[Biochemical characterization of phenytoin-induced hyperplastic human gingival fibroblasts. Non-collagenous proteins...

Phenytoin (PHT), administered as an anticonvulsant, has a side effect gingiva overgrowth in approximately 50% of patients. The present study was attempted to explore the biochemical mechanism on non-collagenous protein biosynthesis as affected by PHT. Responder cells (RES A3, RES C2) of a patient with gingival overgrowth were obtained by the method of Kawase et al. Normal human gingival fibroblasts (Gin-1), purchased from ATCC, were also used. All cells were inoculated at 1 x 10(4) cells/cm2 (12 multi-well plate or 60 mm tissue culture dish), and then cultured for 4, 8 and 12 days with or without PHT (5 micrograms/ml). Prior to harvesting at the indicated times, cells were incubated with 14C-amino acids (1.25 microCi/ml) for 24 hours. The 14C-labeled proteins were isolated from the cell layers including extracellular matrix, following Kurkinen et al. with a minor change. Each 14C-labeled fraction was dissolved in 3 ml of Aquasol-2 and the radioactivity by a liquid scintillation counter. The DNA content of cell layers affected by PHT was increased on Gin-1, RES A3 and RES C2 at the post-confluence, resulting also in an increase in cell number. Two morphologically different phenotypes of responder cells were observed, differing in nuclear and cell sizes. At 12 days culture, RES A3, were stimulated by PHT, showed increased synthesis of both total extractable proteins (EP) and binding proteins (BP) labeled with 14C-amino acids. Therefore, at least two distinct phenotypic responder cells are present in the PHT-induced overgrowth gingiva, alter the synthesis of non-collagenous proteins.