Establishment and characterization of spontaneously immortalized human oral keratinocytes

Objectives: To clarify the biological characteristics of oral carcinogenesis, particularly immortalization process, and to use for the development of a diagnostic kit for the prediction of oral cancer outbreak, spontaneously immortalized human oral keratinocytes (sp-HOK) were established from primary human oral keratinocytes that were cultured from human gingival specimens by serial subculture to the postmitotic stage.

Methods: The in vitro growth characteristics, epithelial origin, in vivo tumorigenicity, the frequency of 'high-risk' human papillomavirus (HPV) infections, and the status of tumor suppressor genes and DNA mismatch repair genes were investigated in the cell line.

Results: Sp-HOK was established from human gingival specimen that originated from the mandibular tooth. The spontaneously immortalized line grew as monolayer and showed (i) 34.2 h of doubling time; (ii) epithelial originality by the presence of desmosome and keratin; and (iii) non-tumorigenic potential in nude mice. This cell line did not contain HPV DNA and not express viral genes. Western blot analysis revealed overexpression of p53 and Rb proteins and decreased expression of p21WAF1/Cip1 in spontaneously immortalized sp-HOK line versus normal human oral keratinocytes. In sp-HOK line and its corresponding gingival tissue, mutations were not identified in highly conserved functional regions of the p53 gene. DCC, a tumor suppressor gene, was not detected from this cell line. The sp-HOK line showed a loss of hMSH2 expression.

Conclusions: To our knowledge, this is the first report demonstrating that the sp-HOK line is a novel cell line, which is spontaneously immortalized from human oral keratinocytes. This well-characterized human oral cell line should serve as useful tool for understanding the biological characteristics of oral carcinogenesis, particularly immortalization process.