Abstract
Purpose: :
To show that telomerase transformed corneal epithelial cells, that have not been cultured on 3T3 feeder layer, before or during transfection, are a good model for studies of cellular differentiation and DNA repair in vitro.Introduction: Primary human corneal epithelial cells (CECs) are a heterogenous mixture of cells with varying proliferation potential and at different stages of differentiation. In vitro the cultures have a short proliferative life span and high propensity to differentiate. It is therefore necessary to establish a cellular model that has very similar characteristics to the primary cells. Most of the current cell lines, with potentially useful life spans such as E6/E7 or SV-40 transformed cells, have inactive Rb and/or p53 proteins, and are heteroploid in nature. We have examined our hTERT immortalized human corneal epithelial cells (TCEC for clonal growth, expression of cell cycle proteins, telomere associated proteins, and differentiation markers are not tumorogenic and DNA repair ability.
Methods: :
Indirect immunofluorescence and western blot analysis were used to determine expression of cell cylcle proteins (p53, Rb, p63) and cytokeratins, telomere associated proteins (Tin 2 ,TRF-1, and Tankyrase), involucrin and cytokeratin (AE5). DNA damage response was studied over time in the presence of adriamycin (0.2µg/ml). Anchorage dependent growth assay and colony forming assay were used to determine tumorogenicity. Karyotyping was done to determine the ploidy.
Results: :
Cell cycle protein expression (p16, p21, p53 and pRb) was similar to that in CECs. The TCECs also had a similar cytokeratin expression profile to that of CECs, and increased involucrin expression in response to serum and elevated calcium. These cells had low expression of Tin 2 and TRF-1 and high expression of tankyrase. The TCECs have a population doubling time of 36 hrs, do not form colonies in an anchorage independent culture, and are diploid in chromosomal content. In response to DNA damage there is a time dependent increase in p53 and p21 expression.
Conclusions: :
TCECs do not have compromised cell cycle protein expression and are not tumorogenic and are diploid. They differentiate in response to serum and elevated calcium. TCECs express low levels of negative regulators of telomere elongation (Tin 2 and TRF-1), high level of the positive regulator (tankyrase) and are capable of DNA repair. Collectively these properties support the view that TCECs are a useful cellular model for a variety of studies related to human corneal epithelial proliferation differentiation and repair .
Keywords: cornea: basic science • cell survival • gene/expression