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 < 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 < 0.05) and 3 (p < 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.
Microbiology and Molecular Genetics
Lora M. Green
Daila S. Gridley
Thomas A. Linkhart
Richard A. Luben
Nathan R. Wall
Doctor of Philosophy (PhD)
Year Degree Awarded
Date (Title Page)
Library of Congress/MESH Subject Headings
Thyroid Gland -- microbiology; Thyroid Gland -- radiation effects; Transforming Growth Factor Beta 1; Thyroxine.
Loma Linda University Libraries
This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.
Burrell, Cheryl G., "The Effect of Radiation and Repeated Sub-culturing on TGF-β1 Signaling in FRTL-5 Cells" (2007). Loma Linda University Electronic Theses, Dissertations & Projects. 1507.
Loma Linda University Electronic Theses and Dissertations
Loma Linda University. Del E. Webb Memorial Library. University Archives
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