Purposes: To transform HPV E6/E7 immortalized human oral epithelial cell line HIOEC cells by benzo(a)pyrene B(a)P and tetradecanoyl phorbol acetate (TPA) in vitro, and establish a carcinogenesis model of oral squamous cell carcinoma.
Methods: HIOEC cells were treated with 0.1 microg/ml -1.2 microg/ml B(a)P for 6 months. Some of these cells were treated with 0.1 microg/ml TPA 24 hours at 4th passage and 10th passage, respectively. The cells were cloned at 18th passage, and then were cultured with DMEM medium contain 10% FBS at 21st passage. The cells were cultured in vitro for 1 year and developed into a malignant cell line HIOEC-B(a)P-TPA. The morphological changes of the cells were observed with differential interference contrast microscope and HE staining. The expression of cytokeratin and vimentin was identified with immunohistochemical staining. The soft agar colonies forming ability and tumorigenesity of the cells were identified to confirm the malignant characteristics of HIOEC-B(a)P-TPA cells.
Results: (1) After HIOEC cells were treated with B(a)P plus TPA for 6 months, HIOEC-B(a)P-TPA cells grew well in DMEM medium containing with physical concentration of calcium and 10% FBS. (2) During HIOEC cells were treated with chemical carcinogens, the morphology of the cells was changed. HIOEC-B(a)P-TPA cells showed as fibroblast-like cells with many atypical mitosis. (3) The expression of cytokeratin decreased in the cells while that of vimentin increased in the cells. (4) HIOEC-B(a)P-TPA cells had strong soft agar colony formation ability and the colony formation ratio was 24.5%. (5) HIOEC-B(a)P-TPA cells have no tumorigenisity till now.
Conclusions: We established a biological factors and chemical carcinogens induced malignant cell line-HIOEC-B(a)P-TPA after a long period. It will provide a good multiple factors, multistage carcinogenesis model of OSCC for further research.