4HPR triggers apoptosis but not differentiation in retinoid sensitive and resistant human embryonal carcinoma cells through an RARγ independent pathway

Retinoids signal biological effects through retinoic acid receptors (RAR) and retinoid X receptors (RXR) and their co-regulators. We previously reported that all-trans retinoic acid (RA) triggers terminal differentiation in the human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1), through an RARγ dependent pathway. RARγ repression in NT2/D1-R1 cells accounts for RA resistance in this line. This report finds RARγ repression is due to selective repression of RARγ but not RARβ transcription in NT2/D1-R1 cells. The repression is neither due to mutations in RARγ nor its promoter containing the RA response element. Prior work was confirmed and extended by demonstrating that an RARγ selective agonist preferentially signals differentiation of NTZ/D1 cells, while RARα/β, RARβ, RXR agonists and an RAR pan-antagonist do not even when NT2/D1 cells are treated with these retinoids at 10 μM dosages. None of these examined retinoids induced differentiation of the RA resistant NT2/D1-R1 cells. In contrast, N-(4-hydroxyphenyl)retinamide (4HPR), a reported transcriptional activator of RARγ was shown to potently induce growth inhibition and apoptosis in both NTZ/D1 and NT2/D1-R1 cells. 4HPR-induced apoptosis was unaffected by co-treatment of both cell lines with equimolar RAR antagonist. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays of total RNA from 4HPR-treated NT2/D1 and NT2/D1-R1 cells did not reveal RARγ induction. Since 4HPR signals in RA-resistant NT2/D1-R1 cells having an RARγ transcriptional block, these results indicate that 4HPR triggers apoptosis but not differentiation through an RARγ independent pathway. Taken together, these findings implicate a therapeutic role for 4HPR mediated apoptosis in germ cell tumors even when a maturation block is present.