DERIVATION AND CHARACTERIZATION OF RETINOID-RESISTANT HUMAN EMBRYONALCARCINOMA-CELLS

The retinoids exert potent growth and differentiation effects on norma l and neoplastic cells through two families of nuclear receptors. Thes e 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 repre sses tumorigenicity of the multipotent human embry onal carcinoma cell line NTERA-2 clone D1 (NT2/D1). Hexamethylene bisacetamide (HMBA) ind uces a phenotype distinct from RA-treated NT2/D1 cells. This study rep orts the derivation and characterization of RA- and HMBA-resistant NT2 /D1 clones. Nine RA-resistant (NT2/D1-R1 through NT2/D1-R9) and one HM BA-resistant (NT2/D1-H1) clones were derived after mutagen treatment o f 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 R AR and RXR pathways, but retained maturation response to HMBA. A repre sentative 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 exhi bit deregulated expression of RARY but not RAR alpha or RAR beta. Sout hern analysis using RAR gamma probes shows no apparent structural diff erences in genomic DNA between NT2/D1 cells and the RA-resistant subcl ones. Pulsed-field gel electrophoresis (PFGE) with RAR gamma probes de monstrated an Mlu-I restriction fragment length polymorphism, but no o ther structural abnormalities in these cells or a panel of germ cell t umor (GCT) cell lines. Full-length RAR gamma 1 coding region cDNAs wer e cloned from NT2/D1 and NT2/D1-R1 cells and these sequences were iden tical, suggesting RA resistance in these cells is due to altered regul ation of RAR gamma. These differentiation-resistant cells are useful t o study RAR gamma target genes or mechanisms engaged by these differen tiation inducing agents in human embryonal carcinomas.