Embryonal carcinoma cell lines stably transfected with mRARbeta2-lacZ: sensitive system for measuring levels of active retinoids.
Embryonal carcinoma cell lines (F9 EC and P19 EC) were stably transfected with 1.8 kb promoter sequence of RARbeta2 coupled to the lacZ gene as a system for measuring active retinoids. These stable transfectants, designated F9-1.8 and P19-1.8, were used as reporter cell lines to investigate different retinoids for their ability to activate the reporter gene. F9-1.8 cells showed similar EC(50) values for the acidic retinoids all-trans retinoic acid (RA), 4-oxo RA, 9-cis RA, and 13-cis RA, in the range of 1-7 nM, while P19-1.8 cells were less sensitive. Retinal showed decreased activity compared to the RA isomers in both lines. However, P19-1.8 cells hardly showed beta-gal activity after treatment with retinol, while the lacZ reporter in F9-1.8 cells was still inducible by this retinoid. In addition, the reporter system was used to investigate RA metabolism and its inhibition by P450 inhibitors. A combination of RA and liarozole showed a 10 times greater induction of the RARbeta2-lacZ reporter in P19-1.8 cells, but not in F9-1.8 cells. The EC(50) value for 4-oxo RA, however, was not altered, indicating that metabolic conversion of RA to 4-oxo RA is the target for inhibition by liarozole in P19-1.8 cells. HPLC analysis revealed nearly complete inhibition of RA metabolism after liarozole treatment in P19-1.8 cells, resulting in higher levels of RA. Finally, the F9-1.8 cells were used to detect active retinoids during different stages of chick limb bud development, demonstrating that it is the limb bud mesenchyme which generates RA and not the epidermis, with a twofold higher level of RA in the posterior half than in the anterior half.
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands.
E
E
Sonneveld
C E
CE
van den Brink
B J
BJ
van der Leede
M
M
Maden
P T
PT
van der Saag
D000818:Animals; D018236:Carcinoma, Embryonal / Q000235:genetics / Q000378:metabolism; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter / Q000235:genetics*; D007093:Imidazoles / Q000494:pharmacology; D007536:Isomerism; D007654:Ketoconazole / Q000494:pharmacology; D018878:Limb Buds / Q000166:cytology / Q000378:metabolism; D008648:Mesoderm / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000235:genetics* / Q000502:physiology; D012176:Retinoids / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D013997:Time Factors; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014212:Tretinoin / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D065607:Cytochrome P-450 Enzyme Inhibitors; D007093:Imidazoles; D018168:Receptors, Retinoic Acid; D012176:Retinoids; C082925:retinoic acid receptor beta; D014212:Tretinoin; D003577:Cytochrome P-450 Enzyme System; C061121:liarozole; D007654:Ketoconazole