SPECIFIC RETINOID RECEPTORS COOPERATE TO SIGNAL GROWTH SUPPRESSION AND MATURATION OF HUMAN EMBRYONAL CARCINOMA-CELLS

This study addresses the contributions of specific retinoid receptors during all-trans-retinoic acid (RA)-mediated differentiation and growt h suppression of human embryonal carcinoma cells. The pleiotropic effe cts of RA are mediated bg retinoic acid receptors (RARs) and retinoid, X receptors (RXRs), members of the nuclear receptor family of transcr iption factors, After RA-treatment the multipotent human embryonal car cinoma cell line NTERA-2 clone D1 (NT2/D1) displays limited proliferat ive potential, reduced tumorigenicity, and morphologic and immunopheno typic neuronal maturation. RAR gamma over-expression in NT2/D1 cells s ignals mesenchymal NT2/D1 terminal differentiation while RAR alpha and RAR beta do not and RAR gamma overcomes retinoid resistance in an NT2 /D1 clone (NT2/D1-R1) having deregulated RAR gamma expression, Since R AR gamma transfectants do not display neuronal maturation, this study sought to identify cooperating retinoid receptors engaged in NT2/D1 di fferentiation. Through gain of function experiments, this report highl ights RXR beta as playing an important role along with RAR gamma in si gnaling differentiation of NTZ/DI cells. Stable over-expression of RXR beta, but not RXR alpha or RXR gamma, was found to signal NT2/D1 grow th suppression and to induce a non-neuronal morphology and immunopheno type pe. Notably, co-transfection of RAR gamma, and RXR beta resulted in marked growth suppression and for the first time, expression of typ ical neuronal markers of NT2/D1 differentiation. To clarify the role o f RXR beta and RAR gamma in this differentiation program, a modified t ransient fibroblast growth factor-4 (FGF4) promoter-enhancer reporter assay that reflects effective RA-mediated differentiation of NT2/D1 ce lls was employed, Transfection of RAR gamma or RXR beta in NT2/D1 cell s augments transcriptional repression of the FGF4 reporter and RAR gam ma and RXR beta co-transfection markedly repressed reporter activity, indicating the combined role of these receptors in RA-induced NT2/D1 d ifferentiation. Taken together, these findings reveal specific retinoi d receptors must cooperate to signal terminal growth suppression and m aturation of NT2/D1 cells. Since the transcriptional repression of FGF 4 is coupled to the effective maturation of human embryonal carcinoma cells, the described co-transfection strategy should prove useful to i dentify genes with positive or negative effects on the differentiation program of these tumor cells.