Establishment of human cementifying fibroma cell lines by transfection with temperature-sensitive simian virus-40 T-antigen gene and hTERT gene

Abstract

Human cementifying fibroma (HCF) is a benign fibro-osseous neoplasm of periodontal ligament (PDL) origin containing varying amounts of mineralized material resembling cementum. In the present study, we established cell lines from HCF, which were detected in the mandible of a 54-year-old Japanese man. To obtain immortalized cell clones, we undertook transfection with temperature-sensitive simian virus-40 (SV40) T-antigen and hTERT into HCF cells. Cells transfected with SV40 T-antigen entered “crisis” state between passages 22 and 35, but activation of telomerase by transfection with hTERT in the SV40-transformed HCF cells resulted in bypass of the crisis and maintenance over passage 200. HCF cell lines decreased the expression of SV40 T-antigen and the activity of cell proliferation at a nonpermissive temperature (39°C) in comparison with that at a permissive temperature (33°C). High activities of alkaline phosphatase and mineralization and the expression of type I collagen, osteocalcin, osteopontin, and bone sialoprotein by reverse transcription-polymerase chain reaction (RT-PCR) were observed in HCF cells at 39°C. Overall, these findings suggest that: (i) HCF cell lines may represent a novel in vitro human cell model for the study of the regulatory mechanism of differentiation and proliferation of the human PDL; and (ii) transfection of plasmids encoding the temperature-sensitive SV40 T-antigen gene and hTERT gene may be useful for obtaining immortalized cell lines from benign human tumor and, probably, nonneoplastic human tissues.