Jean-Claude Heuson

Aromatase, 17β-hydroxysteroid dehydrogenase and intratissular sex hormone concentrations in cancerous and normal glandular breast tissue in postmenopausal women

In a study of the origin of estrogens in patients with breast cancer, the concentrations of estrogens and their androgen precursors, and aromatase and 17 beta-hydroxysteroid dehydrogenase (E2DH) activities were determined in normal glandular and cancerous breast tissue. The correlation between tissue estrogens, precursor concentrations, enzyme activities and plasma levels and/or receptor status were calculated. In both normal glandular and carcinomatous breast tissue, the concentrations of androstenedione (A), dehydroepiandrosterone (DHEA), 5 androstene-3 beta, 17 beta-diol (5-Adiol), estrone (E1), estradiol (E2) and progesterone (P) were significantly higher than plasma concentrations. While testosterone (T) concentrations were similar, dehydroepiandrosterone (DHCA) and estrone sulphate (E1S) concentrations were lower in tissue than in plasma. In carcinomatous tissue androgen concentrations were lower, but estrogen concentrations were higher than in glandular breast tissue. Estradiol (E2) concentration was positively correlated with the receptor concentration with the mean E2 concentration corresponding to an estimated receptor occupancy of about 25%, probably sufficient for a submaximal biological response. Aromatase and E2DH (E2----E1) activities were observed in all breast cancer and glandular breast tissues, activities being higher in carcinoma than in glandular breast tissues; nevertheless, aromatase activity accounts probably only for a small fraction of tissue estrogen concentration. E2DH, but not aromatase activity, was significantly higher in estrogen receptor positive than in estrogen receptor negative tissues and was negatively correlated with tissue dehydroepiandrosterone (DHEA) and its sulphate (DHEAS) concentration; the latter two steroids are non competitive inhibitors of E2DH which inactivates E2 to E1. This effect of DHEA(S) may constitute a mechanism by which these androgens stimulate cancer growth and a rationale (besides suppression of estrogen precursors) for medical or surgical adrenalectomy in hormone sensitive metastatic mammary cancer. E2DH activity might constitute an additional marker of hormone dependency of mammary cancer.

Clinical significance of the quantitative assessment of estrogen receptors in advanced breast cancer

The predictive value of the estrogen receptor (ER) assay with regard to the response to hormonal treatment was analyzed in women with advanced breast carcinoma. The significance of ten clinical variables of putative prognostic value was also investigated. A total of 49 courses of endocrine therapy were available for study. The respective merits of using the receptor information as a qualitative or a quantitative variable were compared. Linear logistic regression analysis showed that the quantitative information was significantly related to the therapeutic response (P < 0.0001) and proved to be superior to the qualitative information. Compared with the clinical variables tested with the logistic model, receptor concentration was by far the most important single predictor of response. Nevertheless, introduction of two of these clinical variables (i.e., age and menopausal status) into the model in addition to receptor concentration improved its predictive value. Presented in graphic form, the improved model provides a simple means to estimate the probability that a given patient will respond to endocrine therapy.

Assay for estrogen and progesterone receptors of breast cancer cell lines in monolayer culture

A whole-cell assay for measuring estrogen (ER) and progesterone (PgR) receptors in monolayer culture of human breast cancer cell lines is described. It is based on the measurement of incorporated tritiated ligands during 50 min of incubation (i.e. [3H]estradiol for ER, [3H]ORG-2058 for PgR). The assay fulfills all criteria of specificity as shown by competitive studies and measurements of the dissociation constants of the binding reactions. Moreover, a subcellular fractionation of MCF-7 labeled cells revealed that the majority of incorporated steroids was associated with the nuclear fraction. This finding is consistent with the concept of nuclear location of steroid-receptor complexes. Cultures in the presence of 10(-8) M estradiol indicated that the methodology is adequate for detecting the well-known estrogenic induction of PgR synthesis. The assay proved suitable for the quantitative assessment of the receptor content of various neoplastic (MCF-7; ZR-75-1, Cama-1, Evsa-T) and non-neoplastic (HBL-100) cell lines. The methodology has the other advantages of being simple and rapid, of requiring small amounts of cells and of allowing histological examination of the latter before, during and after biochemical analysis.

Estrogen receptors in human breast cancer

Abstract Samples from 77 primary and 65 metastatic human breast cancers were assayed for specific cytoplasmic estrogen receptors. Cytosol preparations were incubated with increasing amounts of 3 H-estradiol- 17 β. The unbound radioactivity was removed by charcoal-coated dextran. Saturable high affinity binding sites were detected in 56% of the primary and 37% of the metastatic tumors. The binding constants of these receptor sites for estradiol were quite variable; most ranged from 1·0 to 20 × 10 −10 M, but some were larger (up to 108 × 10 −10 M). The concentrations of binding sites were distributed within a continuous range from 5 to 1330 femtomoles per mg protein. This wide range was not ascribable only to variations in amounts of contaminating serum proteins. Receptors were detected in only 8% of cytosol preparations containing less than 2 mg protein per ml; in contrast they were detected in 53% of cases with higher protein concentration. This indicates that at low protein concentration, false negative results are likely to occur. Detectable amounts of receptors were not found in sera or samples from normal mammary gland, nipple, areola, skin and non-invaded lymph nodes. The reliability of a simplified procedure based on isotopic dilution of the labelled estradiol, to be used with very small tumor tissue samples, was studied. It compared quite well with the other one except in case of very low concentrations of receptor. No relationship was found between the occurrence of receptor and the age of the patient or the histological type of the tumor. There was also no relationship between the occurrence of receptors in the primary tumor and presence or absence of metastatic axillary lymph nodes. However, when present, the latter had the same characteristics with respect to the receptor as the corresponding primary, with only one exception. In cases of metastatic tumors, no correlation was observed with the free interval.

Inhibition of DMBA-Induced Mammary Carcinogenesis in the Rat by 2-Br-α-Ergocryptine (CB 154), An Inhibitor of Prolactin Secretion, and by Nafoxidine (U-11, 100 A), an Estrogen Antagonist

The DMBA-induced mammary carcinogenesis in the Sprague-Dawley rat is known to be both estrogen and prolactin dependent. The experiments presented here show that 2-Br-Α-ergocryptine (CB 154), an inhibitor of prolactin secretion, and nafoxidine (, 100 A), an estrogen antagonist, inhibit the U 11 successive stages of carcinogenesis, namely formation of preneo plastic nodules, transformation of the latter into tumors, and finally growth of the established tumors. Endocrine studies, involving histological examination of organs and serum prolactin determination by radioimmunoassay, show that CB 154 inhibits prolactin secretion and produces peripheral changes consistent with this effect, and that nafoxidine behaves as an antiestrogen in intact animals and in oophorectomized animals given estrogens, that it is devoid of intrinsic estrogenic properties but rather displays progestational-like effects, and that it inhibits prolactin stimulation by estrogens in oophorectomized rats. The antitumor effect of CB 154 can be ascribed to interference with prolactin secretion. That of nafoxidine may result from its antiestrogenic properties at the tumor tissue level, although part of it could be tentatively ascribed to inhibition of prolactin secretion.

Growth inhibition of rat mammary carcinoma and endocrine changes produced by 2-Br-α-ergocryptine, a suppressor of lactation and nidation

Abstract Administration of 2 -Br-α-ergocryptine for 6 weeks to Sprague-Dawley rats bearing DMBA-induced mammary carcinomas increased the number of regressing tumors and decreased the incidence of newly-formed ones. This inhibitory effect on tumor growth was highly significant. In addition, the drug produced the following endocrine changes: shortening of the estrus cycle and accumulation of large numbers of corpora lutea, which seemed inactive with respect to secretion of progestational compounds and did not undergo normal involution. It is suggested that these effects, both on tumor growth and on endocrine functions, probably resulted from interference with prolactin secretion.

Relation between estrogen receptor concentration and clinical and histological factors: their relative prognostic importance after radical mastectomy for primary breast cancer.

After modified radical mastectomy, 490 primary breast cancer patients were followed for a median of 75 months. Bloom grade was measured in 340 patients and ER status in 341. Follow-up of these patients has yielded the following results: (a) The value of traditional indices has been reaffirmed. (Coxs multivariate analysis identified, in order of decreasing importance, the number of invaded lymph nodes, the initial tumor size and the histological grade. Other variables were found to be of lesser importance and were correlated with the three main indices.) (b) The value of ER status disappeared after more than 3 years of follow-up. (c) ER positive patients fared better after recurrence. This was interpreted as being a consequence of their responsiveness to hormonal treatment.

Specific estrogen receptor of the DMBA-induced mammary carcinoma of the rat and its estrogen-requiring molecular transformation☆

Abstract Presence of specific estrogen receptors was observed in 11 cytosol preparations from DMBA-induced mammary tumors. These receptors are characterized by a high affinity for estradiol- 17 β similar to the one reported for the receptors of the estrogen-target tissues. Incubation of cytosol with 3 H-estradiol- 17 β at 18 °C yielded 3·5 S and occasionally 7·5 S receptor in low-ionic sucros gradients. On the contrary, a predominant 7·5 S receptor was found when non-incubated cytosol samples were fractionated by ultracentrifugation and then labelled with the hormone. Conversion of the 7·5 S to the 3·5 S receptor on incubation was obtained only in the presence of estradiol.

Effects of 2-Br-α-ergocryptine, L-dopa and cyclic imides on serum prolactin in postmenopausal women☆☆☆

Abstract The effects of cyclic imide derivatives (CG 603 and CG 809 ), of l -dopa and of 2 -Br-α-ergocryptine (CB 154 ) on serum prolactin levels were studied in postmenopausal women. A single oral dose of l -dopa ( 500 mg) or CB 154 ( 5 mg) was followed by a significant decrease in serum prolactin levels. After l -dopa, this decrease lasted an average of 4 hr and was followed by a rebound at 8 hr. After CB 154 , the decrease lasted at least 8 hr. Single doses of CG 603 ( 660 mg) or CB 809 ( 2 g) failed to influence prolactin levels. The effects of chronic oral administration of either l -dopa or CB 154 were also studied. The pretreatment circadian rhythm of serum prolactin levels, as determined on samples taken every 2 hr, served as control: it was characterized by a high and broad peak occurring during the night. l -dopa, 500 mg given every 6 hr, produced a saw-toothed effect with short-lasting falls after each dose, followed by sharp rebounds. The mean 24 -hr levels on days 9 and 14 of treatment were 94% and 72% of the mean control value. CB 154, 5 mg three times daily, induced a profound and sustained decrease in prolactin levels, with complete abolition of the circadian rhythm. The mean 24 -hr levels on days 13 and 17 of treatment were 27% and 33% of the mean control value. These experiments show that CB 154 is an effective prolactin-inhibiting agent although complete suppression was not achieved. l -dopa was effective though definitely less so than CB 154 . Reported successes of the former and failures of the latter to induce remissions in advanced breast cancer are discussed with regard to the present data.

Interaction of activated estradiol-receptor complex and chromatin in isolated uterine nuclei

Abstract Exposure at 4 °C and 22 °C of purified uterine nuclei to uterine cytosol preincubated with estradiol- 17 β causes a significant increase in their ability to bind 3 H-actinomycin D. This increase reaches almost its maximum within 15 minutes following exposure. It does not occur at 4 °C when the nuclei are exposed to cytosol added with estradiol- 17 β immediately before mixing rather than preincubated with it. It does not occur either when nuclei are exposed to any of the following solutions: cytosol preincubated with estrone, cytosol alone, buffer added with estradiol- 17 β or estrone, or buffer alone. Exposure of nuclei from uterus and from diaphragm to cytosols from these tissues increases the nuclear binding of 3 H-actinomycin D only when uterine nuclei are exposed to uterine cytosol. These data suggest that a change in chromatin structure occurs in isolated nuclei solely from estrogen-target tissue as a specific effect of exposure to estradiol-“activated” uterine cytosol. They are consistent with the hypothesis that the “activated” form of the estrogen receptor induces metabolic changes in target tissue through a direct effect at the chromatin level.