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A. A. Kramerov, A. V. Ljubimov; Effects of Protein Kinase CK2 Inhibition on Cytoskeleton of Cultured Human Astrocytes and Vascular Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3986.
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We previously showed that protein kinase CK2 co-distributed with GFAP-containing intermediate filaments in human cultured astrocytes and proposed that it may be associated with the cytoskeleton. The purpose was to examine the effect of CK2 inhibition on cytoskeleton organization in cultured astrocytes and retinal endothelial cells.
Human cultured embryonic astrocytes (HAST-40), astrocytic glioblastoma cell line U87MG, human brain microvascular (HBMVEC) and bovine retinal (BREC) endothelial cells were used. Specific CK2 inhibitor, tetrabromobenzatriazole (TBB, Calbiochem), dissolved in DMSO was added at 0.01-0.20 mM to the medium one day after passaging the cells. After 1-3 days of treatment, cells were fixed in 4% p-formaldehyde, permeabilized in 0.1% Triton-X100, blocked in 5% normal goat serum and incubated with mouse anti-CK2 antibody, or rabbit anti-GFAP, followed by fluorochrome-conjugated secondary antibodies.
A specific CK2 inhibitor TBB caused dramatic changes in cell shape and adhesion of the cultured cells. Typically, a transformation of attached cells to cells with fewer shortened processes eventually led to rounding up of cells that became loosely attached to the substratum. The co-localization of CK2 and GFAP was preserved upon treatment by TBB further indicating association of CK2 with the cytoskeleton. TBB-induced alterations were completely reversible, indicating that morphological changes were not caused by apoptosis. However, longer treatment or higher TBB concentration did cause detachment from the substratum and apoptosis, in accordance with well-documented anti-apoptotic role of CK2. TBB-induced changes were dependent on serum concentration. The lower the concentration of serum was, the faster and stronger the effects of TBB on cell shape and adhesion were. However, we detected no significant influence of serum concentration on the minimal effective TBB concentration (0.1 mM). Cultured cells tested differed significantly with respect to the time required for morphological changes to occur (3 to 20 hr), whereas an opposite correlation was observed for the time needed for reversal to normal morphology.
CK2 inhibition in cultured human cells causes dramatic changes in cytoskeleton organization, which affect their adhesive properties and migratory ability. Similar changes in retinal astrocytes and/or vascular endothelial cells may cause the earlier reported anti-angiogenic effect of CK2 inhibition in the mouse model of oxygen-induced retinopathy.
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