CHARACTERIZATION AND ASSAY OF PROGESTERONE RECEPTOR IN HUMAN MAMMARY-CARCINOMA

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  • 1 January 1977
    • journal article
    • research article
    • Vol. 37  (2) , 464-471
Abstract
[3H]Pregn-4-ene-3,20-dione ([3H]progesterone)-receptor complexes from human mammary carcinoma were stabilized in the presence of glycerol. The dissociation rate constant was lowered and the equilibrium dissociation constant was decreased (Kd = 3 nM in the absence of glycerol and 1.1 nM in the presence of 30% glycerol), whereas no clear-cut effect on the association rate was observed and no change occurred in the concentration of binding sites. Cortisol competed with [3H]progesterone only at concentrations higher than 1 .mu.M. This made it possible to distinguish [3H]progesterone binding to the receptor from binding to corticosteroid-binding globulin. Synthetic progestins [6-chloro-17-acetoxypregna-4,6-diene-3,20-dione (chlormadinone acetate), 17.alpha.-ethinyl, 17-hydroxyestr-4-en-3-one (norethisterone) and 17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione (R5020)] had a high affinity for the receptor, whereas 5.alpha.-pregnane-3,20-dione had an affinity about 1/2 that of progesterone itself. 5.beta.-Pregnane-3,20-dione, 17.alpha.-hydroxypregn-4-ene-3,20-dione (estradiol), 11.beta.,21-dihydroxypregn-4-ene-3,20-dione (corticosterone), estra-1,3,5(10)-triene-3,17.beta.-diol and 17.beta.-hydroxyandrost-4-en-3-one (testosterone) were weak inhibitors of [3H]progesterone binding. Sedimentation on glycerol gradients showed different patterns in different tumors, i.e., [3H]progesterone specific binding having the characteristics of receptor was found in the 8 S region, the 4.5 S region or both. Activated progesterone-receptor complex from human mammary carcinoma cytosol bound to human DNA. An assay of the receptor based on these binding properties is described. This assay measures the total concentration of cytosol receptor since it makes possible the exchange of endogenous hormone for excess added [3H]progesterone. Of 55 biopsies examined by this method, 35 (64%) had a concentration of progesterone receptor binding sites higher than 10 fmol/mg protein. There was a positive correlation between the amounts of estrogen and progesterone receptors.