Bcl-2 expression in the human cornea

The purpose of this study was to determine the localization of Bcl-2 protei n in the human cornea. Antihuman Bcl-2 monoclonal antibodies (MAbs) against selective Bcl-2 peptide sequences were used to localize Bcl-2 protein immu nocytochemically in fresh eye bank donor human corneas (n = 4). Specificity of each MAb was determined by Western blot analysis of pooled protein extr acted from human corneal epithelium (n = 3). Expression of Bcl-2 protein in apoptotic surface epithelial cells was detected by co-labeling with TUNEL assay and anti-Bcl-2 antibody staining. Two MAbs specific for amino acids r esidues (aa) 41-54 within the loop domain of Bcl-2 protein stained nuclei o f all corneal epithelial cell layers. MAb specific for aa 61-76, also withi n the loop domain, produced faint nuclei and nuclear envelope staining. Occ asional corneal surface epithelial cells however, consistently lacked anti- Bcl-2 nuclear staining with these three MAbs; concomitant TUNEL assay revea led that all TUNEL positive-surface cells were Bcl-2 negative. In the strom a, keratocytes showed similar but weak anti-Bcl-2 staining, All corneal end othelial cells showed intense nuclear staining with MAbs, with no gradient or absence of staining, In summary, Bcl-2 protein can be localized to the n uclei and nuclear envelope of corneal epithelial cells, keratocytes and end othelial cells with the use of MAbs specific for the loop domain of Bcl-2. TUNEL-labeled surface epithelial cells did not stain with MAbs to Bcl-2, su ggesting degradation or epitope masking perhaps by specific phosphorylation of the loop domain during apoptosis, Taken together, these findings sugges t that Bcl-2 protein may play a critical role in modulating apoptotic cell desquamation in the human corneal epithelium. (C) 2001 Academic Press.