Retinoic acid receptor beta2 re-expression and growth inhibition in thyroid carcinoma cell lines after 5-aza-2'-deoxycytidine treatment.
The treatment of both undifferentiated and de-differentiated thyroid tumors, which are unresponsive to radioiodine, represents one of the biggest challenges for thyroidologists. The aim of the present study was to investigate in vitro the methylation status of retinoic acid receptors (RAR)beta2 promoter and the effect of the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR) on 5 human thyroid cancer cell lines. The methylation status of RARbeta2 promoter was analyzed by methylation-specific PCR. The effect of 5-Aza-CdR on cell growth and apoptosis was evaluated by cell counting, enzymelinked immunosorbent assay tests and fluorescence-activated cell sorting analysis, while the effect on the expression of RAR and thyroid-specific genes was measured by qualitative and quantitative RT-PCR. Methylation of RARbeta2 promoter was present only in ARO cells. 5-Aza-CdR determined growth inhibition in all cell lines, probably due to apoptosis in WRO, NPA, and ARO cells, and to inhibition of DNA synthesis in TT cells. Treatment with 5-Aza-CdR induced the expression of RARbeta mRNA in ARO and FRO cells, a slight increase of the expression of Tg, TPO and thyroid trancription factor 1 (TTF-1) mRNA and the new expression of low levels of NIS in TT cells. A significant increase of TTF-1 mRNA in FRO cells was also observed. In this study we demonstrated that RARbeta2 promoter was methylated in ARO cell line. However, the 5-Aza-CdR treatment induced RARbetamRNA expression not only in ARO but also in FRO and TT cell lines, whose RARbeta2 promoter was unmethylated. A significant reduction of cell growth, but not cell re-differentiation, was also observed after 5-Aza-CdR treatment.
Miasaki
F Y
FY
Department of Endocrinology and Metabolism, University of Pisa, 56124 Pisa, Italy.
Vivaldi
A
A
Ciampi
R
R
Agate
L
L
Collecchi
P
P
Capodanno
A
A
Pinchera
A
A
Elisei
R
R
D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000627:therapeutic use; D002277:Carcinoma / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019175:DNA Methylation; D000077209:Decitabine; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D009928:Organ Specificity / Q000187:drug effects / Q000235:genetics; D011401:Promoter Regions, Genetic; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D000072482:Retinoic Acid Receptor alpha; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism; D013964:Thyroid Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D014162:Transfection; D000097768:Retinoic Acid Receptor gamma
D000964:Antimetabolites, Antineoplastic; D001374:Azacitidine; D000077209:Decitabine; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D000097768:Retinoic Acid Receptor gamma; C000607071:RARA protein, human; C533416:RARB2 protein, human