To correlate the differentiation phenotype of two human thyroid cancer cell lines with their expression of various molecular markers, we analyzed the mRNA levels of four thyroid-specific genes, including thyrotropin receptor (TSHR), thyroglobulin (Tg), thyroid transcription factor-1 (TTF-1), and paired-box containing transcription factor-8 (PAX-8) genes. The results showed a differentiation-status-related pattern in which a welldifferentiated cell line (WRO) expressed all the four genes, in contrast to an anaplastic cell line (ARO) that expressed TTF-1 and reduced levels of TSHR, but no Tg or PAX-8 genes. Furthermore, to verify the finding of concomitant loss of subtype thyroid hormone receptor (TR ) and TSHR gene expression in neoplastic thyroid tumors (Bronnegard et al. 1994), we examined the expression levels of TR 1 gene in these cell lines. Whereas the WRO cells produced an abundant amount of TR 1 protein detectable by immunoprecipitation, the ARO cells produced none. This new observation prompted us to investigate whether overexpression of TR 1 protein in ARO cells might produce changes in the differentiation phenotypes. We found that the level of expression of the TSHR gene and the proliferative index of ARO cells were significantly upregulated in the cells stably transfected with wild-type TR 1. These findings suggest that TR 1 protein overexpression can affect the differentiation phenotypes and induce more efficient cell proliferation of the anaplastic ARO cells.

The expression of TSH receptor (TSHR) gene is frequently lost in thyroid cancers during the process of dedifferentiation that involves perturbation of several nuclear transcription factors. We have established that thyroid hormone receptor 1 (TR 1) is associated with the loss of TSHR gene expression in an anaplastic human thyroid cancer cell line, ARO. To demonstrate that TR 1 regulates TSHR gene expression, we performed electrophoresis mobility shift and 3,5,3 -triiodothyronine (T3) transactivation assays. As expected, TR 1 bound the synthesized oligomer containing TSHR promoter sequence by heterodimerizing with retinoid X receptor. When a chimeric reporter pTRCAT5 -146 enclosing the minimal TSHR promoter was applied for T3 transactivation assay, two TR 1-overexpressing transfectants of ARO cells (ARO1 and ARO2) demonstrated higher basal activity than their parental cells. Consequentially, T3 suppressed the reporter gene activity only in ARO1 and ARO2, but not in ARO cells. A point mutation creating a cAMP response element (CRE) in the reporter pTRCAT5 -146 CRE led to T3-induced suppression of the reporter gene in ARO cells without changing the basal or T3-induced activities in ARO1 and ARO2 cells. We conclude that the regulatory effect of T3 on TSHR gene expression is TR-and promoter DNA sequence-determined.