title: Immortalization of human corneal epithelial cells using simian virus 40 large T antigen and cell characterization.
abstract: BACKGROUND
  Primary cultures of human corneal epithelial (HCE) cells usually cease to grow after four or five passages. This result in a small cell yield for experiments such as the eye irritancy test represents a serious problem for human and animal corneal epithelial research. In the present study, we established an HCE cell line immortalized by simian virus 40 (SV40), a polyomavirus, and characterized the inherent morphologic and cytologic cell properties.
  
  
  METHODS
  Primary cultured HCE cells were infected with a SV40 large T antigen (SV40 T)-expressing retrovirus, and were selected using G418 solution, an aminoglycoside antibiotic. To ensure that the immortalized cell lines express SV40 T and cytokeratin-3, a corneal epithelial-specific marker, we conducted reverse-transcription (RT)-PCR and Western blot analysis.
  
  
  RESULTS
  These cell lines continued to grow for more than 50 generations, exhibiting a cobble stone-like appearance similar to normal HCE cells and an increased proliferation rate compared to primary cultured HCE cells. RT-PCR results showed that the immortalized cell lines expressed SV40 T while the primary cultured cells did not. In the Western blot assay, protein levels of phosphorylated (Ser15) p53 protein were significantly decreased in the immortalized cell lines while the expression of total p53 protein was constant. In addition, expression of p21(cip1), a cell cycle protein, was down-regulated in the immortalized cells. Moreover, a cornea epithelium-specific marker, cytokeratin-3 (CK-3), was expressed at equal levels in the immortalized cells and primary HCE cells.
  
  
  CONCLUSIONS
  Taken together, these results indicate that immortalized HCE cell lines were successfully established using the SV40-retroviral vector. These cells may be an excellent model for detecting the adverse effects of standard toxic materials and could replace the traditional eye irritation test as an animal-free alternative method.
authors.affiliation: Laboratory of Biochemistry and Immunology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Cho-Won
authors.initials: CW
authors.lastname: Kim
authors.affiliation: Laboratory of Biochemistry and Immunology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Ryeo-Eun
authors.initials: RE
authors.lastname: Go
authors.affiliation: Laboratory of Biochemistry and Immunology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Geum-A
authors.initials: GA
authors.lastname: Lee
authors.affiliation: Department of Dermatology, School of Medicine, Research Institute for Medical Sciences, Chungnam National University, Daejeon, Republic of Korea.
authors.forename: Chang Deok
authors.initials: CD
authors.lastname: Kim
authors.affiliation: College of Pharmacy, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Young-Jin
authors.initials: YJ
authors.lastname: Chun
authors.affiliation: Laboratory of Biochemistry and Immunology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea. Electronic address: kchoi@cbu.ac.kr.
authors.forename: Kyung-Chul
authors.initials: KC
authors.lastname: Choi
mesh_terms: D000957:Antigens, Viral, Tumor / Q000008:administration & dosage / Q000502:physiology*; D002461:Cell Line, Transformed; D049109:Cell Proliferation / Q000502:physiology; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000502:physiology* / Q000821:virology*; D019573:Epithelium, Corneal / Q000166:cytology / Q000502:physiology* / Q000821:virology*; D006801:Humans; D013539:Simian virus 40 / Q000502:physiology*
chemical_list: D000957:Antigens, Viral, Tumor
keywords: Corneal epithelial cells; Immortalization; Retroviral vector; SV40 large T antigen