Phenytoin is a frequently prescribed drug for the treatment of seizure disorders. Unfortunately it has many adverse side effects, including Phenytoin-induced gingival overgrowth. Studies of fibroblasts grown in culture have shown Phenytoin's effect of increased cell proliferation and protein synthesis. Recent research has shown the presence of functionally heterogeneous and phenotypically stable subpopulations of human gingival fibroblasts. The subpopulations vary greatly in their cell proliferation potential and their protein and collagen synthesizing ability. It is suggested that Phenytoin stimulates a subpopulation of cells that are present in normal gingival tissue in a certain percentage and by stimulating rapid growth rates in this subpopulation, Phenytoin alters the composition of the various subpopulations to produce the overgrowth. It is possible that the pathogenesis of many other diseases of connective tissue occurs by a similar mechanism. One of the major aims of the proposed research is to isolate the responder subpopulation of the human gingival fibroblasts grown in tissue culture by the use of a Fluorescence Activated Cell Sorter (FACS). The FACS is a sophisticated flow cytometer with computer capabilities that individually analyzes each cell for the properties of fluorescence and light scatter. The FACS has the ability to viably sort cells based on a variety of parameters chosen by the operator. Preliminary studies have suggested a parameter that may be used as the basis for sorting the Phenytoin-responsive and nonresponsive subpopulations on the FACS. Since the sorting can be done on viable cells, the subpopulations can be continued in culture and re-examined by the FACS to confirm the isolation of subpopulations. The isolated responder and nonresponder subpopulations will be continued in culture, characterized by their cell proliferation potential and protein synthesizing ability, and then used for further studies of the effects of Phenytoin on nuclear protein phosphorylation. The phosphorylation of nuclear nonhistone proteins has been correlated with the expression of genes including those involved in cell proliferation. Studies in this lab have shown a Phenytoin-induced increase in the phosphorylation of nuclear proteins. Hence the mechanism by which Phenytoin could stimulate cell proliferation in a subpopulation of cells would be by the modification of the phosphorylation of nuclear proteins. This modification would then allow for the transcription of genes necessary for cell proliferation.
