Purpose: To examine the signal transduction pathways involved in the activation of orbital fibroblast effector functions relevant to the pathogenesis of Graves' ophthalmopathy (GO). To determine, using antisense technology, whether the c-myc protooncogene is involved in cell proliferation and glycosaminoglycan (GAG) synthesis in cultured orbital fibroblasts (OF).
Methods: The effects of a 16-mer c-myc antisense phosphorothioate oligodeoxynucleotide (S-ODN) on OF monolayers derived from orbital connective tissue of patients with severe GO (n = 6) and healthy individuals (n = 3) were investigated. Quiescent OF monolayers were treated with serum or cytokines and were exposed to increasing concentrations of a c-myc antisense S-ODN and several control S-ODN. Cell proliferation was quantitated by direct cell counting and by immunocytochemistry for the nuclear Ki-67 antigen. Glycosaminoglycan synthesis was examined by [3H] GAG analysis. The effects of the c-myc antisense S-ODN and control S-ODN on c-myc mRNA and protein product levels were analyzed using reverse-transcriptase polymerase chain reaction, immunocytochemistry, and immunoblotting, respectively.
Results: Transient suppression of c-myc mRNA and the c-myc protein product by a c-myc antisense S-ODN (2 to 8 microM) strongly inhibited cell proliferation and GAG synthesis in OF derived from patients with GO and healthy individuals. These effects occurred in a dose-dependent manner and were specific for the c-myc antisense S-ODN used. Cell morphology or viability were not affected.
Conclusions: The c-myc protooncogene and its protein product are involved in the proliferative and metabolic activities of OF exposed to serum or cytokines in vitro. C-myc appears to be an essential component of at least two OF cellular activities likely to contribute to the orbital tissue alterations in GO.