The Effect And Repair Progress Of Nitrogen Mustard Induced Crosslink Between Methyltransferase MGMT And DNA

BackgroundMustard is a kind of vesicants,which are harmful to skin,eye as well as respiratory tract.Amoung all target organs,respiratory tract damage is hard to prevent and treat,leaving obvious chronic effect.Nitrogen mustard is one of bifunctional alkylating agent,contains two functional N-chloroethyl arms,which can react with nucleophilic sites within DNA or proteins to cause a stable covalent DNA–protein cross-link(DPC).DPC belongs to DNA damage.The bulky adducts and helix-distorting lesions can stall replication,transcription,recombination,and chromatin remodeling,which lead to genotoxicity and cytotoxicity.O6-methylguanine–DNA methyltransferase(MGMT)is a DNA-repair protein that transfers alkyl adducts from the O6-position of guanine.It can repair DNA alkyl damage produced by alkyl agent,and is one of the most important enzyme in DNA repair.However,it is found that MGMT is ineffective in bifunctional alkylating agent damage such as nitrogen mustard,because each alkylating site in these kinds of agent can bind with DNA and MGMT separately.This may form MGMT-DNA crosslink(mDPC).In this situation,MGMT becomes a DNA damage inducer other than DNA damage repair enzyme.There are many DNA damage repair pathways.The most important way to repair DPC in high eukaryotes is homologous recombination(HR)as well as proteolysis,which is reported recently.So far,the effect of nitrogen mustard in inducing mDPC,as well as the influence on m DPC is not clearly understood yet.Besides,there is no research on the repair of nitrogen mustard induced mDPC.Content1.To evaluate the damage effect of mechlorethamine,also called N-methyl-2,2-di(chloroethyl)amine(HN2)on human bronchial epithelial cell(16HBE): To evaluate the cell vitality after HN2 exposure.To evaluate the DNA damage effect,DPC formation under suitable HN2 concentration,especially to evaluate mDPC formation,and its dose-and time-dependent effects.2.To detect the influence factors on m DPC formation: to control the expression of MGMT protein using transcription regulation factors,and to accelerate to degradation of MGMT with specific inhibitor.To observe the influence of these treatment on the formation of mDPC as well as total-DPC(t DPC)in cell.3.To study repair pathway of mDPC: To observe activities of several DNA damage repair pathways as well as the time-dependent effect.To observe the effect of proteolysis on DPC repair,and pay much attention on the newly predicted protein DVC1 in proteolysis after HN2 treatment.To find the m RNA and protein expression characteristic of DVC1;the effect of DVC1 on mDPC repair as well as how it functions;To find the molecular function of DVC1;and to evaluate the effect of MGMT expression level to DVC1.4.To study the degradation of MGMT: To find whether MGMT is degraded via ubiquitin-proteosome system after HN2 treatment;and if yes,the progress of degradation and its regulation mechanism.To find the time-dependent effect.To evaluate the influence of ubiquitination to the progress of m DPC repair as well as t DPC repair.Finally,to find the difference of several ubiquitin inhibitors on DPC repair.Result1.Cell vitality decreased after HN2 treatment.It was dose dependent.Under the concentration of 50 ?M,cell vitality was 80% compared to normal cell.HN2 exposure caused double strand break(DSB),with high expression of m RNA and phosphorylated protein of H2 AX and NF-?B.However,it is opposite that the mRNA and protein level of MGMT decreased after HN2 treatment.HN2 elevated tDPC and m DPC level.The formation of m DPC was dose-dependent.The mDPC was formed 1 h after HN2 treatment.After decreased at 6 h,it was increased again at 24 h after HN2 treatment.Cells after MGMT si RNA transfection formed less m DPC.2.Cells without HN2 treatment produce more ?-H2 AX after MGMT si RNA transfection.However,when treated with HN2,the elevation degree of ?-H2 AX in MGMT deletion cell was lower than that of normal cell,indicating less DSB damage.HN2 exposure had no influence on the mRNA and protein level of c-MYC,the negative transcription regulation factor of MGMT.Treatment of 10058-F4,the specific inhibitor of c-MYC,elevated the expression of MGMT.However,the elevation could be interfered by HN2 exposure.C-MYC inhibition combined with HN2 exposure caused a elevation of ?-H2 AX expression and mDPC formation,but there was no obvious effect on t DPC formation.O6 BG,the specific substrate of MGMT,can inhibit mDPC formation;however,treatment in a short time elevated a compensation production of mDPC.3.mRNAs related with HR pathway were upregulated after HN2 exposure,such as FANCD2?BRCA2 and RAD51.Besides,the protein expression of FANCD2 was also increased 1 h-6 h after HN2 exposure.However,the protein expression of RAD51 had no change 1h-24 h after HN2 exposure.The m RNA of CHK2,which was related with cell cycle regulation and DNA damage checkpoint,also increased.Other DNA damage repair pathways had no obvious change after HN2 exposure.Treatment of MG132,a inhibitor of proteosome,leaded to increase of both tDPC and mDPC formation.The mRNA and protein level of DVC1,a proteolysis related protein predicted by bioinformation,were all elevated after HN2 exposure.DVC1 deletion leaded to elevation of mDPC formation and deceleration of tDPC repair.DVC1 had the activity of 20 S proteosome.The mechanism of DNA repair was p97 dependent.The inhibition of MGMT expression level alleviated the expression of DVC1.4.MGMT had both prototype and ubiquitinated form.The level of ubiquitinated MGMT increased as the time after HN2 exposure.MGMT could also be small ubiquitin-like modified(SUMOylated),and the level of SUMOylated MGMT decreased as the time after HN2 exposure.It was opposite to ubiquitination.Proteosome inhibition caused an elevation of ubiquitinated MGMT level,while as,treatment of PYR-41,an E1 activating enzyme inhibitor,caused a decrease of ubiquitinated MGMT level.DVC1 deletion presented a similar result to proteosome inhibition.The speed of t DPC repair could be interfered by E1,E2 and E3 inhibition,and the effect of E2 and E3 were higher than E1.All three inhibitor could also affect the speed of mDPC repair,but when comparing with t DPC,the influence of E1 was highest on mDPC clearance.ConclusionHN2 could cause obvious cell damage and DNA damage,promote DPC formation,and specifically cross-linking with MGMT.The formation of m DPC was influenced by HN2 concentration and MGMT expression.HN2 exposure activated HR related genes,but other DNA damage repair pathways were inactivated.Proteolysis took part in HN2 induced DPC repair,and DVC1 was a important protein in proteolysis.DVC1 had 20 S proteosome activity,and be of importance in m DPC repair as well as t DPC repair.The repair activity of DVC1 was p97 dependent.The key point of m DPC repair was the degradation of MGMT.MGMT was degraded via ubiquitin-proteosome system.The ubiquitination degree of MGMT increased along with the time after HN2 exposure,and was also dependent on SUMO.The level of ubiquitination and SUMOylation was complementary balanced.Ubiquitination inhibition could slow down the clearance rate of tDPC and mDPC severely.