Extracellular Superoxide Production by Enterococcus faecalis Promotes Chromosomal Instability in Mammalian Cells

doi:10.1053/j.gastro.2006.11.040

Gastroenterology

Volume 132, Issue 2, February 2007, Pages 551-561

https://doi.org/10.1053/j.gastro.2006.11.040 Get rights and content

Background & Aims: We investigated whether Enterococcus faecalis, a Gram-positive intestinal commensal that produces extracellular superoxide, could promote chromosomal instability (CIN) in mammalian cells. Methods: We measured the ability of E faecalis to promote CIN using hybrid hamster cells (A_LN) containing human chromosome 11. Results: E faecalis promoted CIN in A_LN cells with average mutant fractions per 10⁵ survivors (±SD) of 72.3 ± 6.7 at 1 × 10⁹ cfu mL⁻¹ compared with 22.2° ± 4.5 for the no bacteria control. γ-Irradiation at 2 Gray similarly resulted in 74.7 ± 5.7 mutant clones per 10⁵ survivors. Deletions in chromosome 11 consistent with CIN were verified in 80% of mutant clones. E faecalis-treated A_LN cells were protected from CIN by superoxide dismutase, γ-tocopherol, and cyclooxygenase-2 (COX-2) inhibitors. In a dual-chamber tissue culture model designed to mimic stromal-epithelial cell interactions, macrophages pretreated with E faecalis grown on permeable supports increased mutant fractions 2.5-fold for A_LN cells. COX-2 was up-regulated by superoxide from E faecalis and mutant fractions decreased when COX-2 was silenced using short interfering RNA. Escherichia coli, a Gram-negative commensal that produces negligible extracellular superoxide, only modestly promoted CIN in this model. Conclusions: These findings indicate that macrophage COX-2 is induced by superoxide from E faecalis and promotes CIN in mammalian cells through diffusible factors. This mechanism links the oxidative physiology of E faecalis to propagation of genomic instability through a bystander effect, and offers a novel theory for the role of commensal bacteria in the etiology of sporadic colorectal cancer.

Section snippets

Cell and Bacterial Culture

A_LN human-hamster hybrid cells (a gift from Elizabeth McNiel) were maintained in Ham’s F12 Nutrition Mixture containing 4% heat-inactivated FCS and 3% heat-inactivated newborn calf serum, penicillin G (50 units mL⁻¹), streptomycin (50 μg mL⁻¹), G418 (400 μg mL⁻¹), and 20 mmol/L HEPES buffer, pH 7.4, at 37°C in 5% CO₂. The murine macrophage RAW264.7 cell line (American Type Culture Collection, Rockville, MD) was maintained in high glucose DMEM supplemented with 10% fetal bovine serum, penicillin

E faecalis Promotes CIN in A_LN Cells

We initially determined the ability of E faecalis OG1RF to promote CIN in A_LN human–hamster hybrid cells. This engineered cell line contains 1 copy of human chromosome 11 that encodes CD59 surface antigen at 11p13.5, confers neomycin resistance at another locus, and is a validated assay for measuring CIN.²² As a control for comparing E faecalis treatments, we also exposed A_LN cells to γ-irradiation. Both conditions produced cytotoxic effects with decreasing cell survival as doses of E faecalis

Discussion

Plausible theories for sporadic colorectal cancer should successfully address the key questions concerning carcinogenesis for these tumors, ie, the origin of CIN, a role for COX-2, and causal relationships between submucosal cell reactivity and transformed epithelial cells. Our data support a model that mechanistically links these characteristics. In this scheme, extracellular ·O₂⁻ production by E faecalis triggers COX-2 expression in macrophages that drives carcinogenesis in epithelial cells

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: Supported by Department of Veterans Affairs Merit Review Program (to M.M.H.) and the Frances Duffy Endowment.

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Basic–alimentary tractExtracellular Superoxide Production by Enterococcus faecalis Promotes Chromosomal Instability in Mammalian Cells

Section snippets

Cell and Bacterial Culture

E faecalis Promotes CIN in ALN Cells

Discussion

J Nutr

Am J Gastroenterol

Free Radic Biol Med

Free Radic Biol Med

Mutat Res

Arch Biochem Biophys

Am J Pathol

Gastroenterology

J Hosp Infect

Cell

Am J Pathol

J Biol Chem

Mutat Res

Biochem Biophys Res Commun

J Biol Chem

Gastroenterology

Genomic instability and colon cancer

Cancer Metastasis Rev

The role of chromosomal instability in tumor initiation

Proc Natl Acad Sci U S A

The onset and extent of genomic instability in sporadic colorectal tumor progression

Proc Natl Acad Sci U S A

Evidence that genetic instability occurs at an early stage of colorectal tumorigenesis

Cancer Res

Cancer genes and the pathways they control

Nat Med

Intestinal floras of populations that have a high risk of colon cancer

Appl Environ Microbiol

Diet, anaerobic bacterial metabolism, and colon cancer: a review of the literature

J Clin Gastroenterol

Extracellular superoxide production by Enterococcus faecalis requires demethylmenaquinone and is attenuated by functional terminal quinol oxidases

Mol Microbiol

Physiology of enterococci

Enterococcus faecalis produces extracellular superoxide and hydrogen peroxide that damages colonic epithelial cell DNA

Carcinogenesis

Mechanism of radiation-induced bystander effect: role of the cyclooxygenase-2 signaling pathway

Proc Natl Acad Sci U S A

Basic–alimentary tract
Extracellular Superoxide Production by Enterococcus faecalis Promotes Chromosomal Instability in Mammalian Cells

E faecalis Promotes CIN in A_LN Cells