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U.P. Andley, F. Bai, J. Xi, R. Higashikubo; The Expression of A and B-Crystallins in Synchronized Primary Mouse Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):953.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To define the expression of αA- and αB-crystallin with change in the cell cycle phase of lens epithelial cells, and determine whether the expression of the crystallins alters the cell cycle parameters. Methods: αA-/-, αB-/- and αA/αB-/- mice were kindly provided by Dr. Eric Wawrousek, National Eye Institute. Primary cultures of mouse lens epithelial cells were derived from capsule-epithelial explants of wild type (129Sv), αA-/-, αB-/- and αA/αB-/- lenses. Cells were synchronized at the G0/G1 boundary by incubation in 0.5% fetal bovine serum (FBS)-containing medium. Cells were released from the cell cycle block by the addition of 20% serum-containing medium. Every 2 hours after the release from serum-starvation, cultures were pulsed with BrdU, and labeled with propidium iodide. BrdU labeled cells were detected by immunofluorescence. Dual parameter flow cytometric analysis (FACS) was performed to determine the distribution of cells in different cell cycle phases. The cell cycle-dependent expression of αA and αB, and of tubulin in synchronized cells was analyzed by immunofluorescence and immunoblotting, using specific antibodies. Results: Incubation of primary lens epithelial cells in 0.5% serum for 72 hours synchronized the cells (>95%) at the G0/G1border. Wild type cells entered the S phase 14 hours after release from serum starvation. The fraction of cells in the S phase was 0.1 at 14 hours, increased to 0.4-0.5 after 18 hours, and declined after reaching a maximum at 20 hours. This indicates that the S phase in these cells lasts for 6 hours. αA and αB expression increased three-fold as cells entered the S phase, and declined after the cells went through mitosis. αA-/- cells also entered the S phase after release from the cell cycle block at a time comparable to the wild type cells. Immunofluorescence and confocal microscopy using antibodies for αA and ß-tubulin showed that αA was strongly co-localized with the intercellular bridge microtubules during cytokinesis. The mitotic spindle was not properly organized in a significant proportion of the αA-/- and αB-/- cells. Conclusions: Studies on synchronized cells indicate that a major increase in the expression of αA- and αB-crystallin accompanies the entry of cells into the S phase, and their levels decline after the completion of the cell cycle. αA- and αB-crystallins may interact with the microtubule cytoskeleton, and mediate the proper organization of the mitotic spindle. Deletion of the αA-crystallin gene did not affect the rate at which cells enter the S phase, consistent with our previous, in vivo studies.
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