Molecular Mechanisms Of EBV-LMP1 DNAzyme Mediated Radiosensitization

Nasopharyngeal carcinoma(NPC) has a remarkably distinctive ethnic and geographic distribution,more than 80%of which were reported from China,Southeast Asia,and some other Asian countries.A unique feature of NPC is its strong association with Epstein-Barr Virus (EBV).Latent membrane protein 1(LMP1) is a major one with oncogenic properties among EBV encoded proteins.Since NPC is highly radiosensitive,radiotherapy(RT) has always been the main treatment of choice for this cancer.Although overall survival after RT for the patients at early stages is encouraging,there are significant rates of local failure and distant metastases subsequent to RT in the advanced stage disease. Thus,it has been a great challenge to identify biological agents as radiosensitizers that could enhance radiosensitivity for treatment of the EBV-associated NPC.In previous studies,we experimentally demonstrated that the phosphorothioate-modified"10-23"DNAzymes specifically against the LMP1 mRNA could down-regulate the expression of LMP1 in a nasopharyngeal carcinoma cell line CNE1-LMP1 and affect the down-stream pathways activated by LMP1,such as NF-κB, JNK/AP-1 and STAT signaling pathways.When tested in a mouse xenograft model,the DNAzyme was found to inhibit tumor growth and enhance radiosensitivity in vivo.We also demonstrated that when combined with the radiotherapy the DNAzyme could enhance the radiosensitivity both in vivo and in vitro.It is known that the celluar radiosensitivity is closed related to the cell cycle regulation and DNA repairing.To explore the molecular mechanisms underlying the radiosensitizing effect of the LMP1-targeted DNAzyme in nasopharyngeal carcinoma(NPC),we used the NPC cell lines as model to investigate how EBV-LMP1 is involved in the radioresistance via cell cycle control and DNA repair mechanisms.The LMP1-targeted DNAzyme inhibited the expression of LMP1 and affected the cell cycleWe first demonstrated when the expression of LMP1 was inhibited by DNAzyme,the NPC cells was shown to be arrested at the S phase. This cell cycle arrest was accompanied with a decrease of cyclin D1 and cyclin E protein levels at 24 h from the DNAzyme treatment.Moreover, we observed an inhibition of CDK4 activity and a decreased cyclinD1 expression in the complexes immunoprecipitated with CDK4 antibody and the suppression of RB/E2F pathway in DNAzyme treated cells.We also found that,a reduction in cdc2 phosphorylation at Thr161,which partially stands for the cdc2 kinase activity in DNAzyme treated CNE1-LMP1 cells,although the active DNAzyme did not affect cyclinB1 and cdc2 expression.Further,we analyzed that changes in cdc2 kinase activity induced by DNAzyme and found that the down-regulation of the LMP1 expression resulted in a 5-fold reduction of cdc2 kinase activity in CNE1-LMP1,suggesting that G1/S and G2/M checkpoint pathways could contribute to the S phase arrest in LMP1-positive cells induced by the LMP 1-targeted DNAzyme.The LMP1-targeted DNAzyme inhibited the expression of ATM,a key factor of DNA repairing systems through NF-κB pathway.The central role of the ATM protein in DNA damage repair is now well established.Ionizing radiation(IR),not UV radiation,enhances ATM kinase activity and phosphorylates a series of target proteins(e.g. p53,BRCA1,c-ab1,etc.),which are involved in cell cycle control and repair of DNA damage.ATM-deficient cells have impaired ability to efficiently halt proliferation and repair DNA damage.We further examined if the LMP1-targeted DNAzyme could regulate the expression of ATM.Using the induction strategy by LMP1 expression plasmid and the blockage strategy by LMP1-targeted DNAzyme,we confirmed that LMP1 up-regulated the ATM transcriptional activity and the protein level in the reporter gene assay and western blotting assay.Based on the finding that LMP1 could up-regulate the ATM expression,we investigated the signaling pathway involved in the procession.Bioinformatic analysis revealed that there are three putative NF-κB binding sites in ATM promoter region(GenBank Accession GXP₄80587).The first and third binding sites were located in the same location,but in different strands.It implied that NF-κB binding to corresponding sites might be responsible for the modulation of ATM gene expression.By using a specific inhibitor of the NF-κB signaling pathway,the suppression of the ATM up-regulated in CNE1-LMP1 cells by the inhibitor could be achieved in a dose-dependent manner.A stable NPC cell line expressing dominant-negative mutant of IκBα(DNMIκBα) was used to further confirm the role of NF-κB pathway in regulating ATM expression.As verified by Western blotting,the stable expression DNMIκB resulted in a decrease ATM expression in HNE2-LMP1-DNMIκBαcell.On the basis of the findings that NF-κB pathway was involved in LMP1-augmented ATM expression in human NPC cells,we attempt to confirm the precise element which NF-κB binds to.The site-directed mutagenesis by Overlap Extension PCR was used to introduce mutations into two locations of NF-κB in promoter region(the first/third and second sites).From the reporter gene assay we found that the different mutations could suppress the ATM promoter activity in different degrees.Mutations at two locations at the same time downregulate the ATM promoter activity.This suggested that a synergistic effect of the NF-κB binding to two sites may be responsible for the up-regulation of ATM expression mediated by LMP1.To demonstrate the direct binding of NF-κB to the ATM promoter, an electrophoretic mobility supershift assay(EMSA) was further conducted.We showed that the NF-κB DNA-binding activity was much more stronger in CNE1-LMP1 cells than in CNE1 cells,indicating the role of LMP1 in mediation of NF-κB-ATM pathway.The induction of NF-κB DNA binding activity by LMP1 was clearly inhibited by LMP1-targeted DNAzyme.Supershift analysis with antibodies specific for NF-κB family members showed that NF-κB DNA/protein complex composed of p50 subunit in nuclear extracts of CNE1-LMP1 cells,which suggested that at least the NF-κB p50 directly bind to the ATM promoter. Inhibition of the ATM expression could enhance the radiosensitivity in NPC cellsWe have confirmed that the ATM expression could be downregulated by LMP1-targeted DNAzyme through suppressing the transcription factor NF-κB binding to the ATM promoter.It has been demonstrated that silence of the ATM could enhance radiosensitivity in breast cancer and glioma cells.We then investigate if the radiosenstivity caused by LMP1-targeted DNAzyme is through the downregulation of the ATM expression in NPC cells.Using the ATM-targeted siRNA and FACS assay,we found when exposed to 5 Gy,nearly 60%of the ATM-siRNA-treated cells underwent apoptosis.The colony-formation assay showed when the expression of ATM was deceased by the siRNA,the colony-formation ability was reduced.The result suggested that the down-regulation of ATM may suppress the DNA repairing signaling pathway and promote the cellular death,then enhanced the radiosensitivity of NPC cells.Comprehensive analysis of the radiobiological parameters in the single-hit multitarget model and linear quadratic equation further confirmed that the silence of the ATM expression led to sensitizing of the NPC cells to radiation.In conclusion,our finding demonstrated that the LMP1-targeted DNAzyme inhibited the expression of LMP1 and affected the cell cycle; the LMP1-targeted DNAzyme inhibited the expression of ATM;and inhibition of the ATM expression could enhance the radiosensitivity in NPC cells.The data provided solid experimental evidence to support our hypothesis that the radiosensitization of NPC cells by LMP1-targeted DNAzyme is through the impact on cell cycle control and DNA repair systems.The results can provide a basis for the use of the LMP1-targeted DNAzyme as potential radiosensitizer for clinical treatment and supply a new way to treat the EBV-associated radioresistance carcinomas.