, the survival rate of HBV-infected cells after 8 s of UV radiation increased from 30% to 49%. By contrast, HBV infection pre-treatment with 4 Gy of IR radiation reduced the cell survival rate from 31% to 21%, suggesting that survival of UV-treated cells was enhanced by HBV infection (Figure 3C). Based on this result, we analyzed the difference in apoptosis between UV radiation only and combined treatment of UV with HBV infection. Decreased apoptosis was seen in combined treatment cells (Figure 4A and B). Twenty-four percent apoptosis was seen in UV-radiation-only cells, while about 8% apoptosis was detected in cells treated with UV radiation followed by HBV infection. It is known that cell cycle checkpoint triggered by DNA damage response induces cell apoptosis if damaged DNA cannot be repaired. The inhibitory effect of HBV on cell survival was reduced in cells treated with UV, indicating that checkpoint signaling controlled by the ATR DNA damage pathway was at least partially compromised in HBV-infected cells; therefore, the ATR signal pathway did not function completely.

 

DISCUSSION

The induction of cell cycle checkpoints and activation of the ATM/ATR-dependent pathway have been reported to accompany infection by a number of different viruses. Recent reports have shown that viruses have evolved a variety of mechanisms to manipulate DNA damage signaling for their replication and propagation. In this study, HL7702 cells and primarily cultured hepatocytes were inoculated with HBV-positive serum (106 particles per 105 cells), mimicking the HBV infection process. Serum from healthy individuals was used as a non-infected control. We propose that HBV infection induces acute cellular DNA damage response dependent on ATR, as demonstrated by ATR protein and increased phosphorylation of Chk1, p53 and H2AX. Since Chk1 phosphorylation, ATR foci formation, and Mre11 and p21 degradation happened shortly after HBV-positive serum addition, we propose that incoming virion protein and genetic materials triggered this response. p53 and H2AX phosphorylation did not begin to accumulate until 24 h after HBV serum addition, implying HBV replication inside the infected cells may be responsible for these phenomena. Interesting questions are raised and need further investigation.

    Although ATM/ATR kinase phosphorylates Chk2 at Thr-68 and p53 at Ser-15, the ATR kinase predominantly targets Chk1 at Ser-345, leading to increased Chk1 activity. Our results indicate that HBV infection preferentially activates ATR DNA damage response signaling, as is the case with human adeno-associated-virus type 2[30-36].

    As a latent virus, HBV abrogates checkpoint signaling controlled by ATR, to prevent triggering of signals for apoptosis in multiple ways. The mechanism of regulation of apoptosis by HBV was via both p53-dependent and p53-independent pathways. p53-dependent cell cycle checkpoint features p21-mediated inactivation of cdk2/cyclinE; HBV abrogates p53 dependent checkpoint activation by p21 degradation. Chk2 inhibition inhibits cdk2/cyclin E activity by phosphorylation of cdk2 at Tyr-15 in a p53-independent fashion, and the virus-decreased phosphorylation of Chk2 by Mre11 degradation inhibits the p53-independent DNA damage signaling pathway. It is known that ATM and MRN complex function in a common pathway, and the MRN complex can function to activate ATM kinase activity, so degradation of Mre11 protein by virus would inhibit ATM kinase activity, and thus affect phosphorylation of its downstream target Chk2. Therefore, like adenovirus[24,25], HBV appears to have evolved double check mechanisms to block cell cycle checkpoint signaling pathways. Consistent with this, HBV has been reported to express an additional anti-apoptotic gene HBV X that mislocates p53 to the cytoplasm, in order to evade host cellular DNA damage response and modulate apoptosis. HBX has also been reported to sensitize liver cells to environmental carcinogens, including diethylnitrosamine and aflatoxin B and UV. We observed that HBV virus enhanced cell survival upon UV radiation but hypersensitized host cells to IR; this discrepancy may be due to the complex interactions between the virus as a whole and DNA repair machinery, indicating that the virus mainly blocks cellular signaling checkpoint dependent on ATR. The risk of acquiring mutations would be enhanced by compromised cellular DNA repair caused by HBV infection. Accordingly, exposure to other environmental risk factors should act synergistically to favor the carcinogenesis process[37-39].

    In summary, HBV induces cellular DNA damage response dependent on ATR, but escapes the consequences of activation of the DNA damage checkpoint by degradation of checkpoint proteins at different levels. The implication of this is that with time, persistent HBV infection may lead to the accumulation of a variety of mutations which would ultimately give rise to hepatocellular carcinoma.

 

COMMENTS

Background

Eukaryotic cells employ multiple strategies of checkpoint signaling and DNA repair mechanisms to monitor and repair damaged DNA. There are two branches of the checkpoint response pathway, ataxia telangiectasia-mutated (ATM) pathway and ATM-Rad3-related (ATR) pathway. Virus replication presents the host cells with large amounts of exogenous genetic material, including DNA ends and unusual structures. Thus, infected cells recognize viral replication as a DNA damage stress and elicit DNA damage signal transduction, which ultimately induces apoptosis as part of host immune surveillance. However, recent reports have shown that viruses evolve a variety of mechanisms to manipulate DNA damage signaling for their replication and propagation.

Research frontiers

Epstein-Barr virus (EBV) abrogates p53 checkpoint signaling pathway through the interaction of the BZLF1 protein and p53 to avoid apoptosis. Other viruses such as human immunodeficiency virus type 1 (HIV-1), herpes simplex virus type 1 (HSV-1) and human cytomegalovirus can activate and exploit a cellular DNA damage response, which aids viral replication. Adenovirus blocks ATM signaling and concatemer formation through targeting the DNA repair complex of MRN for degradation and mislocalization. Thus, under some circumstances, viruses have co-opted endogenous checkpoint regulators to ensure their own efficient replication.

Innovations and breakthroughs

There was no evidence indicating that the ATM/ATR kinase or their downstream pathways were triggered by hepatitis B virus (HBV) infection. The present study was undertaken by culturing normal hepatocyte cell line HL7702 and primary hepatocytes from a healthy liver donor with HBV-positive serum, mimicking natural HBV infection. We showed, perhaps for the first time, that HBV infection elicited acute cellular DNA damage response dependent on ATR. However, the ATR checkpoint signaling was blocked downstream of p53-dependent and p53-independent pathways to evade apoptosis.

Applications

Since DNA damage response is an acute response that happens quickly after virus infection, we assume that early intervention of the DNA damage pathway will function more efficiently and can be used clinically as HBV infection therapy during its early infectious stage or fulminant HBV infection.

Peer review

In this interesting study the authors investigated whether exposure to HBV infection will upregulate DNA damage checkpoint signaling pathways. They show that ATM-Rad3 is upregulated as well as several downstream targets.

 

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S- Editor  Zhong XY    L- Editor  Li M    E- Editor  Ma WH

 

 

 

 

 

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