3545

Enterococcus faecalis is a human intestinal commensal that produces extracellular superoxide and can damage colonic epithelial cell DNA. To investigate whether E. faecalis can induce chromosomal instability (CIN), the most common form of genomic instability in sporadic human colorectal cancer, we measured the mutagenic effects of E. faecalis strain OG1RF on ALN Chinese hamster ovary cells. These cells contain one copy of human chromosome 11 that encodes CD59, a cell surface marker. Following 2 hour exposure to OG1RF, ALN cells deficient in CD59 were selected by lysing CD59 expressing cells with anti-CD59 antibody and complement. OG1RF caused CIN in a dose dependent manner (average mutant fraction per 105 survivors [± SD] of 22.2 ± 4.5 for no bacteria, and 39.1 ± 9.3, 49.6 ± 6.8, and 72.3 ± 6.7 for OG1RF at 1×107, 1×108, and 1×109 cfu ml-1, respectively). In comparison, 2 Gray of γ irradiation resulted in a mutant fraction of 74.7 ± 5.7 per 105 survivors. Loss of CD59 was verified by PCR. Large deletions in chromosome 11 (12 Mb to 110 Mb) were verified in 4 of 10 randomly selected clones using SNP GeneChip mapping. The mutant fraction for ALN cells exposed to OG1RF at 1×109 cfu ml-1 decreased 50% after addition of MnSOD (p = 0.0005). No further decrease was observed by adding catalase (p = 0.78). OG1RF-induced CIN was augmented by exogenous ω-6 polyunsaturated fatty acids. The mutant fraction increased 20% with 10 μM arachidonic acid (p = 0.003). Furthermore, OG1RF-induced CIN decreased 47% with 100 μM γ-carboxyethyl-hydroxychroman (γ-CEHC), a metabolite of γ-tocopherol and potent COX-2 inhibitor, compared to 42% (p = 0.002) and 34% (p = 0.004) using 200 μM α- and γ-tocopherol, respectively. Reverse transcriptase PCR showed rapid induction of COX-2 in ALN cells following exposure to OG1RF. γ-CEHC may have protected against CIN through this mechanism. This hypothesis was supported by a 57%, decrease in OG1RF-induced CIN using 10 μM celecoxib, another selective COX-2 inhibitor. Finally, to assess the ability of E. faecalis to activate innate immune effector cells and induce CIN indirectly, ALN cells were exposed to murine macrophages (RAW 264.7 cells) grown on a permeable support following infection with OG1RF (i.e., no direct contact between cells). A significantly increased mutant fraction (68.6 ± 13.0 mutants per 105 survivors; p = 0.001) was observed. These data support the hypothesis that E. faecalis directly and indirectly induces CIN in target cells possibly through peroxidation of membrane lipids and/or induction of COX-2 to produce clastogens. These findings suggest that redox-active intestinal microbiota can contribute to the induction of CIN and thereby promote colorectal adenomas and cancer.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]