Tate A C, Greene G L, DeSombre E R, Jensen E V, Jordan V C
Cancer Res. 1984 Mar;44(3):1012-8.
Radiolabeled estrogens 17 beta-[3H]estradiol and diethylstilbestrol ( [3H]DES) and the antiestrogen [3H]monohydroxytamoxifen ( [3H]MHT) all bind with high affinity to the extranuclear estrogen receptor (ER) from the MCF-7 human breast tumor cell line (Kd = 3 X 10(-10), 2 X 10(-10), and 0.63 X 10(-10) M, respectively). A polyclonal antibody raised in a goat to the calf nuclear ER selectively decreased the binding affinity and number of binding sites for 17 beta-[3H]estradiol, but did not appear to affect these binding parameters for [3H]MHT. In the presence of goat antibody, the binding of the nonsteroidal estrogen DES was so perturbed that it was not possible to quantitate the decreased number of binding sites or affinity of this compound as assessed by Scatchard saturation analysis. These results were confirmed in human breast tumor cytosols by sucrose density gradient analysis. The binding of 17 beta-[3H]-estradiol and [3H]DES to the ER was significantly reduced by preincubation with the polyclonal antibody, whereas the binding of [3H]MHT was reduced only when the tumor cytosol was preincubated with a very high concentration of antibody. At these concentrations of antibody, the binding of 17 beta-[3H]estradiol and [3H]DES to the receptor was prevented completely. In contrast, when the antibody was added to the tumor cytosol after the 3H-ligand had bound to the receptor, the binding properties of all 3H-ligands were unaffected. The [3H]MHT-ER antibody complex consistently sedimented as a higher-molecular-weight complex on sucrose density gradients than did the corresponding estrogenic complexes. The decrease in the affinity of estrogenic ligands can be explained in part by an increase in the dissociation rate at 4 degrees of these compounds from the ER. The dissociation rate of MHT was unaffected by the goat antibody. These results imply that there are important differences in the binding of antiestrogen and estrogens to the tumor cytosol ER. A ligand-binding model is proposed that may aid in the understanding of antiestrogen action.
放射性标记的雌激素17β-[3H]雌二醇和己烯雌酚（[3H]DES）以及抗雌激素[3H]单羟基他莫昔芬（[3H]MHT）都能与MCF-7人乳腺癌细胞系的核外雌激素受体（ER）高亲和力结合（解离常数Kd分别为3×10^(-10)、2×10^(-10)和0.63×10^(-10)M）。用山羊制备的针对小牛核ER的多克隆抗体选择性地降低了17β-[3H]雌二醇的结合亲和力和结合位点数，但似乎不影响[3H]MHT的这些结合参数。在山羊抗体存在下，非甾体雌激素DES的结合受到如此大的干扰，以至于通过Scatchard饱和分析无法定量该化合物结合位点数量的减少或亲和力的降低。这些结果通过蔗糖密度梯度分析在人乳腺癌细胞溶质中得到证实。与多克隆抗体预孵育后，17β-[3H]雌二醇和[3H]DES与ER的结合显著降低，而只有当肿瘤细胞溶质与非常高浓度的抗体预孵育时，[3H]MHT的结合才会降低。在这些抗体浓度下，17β-[3H]雌二醇和[3H]DES与受体的结合被完全阻止。相反，当3H配体与受体结合后将抗体加入肿瘤细胞溶质中时，所有3H配体的结合特性均未受影响。[3H]MHT-ER抗体复合物在蔗糖密度梯度上始终比相应的雌激素复合物沉降为更高分子量的复合物。雌激素配体亲和力的降低部分可通过这些化合物在4℃时从ER的解离速率增加来解释。MHT的解离速率不受山羊抗体的影响。这些结果表明抗雌激素和雌激素与肿瘤细胞溶质ER的结合存在重要差异。提出了一个配体结合模型，可能有助于理解抗雌激素作用。
