Modulation of cellular functions in retroorbital fibroblasts using antisense oligonucleotides targeting the c-myc protooncogene.
OBJECTIVE
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.
Heufelder
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AE
Molecular Thyroid Research Laboratory, Ludwig-Maximilians Universität, München, Germany.
Bahn
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RS
D001483:Base Sequence; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D016259:Genes, myc / Q000235:genetics / Q000502:physiology*; D006025:Glycosaminoglycans / Q000096:biosynthesis; D006111:Graves Disease / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D009915:Orbit / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction
D016207:Cytokines; D017931:DNA Primers; D006025:Glycosaminoglycans; D016376:Oligonucleotides, Antisense; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger