Combined oral carcinogenicity of HPV-16 and benzo(a)pyrene: an in vitro multistep carcinogenesis model.

We previously immortalized normal human oral keratinocytes by transfection with recombinant HPV-16 DNA and subsequently exposed the cells to benzo(a)pyrene for 7 days. The exposure to benzo(a)pyrene modified the immortalized cells: the modified cells (HOK-16B-BaP) proliferated in an ordinary culture medium containing physiological calcium level (1.5 mM), but demonstrated only enhanced proliferation capacity without tumor formation in nude mice and failed to show in vitro anchorage-independency. In this study, we further modified the HOK-16B-BaP cells by subculturing the cells in a medium containing benzo(a)pyrene for 6 months. The cells were further modified with a chronic benzo(a)pyrene exposure and were termed HOK-16B-BaP-T cells (1) demonstrated a malignant phenotype in organotypic 'raft' culture, (2) showed in vitro anchorage-independency, (3) developed tumors in nude mice when injected subcutaneously, (4) contained a significantly higher copy number of intact and integrated HPV-16 DNA; (5) contained higher level of HPV-16 E6/E7 messages and E7 protein, (6) were more resistant to transforming growth factor-beta 1 and (7) secreted higher level of vascular endothelial growth factor with molecular weight of 56 kd than parental HOK-16B-BaP cells. However, the levels of p53 and ras proteins and the levels of p53, c-myc and c-fos transcripts in the HOK-16B-BaP-T cells were not different from those in the HOK-16B-BaP cells. The highly conserved coding regions of the p53, c-Ha-ras1, and c-Ki-ras2 genes of the tumor cells were not mutated. These data indicate that the HPV-immortalized human oral keratinocytes can convert to tumorigenic cells by chronic exposure to benzo(a)pyrene. The tumorigenic conversion seems to be associated with (1) the overexpression of viral oncogenes such as E6 and E7 genes, (2) the higher resistance of cells to transforming growth factor-beta 1 and (3) the high secretion of 56 kd vascular endothelial growth factor from the cells.