Overexpression of USF Increases TGF-beta1 Protein Levels, But G1 Phase Arrest was not Induced in FRTL-5 Cells

Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of cellular growth and proliferation by G1 phase arrest or apoptosis. We investigated the association of TGF-beta1 with the anti-proliferative effect of upstream stimulatory factor (USF) in Fischer rat thyroid cell line (FRTL-5) cells. [Methyl-(3)H] thymidine uptake was measured after treatment of FRTL-5 cells with TGF-beta1 to identify its anti-proliferative effect. USF-1 and USF-2 proteins were in vitro translated, and an electrophoretic mobility shift assay was performed to identify the interaction between USF and the TGF-beta1 promoter. FRTL-5 cells were transfected with USF cDNA, and then the expression of TGF-beta1 was examined with Northern and Western blotting. The cell cycle-regulating proteins associated with TGF-beta1 were also measured. TGF-beta1 significantly inhibited [methyl-(3)H] thymidine uptake in FRTL-5 cells. Two specific binding sites for USF were found in the TGF-beta1 promoter: -1,846~-1,841 (CACATG) and -621~-616 (CATGTG). Overexpression of USF increased both the mRNA levels and protein levels of TGF-beta1. However, the expression of cyclin D1, CDK4, cyclin E, and CDK2, and the phosphorylation of retinoblastoma protein remained unchanged. Overexpression of USF in FRTL-5 cells increased the expression of TGF-beta10 through specific binding to TGF-beta1 promoter. However, the USF-induced expression of TGF-beta1 did not cause G1 arrest.

Effects of USF-1, USF-2, PTEN and Thyroid Transcription Factors on the Function and Growth in FRTL-5 Cells / 대한내분비학회지

BACKGROUND: Upstream stimulatory factors (USFs) and PTEN are known to be tumor suppressants. USFs and PAX-8 were reported to be the functional competitors in sodium iodide symporter (NIS) gene expression. We investigated the effects of USF-1, USF-2, PTEN, and thyroid-specific transcription factors (TTF-1, PAX-8) on the function and growth of thyrocytes of FRTL 5 rat thyroid cells. METHODS: Complementary DNAs of the USF-1, USF-2, PTEN, TTF-1 (homeodomain), and PAX-8 were synthesized from RNA extracted from FRTL-5using an RT-PCR kit. Each of them was transiently transfected to the FRTL-5 cells using the lipofectamine after being cloned into the pcDNA3.1 vectors. Stable cell lines, which were transfected by USF-1, PTEN, TTF-1, and PAX-8, were also obtained from the FRTL-5 cells, respectively. Extracellular cAMP concentrations were measured after 24 hours of incubation with varying concentrations of bTSH (0.1~100 mIU/mL). After, [Methyl-3H] thymidine uptake or 5-bromo-2'-deoxyuridine (BrdU) assay was performed. RESULTS: USF-1 and USF-2 significantly increased cAMP levels and decreased thymidine uptake in both transiently and stably transfected cells (p<0.01). PTEN had a tendency to increase both the cAMP levels and BrdU uptake in stable cells, but had a tendency to decrease thymidine uptake in transiently transfected cells. TTF-1 significantly increased the cAMP levels and either thymidine or BrdU uptake in both transiently and stably transfected cells (p<0.05). PAX-8 significantly increased both the cAMP levels and BrdU assay in stable cells, but in transiently transfected cells, it significantly decreased cAMP concentrations (p<0.01). CONCLUSIONS: These results suggested that both the USF-1 and USF-2 play a role in suppressing the growth of thyrocytes but at the same time, they kept the ability to produce cAMP after TSH stimulation. They had opposing effects on TTF-1 and PAX-8 in terms of the proliferation of thyrocytes

Effect of Estrogen on H2O2 Induced Apoptosis of FRTL-5 Cells / 대한내분비학회지

BACKGROUND: Understanding the pathways and controlling mechanisms of thyrocyte apoptosis is important for the elucidation of the pathogenesis of goiter or thyroid cancer. A system for evaluating apoptosis, in FRTL-5 cells, triggered by hydrogen peroxide (H2O2), a highly likely apoptogenic signal in physiologic condition, was be set up to see the effects of TSH and estrogen on H2O2-induced apoptosis. METHOD: DNA laddering was used in the optimization process or the conditions of the set-up of system for the evaluation of apoptosis in the FRTL-5 cells. To quantify the apoptosis under the optimized conditions, histone-bound DNA fragments in the cytoplasm were measured by ELISA. RESULTS: 1) The optimized conditions for induction of apoptosis in the FRTL-5 cells by H2O2 were; observation of DNA laddering 18~24 hrs after the addition of 0.3 mM H2O2 to cells maintained in TSH-free, low serum containing media (5H1 or 5H0 media) for 48 hrs. 2) Exposure of the FRTL-5 cells to TSH (1 mU/L) for more than 48 hrs (6H0 media). before the addition of H2O2 significantly decreased the degree of apoptosis, compared to cells maintained under TSH-free conditions (0.98+/-0.21 vs. 2.27 0.11 arbitrary unit, p<0.05), whereas exposure for 24 hrs. did not. 3) Exposure of the FRTL-5 cells to high dose 17- estradiol (1-100 M) significantly decreased the degree of H2O2-induced apoptosis in a dose dependent manner. The addition of serum (1%) blunted the effects of estrogen on H2O2-induced apoptosis, and TSH totally abrogated the estrogen effect.Physiologic doses of estrogen (10~100 nM) showed no suppressive effects on H2O2-induced apoptosis in FRTL-5 cells. CONCLUSION: A system for evaluating apoptosis in FRTL-5 cells triggered by hydrogen peroxide (H2O2), a highly likely apoptogenic signal in physiologic condition, was set up, and found for the first time that high dose estrogen suppressed the H2O2-induced apoptosis in FRTL-5 cells

Effect of Estrogen on H2O2 Induced Apoptosis of FRTL-5 Cells / 대한내분비학회지

BACKGROUND: Understanding the pathways and controlling mechanisms of thyrocyte apoptosis is important for the elucidation of the pathogenesis of goiter or thyroid cancer. A system for evaluating apoptosis, in FRTL-5 cells, triggered by hydrogen peroxide (H2O2), a highly likely apoptogenic signal in physiologic condition, was be set up to see the effects of TSH and estrogen on H2O2-induced apoptosis. METHOD: DNA laddering was used in the optimization process or the conditions of the set-up of system for the evaluation of apoptosis in the FRTL-5 cells. To quantify the apoptosis under the optimized conditions, histone-bound DNA fragments in the cytoplasm were measured by ELISA. RESULTS: 1) The optimized conditions for induction of apoptosis in the FRTL-5 cells by H2O2 were; observation of DNA laddering 18~24 hrs after the addition of 0.3 mM H2O2 to cells maintained in TSH-free, low serum containing media (5H1 or 5H0 media) for 48 hrs. 2) Exposure of the FRTL-5 cells to TSH (1 mU/L) for more than 48 hrs (6H0 media). before the addition of H2O2 significantly decreased the degree of apoptosis, compared to cells maintained under TSH-free conditions (0.98+/-0.21 vs. 2.27 0.11 arbitrary unit, p<0.05), whereas exposure for 24 hrs. did not. 3) Exposure of the FRTL-5 cells to high dose 17- estradiol (1-100 M) significantly decreased the degree of H2O2-induced apoptosis in a dose dependent manner. The addition of serum (1%) blunted the effects of estrogen on H2O2-induced apoptosis, and TSH totally abrogated the estrogen effect.Physiologic doses of estrogen (10~100 nM) showed no suppressive effects on H2O2-induced apoptosis in FRTL-5 cells. CONCLUSION: A system for evaluating apoptosis in FRTL-5 cells triggered by hydrogen peroxide (H2O2), a highly likely apoptogenic signal in physiologic condition, was set up, and found for the first time that high dose estrogen suppressed the H2O2-induced apoptosis in FRTL-5 cells

Difference of Thyroid Stimulating Antidody Activities Measured in Chinese Hamster Ovary Cells Stably Transfected with Human TSH Receptor and in FRTL-5 Cells in Graves Disease and Its Clinical Correlations / 대한내분비학회지

Background : Thyroid stimulating antibodies result in the development of hyperthyroidism and goiter in Graves disease. However, thyroid stimulating antibody activities do not correlate with the clinical features in many patients with Graves disease. The purpose of this study is to address this discrepancy between thyroid stimulating antibody activities and clinical features of Graves patients. Methods: We measured thyroid stimulating antibody activities simultaneously using human TSH receptor transfected Chinese hamster(hTSHR-CHO) cells and rat thyroid(FRTL-5) cells in 57 untreated patients with Graves disease, and compared their activities with clinical features including thyroid hormone levels. Results : The detection rate of thyroid stimulating antibody measured by hTSHR-CHO cells was 90% in 57 untreated Graves patients and it was higher than that measured by FRTL-5 cells. Thyroid stimulating antibody activity by hTSHR-CHO cells was significantly correlated with that by FRTL-5 cells(r=0.5, p<0.001), however, 18 of 57(32%) patients showed marked discrepancy of thyroid stimulating antibody activity between in hTSHR-CHO and FRTL-5 systems. Thyroid stimulating antibody activity measured by hTSHR-CHO cells was significantly correlated with serum total T3, free T4 levels, and goiter size but not 99mTc-thyroid uptake. On the other hand, thyroid stimulating antibody activity measured by FRTL-5 cells was significantly correlated with goiter size and 99mTc-thyroid uptake but not thyroid hormone levels. The difference between function and goiter size with respect to thyroid stimulating antibody measurement in two cells system is, nevertheless, particularly evident in the free T4/goiter ratio in patients with high hTSHR-CHO and low FRTL-5 cell assay values. Conclusion: These findings suggest that thyroid stimulating antibodies in Graves disease are heterogeneous population in terms of responses to different origin of cells. Further, thyroid stimulating antibody activities measured by FRTL-5 cells tend to correlate better with goiter size and Tc-thyroid uptake, whereas thyroid stimulating antibody activities measured by hTSH-CHO cells correlate better with thyroid hormone levels.