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David Ramsey, Kathryn Ramsey, Demetrios Vavvas; Synchronization of the Clock in RPE Cells Promotes Reentry into the Cell Cycle. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1161.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal pigment epithelium (RPE) plays an essential role in the daily function and maintenance of the adjacent neural retina. The RPE is made up of a cuboidal epithelium of quiescent cells resting in the G0-phase of the cell cycle. A number of ocular pathologies result from disordered proliferation of RPE cells, such as proliferative vitreoretinopathy (PVR), a major cause of surgical failure in retinal detachment repair. ARPE-19 cells, a spontaneously arising human RPE cell line, manifest contact inhibition and form epithelial-like monolayers in culture that recapitulate the quiescent state of the RPE. In the present study, we examine the role of the core clock on cell cycle regulation in ARPE-19 cells using forskolin shock as a means of synchronizing the endogenous circadian oscillator.
ARPE-19 cells were cultured in 1:1 of Dulbecco’s Modified Eagle’s Medium/F12 (DMEM/F12) with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Forskolin shock was performed in fully confluent ARPE-19 cells by changing the medium to DMEM/F12 supplemented with 10 µM forskolin for 30 minutes, followed by replacement with regular medium. Beginning 16 hours later, cells were washed with ice-cold PBS and harvested in 1 ml of TRIzol reagent at 4 hour intervals over the course of 72 hours. These samples were frozen and stored at −70°C until the extraction of whole cell RNA. Transcript levels were determined by real-time PCR.
After approximately 4 days in culture, ARPE-19 cells reach confluence and manifest contact inhibition. Forskolin synchronization of ARPE-19 cells leads to oscillations in the expression of the core clock genes Clock, Bmal1, and Per2. Forskolin shock of ARPE-19 cells also results in oscillation in the expression of several molecular components of the cell cycle network including Cyclin D1 (CCND1), CCND2, and CCNE1.
Following synchronization of the endogenous circadian oscillator, several molecular components of the cell cycle network are upregulated in contact-inhibited RPE cells. The finding that CCND1, a known clock target, and related cell cycle genes required for the G1/S transition are upregulated suggest that a proliferative phenotype might result from nonspecific stimulation of the circadian oscillator.
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