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  • br Acknowledgements We thank Dr Tai Yuan


    Acknowledgements We thank Dr. Tai-Yuan Yu and Miss Chun-Ping Chang for helping with the drug/DNA sequence specificity study. We also thank the Chemical Synthesis Core and the Pathology Core Laboratory of IBMS for synthesizing SL-1 and for performing the pathology analysis, respectively, and the Taiwan Mouse Clinic (MOST 105-2325-B-001-010), which is funded by the National Research Program for Biopharmaceuticals at the Ministry of Science and Technology of Taiwan, for technical support with the blood cells and blood Fomepizole analysis. The mass spectrometry analysis was supported by the Metabolomics Core Facility, Scientific Instrument Center at Academia Sinica. This work was supported in part by grants from the Ministry of Science and Technology, Taiwan (MOST103-2325-B-001-018, MOST104-2325-B-001-001, and MOST 105-2325-B-001-001), and Academia Sinica (AS-102-TP-B13) to T. C. L. and by a grant from the Ministry of Science and Technology (MOST 105-0210-01-13-01) and an intramural grant from Academia Sinica to C. N. S.
    Introduction Initially, nitrogen mustards (NM) are used as an important military vesicant agent. NM can cause skin inflammation, blisters, and ulcers, as well as eye and respiratory tract damage, with no effective treatment. One of the commonly accepted mechanisms of NMs that causes tissue damage is an active DNA alkylating ability. As a kind of bifunctional alkylating agent (BAA), NM contains two functional N-chloroethyl groups, which can react with nucleophilic groups within DNA or proteins to cause a DNA–DNA or DNA–protein cross-link (DPC) (Loeber et al., 2009). Based on the alkylating effect on biomolecules, many kinds of NM derivatives including N-methyl-2.2-di(chloroethyl)amine (HN2), also called mechlorethamine, chlorambucil, and melphalan are widely used clinically against various tumors including lymphoma, leukemia, and multiple myeloma. However, little is known about the details of DPCs in terms of their levels or their relationship with DNA damage repair. Various endogenous and exogenous agents including irradiation, chemotherapy drugs, and cytotoxic agents can induce DNA damage (Loeber et al., 2009). DPC is a type of DNA damage when a protein covalently binds to DNA to form an adduct (Barker et al., 2005). Compared with DNA damages like double-strand break (DSB) and single-strand break (SSB), much less attention has been paid to DPCs and a poor understanding remains because of the low abundance and structural complexity of DPCs (Wong et al., 2012). In fact, bulky adducts and helix-distorting lesions have lethal effects on DNA replication, transcription, recombination, and chromatin remodeling (Barker et al., 2005). In addition, DPC lesions can prevent DNA repair proteins from binding to the damaged nucleobase, thus promoting subsequent failure of the DNA repair process. Both of these effects may lead to the cytotoxicity and genotoxicity of DPCs. However, because of the difficulty in establishing a model uniquely inducing DPC rather than other types of DNA damage and in accurately detecting different kinds of DPCs, the mechanisms of formation, repair, and effect on cell activity of DPCs are not yet well understood (Stingele et al., 2015, Stingele and Jentsch, 2015). O6-methylguanine–DNA methyltransferase (MGMT), also called O-alkylguanine-DNA alkyltransferase (AGT), is a DNA-repair protein that transfers alkyl adducts from the O6-position of guanine to the 145 cysteine residue (Cys145) of MGMT meanwhile irreversibly inactivating itself. Then, the inactivated MGMT is ubiquitinated and degraded by proteasomes (Cabrini et al., 2015). Normally, the MGMT-mediated irreversible alkyl transfer prevents gene mutations and cell apoptosis resulting from alkylating and cross-linking damages commonly induced by environmental toxicants (Srivenugopal et al., 2016). On the other hand, because of the capability of alkyl adduct removal, MGMT may cause drug resistance of tumor cells to chemotherapy by upregulated MGMT expression. However, some recent studies find that MGMT may enhance the cytotoxicity and mutagenicity of several bis-electrophiles, which usually have two symmetrical reactive sites readily for substrate DNA or protein (Loeber et al., 2006, Kalapila and Pegg, 2010, Pegg, 2011). Definite evidence shows that DNA lesions are formed in NM-treated neurons (Kisby et al., 2009). Overexpressing MGMT in CHO cells can aggravate the cytotoxicity of bifunctional alkylating agents (Kalapila and Pegg, 2010, Pegg, 2011). It was proposed that MGMT increases the cytotoxic and mutagenic effects of NM and its analogues by reacting with one active arm of NM at the cysteine site of MGMT to form a half-mustard, which can either be cleaved via proteasome pathway or react further with DNA via another arm of MN to generate a MGMT-DNA cross-link (mDPC) (Pegg, 2011, Casorelli et al., 2012). We proposed that MGMT might play a different role in BAA-treated cells in addition to its original DNA repair function.