The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Commemorative Booklet for CAAT 20th Anniversary Symposium

Development of HPV16-E6/E7 Transduced Human Corneal Epithelial Cell Line: Tight Regulation of Proliferation

Rajiv R. Mohan, Daniel E. Possin, Rahul R. Mohan, and Steven E. Wilson
University of Washington, Seattle

The objective of this study was to develop a tetracycline-responsive HPV16-E6/E7 transduced human corneal epithelial (HCE) cell line having tightly regulated proliferation and normal differentiation.

The retroviral pRevTet-On system was used to generate HPV16-E6/E7-transduced HCE clones. Expression of E6/E7 mRNA and HPV16-E7 protein in selected cell strains in +/- doxycycline (Dox) was examined with RNase protection assay and immunoprecipitation (IP) followed by western blot (WB) analysis, respectively. Expression of HCE cell specific marker, keratin K-3, was tested by IP+WB in +/- Dox in medium. The cell morphology of generated clones in serum-free and serum-containing medium on plates coated with/without collagen type-I, cell proliferation in response to growth factors (hepatocyte growth factor, epidermal growth factor) and cell differentiation at varied ionic calcium concentration in medium in +/- of Dox was observed by phage-contrast microscopy. Light (LM) and Transmission electron microscopy (TEM) were utilized to evaluate cell stratification and ultrastructural features of the clones grown at the air-liquid interface on the collagen-membrane.

Two clones having tight regulation of E6/E7 were identified. The clones expressed HPV16-E6/E7 mRNA and HPV16-E7 protein only in presence of Dox. The selected clones continued to proliferate for a maximum of 11 population doublings (PD) in the presence of Dox (1.0 µg/ml) and < 1 PD on withdrawal of Dox. The newly generated clones exhibited typical HCE morphology. The clones cultured on plates coated with collagen type-1 showed extended life span, better retention of epithelial morphology even at higher PD (=9), and lower population doubling time (30-36 hrs) in serum-free medium compared to serum-containing medium. The growth factors showed dose response effects on cell proliferation in +/- Dox. Ionic calcium concentration in medium altered growth and differentiation of tested cells.

The generated cell line mimics the in vivo HCE model and should provide an excellent in vitro model for toxicity testing and studying normal functions and genes expression in HCE.