The Establishment Of Method For MGMT-DNA Crosslinks Detection And Its Application

Research background and purposesDNA-Protein crosslinkings(DPC)is the high genotoxicity of DNA adducts,and the crosslinked DNA-protein complex via covalent interaction under drug and/or toxicant exposure.Because DPC prevented DNA replication and transcription,it is a serious way of DNA damage.Formaldehyde,radiation,nitrogen mustard and cisplatin can cause the formation of DPC,and induce the damage of actively proliferating cells in tissue,such as bone marrow and tumor and so on,which in turn increases drug sensitivity.But,the law of DPC's formation,repair kinetics and mechanism are still not clear,this is mainly because of complex method of DPC detection,difficult quantification,and difficult to carry out widely.Therefore,the progress of detecting DPC methods is the foundation that promotes the research on the formation of DPC and repair mechanism.Developing a simple,accurate,popularizing DPC detection method are urgent affairs.Nitrogen mustard and sulfur mustard are two kinds of mustard gas poisons,also known as blistering agent,which belongs to the vesicating agent.Vesicant is a kind of chemical agent that can damage cells directly,cause skin inflammation,mucosal necrosis.When absorbed through skin,eyes,mucous membrane of the respiratory tract,this chemical warfare agents can cause systemic injury.The main mechanism of poisoning is alkylation,in which the active chlorine alkyl group of nitrogen mustard cross-links with its sensitive groups in biological molecules.Based on the number of alkyls connected with biological molecules,it is classified as the single and double functional alkylating agents.Mustard gas is a typical kind of bifunctional alkylating agent.Many important biological molecules,such as nucleic acids,cellular proteins,peptides can carry out nucleophilic reaction to form various adducts.Groups in protein,such as amino,imino,and ionized carboxyl and sulfhydryl groups are susceptible to alkylation.The ionized carboxyl in phosphoric acid of nucleic acid and nucleic acid coenzyme,N7 of purine bases and O6 of guanine all can be alkylated.Therefore,mustard gas has complex biological effects.O6-methylguanine-DNA methyltransferase,a 23 k Da protein,and is closely related to the alkylated DNA damage repair,is currently only found in mammalian cells,and can directly remove the alkyl group at the site of O6 alkylation,which is considered that it is important to keep genome stability and prevent tumor formation.MGMT is a limited consumptive enzyme,and its supplement is slow.MGMT has an important role in clinical cancer chemotherapy as well as in the drug development.Some reports,the high expression of exogenous MGMT can increase the tolerance of cells to alkylating agents.This explains that expression level of MGMT and alkylating agent tolerance in cells are closely linked.Our previous study found that MGMT cannot give play to remove the DNA adduct as its normal function after the effect of nitrogen mustard and other bifunctional alkylating agents,because the MGMT is now crosslinked with DNA and forms MGMT-DNA crosslinks(M-DPC)and cannot be released.As other DPC damage,M-DPC has been shown to have multiple effects by damaging replication and inducing DNA mutations.In some MGMT rich tissues or cells,increase of M-DPC caused by the bifunctional alkylating agent is considered to be an important cause of animal and cell toxicity.In this case,detecting the level of M-DPC in cells or tissues has important significance to reflect the degree of cell injury and drug sensitivities.The gene and cell toxicity of various injury factors are closely linked with DPC formation.Based on reported DPC detection method of the existing literature,present study aims to establish an ELISA(enzyme-linked immuno sorbent assay)based analysis on DPC detection,and pay special attention to the formation of MGMT-constituted DPC in terms of its characteristics of m-DPC formation with nitrogen mustard exposure.This method is simple,practical,experimental needs of ordinary conditions,and can be carried out in general laboratories.The application of this method in clinical practice may be an important assay to determine anti-tumor drug sensitivity and evaluate the mechanism of alkylating agents.Research contents: 1.Setting up nitrogen mustard exposed HBE cells model.Cell proliferation activity is confirmed through a microscope observation and CCK 8 analysis on cell proliferation,which determines appropriate HN2 exposure concentration.2.Establishing DPC detection method based on ELISA.By integrating the the methods of cell nuclear extraction,nuclear lysis and Tris-saturated phenol recovery,to create the method of extraction and purification of DPC,Using SYBR Green I-based quantification method to improves the detection of ds DNA in DPC,To simplify the extraction,purification,and quantitative methods of DPC,Based on the principle of ELISA,to establish a detection method for M-DPC and comparing with classical slot blot.3.A preliminary study on the concentration and time effect of M-DPC formation under HN2 exposure.From the perspective of the M-DPC formation dynamics,to reveal the effect of MGMT on DNA damage and repair in the presence of the double functional alkylating agent.4.EGF-stimulated cell proliferation and the proteolytic effect of DVC1 in M-DPC formation and repair were investigated.Results and conclusions:1.The morphological observation on human bronchial mucosa epithelial HBE cells 24 hours after HN2 poison reveals that cell shape altered obviously with the concentration of HN2 increased.Little influence on cell morphology can be seen at 10 ?M HN2 treatment,but 30 ?M and 100 ?M HN2 exposure makes the cell deattachment,rounding,and death,especially at 100 ?M HN2.CCK 8 assay of cell proliferating activity indicates a gradually decreases with the increase of poison doses,such as activity dropped to 60% at 30 ?M and to 20% at 100 ?M.2.SYBR Green I method can proceed accurate quantitation on standard ds DNA in the range of 2 ng-1.5 ?g.Using cell nuclear extraction,nuclear lysis and Tris-saturated phenol recovery method,ds DNA including DPC is successfully isolated.Using SYBR Green I quantitative methods of the ds DNA,the concentration of ds DNA can be calculated from the standard curve.The number of total cells and the amount of total isolated DNA has good linear relationship.3.In the range of 20-100 ng of loading sample,the ELISA method has good color developing reaction,and can be quantitated by compared with the standard curve.The results of ELISA method and slot blot method for M-DPC detection have good consistency.4.MGMT decreased after 10 ?M HN2 poisoning.The addition of O6-BG before poisoning can aggregate MGMT inhibition.The application of MGMT inhibitor O6-BG does not have influence on cell cytotoxic effect of HN2,and cell proliferation activity has no obvious change.MGMT normally is expressed in cytoplasm,and as an enzyme gives play to methyl transfer in the nucleus.After HN2 treatment,MGMT increasingly translocates into the nucleus,and possibly to be ubiquitinized to form a 35 k D band higher than the native 23 k Da MGMT monomer.In the cytoplasm,MGMT exists in prototype,and its level decreased after poisoning,which may be due to the increase of nuclear translocation of MGMT.5.After HN2 exposure,M-DPC increased significantly in a concentration-dependent way in the range of 10-100 ?M HN2 treatment.With 30 ?M of HN2 poison,the time effect shows as M-DPC level increased significantly at 3 hours,and remain at this level until 24 hours.The preliminary observation of HN2 mediated the formation of the M-DPC kinetics characteristics is achieved.6.Cell proliferation and proteolysis were confirmed participating M-DPC formation and repair separately.Present research combined with newly reported methods of DPC preparation and detection,establishes a easy protocol for DPC preparation.An ELISA-based method for DPC quantitation can rapidly,economically and accurately detect M-DPC.Applying the established method,preliminary observation on HN2 mediated the formation of the M-DPC kinetics characteristics is achieved.