Nuclear Overexpression of NADH:Cytochromeb 5 Reductase Activity Increases the Cytotoxicity of Mitomycin C (MC) and the Total Number of MC-DNA Adducts in Chinese Hamster Ovary Cells

Abstract

NADH:cytochrome b 5 reductase (FpD) is an enzyme capable of converting the prodrug mitomycin C (MC) into a DNA alkylating agent via reduction of its quininone moiety. In this study, Chinese hamster ovary (CHO) cells were transfected with a cDNA encoding rat FpD. Despite the demonstrated ability of this enzyme to reduce MC in vitro, a modest 5-fold level of overexpression of FpD activity in CHO cells did not increase the cytotoxicity of the drug over that seen with the parental cell line under either aerobic or hypoxic conditions. When the enzyme, which is predominantly localized in the mitochondria, was instead directed to the nucleus of cells by the fusion of the SV40 large T antigen nuclear localization signal sequence to the amino terminus of an FpD gene that lacked the membrane anchor domain, drug sensitivity was significantly enhanced at all concentrations of MC examined (2-10 M) under both aerobic and hypoxic conditions, with greater cell kill occurring under hypoxia. The marked increase in drug sensitivity under hypoxia at 10 M MC corresponded to a measurable increase in total MC-DNA adducts at the same concentration. The results indicate that the cytotoxicity of MC is modulated by the subcellular location of FpD, with greater cell kill occurring when bioactivation occurs in the proximity of its target, nuclear DNA.Mitomycin C (MC), 1 a natural product antibiotic with antitumor activity, has been used successfully to eradicate hypoxic tumor cells in head, neck, and cervix cancers in combination with ionizing radiation (1-4). As a prodrug, MC can be chemically and enzymatically converted into a reactive species capable of alkylating a variety of nucleophilic molecules, including genomic DNA (5). Specifically, the quinone moiety of MC is reduced to give unstable intermediates that are capable of further transformations to generate a reactive molecule with two electrophilic carbon centers (6). Both monofunctional and bifunctional adducts are formed between MC and DNA (7, 8).The variety of adducts formed between MC and DNA both in vitro and in drug-treated EMT6 cells have been extensively characterized (9 -13). The majority of these lesions are monofunctional alkylations occurring on the guanine-N(2) position. These lesions are considered to be less cytotoxic than the covalent bifunctional cross-link that forms exclusively in the 5Ј-CpG-3Ј sequence between the N-2 amino groups of two guanines in complementary strands of the minor groove. This interstrand MC-DNA cross-link prevents DNA replication, and corresponds directly with cell survival (14, 15).Several enzymes have been shown to catalyze the reduction of MC to reactive intermediates using either a one-electron or a two-electron mechanism. NADPH:cytochrome P-450 oxidoreductase (EC 1.6.2.4) (16, 17), NADH:cytochrome b 5 oxidoreductase (EC 1.6.2.2; FpD) (18, 19), xanthine:oxygen oxidoreductase (EC 1.1.3.22; xanthine oxidase) (16,20), and, more recently, nitric-oxide synthase (EC 1.14.13.39) (21,22) are among the enzymes using a one-electron reduction mechan...

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

“…2B), on the cytotoxicity of MC under aerobic and hypoxic conditions were measured by comparing the survival curves for the CHO transfectants with that of parental cells. In the parental cell line, greater levels of cell kill were obtained in hypoxia than under aerobic conditions, a finding consistent with previous reports (27,29,30). Comparative survival curves for CHO-K1/dhfr Ϫ parental and CYTO-NQO1 transfectants ( Fig.…”

“…CYTO-NQO1 and NLS-NQO1 CHO-K1/dhfr Ϫ cell transfectants that expressed significantly more NQO1 activity (163-fold and 148-fold, respectively) than parental CHO cells were isolated. This level of overexpression in transfected CHO-K1/dhfr Ϫ cells is roughly comparable with previously reported increases in NQO1 activity of 126-and 133-fold over parental CHO cells (30) and is markedly higher than the increase in expression obtained for cells transfected with the one-electron bioreductive enzyme systems NPR (44,45) and NBR (28,29). No significant changes occurred in the NBR and NPR activities of the CYTO-NQO1 and NLS-NQO1 CHO-K1/dhfr Ϫ transfectants and parental cells.…”

“…This separation, known as the "aerobic/hypoxic differential," is reflected in the cytotoxicity profiles for Chinese hamster ovary (CHO) cell transfectants overexpressing NPR (27) and NBR (28,29) but not for those overexpressing NQO1 (30). Thus, although NPR and NBR are not important enzymes in the activation of MC under aerobic conditions, they contribute to the preferential activation of MC in hypoxia.…”

“…Thus, activation of MC in the nucleus close to the DNA target should result in a greater number of DNA cross-links and increased cell kill. 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.…”

Molecular construction of NADH-cytochrome b5 reductase inhibition by flavonoids and chemical basis of difference in inhibition potential: Molecular dynamics simulation study

“…Thus, in addition to the highly reactive DNA-adduct forming metabolites formed by cytochrome P450 isozymes through oxidative pathways, reactive oxygen species generated in the presence of cytochrome b5 reductase may contribute to the carcinogenic effects of heterocyclic amines (Hodnick and Sartorelli, 1993;Holtz et al, 2003). As the above-mentioned studies demonstrate, it appears that the microsomal cytochrome b5 reductase is a clinically and toxicologically important enzyme.…”