Sequential combined tumorigenic effect of HPV-16 and chemical carcinogens

We immortalized oral keratinocytes by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA and established two cell lines, human oral keratinocytes-16A (HOK-16A) and -16B (HOK-16B). These cell lines were morphologically different from the normal counterpart, contained HPV-16 DNA as integrated form and expressed numerous viral genes. However, these cells proliferated only in culture medium containing low calcium (0.15 mM) and are not tumorigenic in nude mice. To test the hypothesis that tumors can be developed by sequential combined effect of human papillomavirus and chemical carcinogens in the oral cavity, these immortalized cell lines were chemically transformed by exposure to either benzo[a]pyrene or methanesulfonic acid ethyl ester. Such transformants proliferated in medium containing physiological calcium levels (1.5 mM) and demonstrated enhanced growth potential in nude mice, whereas primary human oral keratinocytes treated with these chemical carcinogens failed to show any evidence of transformation. Chemically transformed cells contained integrated, intact HPV-16 sequences and transcribed significantly higher amount of HPV-16 E6/E7 messages and transforming growth factor-alpha (TGF-alpha) compared with the immortalized oral keratinocytes. Like the HPV-immortalized cell lines, the chemically transformed oral keratinocytes contained lower levels of newly synthesized, wild-type p53 proteins compared to normal cells, and expressed wild-type c-Ha-ras. These results indicate that this in vitro system is useful for investigating the mechanisms of multistep oral carcinogenesis.