Mechanisms Of XRCC1 In DNA Replication Damage Repair And Anti-tumor Effects Of Oleandrin

DNA replication fork is the basic structure of DNA replication,its integrity is crucial to the maintenance of genomic stability,directly determines the fate of cells.However,it is vulnerable to various internal and external factors.It may lead to DNA replication fork stall,threat the genomic stability,induce apoptosis,necrosis,even cancer.The maintenance of DNA replication fork stability is an intricate process that requires the involvement of multiple DNA damage repair proteins.X-ray repair cross-complementing gene 1(XRCC-1)is an important gene involved in DNA damage repair.Its coding protein,XRCC1,consists of three domains:the NTD domain at the amino terminus,the BRCT II domain at the C-terminus,and the BRCT I domain in the middle.XRCC1 bind with DNA repair enzymes such as DNA polymerase?,DNA ligase 3a and other domains,which is mainly involved in DNA single strand damage repair(SSBR)and base excision repair(BER).Studies have shown that XRCC1 can be gathered at the DNA replication fork.However,the study of XRCC1 on the restart DNA replication has not been reported ever.Poly(ADP-ribose)polymerase(PARP)is a multifunctional protein-modified enzyme that exists in most eukaryotic cells.It is activated by identifying damaged DNA fragments,so it was thought to be receptors of DNA damage.Research have shown that XRCC1 accumulates into the lesion site under the action of PARP enzyme during DNA single chain damage repair.DNA-dependent protein kinase(DNA-PK)is composed of DNA-PKcs,Ku70,Ku80,which is an important enzyme for DNA damage repair,mainly involved in DNA double-strand breaks(double-strand breaks,DSBs)repair through non-homologous end joining(NHEJ).When the DNA double-strand breaks,Ku70,Ku80 rapid identifies and combines with the DSBs,activates of DNA-PKcs,recruiting DNA ligase IV-XRCC4 complex to DNA disconnection.Studies have shown that XRCC1 had to be phosphorylated by DNA-PK,before revolved in DNA single strand repair.Based on the above studies,we propose our assumptions:XRCC1 accumulated into the stalled DNA replication fork under the regulation of PARP or DNA-PK,and restart the stalling replication fork.Homologous recombination(HR)repairs the broken chromosomes.While DNA single strand break repair(SSBR)is achieved primarily by activating the activity of PARP enzyme.Interestingly,HR and SSBR can complement each other.In normal tissue cells,two kinds of repair methods coexist,to protect the survival of cells.Inhibition only of the two repairs does not lead to cells death.However,in some conditions(such as tumor cells),there is one of the two repairs deficient usually;if the rest of the repair method can be selectively inhibited,will lead to the cell death.We call this theory " synthetic lethality".In recent research,small molecular inhibitors targeting DNA damage repair pathways have been developed for the treatment of tumors.Such as Olaparib,a kind of PARP1 inhibitors,has been approved by the FDA for treatment of homologous recombination deficiency cancers.However,due to homologous recombination repair defects are mainly happened in ovarian cancer and breast cancer,and less happened in other tumor cells,which limited the use of PARP1 inhibitors.Oleandrin is a monomer compound extracted from the Chinese herbal oleander,in which is benefit for anti-tumor.Our previous study found that oleandrin could induce the increase of DNA single-stranded structures.Based on these results,we propose our hypothesis:Oleandrin could inhibit homologous recombination repair.If this is established,the combination of Oleandrin and Olaparib will expand the use of PARP inhibitors,and provide a new strategy for the treatment of tumors.There are two parts in our study:(1)To explore the mechanism of XRCC1 on DNA replication fork stall and restart,and the regulation of PARP1 and DNA-PKcs to XRCC1 in this process;(2)To clarify the anti-tumor effect of oleandrin,and find a novel inhibitor of DNA damage repair response.Part 1.Mechanisms of XRCC1 induced DNA replication damage repairObjective:To explore the role of XRCC1?PARP1?DNA-PKcs at stalled DNA replication forks,and the underlying mechanisms.Methods:U20S cells were treated with HU,and detected by immunofluorescence for XRCC1 foci positive cells,EdU and XRCC1 co-location cells,XRCC1 and Cyclin-A co-location cells.GFP-XRCC1-U20S cells were treated with HU and Olaparib,detected for XRCC1 positive cells.DNA fiber assay to detect the stalled DNA replication fork in EM9-V and EM9-XH cells treated with HU.EM9-V and EM9-XH cells treated with HU,and then detected by colony formation assay.U20S?EM9-V?EM9-XH cells were treated with HU,then detected by immunofluorescence and western blot for XRCC1 and RAD51 expression.U20S cells were treated with Mirin,and detected for XRCC1 foci cells;and EM9-V.EM9-XH cells were treated with HU,detected for Mrell foci cells;EM9-V?EM9-XH cells were treated with Mirin,detected by colony formation assay.U20S cells were treated with Mirin and Olaparib,detected by immunofluorescence for XRCC1 positive cells.U20S cells were treated with HU and CK2 inhibitor TBB,XRCC1 protein expression detected by western blot.V3-3 and AA8 cells were treated with HU,and detected by immunofluorescence for XRCC1 positive cells;The DNA-PKcs activity of U20S cells were suppressed by siRNA,then treated with HU,detected by western blot for XRCC1 expression;GFP-XRCC1-U20S cells were treated with HU,then labelled with p-DNA-PKcs antibody,detected for XRCC1 and p-DNA-PKcs co-location cells.siRNA transfection to deplete the DNA-PKcs in EM9-V and EM9-XH cells,the stalled DNA replication fork was detected by DNA fiber assay.Results:Induced by HU,XRCC1 foci increased in U20S cells,and co-located with EdU,only in Cyclin-A positive cells.Interestingly,HU induced increase of XRCC1 foci was conteracted by Olaparib.EM9-V cells have a higher rate of DNA replication fork stalling than EM9-XH cells,and the clongenic ability of EM9-V cells were also inhibited.XRCC1 increased early in HU treated U20S cells,while RAD51 increased later.XRCC1 foci increased in Mirin induced U20S cells;the Mrell foci also increased in HU treated EM9-V cells.When the activity of CK2 was suppressed,HU failed to induce the increase of XRCC1.In DNA-PKcs deficient V-V3 cells,HU failed to induce the increase of XRCC1;besides XRCC1 was found to co-localized with DNA-PKcs;moreover,the DNA replication fork was aggravated following the depletion of DNA-PKcs.Conclusions:XRCC1 is recruited to stalled replication forks by PARP1 and DNA-PKcs;and XRCC1's recruitment to stalled forks is important for restart of stalled replication forks,and survival of cells upon replication stress.It possible that XRCC1 involved in the early repair of stalled replication fork.And CK2 is also required for the recruitment of XRCC1.Part 2.Mechaniams of DNA damage responses in the anti-tumor role of OleandrinObjective:To investigation the anti-tumor role of Oleandrin,and find a new inhibitor for homologous recombination repair.Methods:A549 were treated with oleandrin in different concentrations(0.02 ug/ml,0.04 ug/ml,0.06 ug/ml)for 24 hours;or A549 were treated with oleandrin(0.02 ug/ml)for 6hous,12 hours and 24 hours;and then the apoptosis rate of A549 cells were detected by Flow cytometry.A549 cells or H1299 cells were treated with oleandrin,and then detect by immunofluorescence or western blot for DNA damage related proteins RPA,yH2AX.And then DAN damage repair proteins RAD51 and XRCC1 were also detected by western blot.A549 cells were treated with oleandrin,then cell cycle was detected by Flow cytometry.To further the effect of oleandrin on Homologous Recombination Repair,the XRCC1 gene of A549 cells was deleted by siRNA,and then treated with oleandrin,Flow cytometry was used to detect the apoptosis of A549 cells.Results:Following the treatment of oleandrin,the apoptosis of A549 cells increased significantly.Treated with oleandrin,RPA and ?H2AX expression were increased;and DNA damage repair protein RAD51 was significantly decreased,while XRCC1 was increased.Oleandrin seemed have no effect on cell cycle.When the activity of XRCC1 was suppressed,A549 cells became more sensitive to oleandrin.Conclusion:Oleandrin was effectively in anti-tumor;and this was related with DNA damage repair.Oleandrin was a potential homologous recombination repair inhibitor.