Abstract 4391: 4-Hydroxy-2-nonenal causes tetraploidy through a macrophage-induced bystander effect triggered by a commensal bacterium

Abstract

Abstract Tetraploidy can be an intermediate condition for cells undergoing tranformation from diploidy to aneuploidy. We previously showed that Enterococcus faecalis, a human intestinal commensal, generated tetraploidy and aneuploidy in colonic epithelial cells via a macrophage-induced bystander effect. To explore potential mediators for this effect, supernatants from E. faecalis-infected macrophages were analyzed by HPLC coupled to a coulometric detection system. Significantly increased concentrations of 4-hydroxy-2-nonenal (4-HNE), a breakdown product of polyunsaturated fatty acids, were detected in macrophage supernatants. This system was used to purify macrophage-generated 4-HNE for further experiments. To investigate aneugenic effects of 4-HNE, a near-diploid human colon cancer cell line (HCT116) and non-transformed diploid mouse colonic epithelial cell line (YAMC) were exposed to 4-HNE and ploidy analyzed by FACS. For HCT116 cells the percent tetraploidy significantly increased following exposure to 4-HNE at 2.5 µM compared to untreated controls (9.90% ± 0.09 vs. 0.21% ± 0.08, P < 0.001). In comparison, the percent tetraploidy was increased to 0.68% ± 0.10 for YAMC cells exposed to 1 µM 4-HNE compared to 0.12% ± 0.06 for untreated controls (P < 0.001). This showed that 4-HNE generated tetraploidy in both transformed and non-transformed cells. To address potential mechanisms by which 4-HNE produced tetraploidy, HCT116 cells exposed to 4-HNE were stained with anti-α/β tubulin antibody and microtubules analyzed by confocal microscopy. We found cells exposed to 4-HNE had degraded microtubules and abnormal mitotic patterns, implicating the induction of tetraploidy through inhibition of microtubule assembly. Moreover, for HCT116 and YAMC cells, 4-HNE caused a G2/M cell cycle arrest with increased γH2AX foci suggesting DNA double-strand breaks. Next, we silenced mGSTα4-4, a key enzyme in the detoxification of 4-HNE, by constitutively expressing RNAi in cells. FACS analysis showed that γH2AX positivity remarkably increased in YAMC cells with silenced mGSTα4-4 that were exposed to E. faecalis-infected macrophages compared to non-infected macrophages. No changes were seen in wildtype YAMC cells. Similarly, γH2AX foci increased in mGSTα4-4-silenced YAMC cells exposed to 4-HNE compared to the wildtype control (41.8% ± 1.3 vs. 24.6% ± 6.5, P = 0.01). Finally, percent tetraploidy also significantly increased for mGSTα4-4-silenced YAMC cells exposed to 4-HNE, supporting the hypothesis that 4-HNE can specifically generate tetraploidy. In sum, E. faecalis-infected macrophages produce 4-HNE that, in turn, induces tetraploidy, DNA double-strand breaks and G2/M arrest in colonic epithelial cells. Inactivation of mGSTα4-4, a detoxifying enzyme for 4-HNE, exacerbated these effects. We propose 4-HNE as a mediator for the E. faecalis-infected macrophage bystander effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4391.