A comparative study of telomerase activity and malignant phenotype in multistage carcinogenesis of esophageal epithelial cells induced by human papillomavirus

To examine certain characteristics of multistep carcinogenesis, we studied telomerase activity and malignant phenotypes in the immortal, premalignant and malignant stages of esophageal epithelial cells induced by HPV. An immo rtalized human fetal esophageal epithelial cell line (SHEE) was induced by E6E7 genes of human papillomavirus (HPV) type 18. Cells in the 10th passage , (SHEE10), 31st passage (SHEE31), 61st passage (SHEE61) and SHEE61A which were selected and expanded from anchorage-independent growth colonies of SH EE61, were examined as follows: cell morphology by electron-microscopy; the cell cycle by flow cytometry, telomerase activity by TRAP assay, tumorigen ic detection including anchorage-independent growth by soft agar culture an d tumor formation by inoculating, cells into SCID and nude mice, and detect ion of HPV IS E6E7 oncoprotein by Western blot. The morphology of the SHEE1 0 cells exhibited good differentiation, the SHEE60 and SHEE61A cells were r elatively poorly differentiated, and the SHEE31 cells were differentiated i n two distinct ways. The telomerase was activated in SHEE31, SHEE61 and SHE E61A, but not in SHEE10 cells. SHEE61 and SHEE61A cells were weakened in co ntact-inhibition and increased in anchorage-independent growth. Inoculated into SCID and nude mice, the cells of the earlier two passages could not de velop tumors; the SHEE61 developed one tumor in four SCID mice, but not in nude mice, and the SHEE61A cells developed tumors in both strains of immuno deficient mice. HPV 18 E6E7 DNA detection by Western blotting was positive in all cell passages. In the process of carcinogenesis by HPV, the cells of SHEE31 are in an immortalized state with telomerase activity. The fact tha t SHEE61 cells remained immortalized and also demonstrated anchorage-indepe ndent growth, reveals premalignant character; the cells of SHEE61A exhibite d malignant transformation with tumor formation in mice. The results reveal ed that the telomerase activity, anchorage-independent growth and tumor for mation in nude mice are the indicators for immortalization, premalignancy a nd malignancy, respectively.