We previously found that two cell lines (ARO and FRO) derived from anaplastic thyroid cancer and one (TT) deriving from medullary thyroid cancer did not respond to the retinoic acid (RA) treatment. We supposed that it was related to the lack of the RA receptor RAR beta mRNA expression. It has been suggested that the promoter methylation was one possible cause of loss of RAR beta expression.
We analyzed the methylation status of RAR beta promoter in ARO, FRO, TT, NPA and WRO (deriving from poor differentiated papillary carcinoma and follicular carcinoma) cell lines. We also analyzed the induction of thyroid specific genes expression (NIS, Tg, TSH-R, TPO, Pax-8, TTF-1) and of RAR alpha, beta and gamma genes expression, after treatment with the demethylating drug azacytidine.
The methylation of RAR beta promoter was analyzed by methylation specific PCR (MSP). Thyoid specific genes and RAR alpha, beta and gamma expression was studied by quantitative real time RT-PCR.
We observed that RAR beta promoter was methylated in ARO, but not in FRO and TT cells, which did not express RAR beta, nor in NPA and WRO, which expressed RAR beta mRNA. However, azacytidine treatment induced RAR beta mRNA expression in ARO, FRO and TT. The analysis of thyroid specific gene expression after azacytidine treatment showed the induction of TTF-1 in FRO and TT, TPO and NIS in TT cells.
In conclusion, in this study we showed that although methylation could explain the lack of RAR beta mRNA expression only in ARO, the azacytidine treatment induced RAR beta expression also in FRO and TT cell lines. These results open the possibility that a treatment with both azacytidine and RA may induce the re-expression of thyroid specific genes, not allowed by the treatment of either drug alone.