title: Research on DNA methylation of human osteosarcoma cell MGMT and its relationship with cell resistance to alkylating agents.
abstract: The objective of this study was to explore the O(6)-methylguanine-DNA methyltransferase (MGMT) gene methylation status and its protein expression, as well as the effects of demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) on MGMT gene expression and its resistance to alkylating agents, and to elucidate MGMT expression mechanism and significance in osteosarcoma. The human osteosarcoma cell lines Saos-2 and MG-63 were collected and treated with 5-Aza-CdR for 6 days. The cells not treated with 5-Aza-CdR were set as a negative control. The genomic DNA was extracted from the Saos-2 and MG-63 cells using methylation-specific PCR to detect the promoter CpG island methylation status of the MGMT gene. Cell sensitivity to alkylating agents before and after drug administration was detected by the MTT method. The variation in MGMT gene mRNA and protein was detected by reverse transcription PCR (RT-PCR) and Western blotting. The MGMT promoter gene of normal Saos-2 cells was methylated, with reduced MGMT mRNA and protein expression; the MGMT mRNA and protein expression of Saos-2 cells treated with 5-Aza-CdR was obviously enhanced, and its sensitivity to alkylating agents was reversed. Meanwhile, with promoter CpG island unmethylation of the MGMT gene, MGMT protein was expressed in the normal MG-63 cells and the MG-63 cells treated with 5-Aza-CdR, and both showed resistance to alkylating agents. The methylation status of the MGMT gene promoter in human osteosarcoma cells reflected the cells' ability to induce MGMT protein expression and can be used as a molecular marker to project the sensitivity of cancer tissues to alkylating agent drugs.
authors.affiliation: Department of Orthopedics (307 Hospital of PLA), Affiliated Hospital of Academy of Military Medical Sciences, Beijing 100071, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Guo
authors.forename: Qiu
authors.initials: Q
authors.lastname: Cui
authors.forename: WeiHao
authors.initials: W
authors.lastname: Jiang
authors.forename: Cheng
authors.initials: C
authors.lastname: Liu
authors.forename: DingFeng
authors.initials: D
authors.lastname: Li
authors.forename: Yanjun
authors.initials: Y
authors.lastname: Zeng
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D001859:Bone Neoplasms / Q000378:metabolism; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D012516:Osteosarcoma / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D013997:Time Factors
chemical_list: D000477:Alkylating Agents; D019853:O(6)-Methylguanine-DNA Methyltransferase