An in vitro model of thyroid neoplasia: Permanently transfected FRTL-5 cells with thyroglobulin promoter-cholera toxin A1 subunit minigene

Background. Activating mutations of protein Gsa that stimulates adenylyl cyclase are present in a subset of thyroid adenomas. Cholera loxin A1 subunit (C. T) mimics these mutations via adenosine diphosphate-ribosylation of G_sα. Methods. To test the role of activated G_sα in thyroid neoplasia we developed an in vitro model: With molecular genetic techniques a transgene (TG-CT) in which the thyroglobulin gene (TG) promoter directs expression of CT was made. This transgene was transfected into rat thyroid follicular cells (FRTL-5). Integration of TG-CT transgene and its expression were examined. Results. Polymerase chain reaction of DNA from transfected FR TL-5 cells identified the TG-CT transgene in six cell lines. The TG-CT transfected clones exhibited up to a 65 fold (1.29 ± 0.37 pmols cyclic adenosine monophosphate (cAMP)1jugDNA) increase in cAMP over nonlransfected FRTL-5 cells (0.02 ± 0.001 pmols cAMPIAg DNA). Insulin, a known stimulator of the TG promoter, further induced CT gene expression and led to a 209 fold (10.43 ± 0.10 pmols cAMPIAg DNA) increase in cAMP over nontransfected cells (0.05 ± 0.00 pmols cAMP/μm DNA). Conclusions. By mimicking a known mutation associated with thyroid neoplasms, these permanently transfected FRTL-5 cell lines will serve as a model to examine the long-term potential neoplastic effects of activated G_sα on thyroid follicular cells.