Structure of Adduct X, the Last Unknown of the Six Major DNA Adducts of Mitomycin C Formed in EMT6 Mouse Mammary Tumor Cells

Abstract

Treatment of EMT6 mouse mammary tumor cells with mitomycin C (MC) results in the formation of six major MC−DNA adducts. We identified the last unknown of these (“adduct X”) as a guanine N2 adduct of 2,7-diaminomitosene (2,7-DAM), in which the mitosene is linked at its C-10 position to guanine N2. The assigned structure is based on UV and mass spectra of adduct X isolated directly from the cells, as well as on its difference UV, second-derivative UV, and circular dichroism spectra, synthesis from [8-3H]deoxyguanosine, and observation of its heat stability. These tests were carried out using 17 μg of synthetic material altogether. The mechanism of formation of adduct X involves reductive metabolism of MC to 2,7-DAM, which undergoes a second round of reductive activation to alkylate DNA, yielding adduct X and another 2,7-DAM−guanine adduct (adduct Y), which is linked at guanine N7 to the mitosene. Adduct Y has been described previously. Adduct X is formed preferentially at GpC, while adduct Y favors the GpG sequence. In contrast to MC−DNA adducts, the 2,7-DAM−DNA adducts are not cytotoxic.

DNA-damaging agents cause inactivation of translational regulators linked to mTOR signalling

“…These were mitomycin-C and cis-platinum(II)diammine dichloride (cisplatin), which both cause DNA damage by the formation of bulky adducts (Schweizer et al, 1999;Palom et al, 1998). Such DNA lesions elicit a DNA repair response and cell cycle arrest.…”

Nuclear Overexpression of NAD(P)H:Quinone Oxidoreductase 1 in Chinese Hamster Ovary Cells Increases the Cytotoxicity of Mitomycin C under Aerobic and Hypoxic Conditions

“…MC exhibits a broad spectrum of antitumor activity and is an important component in the combination chemotherapy of malignancies such as early stage head and neck cancer, early stage cervical cancer, and intravesicle therapy of superficial bladder cancer. Specific MC-DNA lesions associated with the action of MC consist of both monofunctional and bifunctional alkylations (2)(3)(4)(5)(6)(7)(8)(9). Monoalkylations initially occur through the linkage of the C-1 position of MC to the amino function in the 2-position of guanine bases in DNA and may proceed to a DNA cross-link through the C-10 position of MC to an amino entity in the 2-position of an adjacent DNA guanine (6).…”

“…Such a prediction was realized in studies with CHO cells transfected with a cDNA encoding rat NBR, in that nuclear localization of the enzyme resulted in greater cell kill and increased numbers of MC-DNA adducts over those occurring with overexpressed NBR localized predominantly in its normal mitochondrial and endoplasmic recticulum locations (29). Since NQO1, being a two-electron reducing system that directly generates MCH 2 (4,7,10,15,22,(31)(32)(33)(34)(35), is considered to be a more important bioactivator of MC than the one-electron activating system, NBR, we have measured the cytotoxicity of MC and the number of MC-DNA adducts formed from this agent in CHO cells overexpressing NQO1 activity in the cytosolic and nuclear compartments under aerobic and hypoxic conditions.…”

Differential Activation of p53 by the Various Adducts of Mitomycin C

“…MC induces DNA damage in the form of DNA cross-links and monofunctional DNA alkylation products in various bacterial and mammalian cells and tissues (2)(3)(4)(5)(6)(7). Treatment of mammalian cells with MC causes an increase in the cellular p53 level (8).…”

“…In cells, MC is enzymatically reduced, yielding reactive species that are capable of producing radicals through redox cycling, as well as a variety of DNA adducts. Six major adducts are formed as shown in mouse mammary tumor cells and their precise molecular structures have been elucidated (4). Very recently, the MC derivative decarbamoyl mitomycin C (DMC; Chart 1) was shown to generate an array of DNA cross-links and monoadducts in EMT6 mouse mammary tumor cells that had similar or identical structures to those formed with MC.…”