Bcl2 Inhibits The Repair Of NNK-induced DNA Damage AP Sites By Down-regulating APE1 Endonuclease Activity

Ojective:Apurinic/apyrimidinic(AP) or abasic sites are the most common form of DNA damage.AP sites are primarily repaired by the DNA base excision repair(BER) pathway.APE1/Ref-1 (apurinic/apyrimidinic endonuclease 1/redox effect factor-1)is the essential enzyme in the BER pathway.Interestingly,APE1 is a multifunctional protein that not only is a DNA repair enzyme,but also functions as a redox factor maintaining transcription factors.Bcl2 is a major cellular oncogenic protein,it's oncogenic activity may result from its inhibitory effects on multiple DNA repair pathways including BER pathway.Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions.However,the mechanism(s) is not fully understood.In order to understand the mechanism(s) that Bcl2 suppresses the repair of NNK-induced abasic sites of DNA lesions,testing for a direct interaction between Bcl2 and APE1 will be researched.Methods:The expression levels of Bcl2,APE1 and XRCC1 proteius will be detected with Western Blotting.Single cell gel electrophoresis and AP site counting will be used to test the DNA damage. Co-immunoprecipitation will be used to analysis the interactions between Bcl₂ and APE1,APE1 and XRCC1.The cellular distributious of Bcl₂ and APE1 proteins will be tested by immunofluorescence staining.Subcellular fractionation will be used to extract the Bcl₂ protein in nucleus and mitochdria to test their expression levels and changes.AP oligonucleotides assay for APE1 endonuclease activity will be used to observe Bcl₂'s effect the APE1.Oendonuclease activity and velationship between Bcl₂'s BH domaius and APE1 endonuclease activity.Bcl₂ siRNA technology will be used to test the effect of APE1 endonuclease activity after the expression of endogenous Bcl₂ will be silent.Results:Many AP sites are produced by NNK-induced DNA damage.After NNK was washed,AP sites in H1299 cells with no Bcl2 plasmid transfected are fall-off,APsites in H1299 cells with wild type(WT)Bcl2 plasmid also decreased,but slower than first group, maintaining the high level.It explains that after washing NNK,AP sites DNA damage in H1299 cells with no Bcl2 plasmid was repaired,the repair of AP site DNA damage H1299 cells with WT Bcl2 plasmid was inhibited.After treates one hour with NNK,the nucleuses of both two H1299 cells groups have trails.It shows that NNK may induce DNA damage in H1299 cells.After washes NNK for 24 hours,the nucleuses of H1299 cells with no Bcl2 plasmid have no trails.It explained that the DNA damage has been repaired.Still have trails,it show that pare of DNA damage is not repaired.It explains again that Bcl2 protein can suppress NNK-induced DNA damage repair.After H₂O₂ and NNK induced DNA damage,Bcl2 protein can be detected with co-immunoprecipitation,It explains Bcl2 and APE1 protein can directly interaction.Red Bcl2 protein and green APE1 protein are observed to accumulate in nucleus.Via immunofluorescence staining.After emergeing,there are some orange spot in nuclus.It explains that Bcl2 and APE1 protein can directly interaction in nucleus. After treated for one hour,Bcl2 protein in H460 nucleus starts increase. After three hours,it will be highest.But Bcl2 protein in mitochondrium has no change.It imports that increased nuclear Bcl2 may not result from a movement from mitochondria into nucleus.APE1 protein can be expressed in all lung cancer cell lines,but endogenous Bcl2 protein is only expressed in H69 and H460 cells.APE1 nuclease activity in H69 and H460 cells is lower than others.It shows that Bcl2 protein can inhibit APE1 nuclease activity.Co-immunoprecipitation in H1299 cells with WT Bcl2 plasmid and deletion mutationâ–³BH₁,â–³BH₂,â–³BH₃,â–³BH₄ plasmid was found that only WT Bcl2 group can be detested Bcl2 protein,but others can not.It explains that the direct interaction between Bcl2 and APE1 proteins is concerned with BH₁,BH₂,BH₃ and BH₄ domains.Assay for APE1 nuclease activity found APE1 nuclease activity of WT group is lower than others.It will be concludes that WT Bcl2 protein can inhibit APE1 nuclease activity.The inhibitory effect is related with four BH domains.Purified WT Bcl2 protein andâ–³BH₁,â–³BH₂,â–³BH₃,â–³BH₄ protein have been tested in vitro.The result is same as that in vivo.When Bcl2 siRNA was transfected in H460 cells,the express level of Bcl2 protein is much low. But there is no change in control group.It was found that the APE1 activities in three Bcl2 siRNA gronps are increased.It show Bcl2 protein can inhibit APE1 nuclease activity. APE1 and XRCC1 DNA damage repain proteins can be expnessed in H1299 and H1299 with WT Bcl2 plasmid lung cells.But Bcl2 protein can only be detected in H1299 cells with WT Bcl2 plasmid.After NNK induced DNA damage,co-immunoprecipitation test can detect XRCC1 protein increased in H1299 cells.But XRCC1 protein in H1299 cells with WT Bcl2 plasmid is lower.It shows that Bcl2 can dissociate the DNA repair complex APE1/XRCC1 in vivo.The different concentration purified Bcl2 protein is tested the same experience in vitro. The result is same as in vivo.Conclusion:1.Bcl2 suppresses the repair of NNK or H₂O₂-induced AP site DNA lessions.2.The expression level of nuclear Bcl2 are increased when NNK induced DNA damage,increased nuclear Bcl2 may not result from a movement from mitochondria into nucleus.3.Bcl2 downregulates APE1 activity via its direct interaction with APE1 at the BH domains,which may contribute retardation of DNA repair,genetic instability and tumorigenesis.4.Bcl2 can dissociate the DNA repair complex APE 1-XRCC1 in vivo and in vitro to inhibite the repair of NNK or H₂O₂-induced AP site DNA lessions.