Mammalian corneal endothelial cells are considered to be non-proliferative due to the arrest of cells at the G1 phase of the cell cycle. The purpose of this study was to determine whether the down regulation of cyclin dependant kinase inhibitors (p21cip1 and p27kip1) levels by Endothelin-1 (ET-1), would overcome the G1 phase arrest and promote cell cycle progression and proliferation in cultured BCECs (Bovine corneal endothelial cells). BCECs were isolated from bovine corneas and cultured in DMEM supplemented with 10% serum. 5-Bromo 2-deoxy Uridine (BrdU) incorporation was determined in serum starved cultures in 24-well plates as a measure of cell proliferation. Confluent serum starved cells grown in T-25 flasks were treated with 100nM Endothelin-1 in DMEM. The control cells were left untreated in serum free medium. Total cellular protein was isolated using RIPA buffer and quantified according to the Peterson modification of the Lowry method. The level of expression of p21cip1 and p27kip1 proteins relative to β-actin was determined by western blotting technique. Immuno fluorescent localization of p27kip1 was performed using polyclonal anti-p27kip1 and anti-p21cip1 antibodies in confluent and growing cells. An increase in cell proliferation was observed in sub-confluent cultures with Endothelin-1 treatment. This evidence was supported by an increase (~18%) in BrdU incorporation in response to Endothelin-1. Densitometry analysis of immunoblots revealed an increase in the expression of p27kip1 in confluent cell cultures when compared to sub-confluent, dividing cells. p21cip1 was almost undetectable in sub-confluent, actively dividing cultures. Immuno fluorescent analysis revealed that the nuclear staining of p27kip1 was apparently decreased with ET-1 treatment. In conclusion, Endothelin-1 treatment resulted in decrease in p27kip1 and p21cip1 expression in confluent cultures that was greatest at 30 hr of post incubation with Endothelin-1. Endothelin-1 appears to promote cell proliferation. Expression of p27kip1 and p21cip1 was greatly reduced in actively dividing BCECs. Endothelin-1 treatment down-regulated these cyclin dependent kinase inhibitors and may promote cell cycle progression via this mechanism.
| Identifer | oai:union.ndltd.org:WKU/oai:digitalcommons.wku.edu:theses-1112 |
| Date | 01 July 2009 |
| Creators | Bollu, Lakshmi Reddy |
| Publisher | TopSCHOLAR® |
| Source Sets | Western Kentucky University Theses |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses & Specialist Projects |
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