To understand the expression and role of thyroid hormone nuclear receptors (TRs) in hepatocarcinogenesis, we characterized the TRs in 16 human hepatocellular carcinoma (HCC) specimens. The full-length cDNAs for the two TR subtypes, alpha1 and beta1, were cloned from several tumors by reverse transcription-polymerase chain reaction. Southern blot analysis indicated that, in addition to the full-length cDNA, truncated TRalpha1 and TRbeta1 cDNAs were present in nine tumors (53%). In addition, point mutations detected by the mismatch RNase cleavage assay in TRalpha1 and TRbeta1 were found in 65% and 76% of the tumors, respectively. The mutations were confirmed by DNA sequencing. Interestingly, most of the TRalpha1 mutations were in amino acid codons 209-228 and 245-256, two hot-spots in HCC patients. However, no hot-spot was detected in TRbeta1. The expression of TRalpha1 and TRbeta1 proteins was determined in the tissue extracts by western blotting. TRbeta1 protein was expressed or elevated in 10 tumors but not in normal livers, whereas the expression of TRalpha1 was variable among tumors. The mutant TR proteins were translated in vitro, and their hormone- and DNA-binding activities were evaluated. Abnormal binding to the thyroid hormone response elements was observed. The proteins' DNA binding activity was either partially impaired or completely lost. The high prevalence of TR mutations found in the tumors of patients with hepatocellular carcinoma suggests that mutant TRs could play an important role in liver carcinogenesis.

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.

Complementary DNAs for two mutant thyroid hormone alpha1 receptors (TR alpha1) were isolated from hepatocellular carcinomas of two patients. Sequence analyses of the complementary DNAs showed a single Val390Ala and double Pro398Ser/Glu350Lys mutations in mutants H and L, respectively. We characterized their hormone-binding, DNA-binding, and dominant negative activities. Mutants H and L did not bind the hormone T3. Their DNA-binding activities were analyzed using three types of thyroid hormone response elements (TREs) in which the half-site binding motifs are arranged in an everted repeat (Lys), an inverted repeat (Pal), or a direct repeat separated by four nucleotides (DR4). Compared with wild-type TR alpha1 (w-TR alpha1), which bound these TREs with different homodimer/monomer ratios, binding of mutant L to the three TREs as homodimers was reduced by approximately 90%. However, binding of mutant H to these TREs was more complex. Although it bound normally to DR4 as homodimers, its binding to Lys as homodimers was reduced by approximately 80%. Surprisingly, its binding to Pal was markedly enhanced compared with w-TR alpha1. The binding of these two mutants to the three TREs as heterodimers with retinoid X receptors (RXR alpha and -beta) was not significantly affected. Consistent with the lack of T3-binding activity, both mutants had lost their trans-activation capacity. Mutants H and L exhibited dominant negative activity, but differed in their TRE dependency. The dominant negative potency of mutant H was in the rank order of Pal > DR4 > Lys, whereas no TRE dependency was observed for mutant L. The present study indicates that mutations of the TR alpha gene do occur in patients and that these novel TR alpha1 mutants provide a valuable tool to further understand the molecular basis of the dominant negative action of mutant TRs.

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To understand the function of thyroid hormone nuclear receptors (TRs) in human hepatocellular carcinoma cells (HCC), we characterized the hormone binding and transactivational activity of TRs in a HCC cell line, J7. TR alpha 1 (J7-TR alpha 1) and TR beta 1 (J7-TR beta 1) complementary DNAs were cloned from this cell line, and the binding activity to the hormone response elements (TREs) and to the thyroid hormone, 3,3',5-triiodo-L-thyronine (T3) of the expressed TR proteins were evaluated. J7-TR alpha 1 and J7-TR beta 1 bound to TREs similarly as the TRs isolated from other tissues. However, J7-TR alpha 1 did not bind to T3, and J7-TR beta 1 bound to T3 with only about 10% the affinity of the wild-type TR beta 1. Sequencing of the complementary DNAs shows a single Met259Ile mutation in J7-TR alpha 1 and Met334Val in J7-TR beta 1. Using reporters containing TREs, we found that J7-TR alpha 1 and J7-TR beta 1 had virtually lost their transactivational activity. Moreover, these two mutants inhibited the transactivational activity of the wild-type TRs by a dominant negative effect not only on the transfected TRs, but also on endogenous TRs in other two HCC cell lines, SK-Hep-1 and HepG2. The potency of the dominant negative effect of these two mutants inversely correlated with the expression level of endogenous TRs. The present studies identified two novel naturally occurring TR mutants that have potent dominant negative action. The identification of both the alpha and beta dominant negative mutants in J7 made this cell line a useful model system to further understand the molecular mechanism of the dominant negative action of TR mutants.