The proper timing of cell division is a major factor contributing to the regulation of normal growth and emerges as a fundamental process in the development of most cancers. The existence in most, if not all, living organisms of many behavioral and physiological events that occur at well-defined and controlled times argues for the presence of time-keeping systems that allow them to adapt to cyclic changes in environmental conditions. Disruption of circadian rhythm has recently emerged as a new potential risk factor in the development of cancer, pointing to the core gene period 2 (per2) as a tumor suppressor. However, it remains unclear how the circadian network regulates tumor suppression. In the present study, we use cDNA microarray analysis to identify Per2 target genes following siRNA knock-down expression of Per2 in mouse hepatocytes AML12 cells. We identified cyclin B1, a key G2/M regulator of the cell cycle, as a downstream target gene that is negatively regulated by Per2 under normal conditions. Quantitative real-time PCR confirmed our observations by showing cyclin B1 expression is enhanced under Per2 knock-down condition. Accordingly, Bmal1 expression, a target gene of Per2, was specifically augmented under Per2 over-expression conditions. Further experiments focused in characterizing the regulation of the human cyclin B1 promoter by Per2 using luciferase reporter assays. Results show cyclin B1 reporter activity is enhanced in siPer2 transfected cells compared to control scrambled siRNA. Promoter analysis defined the region comprising −484 to −284 of human cyclin B1 promoter containing response elements for gene trans-activation while the region between −284-1 is mainly responsible for Per2 regulation. Our results show the canonical E-box between −284-1 is not required for Per2 regulation of cyclin B1, suggesting non-canonical E-boxes might be directly responsible for Per2-mediated expression. FACS analysis revealed that siPer2 treated cells exhibit a reduced G2/M phase with correspondingly higher cyclin B1 protein levels and reduced Tyr-15-cdc2 phosphorylation compared to scrambled siRNA treated cells. Our previous results show Per2 stability is directly controlled by the endogenous levels of heme whose endogenous levels oscillate in a 24 h cycle. Here, we evaluated whether cyclin B1 expression is altered in heme-treated cells. In contrast to its up-regulation in siPer2 treated cells, luciferase reporter assay showed a decrease in cyclin B1 promoter activity in response to heme treatment. These results may reflect a complex pattern of regulation in which Per2 acts as a sensor molecule to monitor changes in both environmental and cellular conditions that will impact cell progression.

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 1070.