Increased DNA Binding of the Estrogen Receptor in an Estrogen-resistant Mammary Cancer

Abstract

In the C3H mouse mammary adenocarcinoma, estradiol cannot induce the progesterone receptor, and the tumor growth rate is not decreased by ovariectomy. To find an explanation for this estrogen resistance, we have compared the estrogen receptor (ER) from this tumor to the ER of uterus and of the mammary tumors induced in rats by dimethylbenz(a)anthracene. Since the ER concentration of the C3H tumor is low (≃20 fmol/mg protein), we have used iodoestradiol of high specific activity to label the receptor.

Several criteria of ER activation were studied. The dissociation rates of estradiol with or without sodium molybdate were similar in all tissues. In metrizamide isopycnic gradients, ER from rat uterus and C3H tumor had a similar density, both in the presence or absence of DNA. The binding of ER to DNA-cellulose was analyzed by incubating to equilibrium a constant amount of ER with a variable amount of DNA, the cellulose concentration being kept constant. The saturation data were plotted according to the method of Scatchard. The apparent affinity for DNA of the cytosol ER was similar for the rat dimethylbenz(a)anthracene tumors and the uterus (K_d ≃ 10 µm) but was significantly higher for the C3H tumor ER (K_d ≃ 2.3 µm). Neither the substitution of estradiol by iodoestradiol, nor the difference in cytosol protein and ER concentrations, nor the nonspecific steroid binding to DNA-cellulose could explain this result. This difference was confirmed when using DNA-agarose or soluble DNA in sucrose gradients. Finally, the salt concentrations necessary to elute ER from DNA-cellulose columns were 0.20 and 0.28 m for uterine and C3H tumor ER, respectively. To conclude, the C3H tumor has a low content of ER which appears to have a higher affinity for DNA than the ER of estrogen-responsive tissue. We suggest that the reason for the inefficiency of ER in the C3H tumor may be related to its increased affinity for nonspecific DNA sites.