Browsing by Author "Yaman, Elif"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
Item Open Access The comparative effects of gene modulators on thyroid-specific genes and radioiodine uptake(Mary Ann Liebert, 2007) Tuncel, M.; Aydın, D.; Yaman, Elif; Tazebay, Uygar H.; Güç, D.; Doğan, A. L.; Taşbasan, B.; Uǧur, Ö.The aim of this study was to comparatively investigate the effects of 5-azacytidine-C (5-Aza), trichostatin-A (TSA), and all-trans retinoic acid (ATRA) on mRNA expressions of Na/I symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO), and thyroid stimulating hormone receptor (TSH-R), and radioiodine (RAI) uptake in cancer (B-CPAP) and normal (Nthy-ori 3-1) thyroid cell lines. Cell lines were treated with 10 ng/mL of TSA, 5 μM of 5-Aza, and 1 μM of ATRA, according to the MTT (methyl-thiazol-tetrazolium) test results. Additionally, recombinant thyroid stimulating hormone (rTSH) was also applied, with a selected dose of 100 ng/mL. Following the treatment, NIS, Tg, TPO, and TSH-R mRNA levels were detected by real-time-polymerase chain reaction (RT-PCR) and RAI uptakes were measured by using a well counter as the counts/cell number. 5-Aza increased TSH-R mRNA expression in both of the cell lines and decreased TPO, NIS, and Tg mRNA levels in the cancer cell line. In the normal thyroid cell line, 5-Aza increased TPO mRNA levels 2-fold and made no differences in NIS and Tg mRNA levels. TSA treatment repressed NIS and Tg mRNA levels, and made no differences on other thyroid specific genes investigated in the cancer cell line. In the normal thyroid cell line, TSA increased TSH-R mRNA levels in 72 hours and created no important differences in other genes. ATRA repressed the TSH-R mRNA levels in the normal thyroid cell line and increased the TPO and Tg mRNA levels slightly in both cell lines. Furthermore, in short-term treatment, ATRA repressed NIS gene expression slightly, but in the long term, this repression turned to basal levels. 5-Aza, TSA, and ATRA did not make any differences in RAI uptake in the cancer cell line, but rTSH increased RAI uptake significantly. In the normal thyroid cell line, TSA and ATRA decreased RAI uptake (to 1/10 and 1/2, respectively), but 5-Aza and rTSH increased RAI uptake significantly (2- and 4-fold, respectively). We have shown an increase in TSH-R gene expression and radioiodine uptake with 5-Aza. Further in vitro and in vivo studies are needed to support our findings and the potential clinical use of this agent.Item Open Access Functional identification of RASGEF1 family of exchange factors as activators of RAP2, and as interacting partners of CCDC124(Bilkent University, 2009) Yaman, ElifCoiled coil domain-124 gene is highly conserved among eukaryotes and the human counterpart encodes a protein with no domain similarities with any previously characterized eukaryotic proteins. In this study, we aimed to identify biological functions and interaction partners of human Ccdc-124. A yeast-two-hybrid analysis carried in this study has revealed that Ccdc-124 interacts with RasGEF1B which was predicted to be a member of Ras guanine exchange factors. The highly conserved RasGEF1 family of proteins contain C-terminal CDC25-homology domain (CDC25- HD) and an N-terminal RasGEF-N domain (Ras Exchange Motif, REM), and is of unknown function and specificity. In this thesis, the interaction of Ccdc-124 and RasGEF1 family of proteins was also established with co-immunoprecipitation and GST pull down assays. On the other hand, by using purified RasGEF1A and RasGEF1B proteins, as well as a large number of Ras family of G-proteins, we established that RasGEF1A and RasGEF1B function as very specific exchange factors for Rap2, a member of the Rap subfamily of Ras-like G-proteins. They do not act on Rap1 or other members of the Ras subfamily. On the other hand, Ccdc-124 protein did not change the stimulatory effect of RasGEF1 family of proteins on any of the tested G proteins in vitro. Furthermore, using reciprocal site-directed mutagenesis, we analyzed residues that allow RasGEF1 proteins to discriminate between Rap1 and Rap2, and we were able to identify Phe39 in the switch I region of Rap2 as a specificity residue. Mutation of the corresponding Ser39 in Rap1 changed the specificity and allowed the nucleotide exchange of Rap1(S39F) to be stimulated by RasGEF1B. This study describes for the first time GEFs that are uniquely specific for Rap2 among Rap family of G-proteins.