MODULATION OF CELLULAR FUNCTIONS IN RETROORBITAL FIBROBLASTS USING ANTISENSE OLIGONUCLEOTIDES TARGETING THE C-MYC PROTOONCOGENE

Purpose, To examine the signal transduction pathways involved in the a ctivation of orbital fibroblast effector functions relevant to the pat hogenesis of Graves' ophthalmopathy (GO). To determine, using antisens e technology, whether the c-myc protooncogene is involved in cell prol iferation and glycosaminoglycan (GAG) synthesis in cultured orbital fi brobrasts (OF). Methods. The effects of a 16-mer c-myc antisense phosp horothioate oligodeoxynucleotide (S-ODN) on OF monolayers derived from orbital connective tissue of patients with severe GO (n = 6) and heal thy individuals (n = 3) were investigated. Quiescent OF monolayers wer e treated with serum or cytokines and were exposed to increasing conce ntrations of a c-myc antisense S-ODN and several control S-ODN. Cell p roliferation was quantitated by direct cell counting and by immunocyto chemistry for the nuclear Ki-67 antigen. Glycosaminoglycan synthesis w as examined by [H-3] 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, immuno cytochemistry, and immunoblotting, respectively. Results. Transient su ppression of c-myc mRNA and the c-myc protein product by a c-myc antis ense S-ODN (2 to 8 mu M) 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 pro duct are involved in the proliferative and metabolic activities of OF exposed to serum or cytokines in vitro. C-myc appears to be an essenti al component of at least two OF cellular activities likely to contribu te to the orbital tissue alterations in GO.