A cytosol protease from the estrogen-resistant C3H mammary carcinoma increases the affinity of the estrogen receptor for DNA in vitro

Abstract

We have previously shown, in the estrogen-unresponsive C3H mouse mammary tumor that the affinity of the estrogen receptor (ER) for calf thymus DNA in vitro is four-times higher than that of uterine ER [Baskevitch, P. P., Vignon, F., Bousquet, C. and Rochefort, H. (1983) Cancer Res. 43,22901. By mixing cytosols from this tumor and uterus, we describe a tumor factor capable of increasing ER affinity for DNA, as assayed by DNA-cellulose chromatography and saturation studies. The activity of this factor was inhibited by a-chymotrypsin-inhibitors such as N-tosylphenylalanylchloromethane and chymostatin. Using the fluorogenic substrate glutarylglycylglycylphenylalanyl-N-naphthylamide, we assayed such a protease in the C3H mammary tumor cytosol. This protease and the factor altering ER-DNA binding were eluted together from chromatography on DEAE-cellulose, AcA 44, and carboline-agarose and were sensitive to the same inhibitors. The partially purified factor decreases the molecular mass of the estrogen receptor as seen by denaturing electrophoresis after covalent labelling of the ER with [3H]tamoxifen aziridine. We suggest that the increase of ER affinity for DNA and the decrease of ER molecular size are due to the same protease with an a-chymotrypsin-like specificity.Systems resistant to steroid hormones are useful in analyzing the sequence of hormone action, particularly the nuclear and DNA binding of the receptor-hormone complex, and in determining their biological significance [I]. In mammary tumors, estrogen resistance is mostly associated with the lack of assayable estrogen receptor sites [2]. However, in humans, 50% of breast cancers which possess estrogen receptor (ER-positive) do not respond to endocrine therapy. One possibility is that the ER are present, and are capable of binding the hormone, but are unable to transduce the message efficiently to chromatin. In this respect, hormone-resistant and ER-positive experimental mammary tumors are particularly interesting.We have investigated the ER system in the C3H mouse mammary carcinoma. This tumor is estrogen-unresponsive since in vivo estradiol is unable to increase the concentration of the progesterone receptor sites, and does not sustain tumor growth [3, 41. In this tumor, the ER is present at moderate concentrations (20 -40 fmol/mg cytosol protein) and, after estrogen binding, is translocated into the nucleus in vivo [3]. Therefore the estrogen-unresponsiveness of the tumor is not due to the absence of nuclear ER. It may therefore be the consequence of a post-receptor defect [5]. We have recently shown that the ER from C3H tumor has a fourfold higher affinity in vitro for non-specific DNA as compared to the ER

The proton-driven dissociation of oestradiol-receptor dimers as a preparative tool. Isolation of a 32 kDa fragment from porcine uteri and assignment of C-terminal origin by partial sequencing

“…The tryptic peptides P1 and P2 have been aligned by sequence comparison with domains E/F. endopeptidases in C3H-mouse mammary tumour [26]. We took advantage of the limited receptor digestion by heparin-Sepharose-bound endogenous proteinase(s) from porcine uteri [4] and arrived at relatively stable, well-defined, fragments.…”

Age-associated changes in the estrogen receptor-active proteases of female rabbit liver

“…Proteolysis has been proposed as a mechanism for the activation of receptors t o forms with increased binding affinity for DNA (Hubbard et al 1984;Baskevitch and Rochefort 1985;Puca et al 1986). Horigome et al (1988) observed that the 54 000 but not the 37 000 fragment of the estrogen receptor of mouse uterus retained the ability to bind to DNA.…”

Colloidal carbon blocks oestrogen-induced migration of eosinophils to the uterus and the uterine water imbibition response

“…These responses are considered to be genomic responses to oestrogen, since they are blocked by inhibitors of RNA and protein synthesis (Ui & Mueller, 1963;Tchernitchin & Galand, 1982;Finlay, Katz, Kirsch et al 1983). This has led to the concept that genomic responses to steroid hormone stimulation are the result of the interaction of hormone-receptor complexes with regulatory DNA sequences (Payvar, Wränge, Carlstedt-Duke et al 1981;Mulvihill, LePennec & Chambón, 1982;Dean, Gope, Knoll et al 1984;Rousseau, 1984;Baskevitch & Rochefort, 1985;Jost, Geiser & Seldran, 1985;Moore, Marks, Buckley et al 1985;Shull & Gorski, 1985). This has led to the concept that genomic responses to steroid hormone stimulation are the result of the interaction of hormone-receptor complexes with regulatory DNA sequences (Payvar, Wränge, Carlstedt-Duke et al 1981;Mulvihill, LePennec & Chambón, 1982;Dean, Gope, Knoll et al 1984;Rousseau, 1984;Baskevitch & Rochefort, 1985;Jost, Geiser & Seldran, 1985;Moore, Marks, Buckley et al 1985;Shull & Gorski, 1985).…”