IDENTIFICATION OF ESTROGEN-REGULATED GENES IN FE33 RAT HEPATOMA-CELLSBY DIFFERENTIAL DISPLAY POLYMERASE CHAIN-REACTION AND THEIR HORMONAL-REGULATION IN RAT-LIVER AND UTERUS

We applied the differential display RT-PCR (ddRT-PCR) technology to id entify estrogen-regulated hepatic genes in the estrogen receptor expre ssing rat hepatoma cell line Fe33. Three genes of known sequences were detected by the ddRT-PCR approach: IGF binding protein-1 (IGFBP-1), v itamin D-dependent calcium-binding protein (CaBP9k) and major acute ph ase protein (MAP). Effects of ethinyl estradiol on the mRNA levels of these genes were confirmed by ''Northern-blot'' analysis. If given in combination with dexamethasone and glucagon, ethinyl estradiol caused 40-, 15- and ii-fold increases in the mRNA steady state level of IGFBP -1, CaBP9k and MAP, respectively, in Fe33 cells 24 h after addition of hormone. Besides ethinyl estradiol, the partial estrogen agonist OH-t amoxifen caused dose dependent effects on expression of MAP and IGFBP- 1. Estrogen regulation of the respective genes and the modulatory effe cts of progesterone (10 mg/animal/day) were studied in ovariectomized rats treated subcutaneously for 14 days with 1 mu g/animal/day estradi ol. ''Northern-blot'' analysis of liver RNA revealed a 6-fold stimulat ion of IGFBP-1 mRNA levels in estradiol-treated compared to vehicle-tr eated rats and a weak but detectable increase of MAP mRNA steady state level (1.6-fold) upon estradiol administration. No effect of estradio l treatment could be monitored for CaBP9k in rat liver. Modulatory eff ects of progesterone on estradiol-stimulated expression in the liver c ould be monitored for IGFBP-1 only. In an extension of our investigati on on the expression of the three genes in rat liver, we determined th eir expression and hormonal regulation in the uterus of the same anima ls. In the uterus, estradiol caused an increase in CaBP9k mRNA. In con trast, IGFBP-1 mRNA levels increased dramatically upon progesterone ad ministration, whereas no effect of estradiol treatment could be detect ed. MAP mRNA levels increased only after coadministration of estradiol and progesterone. In conclusion, the ddRT-PCR proved to be a powerful method to identify estrogen-regulated genes. The study on the hormona l regulation of three genes stimulated by estrogen in Fe33 cells revea led similarities and differences in their regulation in vivo and in vi tro.