Nuclear [³H]4-hydroxytamoxifen (4-OHTAM)- and [³H]estradiol (E₂)-estrogen receptor complexes in the MCF-7 breast cancer and GH₃ pituitary tumor cell lines

Nuclear [³H]4-OHTAM-ER complexes extracted by 0.6 M KC1 from the MCF-7 human breast cancer and the GH₃ rat pituitary tumor cell lines, sedimented as a 5S form on sucrose gradients after 1 to 24 h exposure to ligand (20 nM). The nuclear [³H]4-OHTAM-ER from MCF-7 cells increased from 2 + 0.2 pmoles/mg DNA at l h to approximately 4 + 0.1 pmoles/mg DNA at 6 and 24 h. In the GH₃ cells the nuclear binding of [³H]4-OHTAM increased rapidly, and there was no significant difference in the receptor levels at 1 and 6 h (10 ± 1.3 and 8.9 ± 1.1 pmoles/mg DNA, respectively). At 24 h there was a decrease in [³H]4-OHTAM-ER levels (7.0 ± 0.2 pmoles/mg DNA); however, this was due primarily to an increase in DNA synthesis, which occurred in these cells by 24 h. It appears that in both cell lines there is no processing of nuclear [³H]4-OHTAM-ER, and it is not associated with changes in sedimentation coefficient of the complex. When both cell lines were incubated with[³H]E₂ (20 nM), processing of the nuclear [³H]E₂-ER occurred over 6 h. In the MCF-7 cells there was a decrease in receptor content within 6 h from 3.2 ± 0.2 to 0.9 ± 0.2 pmoles/mg DNA. The nuclear [³H]E₂-ER in the GH₃ cells decreased from 7.3 ± 0.5 to 2.8 + 0.3 pmoles/mg DNA. Although these cell lines appeared to process the [³H]E₂-ER complex, the nuclear forms of [³H]E₂-ER were different. In the GH₃ cells the nuclear [³H]E₂-ER consistently sedimented as a 5S complex whereas the nuclear receptor from MCF-7 cells sedimented mainly as a 4S complex. Occasionally (20% incidence) both 4S and 5S [³H]E₂-ER forms were observed which were recognized by a monoclonal antibody (D547) raised to the ER. These data suggest that the activity of estradiol and 4-OHTAM in vitro may be more related to nuclear processing, or lack of it, than to the physical form of the ligand-receptor complex.