Gastroenterology

Gastroenterology

Volume 142, Issue 3, March 2012, Pages 543-551.e7
Gastroenterology

Original Research
Basic and Translational—Alimentary Tract
4-Hydroxy-2-Nonenal Mediates Genotoxicity and Bystander Effects Caused by Enterococcus faecalis–Infected Macrophages

https://doi.org/10.1053/j.gastro.2011.11.020Get rights and content

Background & Aims

Enterococcus faecalis is a human intestinal commensal that produces extracellular superoxide and promotes chromosome instability via macrophage-induced bystander effects. We investigated the ability of 4-hydroxy-2-nonenal (4-HNE), a diffusible breakdown product of ω-6 polyunsaturated fatty acids, to mediate these effects.

Methods

4-HNE was purified from E faecalis–infected macrophages; its genotoxicity was assessed in human colon cancer (HCT116) and primary murine colon epithelial (YAMC) cell lines.

Results

4-HNE induced G2–M cell cycle arrest, led to formation γH2AX foci, and disrupted the mitotic spindle in both cell lines. Binucleate tetraploid cells that formed after incubation with 4-HNE were associated with the activation of stathmin and microtubule catastrophe. Silencing glutathione S-transferase α4, a scavenger of 4-HNE, increased the susceptibility of epithelial cells to 4-HNE–induced genotoxicity. Interleukin-10 knockout mice colonized with superoxide-producing E faecalis developed inflammation and colorectal cancer, whereas colonization with a superoxide-deficient strain resulted in inflammation but not cancer. 4-HNE–protein adducts were found in the lamina propria and macrophages in areas of colorectal inflammation.

Conclusions

4-HNE can act as an autochthonous mitotic spindle poison in normal colonic epithelial and colon cancer cells. This finding links the macrophage-induced bystander effects to colorectal carcinogenesis.

Section snippets

Cell Lines and Bacteria

The near-diploid HCT116 human colon cancer cell (American Type Culture Collection, Manassas, VA), YAMC primary mouse colon epithelial cell (Ludwig Institute for Cancer Research, New York, NY), and RAW264.7 murine macrophage cell (American Type Culture Collection) lines were grown as previously described.12, 25 E faecalis strain OG1RFSS was spontaneously derived from OG1RF, a gram-positive human commensal, and expressed high-level resistance to spectinomycin and streptomycin.26 menB was deleted

E faecalis–Infected Macrophages Produce 4-HNE

We initially assayed E faecalis–infected RAW264.7 cells for 4-HNE production and confirmed its structure by 2-dimensional nuclear magnetic resonance. 1H and 13C shifts were consistent with trans-4-hydroxy-2-nonenal (Figure 1A). Compared with uninfected controls, RAW264.7 cells infected with OG1RF at an MOI of 1000 had a 2-fold increase in 4-HNE concentration in supernatants at 24 to 72 hours postinfection (P < .01) (Figure 1B). No significant increase was noted for supernatants from RAW264.7

Discussion

We previously reported that E faecalis–infected macrophages generate bystander effects that cause G2–M cell cycle arrest and CIN in epithelial cells.12, 13 In this study, we show that bystander effects induced by E faecalis are associated with increased production of 4-HNE by infected macrophages. This aldehyde is a breakdown product of lipid peroxidation16 and acts as a diffusible clastogen19 that causes γH2AX deposition due to DNA double-strand breaks. It also induces G2–M cell cycle arrest

Acknowledgments

The authors thank Jim Henthorn in the Flow Cytometry Laboratory and the Rodent Barrier Facility at the University of Oklahoma Health Sciences Center, Robert Whitehead of Vanderbilt University for YAMC cells, and Graça Dores for helpful discussions.

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    Conflicts of interest The authors disclose no conflicts.

    Funding Supported by National Institutes of Health grant CA127893 (to M.M.H.), Oklahoma Center for the Advancement of Science and Technology grant HR10-032 (to X.W.), National Science Foundation grant 0639199 (to S.L.N.), and funds from the Frances Duffy Endowment.

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