Characterization of the Optimal Stimulatory Effects of Graves’ Monoclonal and Serum Immunoglobulin G on Adenosine 3′,5′-Monophosphate Production in FRTL-5 Thyroid Cells: A Potential Clinical Assay

Abstract

Immunoglobulin G (IgG) preparations derived from the sera of patients with hyperthyroidism due to Graves' disease (TSAb) as well as a monoclonal IgG derived from heterohybridoma fusions of Graves' lymphocytes augmented cAMP levels in a continuous strain of functioning rat thyroid cells (clone FRTL-5) in culture. Optimal stimulation was the same for both types of IgG preparations when measured after 2 h of incubation with 5 X 10(4) cells/well and using cells maintained in a nongrowth, TSH-deficient medium for 7 days. At low IgG concentrations, the stimulatory activities of both preparations exhibited a linear dependence on concentration and similar Ka values (approximately 4 X 10(-8) M) despite the fact that 65% of the Graves' serum IgG preparations had a significantly better ability to inhibit TSH binding to membrane preparations. The Ka value for TSH in the same assay was about 5 X 10(-12) M. Using this cell assay, 90% of a series of hyperthyroid Graves' IgG preparations exhibited stimulating activity, a value comparable to the frequency of positive results found by ourselves and others using human thyroid cell and slice systems. In contrast, only 10% of patients who were euthyroid 1 yr after antithyroid drug withdrawal (n = 21) exhibited stimulating activity, and no stimulating activity was detected in patients with nontoxic nodular goiter (n = 11), toxic adenoma (n = 5), or thyroid carcinoma (n = 6). The studies suggest that an optimized rat FRTL-5 thyroid cell system is a clinically useful and convenient alternative to human thyroid cell and slice systems for detecting TSAbs.

Thyrotropin effect on the availability of Ni regulatory protein in FRTL-5 rat thyroid cells to ADP-ribosylation by pertussis toxin

“…9, exposure of cells to pertussis toxin in the presence of thyrotropin, reduces the level of cAMP (Table 1). This result is quite different from adding Thyrotropin increases cAMP levels in FRTL-5 cells [27]; 1 pM forskolin in the presence of thyrotropin results in a synergistic effect on cAMP levels (Fig. 7) as previously dem- forskolin.…”

“…The intracellular cAMP content of FRTL-5 thyroid cells was measured by adapting the method previously reported [27]. In brief, FRTL-5 cells were grown in 24-well Costar plates for 2 -7 days in 6H medium before being switched to a 5H medium.…”

Elevated levels and mitogenic activity of lysophosphatidylinositol in k‐ras‐transformed epithelial cells

“…Stimulants were then added for 24 h, the pulse of VHIthymidine (1 pCi/well) was applied, in this case for 10 h prior to stopping the reaction by washing as before. The [3H]thymidine incorporated into trichloroacetic-acid-insoluble material was evaluated as previously described [20]. To confirm that the [3H]thymidine incorporation indeed represented cell proliferation, counting of cells by a Coulter Channelyzer 256 (Coulter Electronics Ltd.) was performed in parallel under identical experimental conditions, for both FRTL5 and KiKi cells (see above).…”

Detection of Thyroid-Stimulating Antibody Using Frozen Stocks of Chinese Hamster Ovary Cells Transfected with Cloned Human Thyrotropin Receptor.

“…TSAb have been most commonly measured by using the rat thyroid cell line . This cell line is a non-transformed, differentiated but continuous cell line that has been well characterized in many reports [2,3], but many factors make these cells less than ideal for the assay of TSAb. First, they are slow in growing and it is more difficult to be cultured than other fast growing cell lines [4].…”