◥There is accumulating evidence for a link between circadian clock disruption and cancer progression. In this study, the circadian clock was investigated in cervical and esophageal cancers, to determine whether it is disrupted in these cancer types. Oncomine datamining revealed downregulation of multiple members of the circadian clock gene family in cancer patient tissue compared with matched normal epithelium. Real-time RT-PCR analysis confirmed significant downregulation of CLOCK, PER1, PER2, PER3, CRY1, CRY2, REV-ERBa, and RORa in esophageal tumor tissue. In cell line models, expression of several circadian clock genes was significantly decreased in transformed and cancer cells compared with noncancer controls, and protein levels were dysregulated. These effects were mediated, at least in part, by methylation, where CLOCK, CRY1, and RORa gene promoter regions were found to be methylated in cancer cells. Overexpression of CLOCK and PER2 in cancer cell lines inhibited cell proliferation and activation of RORa and REV-ERBa using agonists resulted in cancer cell death, while having a lesser effect on normal epithelial cells. Despite dysregulated circadian clock gene expression, cervical and esophageal cancer cells maintain functional circadian oscillations after Dexamethasone synchronization, as revealed using real-time bioluminescence imaging, suggesting that their circadian clock mechanisms are intact.Implications: This study is a first to describe dysregulated, yet oscillating, circadian clock gene expression in cervical and esophageal cancer cells, and knowledge of circadian clock functioning in these cancer types has the potential to inform chronotherapy approaches, where the timing of administration of chemotherapy is optimized on the basis of the circadian clock.
<p>CpG island prediction software, MethPrimer (www.urogene.org), identifies CpG islands in the 5' regulatory sequences of circadian clock genes, CLOCK, RORα, CRY1 and PER2. The regions from -2000 to +2000 were analysed, relative to the transcription start site of +1. In the linear scale depicted, 0 refers to 2000 bases upstream and 4000 refers to 2000 bases downstream of the transcription start site.</p>
<p>Relative differences in expression of circadian clock genes between cell lines remains the same at different time points after synchronisation. A. Western blot analysis showing similarly downregulated CLOCK and CRY1 protein levels in HeLa cells compared to ARPE19 cells, as well as upregulated PER2 protein levels, at 0, 12 and 24 hours post-synchronisation with Dexamethasone. Numbers represent quantified band intensities relative to p38, as determined using Image J software. B. Real-time RT-PCR analysis showing similarly downregulated PER2 mRNA expression in SVWI38 cells compared to WI38 cells, at 0, 12 and 24 hours post-synchronisation with Dexamethasone.</p>
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