title: Measurement of O(6)-alkylguanine-DNA alkyltransferase activity in tumour cells using stable isotope dilution HPLC-ESI-MS/MS.
abstract: The repair of DNA mediated by O(6)-alkylguanine-DNA alkyltransferase (AGT) provides protection against DNA damage from endogenous or exogenous alkylation of the O(6) position of guanine. However, this repair acts as a double-edged sword in cancer treatment, as it not only protects normal cells from chemotherapy-associated toxicities, but also results in cancer cell resistance to guanine O(6)-alkylating antitumour agents. Thus, AGT plays an important role in predicting the individual susceptibility to guanine O(6)-alkylating carcinogens and chemotherapies. Accordingly, it is necessary to establish a quantitative method for determining AGT activity with high accuracy, sensitivity and practicality. Here, we describe a novel nonradioactive method for measuring AGT activity using stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). This method is based on the irreversibility of the removal of the O(6)-alkyl group from guanine by AGT and on the high affinity of O(6)-benzylguanine (O(6)-BG) as an AGT substrate. HPLC-ESI-MS/MS was used to measure the AGT activities in cell protein extracts from eight tumour lines, demonstrating that AGT activity was quite variable among different cell lines, ranging from nondetectable to 1021 fmol/mg protein. The experiments performed in intact tumour cells yielded similar results but exhibited slightly higher activities than those observed in cell protein extracts. The accuracy of this method was confirmed by an examination of AGT expression levels using western blotting analysis. To our knowledge, this method is the first mass spectrometry-based AGT activity assay, and will likely provide assistance in the screening of cancer risk or the application of chemotherapies.
authors.affiliation: Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, PR China. Electronic address: sunguohui@emails.bjut.edu.cn.
authors.forename: Guohui
authors.initials: G
authors.lastname: Sun
authors.affiliation: Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, PR China. Electronic address: zhaolijiao@bjut.edu.cn.
authors.forename: Lijiao
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, PR China. Electronic address: fantengjiao2014@emails.bjut.edu.cn.
authors.forename: Tengjiao
authors.initials: T
authors.lastname: Fan
authors.affiliation: Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, PR China. Electronic address: renting@bjut.edu.cn.
authors.forename: Ting
authors.initials: T
authors.lastname: Ren
authors.affiliation: Beijing Key Laboratory of Environmental & Viral Oncology, College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, PR China. Electronic address: lifesci@bjut.edu.cn.
authors.forename: Rugang
authors.initials: R
authors.lastname: Zhong
mesh_terms: D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006147:Guanine / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D057230:Limit of Detection; D016014:Linear Models; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000032:analysis / Q000378:metabolism*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006147:Guanine; D019853:O(6)-Methylguanine-DNA Methyltransferase
keywords: AGT activity; HPLC-ESI–MS/MS; O(6)-alkylguanine-DNA alkyltransferase; O(6)-benzylguanine; Tumour cells