Single cell Ras-GTP analysis reveals altered Ras activity in a subpopulation of neurofibroma Schwann cells but not fibroblasts

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized b y multiple neurofibromas, peripheral nerve tumors containing mainly Schwann cells and fibroblasts. The NF1 gene encodes neurofibromin, a tumor suppres sor postulated to function in part as a Ras GTPase-activating protein. The roles of different cell types and of elevated Ras-GTP in neurofibroma forma tion are unclear. To determine which neurofibroma cell type has altered Ras -GTP regulation, we developed an immunocytochemical assay for active, GTP-b ound Ras. In NIH 3T3 cells, the assay detected overexpressed, constitutivel y activated K-, N-, and Ha-Ras and insulin-induced endogenous Ras GTP. In d issociated neurofibroma cells from NF1 patients, Ras-GTP was elevated in Sc hwann cells but not fibroblasts. Twelve to 62% of tumor Schwann cells showe d elevated Ras-GTP, unexpectedly revealing neurofibroma Schwann cell hetero geneity. Increased basal Ras-GTP did not correlate with increased cell prol iferation. Normal human Schwann cells, however, did not demonstrate elevate d basal Ras activity. Furthermore, compared with cells from wild type litte rmates, Ras-GTP was elevated in all mouse Nf1(-/-) Schwann cells but never in Nf1(-/-) mouse fibroblasts. Our results indicate that Ras activity is de tectably increased in only some neurofibroma Schwann cells and suggest that neurofibromin is not an essential regulator of Ras activity in fibroblasts .