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In mammary cancers, and more especially at the stage of generalization, the detection of estrogen receptors plays a prominent role in the choice of treatment. Recent studies indicate that nearly half of cancers containing receptors are responsive to various hormone treatments while only a minority of cancers not containing receptors are responsive to the same treatments. The present review emphasizes the benefits of a quantitative dosage of these receptors compared to their simple detection. It also evaluates the predictive value of progesterone receptors as an additional test for hormonodependence. Finally, it analyses the part played by receptors dosage in the current therapeutic strategy as well as in the development of new clinical protocols.

As part of a study into nonisomerizable antiestrogens, the diastereoisomeric dihydrotamoxifens 7 and 8 were prepared by catalytic transfer hydrogenation of (Z)- and (E)-tamoxifen and were shown by NMR spectrometry to exist in preferred conformations with hydrogen atoms in an antiperiplanar relationship. The corresponding 4-hydroxy derivatives 9 and 10 were prepared from hydrogenated precursors of (Z)- and (E)-4-hydroxytamoxifen. The relative binding affinities (RBA) of the compounds to estrogen receptors are consistent with the assigned conformations and parallel reported data on derivatives of the nonsteroidal estrogen hexestrol. The growth-inhibitory activity against the MCF-7 human breast cancer cell line in vitro was for 10 comparable to that of 4-hydroxytamoxifen, although increasing the concentration from 10(-8) to 10(-6) M did not significantly improve the growth inhibition. The derivative 9 analogous to (E)-4-hydroxytamoxifen antagonized the growth-stimulating effect of added estradiol and is therefore also an antiestrogen but at low concentration (10(-8) M) in the absence of estradiol, MCF-7 cell growth was stimulated, indicating an estrogenic influence. The enantiomers of the dihydrotamoxifen 8 were individually prepared from the resolved enantiomers of 2-phenylbutanoic acid, the key reaction step being a lithium-ammonia reduction of the 1-(4-methoxyphenyl)-1,2-diphenyl-1-butanol to generate the triphenylbutane. The enantiomers of 8 gave identical RBA values in cytosol.

MCF-7 cells were incubated with [3H]estradiol, unlabeled estradiol and various estrogens or antiestrogens to measure their relative binding affinity (whole-cell assay). Comparison of the values with those previously established on uterine cytosol with a DCC assay revealed a good parallelism for both steroid and diphenolic diethylstilbestrol based estrogens. On the contrary, in the whole-cell assay, antiestrogens and weak estrogens of the triphenyl- and gem-diphenylethylene categories always displayed low values which were in the order of magnitude found with weak steroid estrogens. This property was not due to a reduction of binding capacity, nor to the presence in some compounds of an ethoxyaminoalkyl side-chain (source of antiestrogenicity). In view of all these data, it was concluded that the present test would provide an estimate of the ability of a given compound to transform the receptor in a form which interacts with genomic sites involved in the regulation of "estrogenic-induced products" ("activation").

Phagocyte activity in mice bearing neuroblastoma (NB) has been assayed using a chemiluminescence (CL) technique and proves to be dependent on the pretreatment of the NB cells used to induce the tumor. Indeed, original NB C 1300 tumor, which is maintained by serial in vivo passages induces highly triggered peritoneal phagocytes, producing large amounts of reactive oxygen intermediates (ROI). On the contrary, C 1300 cells cultured in vitro for a few passages and established lines as well as clones derived from the original tumor fail to induce this triggering. However, this decreased phagocyte stimulation can be regained at full extent after a few in vivo passages of the tumor cells.

A high proportion of human mammary cancers contain a measurable amount of estrogen receptors. The presence of estrogen receptors correlates well with the response of patients to endocrine therapy, but not with their response to chemotherapy. However, receptor-mediated chemotherapy should result in more selective and efficient treatment of receptor-positive tumors. Several agents containing an estrogenic and a cytotoxic moiety have been synthesized in the past. It is not surprising that the affinity of these compounds for the estradiol receptor is very low, since most of them were synthesized before our knowledge of hormone receptor mechanisms. An international workshop on cytotoxic estrogens was held on September 27-28, 1979, in Diepenbeek, Belgium, to celebrate the opening of the Dr. L. Willems-Instituut. The purpose of the workshop was to determine the structural requirements for binding to the estrogen receptor and to obtain a consensus on screening techniques for selecting potential drugs of this type.

Metabolism of tamoxifen by rat hepatocytes and hydrolysis of the resulting polar metabolites corresponding to conjugates with beta-glucuronidase gave a major component which was identified as 1-[4-(2-hydroxyethoxy)phenyl]-1-(4-hydroxyphenyl)-2-phenyl-1-butene by comparison of mass spectral properties with those of synthetic material. This compound, which was not observed as a phase I metabolite, is believed to have been found previously in rat bile and in human faeces (metabolite F) but its structure had been incorrectly assigned. Its binding affinity for the estrogen receptor was greater than that of tamoxifen but less than that of 4-hydroxytamoxifen, and it possessed a corresponding degree of antitumour activity against the MCF-7 breast cancer cell line. By carrying out the hepatocyte incubation separately under oxygen and air, it has been shown that the N-oxidation of tamoxifen is favoured by a high concentration of oxygen during in vitro metabolism but that the rate of 4-hydroxylation is not dependent on oxygen availability.

A bioluminescent technique was used to show that murine neuroblastoma (NB) cells or cell-free extracts (H variant) were able to enhance the release of reactive oxygen intermediates (ROI) from peritoneal macrophages in vitro. L-variant NB cells were ineffective. Physiological concentrations of met-enkephalin produced the same effect in vitro but not leu-enkephalin. When both H- and L-variant cells, or their extracts, were incubated together with macrophages, ROI production was not increased. Similar findings were detectable when met- and leu-enkephalin were cultured together with macrophages. In vivo, preliminary studies gave the same results. The concentration rate of met- to leu-enkephalin was higher in H-variant than in L-variant NB cells. We conclude from our results that met-enkephalin can enhance the release of ROI from peritoneal macrophages. The difference in the effects produced by the H and L variants is due to differences in the concentrations of enkephalins released.

Eleven samples from 7 primary and 4 metastatic male breast cancers were analysed for cytoplasmic oestrogen receptors. Two samples of metastatic cancers were obtained from a same patient 6 months apart. Receptors were assayed by measuring the binding affinity of cytosol fractions for 3H-oestradiol- 17beta. Saturable binding sites were obtained in 6 primary and 1 metastatic cancers. The dissociation constant of the binding reactions were within the range reported for the specific oestrogen receptors from female breast cancers. When present, receptor concentrations varied from 59 to 532 fmoles/mg tissue protein. Competition studies of the binding of 3H-oestradiol- 17beta by excess amounts of unlabelled steroid hormones or antihormones revealed that the receptors were specific for oestrogens and antioestrogens. By all these criteria, receptors from male mammary cancer were identical to those from female breast cancer. Chromosome analysis was performed in 6 patients. All had normal 46 XY male pattern indicating that no conspicuous genetic abnormalities such as those of the Klinefelter syndrome seemed responsible for the presence of the receptors.

The role of cytokines in early T cell development was evaluated in a thymic organ culture system. Fetal thymic lobes from 14 day old mouse embryo's were cultured in the presence of antibodies neutralizing either IL-4, IL6 or TNF. In addition antibodies neutralizing TNF were added to cultures supplemented with human recombinant IL-2. The influence of these different treatments were evaluated by analyzing the different subsets generated after 12 days of culture with a panel of monoclonal antibodies. The antibody treatment did not result in dramatic changes in the cell populations nor did the anti-TNF inhibit the significant changes that are induced by the IL-2 treatment. These results show that based on the cellularity and thymocyte subsets no influence can be shown by inhibiting these cytokines. Other criteria, e.g. repertoire specificity have to be evaluated to address the influence of these treatments on early T cell development.

The target cell specificity of LAK cells generated by addition of rIL-2 to thymic rudiments of 14 day old BALB/C embryo's was compared to the lytic potential of adult BALB/C splenic LAK cells. We used four tumor cell lines as targets, one NK-susceptible (YAC-1) and three NK-resistant tumors (C1300, P815 and EL4), which were all killed by splenic LAK cells. It is shown that fetal thymic LAK cells display a lower lytic activity, while EL4 tumor cells are not killed at all. By elimination of the CD8 positive cells from both the adult splenic and the fetal thymic LAK cells, we demonstrate that the fetal thymic LAK activity is mainly exerted by CD4-CD8- cells, whereas part of the cytotoxicity of splenic LAK cells is due to CD8+ cells.

With the erythro-hexestrol derivative 2 as the starting material, a variety of cytotoxic linked hexestrol (HEX) compounds were prepared including the HEX-N-lost derivative 36, the HEX-(chloroethyl)nitrosourea 38, the HEX-cyclophosphamide 44, and the HEX-epoxide 68. Relative binding affinity to estradiol receptors were in the magnitude of 1%, similar to that of comparable diethylstilbestrol compounds. HEX derivatives with long polyether spacers (64, 65, 70, 71) showed no significant decrease in binding affinity in contrast to derivatives with other bulky side chains.

Breast cancer cell lines in monolayer culture are helpful in evaluating the potential antitumor activity of antiestrogens. A new method has been developed to rapidly measure the inhibitory potency of a given compound without any cell harvest and counting. Cells are incubated in 96-multiwell culture dishes in the absence or presence of an antiestrogen at 3 concentrations. At the end of incubation, cells are fixed with ethanol and colored with hematoxylin. The intensity of the coloration, which is measured with a multiscan spectrophotometer, gives an accurate evaluation of the cell number and, thereby, an estimate of the inhibitory potency of the antiestrogen. Addition of estradiol to the culture allows to evaluate the antagonistic effect of this hormone towards the inhibition of the antiestrogen. Because of its rapidity and low cost, our method should offer a new dimension in the in vitro screening of antiestrogens and probably most other antineoplastic drugs.

Estrogens stimulate growth of MCF-7 breast cancer cells in monolayer culture. Possible interference of serum factors leading to an estrogen-insensitive cell growth was analyzed in various experiments carried out on serum batches producing no estradiol stimulation. Out of five estrogen conjugates, only 3-glucurono-estradiol partly suppressed the inhibition of hydroxytamoxifen; the conjugate also reduced the estrogen receptor content of the cells, probably by a down regulation process ("processing"). Moreover, prolonged subcultures in dextran-coated charcoal-treated serum attempting to remove possible intracellular estrogens produced no growth stimulation. Interference by hormone carriers of the serum was ruled out by the fact that two strong synthetic estrogens, moxestrol and diethylstilbestrol with weak binding affinity for these carriers, were unstimulatory. Reduction of the carrier concentration also failed to confer any estrogen sensitivity. This lack of effect of most estrogen conjugates and serum carriers seems to contradict the hypothesis of their interference leading to an estrogen-insensitive growth. Presence in the serum of potential inhibitors towards estrogen action was also examined. Dilution of sera inducing an estrogenic stimulated growth failed to show any growth increase, either in the absence or presence of estradiol, thus excluding the possibility of a major influence of an antagonism on growth control. Moreover, clonogenic assays in soft agar eliminated the hypothesis that a difference between "active" (stimulatory with estradiol) and "inactive" serum batches may result from distinct adherence properties rather than from real growth stimulation. All of these data are consistent with the concept that serum factors which are not of estrogenic nature mediate the trophic effect of estradiol; their absence in some serum batches may lead to an estrogen-insensitive cell growth.

Cytochemical steroids binding activity of human mammary cancer cells is demonstrated in cryostat frozen sections using hydrophilic fluorescent steroid hormone ligands. The cytochemical results indicate that mammary carcinomas are composed of heterogeneous populations of positive and negative cancer cells in varying proportions. A comparison with biochemical data was made. There exists no correlation between the results obtained with the histochemical and the biochemical techniques. The specificity of this new tool of investigation is discussed.

A range of tamoxifen derivatives substituted in the 4-position of the 1-phenyl ring are described. The key steps in the synthesis of 4-iodo-, 4-bromo-, and 4-(methylthio)tamoxifen were reactions of 1,2-diarylbutanones with the (4-halogenophenyl)lithium or [4-(methylthio)phenyl]magnesium bromide. Oxidized precursors of 4-(methylthio)tamoxifen were used to prepare the methylsulfinyl and methylsulfonyl derivatives. Further derivatives (formyl, hydroxymethyl, oxirane, mercapto) were prepared from 4-bromotamoxifen via the 4-lithio derivative. Several of the derivatives (Br, I, SMe, SOMe, SO2Me, oxirane, CHO, CH2OH) displayed a higher affinity for estrogen receptors (ER) of calf uterine cytosol than did tamoxifen, but there was no relationship between affinity to ER and the ability to inhibit the growth of the MCF-7 breast cancer cell line in vitro.

Three types of non-isomerisable antiestrogens analogous to tamoxifen and 4-hydroxytamoxifen are described. Advantages of non-isomerisability are simplified synthesis, simplified metabolism profile, and that structure-activity relationships become more meaningful. The compounds described differ from tamoxifen by having an extra ring methyl group, a fused seven-membered ring system, or the central ethylene linkage saturated. These compounds retained both binding affinity to estrogen receptors and growth inhibition of MCF-7 human breast cancer cells in vitro.

Two lines of research intending to achieve synergism between cytotoxic agents and estrogens for breast cancer treatment are pursued in our laboratory. According to a screening procedure we select cytotoxic-linked estrogens which bind to estrogen receptors and thereby would be specifically concentrated into the tumor cells. A mesylate derivative of estrone has emerged from our investigations. This compound displays a very strong binding affinity for the receptors and inhibits the growth of the receptor-positive MCF-7 breast cancer cell line. The lack of growth inhibition in the receptor-negative line Evsa-T indicates that it is devoid of major non-specific cytotoxicity. We attempt to enhance the vulnerability of the tumor cells by producing an estrogen-induced modification of their chromatin. The in vitro exposure of isolated uterine nuclei to uterine cytosol preincubated with estradiol increases their ability to bind [3H]actinomycin D. Identical results are obtained with MXT mouse mammary tumors. Experiments are in progress to settle whether these changes are associated with enhanced cell killing.

The 2-methyl derivatives of tamoxifen (2-methyltamoxifen and 2-methyl-4-hydroxytamoxifen) were extracted from a cell culture medium at pH 5.4 (Earle's Minimum Essential Medium) with an internal standard (tamoxifen) on a phenyl sorbent cartridge. The compounds were then separated by high-performance liquid chromatography on a nitrile column eluted with acetonitrile-methanol-0.05 M sodium dihydrogenphosphate (19:4:11.6:69,v/v) containing 0.11 mmol/l disodium EDTA and determined by electrochemical detection at +1.1 V vs. Ag/AgCl/3 M NaCl. The absolute detection limits were 50 pg for 2-methyl-4-hydroxytamoxifen and 100 pg for tamoxifen and 2-methyltamoxifen at a sensitivity of 1 nA/V.

Derivatives of tamoxifen (1) and 4-hydroxy-2-methyltamoxifen (2) in which the basic side chain has been modified by N-oxidation or by quaternization have been investigated with respect to the effects on affinity for the oestrogen receptor and on cytostatic activity towards the MCF-7 cell line in vitro. In addition to the conventional cytosol assay for receptor binding affinity (RBA) a recently developed whole-cell assay was employed. N-oxidation (e.g. 2----3) produced no significant alteration in RBA value either in cytosol or in whole cells, nor in activity towards the MCF-7 line. Quaternization with methyl iodide (1----4, 2----6) or ethyl bromide (1----5, 2----7b: the cis isomer 7a also formed) did not alter receptor binding in the cytosol assay but almost abolished binding in the whole cell and cytostatic activity. The whole-cell RBA values for 2 (0.45) and 3 (0.5) were lower than those for 4-hydroxytamoxifen (2.9), suggested to be due to the lower oestrogenicity of the 2-methyl derivatives since activity against MCF-7 cells was unimpaired. The even lower values of whole-cell RBA (0.01-0.02) for the quaternary ethyl bromide derivatives 7a and 7b were ascribed to poor penetration into the cell since these compounds had minimal cytostatic activity.

Since the binding sites of hormone receptors may be similar to those of antihormone antibodies, we wondered whether the former might not be recognized by the idiotypic network. To test this hypothesis we investigated the interaction of plasma immunoglobulin G (IgG) with the binding sites of estrogen receptors (ER) from uterine or mammary tissue. Using ER isolated from uterine cytosol we found that IgG from normal subjects shifted the position of purified receptor in sucrose gradients and displaced [3H]estradiol (E2) from its receptor-binding sites. Equilibrium studies revealed competitive inhibition by IgG of E2 binding to the ER. IgG isolated by adsorption on a rat uterine cytosol-Blue B matrix gel column also bound to the ER, and this binding was inhibited by an excess of E2. After an 18-h exposure of MCF-7 mammary carcinoma cells in monolayer culture to IgG (2 mg/ml), Scatchard analysis of [3H]E2 binding revealed a reproducible decrease in the available receptor sites from 2.52 +/- 0.56 (+/- SEM) to 0.68 +/- 0.48 fmol/microgram DNA (n = 10). This effect was selective, since enriched anti-ER IgG obtained by adsorption on purified receptor was 20 times more potent than total IgG, whereas IgG identically prepared but not retained by affinity chromatography had no activity. Exposure of the cells to the IgG for 45 min also revealed, as with isolated ER, specific competition of the IgG with E2 for the E2-binding sites; the Kd increased from 10.5 +/- 1.6 to 27.5 +/- 7.2 X 10(-11) M (n = 7). Enriched antireceptor IgG was a 20 times more effective competitor, and the IgG not retained by affinity chromatography had no activity. In conclusion, our observations indicate the presence of ER on the cell surface, interaction of ER with IgG from plasma of normal subjects, and competitive antagonism of these IgG with E2 uptake leading to a decrease in effective ER concentrations.

The MXT mouse transplantable mammary cancer is characterized by significant amounts of steroid hormone receptors. Moreover this model, as other solid tumors, contains diverse subpopulations of neoplastic cells. In the present study we have examined this pattern as well as the influence of exogenous physiologic doses of estradiol on tumor cell subpopulations. Their identification was undertaken at the morphological level employing incubation of tritiated actinomycin D with fixed material followed by autoradiography. This semiquantitative cytochemical method has proved itself to be a useful tool in order to assess the cellular heterogeneity of carcinoma cells.

Estradiol mustard (EM) is the 3,17beta-diester of estradiol-17beta (E2) with the nitrogen mustard derivative chlorphenacyl. The ability of EM to bind to cytoplasmic estrogen receptors was tested by inhibition of the binding of 3H-E2 to rat uterine cytosol at 18 degrees C and 30 degrees C. At both temperatures an inhibition curve was observed in the presence of a large excess of drug, suggesting that the latter has a very weak binding affinity (100,000 times lower than E2). Incubation of uterine cytosol with increasing amounts of 3H-E2 in the presence and absence of an excess of EM indicated that the drug interacted with the receptors at the same sites as E2 (competitive inhibition). Preincubation of uterine cytosol at 18 degrees C with EM induced a progressive reduction of 3H-E2 binding capacity. This reduction also occurred, although to a lesser extent, when long-term incubation of the cytosol with EM was performed in the presence of labelled E2 from the start. The process was faster at 18 degrees C than at 4 degrees C and did not occur with EM preincubated in homogenization buffer. Exchange assays by 3H-E2 of uterine receptors preincubated with labelled E2 and excess EM indicated that the drug-induced inhibition of binding capacity was reversible and produced no apparent alteration of the receptors. Furthermore, the rate of exchange was similar to that observed with receptors previously filled with unlabelled E2. In 9 "receptor-positive" cytosols from human breast cancers, time-course study of the binding of 3H-E2 in the presence of excess of EM yielded similar results as those obtained with rat uterine cytosol. These results show that EM has a very low binding affinity for the extrogen receptors and that it is metabolized into one or several compounds of higher binding affinity. They suggest that EM is probably not significantly concentrated by the estrogen target tissues such as mammary cancers. Therefore, the drug is unlikely to be very valuable in the treatment of breast cancer through a specific mechanism involving concentration by the estrogen receptors.

Trilostane (4-alpha-5-epoxy-17 beta-hydroxy-3-oxo-5-alpha-androstan-2-carbonitrile) is a modified steroidal molecule. In vitro and in vivo studies in rats have shown that it inhibits adrenal, ovarian and placental steroid synthesis. It seems to act by exerting a selective blockade on 3 beta-hydroxysteroid dehydrogenase. In this study, we investigated whether this molecule interacts with hormone receptors for estrogen, androgen or progesterone. We also tried to demonstrate the effect which Trilostane may have on cellular cultures of human mammary carcinoma (MCF-7 Evsa-T). We also studied hormonal modifications in a series of 12 patients treated with different doses of Trilostane, since this drug is supposed to inhibit the production by the adrenal glands of mineralocorticoids, of glucocorticoids and of the precursors of estrogens. Our results indicate that Trilostane does not react with any of the main hormonal sex steroid receptors, nor does it interfere with cultures of human mammary cancer cells either containing estrogen receptors and therefore allegedly hormone-dependent (MCF-7 line), or estrogen receptor-negative cells, presumably independent of hormonal manipulations (Evsa-T cell line). Finally, endocrine studies on postmenopausal women with advanced breast cancer show that Trilostane significantly reduces the plasma levels of estrone and of its major androgen precursor (androstenedione). However, the latter inhibition is no different from that exerted by hydrocortisone acetate administered alone at a dose of 40 mg/day. The results of clinical trials comparing hydrocortisone alone with hydrocortisone plus Trilostane are awaited.

Two days after inoculation of A/J mice with in vivo propagated C1300 neuroblastoma (NB) cells, a 4-5 fold increase of the splenic natural killer (NK) activity is noticed. A similar rise of NK activity is also displayed by the host lymph node, bone marrow and peritoneal cells, reflecting a general stimulation of NK cells. These effector cells only lyse classical NK target cells. They are of low specific gravity, nylon wool non-adherent and insensitive to anti-Thy treatment, suggesting that they are indeed NK cells. In spite of the ability of in vivo maintained NB cells to increase the NK activity, they are not lysed by NK cells, nor do they have NK-recognizable target structures as demonstrated by competition studies. In contrast with the previously mentioned experiments, NB cells that have been cultured in vitro for at least one week, do not increase the NK activity. During further growth of both tumor variants, the NK activity gradually decreases. In vivo experiments revealed that the presence of a large NB tumor load exerts a negative effect on the induction of active NK effectors. This effect is not due to the intervention of splenic suppressor cells.