Original ContributionOverexpression of human NOX1 complex induces genome instability in mammalian cells
Section snippets
Vector construction
The EGFP/hNOX1 fusion plasmid was obtained by subcloning into the mammalian expression vector pEFGP-C2 (Clontech Laboratories, Inc.) the EcoRI–KpnI fragment containing the cDNA of the human NOX1 downstream of the EGFP gene. Plasmids pBlueScript/Mox1, pCMV Sport 6/hNOXA1, and pcDNA 3.1/hNOXO1 have already been described [30].
Cell culture and DNA transfection
All cells were grown routinely in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 g/ml) (Gibco
Overexpression of the hNOX1 complex in human HeLa cells
Because hNOX1 acts in a complex with two cofactors, hNOXO1 and hNOXA1, their genes were also transfected into HeLa cells to achieve maximal activation of the complex [30], [34]. In two clones, hNOX1 C1 and hNOX1 C10, ROS levels were five- to sevenfold higher than those of the parental HeLa cells as measured by fluorescence produced from the CM-H2DCFDA dye (p = 0.008 for hNOX1 C1 and p = 0.001 for hNOX1 C10, t test) (Fig. 1A). The presence of increased numbers of hNOX1 transcripts in both clones was
Discussion
Our data demonstrate a direct link between chronic oxidative stress and DNA base damage. Because we have previously shown that MMR plays a major role in limiting the detrimental consequences of incorporation of oxidized precursors into DNA, we experimentally induced oxidative stress in both MMR-proficient and MMR-deficient cells. Expression of the hNOX1 complex increased ROS/RNS levels—presumably via the production of superoxide—in both MMR-proficient HeLa cells and MMR-defective Msh2−/− MEFs.
Acknowledgments
We thank C. D'Ascoli for technical support, Annapaola Franchitto for essential help in the comet assay, and Dr. P. Karran for helpful discussions. This work was supported by a fellowship from the FIRB to F.C. and grants from the ISS/NIH and AIRC to M.B.
References (52)
- et al.
Reactive oxygen and nitrogen species as signaling molecules regulating neutrophil function
Free Radic. Biol. Med.
(2007) - et al.
Free radicals and antioxidants in normal physiological functions and human disease
Int. J. Biochem. Cell Biol.
(2007) - et al.
The interacting pathways for prevention and repair of oxidative DNA damage
Mutat. Res.
(2003) - et al.
8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G→T and A→C substitutions
J. Biol. Chem.
(1992) - et al.
Functional cooperation of MutT, MutM and MutY proteins in preventing mutations caused by spontaneous oxidation of guanine nucleotide in Escherichia coli
Mutat. Res.
(1995) - et al.
Mutational specificity of mice defective in the MTH1 and/or the MSH2 genes
DNA Repair (Amsterdam)
(2002) - et al.
Cloning and expression of cDNA for a human enzyme that hydrolyzes 8-oxo-dGTP, a mutagenic substrate for DNA synthesis
J. Biol. Chem.
(1993) - et al.
The mammalian mismatch repair pathway removes DNA 8-oxodGMP incorporated from the oxidized dNTP pool
Curr. Biol.
(2002) - et al.
Enhancement of catalase activity by repetitive low-grade H2O2 exposures protects fibroblasts from subsequent stress-induced apoptosis
Mutat. Res.
(2003) - et al.
Transient adaptation of oxidative stress in mammalian cells
Arch. Biochem. Biophys.
(1995)
Novel homologs of gp91phox
Trends Biochem. Sci.
The Nox family of NAD(P)H oxidases: host defense and beyond
J. Biol. Chem.
Nox1-dependent reactive oxygen generation is regulated by Rac1
J. Biol. Chem.
Proteins homologous to p47phox and p67phox support superoxide production by NAD(P)H oxidase 1 in colon epithelial cells
J. Biol. Chem.
Overexpression of a novel superoxide-producing enzyme, NADPH oxidase 1, in adenoma and well differentiated adenocarcinoma of the human colon
Cancer Lett.
Nox3 regulation by NOXO1, p47phox, and p67phox
J. Biol. Chem.
Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases
J. Biol. Chem.
HIF-1alpha induces genetic instability by transcriptionally downregulating MutSalpha expression
Mol. Cell
The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B
Chem. Biol.
Endogenous reactive intermediates as modulators of cell signaling and cell death
Chem. Res. Toxicol.
Oxidative stress and cancer: have we moved forward?
Biochem. J.
Nitric oxide, cell bioenergetics and neurodegeneration
J. Neurochem.
Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases
Nature
Repair and genetic consequences of endogenous DNA base damage in mammalian cells
Annu. Rev. Genet.
Accumulation of the oxidative base lesion 8-hydroxyguanine in DNA of tumor-prone mice defective in both the Myh and Ogg1 DNA glycosylases
Cancer Res.
The oxidized deoxynucleoside triphosphate pool is a significant contributor to genetic instability in mismatch repair-deficient cells
Mol. Cell. Biol.
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These authors equally contributed to this work.