RET/PTC1-mediated phosphotyrosine signaling pathways involved in thyroid cell transformation

The RET/PTC family of oncogenes are detected in human papillary thyroid carcinomas. RET/ PTC1 is the most common form of RET/PTC that is detected in the general population. 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. Previously, site directed mutagenesis was performed to generate single tyrosine 404 or 451 to phenylalanine (F) mutants, thus abolishing phosphorylation at Y404 or Y451, and consequent downstream signaling. In this study, PC Cl 3 cell lines stably expressing either RET/PTC1, RET/PTC1 Y294F, RET/PTC1 Y404F or RET/PTC1 Y451F 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 interfere with the thyroid stimulating hormone (TSH) mediated cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway in PC Cl 3 rat thyroid cells by inhibiting cPKA nuclear accumulation. 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, reduction of NIS protein expression and function and TSH-independent proliferation was investigated. Y/F mutagenesis of pY451 reduced RET/PTC1 plasma membrane localization while pY Y404 mediated PKC-Raf-MEK signaling was found to be important for RET/PTC1 mediated NIS reduction. Interestingly, further stimulation of PKC activation in RET/PTC1 expressing cells was sufficient to reduce forskolin mediated cPKA nuclear accumulation, NIS protein expression and function.;Microarray analysis was performed to compare gene expression profiles between PC Cl 3 parental, RET/PTC1, RET/PTC1 Y/F expressing cell lines and to identify novel genes that are dysregulated by RET/PTC1 or RET/PTC1 expression. Osteonectin, HIF-1alpha, aquaporin 5 and alpha crystallin B were identified as novel genes upregulated by RET/PTC1. Interestingly, RET/PTC1 Y294F which has reduced tyrosine kinase activity showed reduced expression of genes involved in tumor progression. (Abstract shortened by UMI.)...