PERK promotes cancer cell proliferation and tumor growth by limiting oxidative DNA damage.

To proliferate and expand in an environment with limited nutrients, cancer cells co-opt cellular regulatory pathways that facilitate adaptation and thereby maintain tumor growth and survival potential. The endoplasmic reticulum (ER) is uniquely positioned to sense nutrient deprivation stress and subsequently engage signaling pathways that promote adaptive strategies. As such, components of the ER stress-signaling pathway represent potential antineoplastic targets. However, recent investigations into the role of the ER resident protein kinase, RNA-dependent protein kinase (PKR)-like ER kinase (PERK) have paradoxically suggested both pro- and anti-tumorigenic properties. We have used animal models of mammary carcinoma to interrogate the contribution of PERK in the neoplastic process. The ablation of PERK in tumor cells resulted in impaired regeneration of intracellular antioxidants and accumulation of reactive oxygen species triggering oxidative DNA damage. Ultimately, PERK deficiency impeded progression through the cell cycle because of the activation of the DNA damage checkpoint. Our data reveal that PERK-dependent signaling is used during both tumor initiation and expansion to maintain redox homeostasis, thereby facilitating tumor growth.

Validation for high-throughput screening of a VanRS-based reporter gene assay for bacterial cell wall inhibitors.

AIMS: The present study was undertaken to validate, for antibiotic discovery, a reporter gene assay based on a Bacillus subtilis strain expressing the Enterococcusfaecium vanRS genes and a vanH-lacZ fusion, which produced beta-galactosidase activity in the presence of cell wall inhibitors (CWI) and lysozyme. METHODS AND RESULTS: The reporter assay was miniaturized, automated and validated with antibiotics and tested against portions of chemical and microbial extract libraries. The assay is simple, fast and reproducible and can detect all CWI, sometimes at concentrations lower than those necessary to inhibit bacterial growth. However, some membrane-interfering compounds also generate comparable signals. While most CWI elicit a signal that is transcription-dependent and abolished in an osmoprotective medium, transcription is not required for beta-galactosidase activity brought about by the membrane-interfering compounds. CONCLUSIONS: At least two distinct mechanisms appear to lead to enzymatic activity in the reporter strain. Effective counterscreens can be designed to discard the undesired classes of compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: Extensive validation is required before introducing a reporter assay in high-throughput screening. However, the ease of operation and manipulation makes the reporter assays powerful tools for antibiotic discovery.