BCL–2 alpha in Human Telomerized Corneal Epithelial Cells

Abstract

Abstract: : Purpose: In the human corneal epithelium, the proto–oncogene BCL–2 exhibits a gradient pattern of expression decreasing from limbus to central cornea and basal to superficial layer, with a loss of expression in surface epithelial cells prior to desquamation. The purpose of this experiment is to examine the expression of BCL–2 in a normally differentiating corneal epithelial cell line to validate this cell line as a viable model for studying surface cell shedding in vitro. Methods: Human Telomerized Corneal Epithelial (hTCEpi) cells immortalized with human telomerase reverse transcriptase were grown on collagen coated culture inserts (Corning) submerged in KGM–2 culture medium (Clonetics) containing 1.15 mM calcium for 7 days. Cells were then air–lifted to induce differentiation and examined at day 0, 7 and 10. Western Blotting using an anti–Keratin K3 antibody (Biogenesis) was used to assess epithelial differentiation. Levels of Bcl–2 expression were determined using an anti–BCL–2 monoclonal antibody (Ancell). RT PCR to generate a 128 bp fragment crossing the intron/exon border of BCL–2 was used to confirm the protein was splice variant alpha. Results: Consistent with previously reported findings, following 7 days of air–lifted culture, hTCEpi constructs differentiate in vitro similar to the human cornea in vivo demonstrated by K3 expression. Western Blotting confirmed that the 26 kD BCL–2 protein is expressed at all time points. Primers specific for splice variant alpha confirmed that the 26 kD protein was BCL–2 alpha. Conclusions:DISCUSSION: These data suggest that hTCEpi cells express the full length BCL–2 transcript (splice variant alpha) containing four conserved homology domains, a regulatory loop, and a transmembrane domain; and that the BCL –2 protein is expressed in hTCEpi cell constructs at all stages of differentiation. These findings are in agreement with previously reported immunohistochemistry demonstrating BCL–2 in both the normal human cornea and our hTCEpi cell line. Taken together, these findings demonstrate a normal pattern of BCL–2 gene expression in the hTCEpi cell line, validating it as viable model for studying surface cell shedding in vitro.

Keywords: cornea: epithelium • apoptosis/cell death • gene/expression