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Danielle M. Robertson, Su-Inn Ho, H. Dwight Cavanagh; C-Terminal Cleavage of ΔNp63α Is Associated with TSA-Induced Apoptosis in Immortalized Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(8):3977-3985. doi: 10.1167/iovs.09-4919.
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In the central human corneal epithelium, loss of ΔNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for ΔNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for ΔNp63 isoforms in caspase-mediated apoptosis in a telomerase-immortalized corneal epithelial cell line.
For in vitro studies, hTCEpi cells were cultured in KGM-2 serum-free culture media containing 0.15 mM calcium. To assess dynamic protein interactions among individual ΔNp63 isoforms, ΔNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 μM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous ΔNp63α, ΔNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA.
Transient expression of ΔNp63-EGFP α and β isoforms resulted in the formation of a smaller isoform similar in size to ΔNp63γ-EGFP. FRAP demonstrated that ΔNp63α-EGFP has greater immobile fraction than β or γ. TSA induced caspase-mediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous ΔNp63α and p53. TSA upregulated ΔNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of ΔNp63α-EGFP. siRNA knockdown of ΔNp63α correlated with a reduction in p53 independently of TSA.
ΔNp63α is the dominant active isoform in corneal epithelial cell nuclei. Loss of ΔNp63α occurs during apoptotic signaling by cleavage at the C terminus. The corresponding loss of p53 suggests that a significant relationship appears to exist between these two regulatory proteins.
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