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

Retinoids signal biological effects through retinoic acid receptors (RAR) a nd retinoid X receptors (RXR) and their co-regulators. We previously report ed that all-trans retinoic acid (RA) triggers terminal differentiation in t he human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1), through a n RAR gamma dependent pathway. RAR gamma repression in NT2/D1-R1 cells acco unts for RA resistance in this line. This report finds RAR gamma repression is due to selective repression of RAR gamma but not RAR beta transcription in NT2/D1-R1 cells. The repression is neither due to mutations in RAR gamm a nor its promoter containing the RA response element. Prior work was confi rmed and extended by demonstrating that an RAR gamma selective agonist pref erentially signals differentiation of NT2/D1 cells, while RAR alpha/beta, R AR beta, RXR agonists and an RAR pan-antagonist do not even when NT2/D1 cel ls are treated with these retinoids at 10 mu M dosages. None of these exami ned retinoids induced differentiation of the RA resistant NT2/D1-R1 cells. In contrast, N-(4-hydroxyphenyl)retinamide (4HPR), a reported transcription al activator of RAR gamma was shown to potently induce growth inhibition an d apoptosis in both NT2/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 RAR gamma induction. Since 4HPR signals in RA-resistant NT2/D1-R1 ce lls having an RAR; transcriptional block, these results indicate that 4HPR triggers apoptosis but not differentiation through an RAR gamma independent pathway. Taken together, these findings implicate a therapeutic role for 4 HPR mediated apoptosis in germ cell tumors even when a maturation block is present.