DNA Adducts of Decarbamoyl Mitomycin C Efficiently Kill Cells without Wild-Type p53 Resulting from Proteasome-Mediated Degradation of Checkpoint Protein 1

Abstract

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-β-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. We find the increase in mitosene 1-β-adduct formation correlates with a condensed nuclear morphology and increased cytotoxicity in human cancer cells with or without p53. DMC caused more DNA damage than MC in the nuclear and mitochondrial genomes. Checkpoint 1 protein (Chk1) was depleted following DMC, and the depletion of Chk1 by DMC was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against Chk1 depletion. Gene silencing of Chk1 by siRNA increased the cytotoxicity of MC. DMC treatment caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity, suggesting that DMC mediated DNA adducts facilitate signal transduction to a pathway targeting cellular proteins for proteolysis. Thus, the mitosene-1-β stereoisomeric DNA adducts produced by the DMC signal for a p53-independent mode of cell death correlated with reduced nuclear size, persistent DNA damage, increased ubiquitin proteolysis and reduced Chk1 protein.

C. elegans CEP-1/p53 and BEC-1 Are Involved in DNA Repair

“…We previously showed that DMC induces p53-independent cell death and DNA damage in human cancer cells [22], [23]. Very few pharmacological agents effectively enter into C. elegans, because of the thick cuticle of the animal, coupled with the animal’s extensive xenobiotic efflux pumps [33].…”

Exposure to Mitochondrial Genotoxins and Dopaminergic Neurodegeneration in Caenorhabditis elegans

“…Furthermore, non-genotoxic mitochondrial toxins may indirectly cause mtDNA damage by disrupting oxidative phosphorylation [18] – [20] . A number of genotoxins cause more mtDNA than nDNA damage [21] – [24] but the opposite is true for other chemicals [23] , [25] , [26] , and relatively few chemicals have been tested for their potency in damaging the mitochondrial genome (reviewed in Meyer et al [4] ).…”

The QPCR assay for analysis of mitochondrial DNA damage, repair, and relative copy number

“…Examples include: rotenone (inducing mtDNA but not nucDNA damage in human cells) (15), methyl methanesulfonate (MMS) (causing nucDNA damage in Saccharomyces cerevisiae) (16), decarbomoyl mitomycin C (inducing mtDNA and nucDNA damage in human cancer cells) (17), menadione (resulting in mtDNA damage in murine myoblasts) (18), benzo[a]pyrene (producing mtDNA and nucDNA damage in killifish) (19, 20), hydrogen peroxide (causing mtDNA but not nucDNA damage in human cells) (9), UV (nucDNA damage induced in mice, C. elegans , adenovirus and zebrafish) (3, 21, 22) (this manuscript, Section 3), zidovudine (3′-azido-3′-deoxythymidine or AZT), lamivudine ((-)2′,3′-dideoxy-3′-thiacytidine or 3TC) and Combivir (zidovudine and lamivudine) (inducing mtDNA in mice) (23), photoactivated hypericin (24), cisplatin, chlorambucil, and psoralen (2), lipopolysaccharide (25), and a complex mixture of environmental contaminants (20). …”