The Effect of Radiation and Repeated Sub-culturing on TGF-β1 Signaling in FRTL-5 Cells

From our ongoing in vitro studies using the Fisher Rat Thyroid cell line-5 (FRTL-5) we recorded accelerated growth, reduced follicularization and reduction in thyroxin release that occurred as the cells were repeatedly sub-cultured. We also recorded that these changes occurred earlier and more rapidly following radiation exposure. We determined that TGF-β1 production increased under both conditions. We hypothesized that alteration in the TGF-β1 signaling pathway contributed to the changes observed in the cellular properties of FRTL-5 cells. Our objective was to examine some of the players in the TGF-β1 signaling pathway to determine whether radiation and/or repeated subculturing promoted changes in their levels and/or activity. We quantified changes in cellular growth rate using the MTS cell proliferation assay. TGF-β1 ligand and receptor levels were quantified via ELISA, immunocytochemistry and western blot analysis respectively. The levels and activity of Smads 2, 3, 4 and 7, downstream effectors in the TGF-β1 signaling pathway were also measured via western blotting. Lastly, we examined whether TGF-β1 was correctly regulating the expression of its response genes; cyclin A and plasminogen activator inhibitor-1 (PAI-1) under our experimental conditions. To determine this we used luciferase reporter constructs containing promoters for cyclin A and PAI introduced to the cultures by transfection. PAI-1 production in response to exogenously added TGF-β was also tested using a PAI-1 specific ELISA. We observed an acceleration of growth that occurred earlier in irradiated cells than it did in cells subjected to repeat sub-culturing (p \u3c 0.05). The TGF-β1 receptor levels remained unchanged as a result of radiation and continual passage. We, however, observed decreases in the TGF-β1 induced phosphorylation levels of Smads2 (p \u3c 0.05) and 3 (p \u3c 0.05) after radiation and repeated subculturing. However, no differences in the inherent un-activated levels of Smads2, 3, 4 and 7 were observed. Alterations were observed in TGF-β1 ability to control the expression of cyclin A and PAI-1. Collectively, these results support that alterations in the TGF-β1 signaling pathway were contributing to the changes in cellular properties that we measured in our cell line, FRTL-5. These alterations were evident at the level of Smad signaling and transcription initiation