The RET/PTC family of oncogenes are detected in human papillary thyroid carcinomas. The overall aim of this study is to investigate the phosphotyrosine signaling pathways downstream of RET/PTC1 oncoprotein that mediate dedifferentiation in PC Cl 3 immortalized rat thyroid cells. Site directed mutagenesis was performed to generate single tyrosine 404 or 451 to phenylalanine (F) mutants. In this study, PC Cl 3 cell lines stably expressing either RET/PTC1 or RET/PTC1 Y/F were generated to 1) investigate the mechanism of RET/PTC1 mediated NIS reduction, 2) investigate the role of pY404 or pY451 on NIS reduction and TSH-independent proliferation and 3) compare global gene expression profiles expression by microarray analysis and identify novel genes that are dysregulated by RET/PTC1 or RET/PTC1 Y/F expression.
RET/PTC1 expression was demonstrated to decrease NIS expression and function in PC Cl 3 cells. Stimulation of the cAMP-PKA pathway by forskolin, 8-Br-cAMP or catalytic PKA expression in the nucleus were able to reverse the effect of RET/PTC1 on NIS expression and function.
In chapter 3, the effect of single Y/F mutagenesis on RET/PTC1 subcellular localization, catalytic PKA nuclear localization and reduction of NIS protein expression and function were investigated. Phosphotyrosine 451 was important for mediating RET/PTC1 plasma membrane localization while pY Y404 mediated signaling was found to be important for RET/PTC1 mediated NIS reduction.
Microarray analysis was performed to compare gene expression profiles between PC Cl 3 parental, RET/PTC1, RET/PTC1 Y/F expressing cell lines. Osteonectin, HIF-1α, aquaporin 5 and alpha crystallin B were identified as novel genes upregulated by RET/PTC1. In order to investigate the role of onset and expression level of RET/PTC1 on thyroid tumorigenesis, PC Cl 3 CMV-Tet-On TRE-RET/PTC1 cell lines which have doxycycline (dox) inducible RET/PTC1 expression were generated and characterized. TRE-RET/PTC1 transgenic mice were also generated and characterization of the mice for dox-inducible RET/PTC1 expression are currently underway. A bi-directional TRE construct expressing RET/PTC1 and luciferase was generated as a first step towards generating bi-transgenic mice with thyroid targeted expression and dox-inducible RET/PTC1 as well as luciferase expression which will serve as a reporter gene for RET/PTC1 expression.