Isolation and Characterization of Human Conjunctival Cell Lines From Hereditary Benign Intraepithelial Dyskeratosis Lesions

Abstract

Abstract: : Purpose: Hereditary benign intraepithelial dyskeratosis (HBID) is an autosomal dominant cell proliferation disorder characterized by opaque epithelial plaques of the conjunctiva and oral mucosa. Found primarily among a Native American tribe in North Carolina and their relatives, HBID plaques appear at birth or in early childhood. Involvement typically includes the conjunctiva, cornea, and buccal mucosa, and loss of vision secondary to corneal involvement can occur. This disorder has been linked to chromosome 4q35 and a near–telomeric DNA duplication in this region (Allingham RR, et al. Am J Hum Genet 2001). In order to examine cell proliferation differences, gene expression and the underlying chromosomal duplication associated with this disease, we established and characterized cell lines from HBID lesions and normal conjunctival tissue. Methods: HBID lesion material and normal conjunctival tissue were obtained and cultured as described in Risse Marsh et al. (Exp Eye Res 2002). Cell morphology and growth characteristics including growth rate, population doublings and passages until senescence were compared. Total RNA was extracted from all cell lines, and first strand cDNA synthesis was performed on these RNAs. Polymerase chain reaction (PCR) using published mucin primers and designed keratin primers were performed. In addition, immunocytochemical characterization of cell lines was performed using primary antibodies to various mucins and keratins. Results: Two HBID lesion cell lines (HCj1 and HCj2) and two control conjunctival cell lines have been established. Morphologically, the two control lines (CCj1 and CCj2) and HCj1 were similar. The second HBID cell line (HCj2) displayed morphology similar to fibroblasts. In addition, HCj2 demonstrated growth characteristics not similar to any of the other cell lines. The HCj2 cell line did express genes associated with epithelial cells, such as mucin 1 and keratin 19; however this cell line also demonstrated message and protein for genes typically expressed by fibroblasts. Conclusions: We have established multiple cell lines from normal conjunctival tissue and HBID lesion material, and have characterized them based on growth characteristics and gene expression. These characterized cell lines will allow us to perform candidate gene transfection and expression studies, and will aid us in characterizing the chromosomal duplication present in HBID patients.

Keywords: conjunctiva • genetics • gene/expression