Retroviral transduction of human periodontal cells with a temperature-sensitive SV40 large T antigen

The periodontal ligament (PDL) is considered to contain subpopulations of c ells responsible for the development, repair and regeneration of the period ontium. Cell cultures have been used as model systems in order to understan d the complex cellular and biochemical events underlying these processes, I n order to obtain long-term cultures of these cells that can be cloned and characterized, primary cultures of PDL and gingival cells were infected wit h an amphotropic retroviral construct encoding a temperature-sensitive SV40 large T antigen (tsT). After selection for drug resistance, the cells expr essed the T antigen and proliferated at 34 degrees C for more than 40 passa ges. However, when the T antigen was inactivated by incubation at 39 degree s C, the cultures became growth-arrested and the granularity of the cells i ncreased, possibly as a result of differentiation. Reverse transcribed-poly merase chain reaction and flow cytometry showed that the tsT-transduced cel ls expressed a number of soft and hard connective-tissue antigens, includin g osteocalcin, osteonectin, osteopontin, collagen type I and alkaline phosp hatase. Moreover, incubation of the transduced PDL cells at 39 degrees C wa s found to upregulate the expression of osteocalcin, osteopontin and collag en type I, but downregulate osteonectin. At this temperature, the presence of the dexamethasone downregulated type I collagen, while vitamin D-3 had n o effect on the expression of any of the antigens examined. Under all cultu re conditions, antigen expression was far higher in the transduced PDL cell s than the gingival cells. The findings thus show that growth of the tsT-tr ansduced PDL and gingival cells is temperature-dependent and that the prese nce of the T antigen increases their lifespan but does not ablate the expre ssion of certain of their characteristic phenotypic and functional features . (C) 1999 Elsevier Science Ltd. All rights reserved.