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 G2M cell cycle arrest, led to formation γH2AX foci, and disrupted the mitotic spindle in both cell lines. Binucleate tetraploid cells that formed following incubation with 4-HNE were associated with the activation of stathmin and microtubule catastrophe. Silencing glutathione S-transferase α-4, a scavenger of 4-HNE, increased 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.

ObjectiveCommensal bacteria and innate immunity play a major role in the development of colorectal cancer (CRC). We propose that selected commensals polarise colon macrophages to produce endogenous mutagens that initiate chromosomal instability (CIN), lead to expression of progenitor and tumour stem cell markers, and drive CRC through a bystander effect.DesignPrimary murine colon epithelial cells were repetitively exposed to Enterococcus faecalis-infected macrophages, or purified trans-4-hydroxy-2-nonenal (4-HNE)—an endogenous mutagen and spindle poison produced by macrophages. CIN, gene expression, growth as allografts in immunodeficient mice were examined for clones and expression of markers confirmed using interleukin (IL) 10 knockout mice colonised by E. faecalis.ResultsPrimary colon epithelial cells exposed to polarised macrophages or 4-hydroxy-2-nonenal developed CIN and were transformed after 10 weekly treatments. In immunodeficient mice, 8 of 25 transformed clones grew as poorly differentiated carcinomas with 3 tumours invading skin and/or muscle. All tumours stained for cytokeratins confirming their epithelial cell origin. Gene expression profiling of clones showed alterations in 3 to 7 cancer driver genes per clone. Clones also strongly expressed stem/progenitor cell markers Ly6A and Ly6E. Although not differentially expressed in clones, murine allografts positively stained for the tumour stem cell marker doublecortin-like kinase 1. Doublecortin-like kinase 1 and Ly6A/E were expressed by epithelial cells in colon biopsies for areas of inflamed and dysplastic tissue from E. faecalis-colonised IL-10 knockout mice.ConclusionsThese results validate a novel mechanism for CRC that involves endogenous CIN and cellular transformation arising through a microbiome-driven bystander effect.

Abstract-Ever-increasing integrated circuit (IC) power densities and peak temperatures threaten reliability, performance, and economical cooling. To address these challenges, thermal analysis must be embedded within IC synthesis. However, this requires accurate three-dimensional chip-package heat flow analysis. This has typically been based on numerical methods that are too computationally intensive for numerous repeated applications during synthesis or design. Thermal analysis techniques must be both accurate and fast for use in IC synthesis.This article presents a novel, accurate, incremental, spatially and temporally adaptive chip-package thermal analysis technique, called ISAC, for use in IC synthesis and design. It is common for IC temperature variation to strongly depend on position and time. ISAC dynamically adapts spatial and temporal modeling granularity to achieve high efficiency while maintaining accuracy. Both steady-state and dynamic thermal analysis are accelerated by the proposed heterogeneous spatial resolution adaptation and asynchronous thermal element time-marching techniques. Each technique enables orders of magnitude improvement in performance while preserving accuracy when compared with other state-of-the-art adaptive steady-state and dynamic IC thermal analysis techniques. Experimental results indicate that these improvements are sufficient to make accurate dynamic and static thermal analysis practical within the inner loops of IC synthesis algorithms. ISAC has been validated against reliable commercial thermal analysis tools using industrial and academic synthesis test cases and chip designs. It has been implemented as a software package suitable for integration in IC synthesis and design flows and will be publicly released.

Intestinal commensal bacteria have recently been shown to trigger macrophages to produce diffusible clastogens (or chromosome-breaking factors) through a bystander effect (BSE) that mediates DNA damage and induces chromosomal instability in neighboring cells. Colon macrophages appear central to colon carcinogenesis and BSE through the expression of tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2). The former induces netrin-1, a regulator of intestinal epithelial cell apoptosis, and the latter generates trans-4-hydroxy-2-nonenal (4-HNE), an endogenous mutagen. To test whether colon macrophages are key effectors for BSE, we depleted these cells in interleukin-10 knockout mice colonized with Enterococcus faecalis using encapsulated liposomal clodronate (ELC), a bisphosphonate that causes macrophage apoptosis. We observed that E. faecalis polarizes colon macrophages to an M1 phenotype. In addition, depleting these cells suppressed COX-2 and TNF-α, blocked the formation of 4-HNE protein adducts, and inhibited up-regulation of netrin-1-all markers for BSE. Finally, treatment with ELC prevented colitis, β-catenin activation, and cancer formation. These results show that selected human commensals can polarize colon macrophages to the M1 phenotype and, when activated, serve as the key effector for bacterial-induced BSE. Our findings suggest that depleting M1-polarized macro-phages is a mechanism for the chemopreventive activity of bisphosphonates and that it represents a new strategy for preventing colon cancer induced by intestinal commensals.