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 growth suppression of human embryonal carcinoma cells. The pleiotropic effects 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. RARgamma over-expression in NT2/D1 cells signals mesenchymal NT2/D1 terminal differentiation while RARalpha and RARbeta do not and RARgamma overcomes retinoid resistance in an NT2/D1 clone (NT2/D1-RI) having deregulated RARgamma expression. Since RARgamma transfectants do not display neuronal maturation, this study sought to identify cooperating retinoid receptors engaged in NT2/D1 differentiation. Through gain of function experiments, this report highlights RXRbeta as playing an important role along with RARgamma in signaling differentiation of NT2/D1 cells. Stable over-expression of RXRbeta, but not RXRalpha or RXRgamma, was found to signal NT2/D1 growth suppression and to induce a non-neuronal morphology and immunophenotype. Notably, co-transfection of RARgamma and RXRbeta resulted in marked growth suppression and for the first time, expression of typical neuronal markers of NT2/D1 differentiation. To clarify the role of RXRbeta and RARgamma in this differentiation program, a modified transient fibroblast growth factor-4 (FGF4) promoter-enhancer reporter assay that reflects effective RA-mediated differentiation of NT2/D1 cells was employed. Transfection of RARgamma or RXRbeta in NT2/D1 cells augments transcriptional repression of the FGF4 reporter and RARgamma and RXRbeta co-transfection markedly repressed reporter activity, indicating the combined role of these receptors in RA-induced NT2/D1 differentiation. Taken together, these findings reveal specific 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 effective maturation of human embryonal carcinoma cells, the described co-transfection strategy should prove useful to identify genes with positive or negative effects on the differentiation program of these tumor cells.
Department of Medicine, Memorial Hospital, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
M J
MJ
Spinella
S
S
Kitareewan
B
B
Mellado
D
D
Sekula
K S
KS
Khoo
E
E
Dmitrovsky
D018236:Carcinoma, Embryonal / Q000378:metabolism*; D002454:Cell Differentiation; D002455:Cell Division; D019281:Dimerization; D051519:Fibroblast Growth Factor 4; D005346:Fibroblast Growth Factors / Q000096:biosynthesis / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006131:Growth Inhibitors; D006801:Humans; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D047488:Retinoid X Receptors; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*
C496445:FGF4 protein, human; D051519:Fibroblast Growth Factor 4; D006131:Growth Inhibitors; D011518:Proto-Oncogene Proteins; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014212:Tretinoin; D005346:Fibroblast Growth Factors