Differential toxicity of DNA adducts of mitomycin C.

The clinically used antitumor agent mitomycin C (MC) alkylates DNA upon reductive activation, forming six covalent DNA adducts in this process. This paper focuses on differential biological effects of individual adducts in various mammalian cell cultures, observed in the authors' laboratories. Evidence is reviewed that various adducts are capable of inducing different cell death pathways in cancer cells.

Repair of mitomycin C mono- and interstrand cross-linked DNA adducts by UvrABC: a new model.

Mitomycin C induces both MC-mono-dG and cross-linked dG-adducts in vivo. Interstrand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation. In Escherichia coli cells, UvrABC repairs ICL lesions that cause DNA bending.

DNA adducts of decarbamoyl mitomycin C efficiently kill cells without wild-type p53 resulting from proteasome-mediated degradation of checkpoint protein 1.

The mitomycin derivative 10-decarbamoyl mitomycin C (DMC) more rapidly activates a p53-independent cell death pathway than mitomycin C (MC). We recently documented that an increased proportion of mitosene1-beta-adduct formation occurs in human cells treated with DMC in comparison to those treated with MC. Here, we compare the cellular and molecular response of human cancer cells treated with MC and DMC.

Recruitment of fanconi anemia and breast cancer proteins to DNA damage sites is differentially governed by replication.

Fanconi anemia (FA) is characterized by cellular hypersensitivity to DNA crosslinking agents, but how the Fanconi pathway protects cells from DNA crosslinks and whether FA proteins act directly on crosslinks remain unclear. We developed a chromatin-IP-based strategy termed eChIP and detected association of multiple FA proteins with DNA crosslinks in vivo. Interdependence analyses revealed that crosslink-specific enrichment of various FA proteins is controlled by distinct mechanisms.

Mapping DNA adducts of mitomycin C and decarbamoyl mitomycin C in cell lines using liquid chromatography/ electrospray tandem mass spectrometry.

The antitumor antibiotic and cancer chemotherapeutic agent mitomycin C (MC) alkylates and crosslinks DNA, forming six major MC-deoxyguanosine adducts of known structures in vitro and in vivo. Two of these adducts are derived from 2,7-diaminomitosene (2,7-DAM), a nontoxic reductive metabolite of MC formed in cells in situ. Several methods have been used for the analysis of MC-DNA adducts in the past; however, a need exists for a safer, more comprehensive and direct assay of the six-adduct complex.

Synthesis of an oligodeoxyribonucleotide adduct of mitomycin C by the postoligomerization method via a triamino mitosene.

The cancer chemotherapeutic agent mitomycin C (MC) alkylates and cross-links DNA monofunctionally and bifunctionally in vivo and in vitro, forming six major MC-deoxyguanosine adducts of known structures. The synthesis of one of the monoadducts (8) by the postoligomerization method was accomplished both on the nucleoside and oligonucleotide levels, the latter resulting in the site-specific placement of 8 in a 12-mer oligodeoxyribonucleotide 26. This is the first application of this method to the synthesis of a DNA adduct of a complex natural product.

Mitomycin-DNA adducts induce p53-dependent and p53-independent cell death pathways.

10-Decarbamoyl-mitomycin C (DMC), a mitomycin C (MC) derivative, generates an array of DNA monoadducts and interstrand cross-links stereoisomeric to those that are generated by MC. DMC was previously shown in our laboratory to exceed the cytotoxicity of MC in a human leukemia cell line that lacks a functional p53 pathway (K562). However, the molecular signal transduction pathway activated by DMCDNA adducts has not been investigated.

DNA adduct of the mitomycin C metabolite 2,7-diaminomitosene is a nontoxic and nonmutagenic DNA lesion in vitro and in vivo.

Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates and cross-links DNA. These effects are dependent on reductive bioactivation of MC. 2,7-Diaminomitosene (2,7-DAM) is the major metabolite of MC in tumor cells, generated by the reduction of MC.

Nuclear localization of NADPH:cytochrome c (P450) reductase enhances the cytotoxicity of mitomycin C to Chinese hamster ovary cells.

Overexpression of endoplasmic reticulum-localized NADPH: cytochrome c (P450) reductase (NPR) in Chinese hamster ovary cells increases the hypoxic/aerobic differential toxicity of the mitomycins. Because considerable evidence indicates that DNA cross-links are the major cytotoxic lesions generated by the mitomycins, we proposed that bioactivation of the mitomycins in the nucleus close to the DNA target would influence the cytotoxicity of these drugs. The simian virus 40 large T antigen nuclear localization signal was fused to the amino-terminal end of a human NPR protein that lacked its membrane anchor sequence.

Mitomycin dimers: polyfunctional cross-linkers of DNA.

The three dimers 3, 4, and 5 of mitomycin C (MC), a natural antibiotic and cancer chemotherapeutic agent, were synthesized in which two MC molecules were linked with -(CH(2))(4)-, -(CH(2))(12)-, and -(CH(2))(3)N(CH(3))(CH(2))(3)- tethers, respectively. The dimeric mitomycins were designed to react as polyfunctional DNA alkylators, generating novel types of DNA damage. To test this design, their in vitro DNA alkylating and interstrand cross-linking (ICL) activities were studied in direct comparison with MC, which is itself an ICL agent.

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.

The effects of the subcellular localization of overexpressed bioreductive enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) on the activity of the antineoplastic agent mitomycin C (MC) under aerobic and hypoxic conditions were examined. Chinese hamster ovary (CHO-K1/dhfr(-)) cells were transfected with NQO1 cDNA to produce cells that overexpressed NQO1 activity in the nucleus (148-fold) or the cytosol (163-fold) over the constitutive level of the enzyme in parental cells. Subcellular localization of the enzyme was confirmed using antibody-assisted immunofluorescence.

Relative toxicities of DNA cross-links and monoadducts: new insights from studies of decarbamoyl mitomycin C and mitomycin C.

Mitomycin C (MC), a cytotoxic anticancer drug and bifunctional DNA DNA alkylating agent, induces cross-linking of the complementary strands of DNA. The DNA interstrand cross-links (ICLs) are thought to be the critical cytotoxic lesions produced by MC. Decarbamoyl mitomycin C (DMC) has been regarded as a monofunctional mitomycin, incapable of causing ICLs.

Nuclear overexpression of NADH:cytochrome b5 reductase activity increases the cytotoxicity of mitomycin C (MC) and the total number of MC-DNA adducts in Chinese hamster ovary cells.

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.

Differential activation of p53 by the various adducts of mitomycin C.

Mitomycin C (MC) is a cytotoxic chemotherapeutic agent that causes DNA damage in the form of DNA cross-links as well as a variety of DNA monoadducts and is known to induce p53. The various DNA adducts formed upon treatment of mouse mammary tumor cells with MC as well as 10-decarbamoyl MC (DMC) and 2,7-diaminomitosene (2,7-DAM), the major MC metabolite, have been elucidated. The cytotoxicity of DMC parallels closely that of MC in a number of rodent cell lines tested, whereas 2,7-DAM is relatively noncytotoxic.