Embryonal carcinoma cell lines stably transfected with mRAR beta 2-lacZ: Sensitive system for measuring levels of active retinoids

Embryonal carcinoma cell lines (F9 EC and P19 EC) were stably transfected w ith 1.8 kb promoter sequence of RAR beta 2 coupled to the lacZ gene as a sy stem for measuring active retinoids. These stable transfectants, designated F9-1.8 and P19-1.8, were used as reporter cell lines to investigate differ ent retinoids for their ability to activate the reporter gene. F9-1.8 cells showed similar EC50 values for the acidic retinoids all-trans retinoic aci d (RA), 4-oxo RA, 9 cis RA, and 13-cis RA, in the range of 1-7 nM, while P1 9-1.8 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 sy stem was used to investigate RA metabolism and its inhibition by P450 inhib itors. A combination of RA and liarozole showed a 10 times greater inductio n of the RAR beta 2-lacZ reporter in P19-1.8 cells, but not in F9-1.8 cells . The EC50 value for 4-oxo RA, however, was not altered, indicating that me tabolic conversion of RA to 4-oxo RA is the target for inhibition by liaroz ole in P19-1.8 cells. HPLC analysis revealed nearly complete inhibition of RA metabolism after liarozole treatment in P19-1.8 cells, resulting in high er levels of RA Finally, the F9-1.8 cells were used to detect active retino ids during different stages of chick limb bud development, demonstrating th at 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 anterio r half. (C) 1999 Academic Press.