Specific retinoid receptors cooperate to signal growth suppression and maturation of human embryonal carcinoma cells

Abstract

This study addresses the contributions of speci®c retinoid receptors during all-trans-retinoic acid (RA)-mediated dierentiation and growth suppression of human embryonal carcinoma cells. The pleiotropic eects of RA are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs), members of the nuclear receptor family of transcription factors. After RA-treatment the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) displays limited proliferative potential, reduced tumorigenicity, and morphologic and immunophenotypic neuronal maturation. RARg over-expression in NT2/D1 cells signals mesenchymal NT2/D1 terminal dierentiation while RARa and RARb do not and RARg overcomes retinoid resistance in an NT2/D1 clone (NT2/D1-R1) having deregulated RARg expression. Since RARg transfectants do not display neuronal maturation, this study sought to identify cooperating retinoid receptors engaged in NT2/D1 dierentiation. Through gain of function experiments, this report highlights RXRb as playing an important role along with RARg in signaling dierentiation of NT2/D1 cells. Stable over-expression of RXRb, but not RXRa or RXRg, was found to signal NT2/D1 growth suppression and to induce a non-neuronal morphology and immunophenotype. Notably, co-transfection of RARg and RXRb resulted in marked growth suppression and for the ®rst time, expression of typical neuronal markers of NT2/D1 dierentiation. To clarify the role of RXRb and RARg in this dierentiation program, a modi®ed transient ®broblast growth factor-4 (FGF4) promoter-enhancer reporter assay that re¯ects eective RA-mediated dierentiation of NT2/D1 cells was employed. Transfection of RARg or RXRb in NT2/D1 cells augments transcriptional repression of the FGF4 reporter and RARg and RXRb co-transfection markedly repressed reporter activity, indicating the combined role of these receptors in RA-induced NT2/D1 dierentiation. Taken together, these ®ndings reveal speci®c retinoid receptors must cooperate to signal terminal growth suppression and maturation of NT2/D1 cells. Since the transcriptional repression of FGF4 is coupled to the eective maturation of human embryonal carcinoma cells, the described co-transfection strategy should prove useful to identify genes with positive or negative eects on the dierentiation program of these tumor cells.

The secreted glycoprotein CREG enhances differentiation of NTERA-2 human embryonal carcinoma cells

“…To our knowledge, this is the ®rst study examining the e ects of overexpressing a secreted protein that is up-regulated during NTERA-2 di erentiation. Interestingly, the phenotype observed when CREG is overexpressed in these cells is di erent from the e ects of constitutive expression of other proteins implicated in the control of NTERA-2 cell growth and di erentiation (Baselga et al, 1993;Maerz et al, 1998;Moasser et al, 1995;Spinella et al, 1998). We have found that CREG increases the number of cells that di erentiate both in the absence of an inducer and in response to RA.…”

“…Previous studies have shown that overexpression of either RARg or RXRb in NTERA-2 cells leads to high levels of di erentiation and a concomitant inhibition of proliferation in the absence of RA (Moasser et al, 1995;Spinella et al, 1998). Growth arrest and morphologic di erentiation was observed several weeks after induction of RXRb overexpression .…”

p53 in human embryonal carcinoma: identification of a transferable, transcriptional repression domain in the N-terminal region of p53

“…This finding is notable because it suggests that RA-mediated reversal of p53-NRD repression is RAR dependent. Furthermore, the relief of p53-NRD-mediated repression in the presence of RA and RARg is consistent with the ability of restored RARg expression to restore RA activation of p53 in NT2/D1-R1 cells (Curtin et al, 2001), and the established ability of RARg to reverse RA resistance in NT2/D1-R1 cells (Moasser et al, 1995;Spinella et al, 1998).…”

“…NT2/D1-R1 cells are deficient in the expression of RARg, and engineered expression of RARg can restore the ability of RA to signal growth suppression and differentiation in these cells (Moasser et al, 1995(Moasser et al, , 1996Spinella et al, 1998) and RARg restored RAmediated activation of full-length p53 (Curtin et al, Figure 5 p53-NRD residues 100-116 are important for p53-NRDmediated repression in human EC. (a) Alanine substitutions along the p53-NRD have minimal effects on p53-NRD-mediated repression.…”

“…RA-resistant NT2/D1-R1 cells do not undergo growth arrest, differentiation, or loss of tumorigenicity in response to RA due to a defect in RARg expression (Moasser et al, 1996). However, restoration of RARg expression restores RA-sensitivity to these cells (Moasser et al, 1995;Spinella et al, 1998) Restoration of RAR expression led to restoration of p53 activation, suggesting that activation of p53 by RA is RAR-dependent (Curtin et al, 2001). Furthermore, RAresistant NT2/D1-R1 cells displayed a striking crossresistance to cisplatin treatment (B7-10-fold) relative to parental NT2/D1 cells (Curtin et al, 2001).…”