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Modulation of cellular functions in retroorbital fibroblasts using antisense oligonucleotides targeting the c-myc
1Molecular Thyroid Research Laboratory, Ludwig-Maximilians Universität, München, Germany.
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.
Insights
The c-myc protooncogene is crucial for orbital fibroblast proliferation and glycosaminoglycan synthesis in Graves' ophthalmopathy. Inhibiting c-myc with antisense technology effectively reduced these key disease-related functions in vitro.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Biology
Background:
- Graves' ophthalmopathy (GO) involves orbital fibroblast (OF) activation.
- Signal transduction pathways in OF pathogenesis are not fully understood.
- The role of the c-myc protooncogene in OF function requires investigation.
Purpose of the Study:
- To investigate signal transduction pathways in OF activation relevant to GO pathogenesis.
- To determine the involvement of the c-myc protooncogene in OF cell proliferation and glycosaminoglycan (GAG) synthesis using antisense technology.
Main Methods:
- Cultured OF from GO patients and healthy individuals were treated with serum or cytokines.
- The effects of c-myc antisense phosphorothioate oligodeoxynucleotide (S-ODN) on cell proliferation and GAG synthesis were assessed.
- c-myc mRNA and protein levels were analyzed using molecular techniques.
Main Results:
- A c-myc antisense S-ODN significantly inhibited OF proliferation and GAG synthesis in a dose-dependent manner.
- These inhibitory effects were specific to the c-myc antisense S-ODN.
- Cell morphology and viability remained unaffected, indicating targeted action.
Conclusions:
- The c-myc protooncogene and its protein product are integral to OF proliferative and metabolic activities in vitro.
- C-myc plays an essential role in OF cellular functions contributing to orbital tissue changes in GO.
- Targeting c-myc may offer a therapeutic strategy for Graves' ophthalmopathy.