Original Contribution
Overexpression of human NOX1 complex induces genome instability in mammalian cells

https://doi.org/10.1016/j.freeradbiomed.2007.09.018Get rights and content

Abstract

The production of reactive oxygen species (ROS) in mammalian cells is tightly regulated because of their potential to damage macromolecules, including DNA. To investigate possible links between high ROS levels, oxidative DNA damage, and genomic instability in mammalian cells, we established a novel model of chronic oxidative stress by coexpressing the NADPH oxidase human (h) NOX1 gene together with its cofactors NOXO1 and NOXA1. Transfectants of mismatch repair (MMR)-proficient HeLa cells or MMR-defective Msh2−/− mouse embryo fibroblasts overexpressing the hNOX1 complex displayed increased intracellular ROS levels. In one HeLa clone in which ROS were particularly elevated, reactive nitrogen species were also increased and nitrated proteins were identified with an anti-3-nitrotyrosine antibody. Overexpression of the hNOX1 complex increased the steady-state levels of DNA 8-oxo-7,8-dihydroguanine and caused a threefold increase in the HPRT mutation rate in HeLa cells. In contrast, additional oxidatively generated damage did not affect the constitutive mutator phenotype of the Msh2−/− fibroblasts. Because no significant changes in the expression of several DNA repair enzymes for oxidative DNA damage were identified, we suggest that chronic oxidative stress can saturate the cell's DNA repair capacity and cause significant genomic instability.

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.

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