The effect of estradiol (E2) on the [3H]-acetylation of nuclear histones was studied in the MCF-7 human mammary cancer cell line in culture. Cells (approximately 10(8) were incubated with 8 x 10(-6) M [3H]-acetate in the absence (control) or in the presence of estradiol (10(-5)-10(-8) M). After 20 min incubation, the nuclear histones were extracted and separated by electrophoresis, and each histone band was measured and calculated in DPM/mg protein. It was observed that only the H2 + H3 and H4 histones were associated with the [3H]-acetate. Estradiol (10(-6)-10(-8) M) provoked a significant inhibition in the incorporation of the acetate. The negative effect, in percentage of the non-treated cell value, was as follows: in E2 (10(-6) M): -25 +/- 10 (SE) for H2 + H3 and -26 +/- 5 for H4; in E2 (10(-7) M): -35 +/- 9 and -39 +/- 10; and in E2 (10(-8) M): -56 +/- 22 and -30 +/- 13 respectively. It is concluded that estradiol has a negative effect in the acetylation of H2, H3 and H4 histones of this mammary cancer cell; no acetylation or effect is observed in H1 histones. The relationship of this finding to the biological responses of the hormone is to be explored.
The 9S nonactivated oligomeric estrogen receptor from fetal guinea pig uterus interacts with the H222 monoclonal antibody (whose epitope is located in the hormone-binding domain) to yield an 11S complex and with the H226 monoclonal antibody (whose epitope is located in the A/B region, just N-terminal to the DNA-binding domain) to yield a 9.4S complex. No reaction was detected with the D547 monoclonal antibody (whose epitope is located between the hormone-binding and DNA-binding domains). In high salt gradients, the 11S oligomeric receptor-H222 complex dissociates to a 8S monomer-H222 complex and the 9.4S oligomeric receptor-H226 complex to a 7S monomer-H226 complex plus the 4.5S monomer receptor not bound to the antibody. These observations suggest that the nonactivated oligomeric receptor contains more than one estradiol-binding subunit with a structure such that the H222 epitopes are fully accessible, the H226 epitopes are partially accessible, and the D547 epitopes are masked. The temperature-activated receptor reacts with the H226 antibody to yield two complexes that sediment at 7S and 9S in high salt gradients. The 9S complex corresponds to a receptor form complexed with more than one antibody molecule; therefore, this suggests the formation of a receptor homodimer where the two H226 epitopes are exposed. However, a single 8S peak is observed when the H222 antibody reacts with the activated receptor, suggesting that only one H222 epitope is accessible in the dimeric receptor. In addition, binding to the H222 antibody before activation prevents dimerization. Thus, the H222 appears to be close to the dimerization domain. The activated receptor reacts with the D547 antibody to yield a 8S complex that apparently contains only one antibody molecule. On the other hand, the receptor extracted from nuclei was found to be a single 5.5S form with the same immunological characteristics as the receptor activated in cytosol. In conclusion, interaction of these monoclonal antibodies with the different forms of the estrogen receptor reveals a structural transformation during activation, with a concomitant change in the exposure of the receptor functional domains.
Estrogen and progesterone receptors were characterized in the fetal and newborn vagina of guinea pig. The concentrations of estrogen receptor (total sites, cytosol plus nuclei) which were very high [7090 +/- 1700 (+/- SD) fmol/mg DNA] in the fetal vagina (62-64 days gestation) decreased significantly after birth and increased slightly at 2 weeks of age. Tamoxifen (TAM) and estradiol (E2) or the combined treatment (TAM + E2) induced a great increase in weight and DNA content in both the fetal and newborn vagina. After a 12-day treatment to pregnant or newborn guinea pigs, the fetal vagina wet weights (milligrams +/- SD) were as follows: control animals, 87 +/- 14; +TAM, 254 +/- 38; +E2, 177 +/- 19; +(TAM+E2), 218 +/- 41. The values in newborn vaginas were: 155 +/- 40, 434 +/- 75, 477 +/- 49, and 512 +/- 76, respectively. The DNA contents per vagina (in micrograms per +/- SD) in the fetal tissues were as follows: control, 187 +/- 64; +TAM, 563 +/- 74; +E2, 650 +/- 100; and +(TAM+E2), 776 +/- 113; in the newborn vagina these values were: 592 +/- 75, 880 +/- 113; 781 +/- 75, and 901 +/- 15, respectively. Histological studies showed drastic morphological alterations after each of the treatments, particularly in the epithelial cells. Similarly, the ultrastructural examination with transmission electron microscopy showed the alteration of mitochondria, the development of the rough endoplasmic reticulum, and the formation of numerous vacuoles and secretory granules. TAM stimulated the number of specific sites for progesterone, but less intensely than did E2. However, in the combined experiment (TAM+E2), TAM did not block the action of E2. It is concluded that TAM acts in the fetal and newborn vagina of guinea pigs as a real estrogen agonist.
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