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Derivation and characterization of retinoid-resistant human embryonal carcinoma cells
M M Moasser1, K S Khoo, W J Maerz
1Laboratory of Molecular Medicine, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Abstract:
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.
Insights
Retinoid resistance in human embryonal carcinoma cells was studied. Resistant clones showed deregulated retinoic acid receptor gamma expression, impacting differentiation and tumor suppression.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Retinoids, including all-trans retinoic acid (RA), regulate cell growth and differentiation via nuclear receptors like retinoic acid receptors (RARs) and retinoid-X receptors (RXRs).
- RA induces neuronal differentiation and represses tumorigenicity in NT2/D1 human embryonal carcinoma cells, while hexamethylene bisacetamide (HMBA) induces a distinct phenotype.
Purpose of the Study:
- To derive and characterize retinoic acid (RA)- and hexamethylene bisacetamide (HMBA)-resistant NT2/D1 cell clones.
- To investigate the molecular mechanisms underlying retinoid resistance, focusing on RAR gamma expression and function.
Main Methods:
- Mutagenesis of NT2/D1 cells followed by selection for resistance to RA or HMBA.
- Cross-resistance studies with 9-cis retinoic acid (9-cis RA).
- Assessment of antitumorigenic effects in athymic mice.
- Analysis of RAR gamma expression and genomic DNA structure using Southern blotting and pulsed-field gel electrophoresis (PFGE).
- Cloning and sequencing of RAR gamma 1 coding region cDNAs.
Main Results:
- Nine RA-resistant (NT2/D1-R) and one HMBA-resistant (NT2/D1-H1) clone were established.
- RA-resistant cells were cross-resistant to 9-cis RA but retained HMBA sensitivity; NT2/D1-H1 cells were resistant to both RA and 9-cis RA.
- RA-resistant clone NT2/D1-R1 overcame RA's antitumorigenic effects in vivo.
- All resistant cells exhibited deregulated RAR gamma expression, without structural genomic alterations in RAR gamma.
- RAR gamma 1 cDNA sequences were identical in sensitive and resistant cells, suggesting regulatory alterations.
Conclusions:
- Retinoid resistance in NT2/D1 cells is associated with deregulated RAR gamma expression, not structural mutations.
- These resistant cell lines serve as valuable tools for studying RAR gamma target genes and differentiation mechanisms in human embryonal carcinomas.