June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Synchronization of the Clock in RPE Cells Promotes Reentry into the Cell Cycle
Author Affiliations & Notes
  • David Ramsey
    Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, MA
  • Kathryn Ramsey
    Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL
  • Demetrios Vavvas
    Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, MA
  • Footnotes
    Commercial Relationships David Ramsey, None; Kathryn Ramsey, None; Demetrios Vavvas, MEEI (P), Kala pharmaceuticals (C), Roche (C), Genentech (C)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1161. doi:
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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)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 701 retinal pigment epithelium • 458 circadian rhythms • 654 proliferation  
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