Abstract B57: Long-term exposure of colonic epithelial cells to Enterococcus faecalis-infected macrophages causes cellular transformation
Authors: Wang Xingmin , Huycke Mark M. 
Published: June 2012

Abstract
Enterococcus faecalis is a human colonic commensal that contributes to early mutagenic events of colorectal carcinogenesis via bystander effects. The macrophage-mediated bystander effect is a long-term process that causes the accumulation of mutations that lead to cancer. We previously showed that E. faecalis-infected macrophages produce 4-hydroxy-2-nonenal (4-HNE), a breakdown product of polyunsaturated fatty acids. 4-HNE damages mitotic spindles, generates tetraploidy, and is a mediator of the bystander effect. We show in this study that long-term exposure of non-transformed murine colonic epithelial cells (YAMC) to E. faecalis-infected macrophages or purified 4-HNE induces chromosomal instability (CIN) and cellular transformation.
We initially exposed YAMC cells to E. faecalis-infected macrophages, or 4-HNE, and used the tsA58 gene as an indicator for mutagenesis. YAMC cells are derived from a transgenic mouse harboring the H-2Kb class I gene promoter fused to the SV40 tsA58 early region. As a consequence, YAMC cells die by senescence when grown at the non-permissive temperature of 39.5°C. Cells with acquired mutations in tsA58 can grow at this temperature to form colonies. YAMC cells were co-cultured with E. faecalis-infected macrophages for 72 hrs and then allowed to recover for 96 hrs. This process was performed for a total of 16 treatment cycles. The average mutant fraction for tsA58 significantly increased following 2–10 treatments with a peak after 10 treatments (56.8 ± 10.6 per 1 × 105 cells) compared to cells co-cultured with uninfected macrophages (10.7 ± 1.9 per 1 × 105 cells). To confirm a role for 4-HNE in this effect, YAMC cells were treated for 1 hr with 1 μM 4-HNE and cells allowed to recover for 1 week. Of note, compared to untreated YAMC cells, the mutant fraction for tsA58 increased to 128.0 ± 16.0, 247.0 ± 14.5, and 311.3 ± 18.3 per 1 × 105 cells after 8, 10, and 12 treatments, respectively.
Next, YAMC cells were treated with E. faecalis-infected macrophages, or 4-HNE, and analyzed for ploidy by fluorescence-activated cell sorting (FACS). The percentage of aneuploid cells increased remarkably after 8 exposures to E. faecalis-infected macrophages (P &lt; 0.001). Notably, for cells treated with 4-HNE, the percentage of aneuploid cells significantly increased after only 2 treatments compared to untreated YAMC cells and was sustained through 10 treatments (P &lt; 0.01). Furthermore, to confirm production of heritable CIN, cells were grown at 39.5°C and colonies isolated from each group. FACS analysis showed that 6 of 10 clones (60%) from the group exposed to E. faecalis-infected macrophages, and 8 of 15 clones (53%) from the group treated with 4-HNE, maintained CIN. In addition, fluorescence in situ hybridization (FISH) showed numerous chromosome translocations in clones, supporting the conclusion that these cells expressed heritable CIN. Finally, to assess whether long-term exposure of YAMC cells to E. faecalis-infected macrophages led to cellular transformation, we tested cells for growth in soft agar. Untreated YAMC cells failed to grow soft agar while anchorage-independent growth was clearly evident for cells exposed to E. faecalis-infected macrophages and 4-HNE, further supporting the conclusion that long-term exposure to E. faecalis-infected macrophages or 4-HNE is tranformative.
In summary, continuous exposure of colonic epithelial cells to E. faecalis-infected macrophages and 4-HNE induces accumulation of mutations, CIN, and cellular transformation. These findings provide a novel mechanism for colorectal carcinogenesis and should permit the development of new chemopreventive strategies.
Citation Information: Cancer Prev Res 2011;4(10 Suppl):B57.