Protective effect of O6-methylguanine-DNA-methyltransferase on mammalian cells.
- Author:
Dong-Bo LI
1
;
Ji-Shi WANG
;
Qin FANG
;
Hai-Yang SUN
;
Wei XU
;
Wei-da LI
Author Information
- Publication Type:Journal Article
- MeSH: Blotting, Western; Cell Survival; drug effects; genetics; physiology; Cells, Cultured; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Green Fluorescent Proteins; genetics; metabolism; Hepatocytes; cytology; drug effects; metabolism; Humans; K562 Cells; Leukocytes, Mononuclear; cytology; drug effects; metabolism; Microscopy, Fluorescence; Nitrogen Mustard Compounds; pharmacology; O(6)-Methylguanine-DNA Methyltransferase; genetics; metabolism; physiology; RNA, Messenger; genetics; metabolism; Reverse Transcriptase Polymerase Chain Reaction; Transfection
- From: Chinese Medical Journal 2007;120(8):714-717
- CountryChina
- Language:English
-
Abstract:
BACKGROUNDO(6)-methylguanine-DNA-methyltransferase (MGMT) is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells.
METHODSMammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells (PBMCs) via liposome, then assayed for gene expression at RNA and protein levels. MTT assay was used to check the drug resistance of cells transfected with MGMT gene.
RESULTSMGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively.
RESULTSof Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC(50) values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC.
CONCLUSIONThe alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.
