Derivation and characterization of retinoid-resistant human embryonal carcinoma cells.

The retinoids exert potent growth and differentiation effects on normal and neoplastic cells through two families of nuclear receptors. These are the retinoic acid receptors (RAR alpha, RAR beta, RAR gamma) and the retinoid-X receptors (RXR alpha, RXR beta, RXR gamma). All-trans retinoic acid (RA) induces terminal neuronal differentiation and represses tumorigenicity of the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1). Hexamethylene bisacetamide (HMBA) induces a phenotype distinct from RA-treated NT2/D1 cells. This study reports the derivation and characterization of RA- and HMBA-resistant NT2/D1 clones. Nine RA-resistant (NT2/D1-R1 through NT2/D1-R9) and one HMBA-resistant (NT2/D1-H1) clones were derived after mutagen treatment of NT2/D1 cells and selection in RA or HMBA. NT2/D1-R cells were cross-resistant to 9-cis retinoic acid (9-cis RA), a ligand activating the RAR and RXR pathways, but retained maturation response to HMBA. A representative RA-resistant clone, NT2/D1-R1, overcame the antitumorigenic actions of RA as assessed in athymic mice. NT2/D1-H1 cells were dually resistant to RA and 9-cis RA. All these retinoid resistant cells exhibit deregulated expression of RAR gamma but not RAR alpha or RAR beta. Southern analysis using RAR gamma probes shows no apparent structural differences in genomic DNA between NT2/D1 cells and the RA-resistant subclones. Pulsed-field gel electrophoresis (PFGE) with RAR gamma probes demonstrated an Mlu-I restriction fragment length polymorphism, but no other structural abnormalities in these cells or a panel of germ cell tumor (GCT) cell lines. Full-length RAR gamma 1 coding region cDNAs were cloned from NT2/D1 and NT2/D1-R1 cells and these sequences were identical, suggesting RA resistance in these cells is due to altered regulation of RAR gamma. These differentiation-resistant cells are useful to study RAR gamma target genes or mechanisms engaged by these differentiation inducing agents in human embryonal carcinomas.