title: Bifunctional alkylating agent-mediated MGMT-DNA cross-linking and its proteolytic cleavage in 16HBE cells.
abstract: Nitrogen mustard (NM), a bifunctional alkylating agent (BAA), contains two alkyl arms and can act as a cross-linking bridge between DNA and protein to form a DNA-protein cross-link (DPC). O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme for alkyl adducts removal, is found to enhance cell sensitivity to BAAs and to promote damage, possibly due to its stable covalent cross-linking with DNA mediated by BAAs. To investigate MGMT-DNA cross-link (mDPC) formation and its possible dual roles in NM exposure, human bronchial epithelial cell line 16HBE was subjected to different concentrations of HN2, a kind of NM, and we found mDPC was induced by HN2 in a concentration-dependent manner, but the mRNA and total protein of MGMT were suppressed. As early as 1h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24h. Quick total DPC (tDPC) and γ-H2AX accumulation were observed. To evaluate the effect of newly predicted protease DVC1 on DPC cleavage, we applied siRNA of MGMT and DVC1, MG132 (proteasome inhibitor), and NMS-873 (p97 inhibitor) and found that proteolysis plays a role. DVC1 was proven to be more important in the cleavage of mDPC than tDPC in a p97-dependent manner. HN2 exposure induced DVC1 upregulation, which was at least partially contributed to MGMT cleavage by proteolysis because HN2-induced mDPC level and DNA damage was closely related with DVC1 expression. Homologous recombination (HR) was also activated. Our findings demonstrated that MGMT might turn into a DNA damage promoter by forming DPC when exposed to HN2. Proteolysis, especially DVC1, plays a crucial role in mDPC repair.
authors.affiliation: Institute of Toxicology, School of Preventive Medicine, the Third Military Medical University, 30 Gaotanyan Avenue, Shapingba District, Chongqing 400038, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Institute of Toxicology, School of Preventive Medicine, the Third Military Medical University, 30 Gaotanyan Avenue, Shapingba District, Chongqing 400038, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Ye
authors.affiliation: Institute of Toxicology, School of Preventive Medicine, the Third Military Medical University, 30 Gaotanyan Avenue, Shapingba District, Chongqing 400038, China.
authors.forename: Guorong
authors.initials: G
authors.lastname: Dan
authors.affiliation: Institute of Toxicology, School of Preventive Medicine, the Third Military Medical University, 30 Gaotanyan Avenue, Shapingba District, Chongqing 400038, China.
authors.forename: Yuanpeng
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Institute of Toxicology, School of Preventive Medicine, the Third Military Medical University, 30 Gaotanyan Avenue, Shapingba District, Chongqing 400038, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.affiliation: Institute of Toxicology, School of Preventive Medicine, the Third Military Medical University, 30 Gaotanyan Avenue, Shapingba District, Chongqing 400038, China.
authors.forename: Jiqing
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Institute of Toxicology, School of Preventive Medicine, the Third Military Medical University, 30 Gaotanyan Avenue, Shapingba District, Chongqing 400038, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Sai
authors.affiliation: Institute of Toxicology, School of Preventive Medicine, the Third Military Medical University, 30 Gaotanyan Avenue, Shapingba District, Chongqing 400038, China. Electronic address: zouzhmin@yahoo.com.
authors.forename: Zhongmin
authors.initials: Z
authors.lastname: Zou
mesh_terms: D000477:Alkylating Agents / Q000633:toxicity*; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D008466:Mechlorethamine / Q000633:toxicity*; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism*; D059748:Proteolysis
chemical_list: D000477:Alkylating Agents; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; C574636:SPRTN protein, human; D008466:Mechlorethamine; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase
keywords: Bifunctional alkylating agent; DNA damage and repair; DNA-protein cross-link; MGMT; Nitrogen mustard; Proteolysis