Establishment and characterization of cultured human gingival keratinocytes immortalized by Transfection of origin (−) SV40 DNA and c-fos gene

Abstract

In order to establish human oral epithelial cell lines, secondary cultures of human oral keratinocytes from normal gingival tissues obtained from 20–29-yr-old females were transfected with origin (−) SV40 DNA (pRNS-1) and human c-fos gene (pBK 28) using Transfectam reagent® (Promega, WI). Out of 20 cultures transfected with pRNS-1, only one culture escaped senescence. After the transfection with origin(−) SV40 (87 days in culture), human c-fos (pBK 28) was introduced into ori− cells. Ori− cells and ori− cells transfected with c-fos, designated NDUSD-1 cells, were subjected to G418 selection (25μg/ml) for 125 days of culture. NDUSD-1 cells were immortal with continious growth for more than 1000 days in culture. Southern blotting analysis demonstrated that both transfected DNAs were integrated into cellular DNA of the NDUSD-1 cells. Ori− and NDUSD-1 cells exhibited persistent production of large T antigen at similar levels to WI38VA13 cells, a SV40 virus transformed cell line, and production of keratin with molecular weights of 64 and 68kD. Chromosomal analysis demonstrated that the number of chromosomes of NDUSD-1 cells (passage 25, 257 days in culture) was mainly distributed in the hypodiploid range (42–46) with a modal number of 44 (63%). NDUSD-1 cells (passage 28, 281 days in culture) were nontumorigenic in nude mice.