[Urinary excretion of 6-sulphatoxymelatonin in normal subjects: statistical approach to the influence of age and sex]. / Approche statistique de l'influence de l'âge et du sexe sur l'excrétion de 6-sulfatoxymélatonine urinaire (a-MT6s) chez l'individu normal.

A radioimmunoassay of urinary 6-sulphatoxymelatonin (a-MT6s) was performed in 90 normal subjects: 44 males and 46 females (17-67 years). Patients treated with betablokers or antidepressants were not included in this study. Urine samples were collected over three periods of time: 7 to 11 p.m., 11 p.m. to 7 a.m., and 7 to 11 a.m. Between 11 p.m. and 7 a.m., the subjects slept in their normal environment and had not ingested alcohol for 24 hours. We searched for a possible relation between urinary a-MT6s excretion (expressed in ng/l/h) and age. From 7 to 11 p.m. and from 7 to 11 a.m. no significant relation could be found. On the contrary, between 11 p.m. and 7 a.m. there was a significant relation indicating decrease of a-MT6s secretion with increasing age. Several linear or non-linear curve patters were tested: Boltzmann sigmoid (1(st), 2(nd), and 3(rd) degree), polynomial curves. The Boltzmann sigmoid showed the best fit judging by the r-squared value (0.152) and the runs test (p=0.64). On this curve the inflection point was located at 53 4 years (SDM, standard deviation of the mean). From 19 to 45 years, the upper sigmoid plateau was located at 1381 91 ng/l/h (SDM). The decrease was found between 45 and 60 years and the lower sigmoid plateau then stabilized at 467 370 ng/l/h (\SDM). In the study group, there was no significant difference between men and women according to the Mann-Withney test. Finally, use of oral contraceptives did not affect urinary a-MT6s (Mann-Withney).

Novel plasma extraction procedure and development of a specific enzyme-immunoassay of oxytocin: application to clinical and biological investigations of small cell carcinoma of the lung.

Paraneoplastic secretion of the lactation-inducing hormone oxytocin (OT) has been reported in about 30% of cases of small cell carcinoma of the lung (SCCL). In order to investigate the role of OT in the biology of SCCL tumours, a specific enzyme-immunoassay (EIA) for OT, which can be applied to both human plasma and culture medium, has been developed. OT EIA is performed on 96-well microtiter plates coated with a rabbit polyclonal antibody (Ab) anti-OT (04). This antibody does not exhibit any significant cross-reactivity either with vasopressin (VP) or with vasotocin (VT). The immunological reaction involving Ab anti-OT is a competition between the tracer (biotinylated OT) and synthetic OT (standard curve) or OT present in biological samples. In order to limit interference induced by plasma proteins, plasma samples are filtrated by a one-step centrifugation on centricon YM-3 (cut-off 3000 Da). After plasma filtration, 90.7 +/- 5.1 (SD) % (n = 22) immunoreactive (IR) OT is recovered. The sensitivity of OT EIA is 1 pmol/L, while intra- and inter-assay coefficients of variation (CV) are around 3.41% and 2.84%, respectively. In healthy volunteers, plasma IR OT is 7.28 +/- 4.49 (SD) pmol/L (n = 32) with no gender difference. As shown by the data both from plasma of SCCL patients and from supernatants and cell contents of SCCL cell lines, this EIA procedure offers a novel, reproducible, specific and sensitive method for the measurement of IR OT.

Transforming growth factor-beta receptor types I and II in cultured porcine leydig cells: expression and hormonal regulation.

The steroidogenic activity of testicular Leydig cells is controlled both by the pituitary hormone (LH) and by growth factors such as transforming growth factor-beta peptides (TGFbeta1, -2, and -3; inhibin/activin; and anti-Mullerian hormone). By using primary cultures of porcine Leydig cells as a model, the aim of the study was to identify and characterize the TGFbeta receptors and to study their regulation by LH/hCG. TGFbeta receptors have been identified and characterized through three different approaches, including cross-linking experiments and Western and Northern blotting analyses. In cross-linking experiments, labeled TGFbeta was shown to bind to three different molecular species of 300, 80, and 53 kDa, which may correspond to the protein betaglycan (also known as TGFbeta type III receptor) and TGFbeta type II and I receptors (TGFbetaRII and TGFbetaRI), respectively. The presence of TGFbetaRI and -RII was further demonstrated by Western blotting analysis using specific polyclonal antibodies. Finally, the expression of betaglycan, TGFbetaRII, and TGFbetaRI messenger RNAs, was confirmed by Northern blotting analysis, as shown by the presence of 6.4-, 4.6-, and 5.8-kb messenger RNAs, respectively. By using a RT-PCR approach, the mediators of the TGFbeta signal, Smads 1-7, were also detected in cultured Leydig cells. TGFbetaRI and TGFbetaRII protein levels were enhanced by hCG/LH in a dose-dependent (maximal effect with 0.3 ng/ml hCG) and time-dependent (maximal effect observed after 48 h of hCG treatment) manner. Furthermore, to determine whether the stimulatory effect of LH/hCG was mediated by testosterone, use was made of aminogluthetimide, an inhibitor of cytochrome P450scc. The inhibition oftestosterone formation did not affect the stimulatory effect of LH/hCG on TGFbetaRI and -RII levels, suggesting that the gonadotropin action is not mediated by the steroid hormone. Together, the present findings demonstrate that the TGFbeta receptors are expressed and are under hormonal (gonadotropin) control in cultured porcine Leydig cells.

Cellular distribution of transforming growth factor betas 1, 2, and 3 and their types I and II receptors during postnatal development and spermatogenesis in the boar testis.

Transforming growth factor betas (TGF betas) 1, 2, and 3 and their types I and II receptors (TGF betas RI and RII) were immunolocalized 1) during testicular development from the perinatal to the adult period and 2) in maturing germ cell populations at successive seminiferous epithelium stages. In the perinatal testis, TGF beta isoforms and receptors were both preponderant in Leydig cells and in spermatogonia. At prepuberty, their expression appeared in Sertoli cells, while germ cells showed specific TGF beta1 and TGF betaRI staining in the spermatocytes. In the adult testis, TGF beta ligands exhibited a preferential tubular distribution. TGF beta1 was mainly detected in young spermatocytes, TGF beta2 in Sertoli cells, and TGF beta3 in Sertoli and premeiotic germ cells. Although the two receptors were systematically observed together in various cells, our data indicate a predominance of one in comparison with the other depending on the cell type. TGF betaRI was predominant in meiotic and differentiated germ cells and TGF betaRII in somatic cells. Finally, in the adult testis, TGF betas 1, 3, and RI showed a germ-cell pattern that depended upon the stage of the seminiferous epithelium cycle. Specifically, staining for the ligands was predominant before meiosis, and TGF betaRI was present particularly during meiosis and spermiogenesis. Together, the temporal and spatial distribution of the TGF beta system components suggests that these signaling molecules may play a crucial role during specific steps of testicular development and during different waves of seminiferous epithelium maturation leading to spermatogenesis.

Transforming growth factor beta receptor expression in cultured porcine granulosa cells.

By using primary cultures of porcine granulosa cells as a model, TGF beta receptors have been identified and characterized through three different approaches including cross-linking experiments, Western- and Northern-blotting analysis. In cross-linking experiments, labeled TGF beta was shown to bind to four different molecular species of 300, 168, 72 and 58 kDa. The 300-kDa species may correspond to beta-glycan, while the 72- and 58-kDa correspond to TGF beta type II and I receptors, respectively. The presence of these receptors was further demonstrated by Western-blotting analysis using specific polyclonal antibodies. Finally, both the expression of beta-glycan, type II and type I mRNA, was confirmed through Northern-blotting analysis as shown by the presence of 6.4, 4.6 and 5.8 kb mRNA, respectively. Additionally, we detected another TGF beta binding protein of 168 kDa which remains to be identified. Together, our present data indicate that the regulatory action of TGF beta on cultured granulosa cells previously reported in several laboratories may be mediated through the receptors identified here.

Absence of antisteroid antibodies in oral contraceptive users presenting with vascular events.

Previous reports speculated that vascular events could be related to the development of antibodies against synthetic steroids contained in oral contraceptives or other hormonal treatments. This study describes original immunoassays designed to detect antisynthetic steroid antibodies. In a first step, the assays were characterized and validated using animal-raised antisteroid antibodies. In a second step, a population of 88 oral contraceptive users, 47 of them having developed a vascular thrombosis during synthetic steroid use and 41 serving as healthy control users, were tested. Detection of antibodies against ethinylestradiol, levonorgestrel, norethisterone, cyproterone acetate, and gestodene showed that the values obtained in normal oral contraceptive users as well as thrombosis patients are very low, and show no statistically significant difference between the two groups tested. Taken together, these data indicate that the "immunological hypothesis" related to antisteroid antibodies is unlikely to explain the pathogenesis of vascular events in oral contraceptive users.

Immunolocalization of transforming growth factor-beta 1 and transforming growth factor-beta 2 in the mouse ovary during gonadotrophin-induced follicular maturation.

An immunohistochemical approach was utilized to evaluate the cellular distribution of transforming growth factor-beta 1 (TGF beta 1) and transforming growth factor beta 2 (TGF beta 2) at different stages of follicle development in the prepubertal mouse ovary under the following conditions: (i) after pregnant mare's serum gonadotrophin (PMSG) treatment; (ii) after PMSG and human chorionic gonadotrophin (HCG) treatment; (iii) after PMSG and HCG treatment plus mating. In the immature ovary, TGF beta 1 and TGF beta 2 immunoreactivities are localized in theca and granulosa cells and in oocytes. After PMSG treatment, TGF beta 1 and TGF beta 2 immunoreactivities are localized in granulosa cells; in addition, TGF beta 2 staining is noted in the matrix surrounding antral cells. Staining for both TGR beta 1 and TGF beta 2 drops in the theca but persists in the oocyte. PMSG plus HCG treatment results in a significant increase in TGF beta 1 and TGF beta 2 immunoreactivity in the theca and in the maintenance of TGF beta 1 staining in both basal granulosa cells and cumulus cells whereas TGF beta 2 immunoreactivity is essentially localized in the matrix surrounding cumulus cells. Staining for TGF beta 1 and TGF beta 2 persists in the oocyte. Following PMSG plus HCG treatment and mating, TGF beta 1 immunoreactivity is localized in the luteal cells of corpora lutea and TGF beta 2 shows a similar localization pattern. This study provides evidence that TGF beta 1 and TGF beta 2 peptides are expressed in specific cell types during induced follicular maturation in the mouse ovary.

Antisteroid immune complexes and vascular thrombosis during steroid hormone therapy.

To test an immunological hypothesis proposed to explain the pathogenesis of cerebrovascular thrombosis in steroid users, circulating immune complexes were assayed in the sera from 6 control subjects, 14 ever users of oral contraceptive having developed a neurological ischaemic accident, and 7 patients with the same clinical history during use of other sex steroid not containing ethinylestradiol. Beaumont's ammonium sulfate and polyethylene glycol precipitation methods, together with a specific method of isolation of circulating immune complexes using affinity chromatography on Protein A, were used. Radioactivity from labeled ethinylestradiol added to the sera before precipitation was monitored in the precipitates to detect anti-ethinylestradiol antibodies. There were no significant differences for these parameters in the three groups. However, protein content and 3H-EE activity in the precipitates were equally and dramatically reduced after affinity chromatography in the three groups. These latter results do not support the presence of antibodies against ethinylestradiol in steroid users with cerebrovascular thrombosis. Moreover, our data suggest a lack of specificity of Beaumont's method for the isolation of immune complexes containing anti-ethinylestradiol antibodies.

Sites of interaction between epidermal growth factor and transforming growth factor-beta 1 in the control of steroidogenesis in cultured porcine Leydig cells.

The present study examines how the hormonal action of gonadotropin is modulated by transforming growth factor-beta 1 (TGF beta 1) and epidermal growth factor (EGF) in primary cultures of purified porcine Leydig cells. Although TGF beta 1 (1 ng/ml; 48 h) and EGF (10 ng/ml; 72 h) individually enhanced hCG-stimulated testosterone formation, the effects of EGF were more pronounced than those of TGF beta 1. When studied in combination, the effects of maximal concentrations of TGF beta 1 and EGF were additive on gonadotropin hormonal action. In the present study we demonstrate that their additive effects resulted from a complex interaction occurring at the levels of cholesterol substrate availability in the mitochondria and of 3 beta-hydroxysteroid dehydrogenase/isomerase activity (3 beta HSDI). First, TGF beta 1 (1 ng/ml; 48 h) and EGF (10 ng/ml; 72 h) were, respectively, shown to reduce and enhance dehydroepiandrosterone (DHEA) formation (evaluated in the presence of 10(-5) M WIN 24540, an inhibitor of 3 beta HSDI) in Leydig cells when acutely (3 h) stimulated with hCG (0.01-1 ng/ml), but not when incubated with 22R-hydroxycholesterol (3 micrograms/ml). Such findings indicate that TGF beta 1 and EGF did not affect cholesterol side-chain cleavage cytochrome P450 activity, but, respectively, decreased and increased cholesterol substrate availability for this enzyme in the mitochondria. Furthermore, when Leydig cells were treated with the combined factors, the formation of delta 5-steroid intermediates (such as DHEA) in untreated (control) and EGF-plus TGF beta 1-treated cells was not significantly different whether the cells were acutely stimulated with the gonadotropin or incubated with 22R-hydroxycholesterol. Such findings indicate that the effects of EGF and TGF beta 1 on cholesterol substrate availability in the mitochondria are antagonistic. Second, EGF, TGF beta 1, and EGF plus TGF beta 1 significantly (P less than 0.001) increased delta 5-steroid intermediate (i.e. pregnenolone and DHEA), but not delta 4-steroid intermediate (i.e. progesterone and androstenedione), conversion into testosterone, indicating that the growth factors increased, individually or in combination in an additive manner, 3 beta HSDI activity (respectively, 90.7 +/- 0.6%, 80.6 +/- 2.6%, and 164 +/- 4.5% increase in the presence of EGF, TGF beta 1, and EGF plus TGF beta 1). Together, the reciprocal suppression of the effects of TGF beta 1 and EGF on the mitochondrial cholesterol substrate availability coupled to their stimulatory additive actions on 3 beta HSDI activity provide an explanation of the additive actions of the two growth factors on gonadotropin-induced testicular androgen formation.

Effects of alpha-methyl-para-tyrosine on monoamine levels in the Japanese quail: sex differences and testosterone effects.

Experiments were performed to obtain more information on the regulation by steroids of catecholaminergic systems in the brain of Japanese quail. Dose-response and time-response experiments were first performed to determine optimal conditions for measuring turnover in the quail brain. The norepinephrine and dopamine turnover were then estimated in microdissected brain nuclei of birds that were either sexually mature or gonadectomized or gonadectomized and treated with testosterone. Two major facts that bear direct relationship with the control of masculine reproductive behavior were demonstrated. On one hand, the dopamine turnover in the medial preoptic nucleus, a sexually dimorphic brain structure which is critically implicated in the control of copulatory behavior was much higher in male than in female quail irrespective of the hormonal condition of the birds. On the other hand, norepinephrine concentrations appeared to be higher in several nuclei of the female brain by comparison with males. These sex differences might represent part of the causal factors that underlie the sex dimorphism in reproductive behavior in quail.

The induction by testosterone of aromatase activity in the preoptic area and activation of copulatory behavior.

A series of 4 experiments was designed to study the relationships between the activity of the aromatase (AA) in the preoptic area (POA) and the activation by testosterone (T) of copulatory behavior in gonadectomized male and female Japanese quail. The induction of AA by T in the POA is dose- and time-dependent. Levels of AA seen in sexually mature males are restored in castrated birds by a treatment with 20 to 40 mm silastic T capsules which produce physiological levels of steroid in the plasma. The minimal dose of T (10 mm implant) which reliably restores copulatory behavior approximately doubles the AA in the POA. The induction of AA is significantly larger in males than in females. A significant increase in AA is observed within 16 hours after the start of the treatment with T and the induction is maximal after 48 hours. Activation of copulatory behavior follows a similar time course but occurs with a delay of 24-48 hours. These results thus suggest that, in male quail, the activity of the aromatase in the POA is a limiting factor in the activation of copulatory behavior. This idea is confirmed by direct experimentation using an aromatase inhibitor, androstatrienedione (ATD). If T-treated birds receive at the same time silastic implants filled with ATD, the activation of behavior is suppressed for at least one week. This behavioral inhibition is, as expected, accompanied and very probably caused by the inhibition of the aromatase activity in the preoptic area and anterior hypothalamus. No increase of enzyme activity over the level seen in castrates was actually detected during the first 8 days of exposure to T. A moderate increase in AA was seen on day 16 and is probably responsible for the behavioral activation which was observed at the end of the experiment.

Sexual differentiation in quail: critical period and hormonal specificity.

There is a discrepancy between results showing that male quail are demasculinized by exogenous estrogens only if the treatment is given before Day 12 of egg incubation and results showing that ovariectomy of females after hatching still affects their sexual differentiation which leads to the conclusion that female demasculinization by ovarian estrogens is a continuing process extending into posthatching life. The first experiment was performed to test different models which have been proposed to reconcile these apparently contradictory results. Male and female quail were treated with 0, 5, or 25 micrograms of estradiol benzoate (EB) on either Day 9 or Day 14 of embryonic life. Birds were castrated at the age of 4 days to avoid the confounding effects of postnatal gonadal hormones and were treated as adults with testosterone (T). Whereas EB-treatment demasculizined sexual behavior and cloacal gland growth of males when administered on Day 9, it was without effect on Day 14. This result confirms the presence of a "critical period" for sexual differentiation of behavior in embryonic life. However, the time course of sexual differentiation and the sensitivity to the demasculinizing actions of estrogens were not the same for different behavioral and morphological characteristics. Some dependent variables such as plasma levels of luteinizing hormone and crowing were still affected by the EB treatment on Day 14. These results show that the whole process of demasculinization is not retricted to the "critical period" ending on Day 12 of incubation. A second experiment was performed to determine if 5 beta-dihydrotestosterone (5 beta-DHT), a metabolite of testosterone, also exerts demasculinizing effects during embryonic life. A large dose of 5 beta-DHT (2 mg/egg) had no effects on behavior and morphology in males if administered on Day 9 of egg incubation. This suggests that 5 beta-DHT, which is a steroid devoid of behavioral effects in the adult bird, is also an inactive compound as far as sexual differentiation of the quail is concerned. The high 5 beta-reductase activity which was previously identified in the hypothalamus of the embryonic quail thus probably plays a protective role. By transforming testosterone into inactive nonaromatizable androgens, it prevents male embryos from being demasculinized by their endogenous testosterone acting through aromatization.

Epithelial membrane antigen (EMA) distribution in various biological fluids.

Different human biological fluids, namely breast cyst fluids (five), milks (four), sera (five), were submitted to molecular sieving chromatography on Sepharose CL6B. Global protein contents of the eluted fractions were estimated by the Bradford method. Epithelial membrane antigen (EMA) was assayed by two different ELISA techniques using polyclonal and monoclonal antibodies. Various molecular species reacting with EMA (15) were found in the chromatographies with molecular weights ranging from 35 to 1500 kd. But the total amount of antigens detected using polyclonal or monoclonal antibodies was quite similar. Moreover no significant difference was found between the sera from two lactating women and the sera from three women with adenocarcinoma with respect to the molecular distribution of different molecular species of EMA.

Assay of epithelial membrane antigen (EMA) in human serum by ELISA.

An enzyme linked immuno sorbent assay (ELISA) for an epithelial membrane antigen (EMA) was described. Possible cross reactions with various antigens were investigated. In sera, EMA has been found in all the subjects studied. In normal population, levels were in the range of 500 ng/ml +/- 125 (S.D.) A significant increase was observed at the end of pregnancy and during lactation. A large number of patients suffering various benign and cancerous diseases were studied. The elevated levels found in breast and pulmonary pathology indicated that this assay could be useful in the follow-up of patients suffering from these diseases.

Antigenic variants of the nonspecific cross-reacting antigen (NCA).

Monoclonal antibodies (Mab) were prepared against nonspecific cross-reacting antigen (NCA) and were selected on the basis of their absence of reactivity with carcinoembryonic antigen (CEA). Four Mab were found which allowed the characterization on CEA of three epitopes, defined A, B, and C. These epitopes were all located on the peptidic moiety of this highly glycosylated antigen and were present on NCA molecules of heterogeneous m.w. (greater than 100,000, 80,000, and 48,000 m.w., the latter being the most abundant). The amount of NCA was estimated in 251 human sera both by a conventional RIA, using a rabbit antiserum, and by EIA, using different Mab: Mab 4, 18, and 33, which reacted, respectively, with epitopes A, B, and C. Each assay gave a different value of the absolute concentration of NCA in the serum. On the whole, Mab 4 gave lower values, whereas Mab 18 and 33 gave higher values as compared to RIA. Furthermore, whereas all of the human sera contained NCA which was measurable by RIA, 67 sera typed negative in EIA when using Mab 4 or 18. Eight additional sera were negative in more than one EIA. Negativity when using Mab 33 was observed in only one serum, which was also negative with Mab 4 and 18. Twenty-five of 30 sera which were negative with Mab 4 came from cancer patients, and 32 of 37 sera negative with Mab 18 came from normal subjects and noncancer patients, giving a statistically highly significant difference between the two groups of sera (p less than 0.001). Analysis of tissue perchloric extracts and NCA samples purified from these extracts gave similar results. Three extracts (one from lung, two from cancer tissue) and the corresponding NCA samples were negative with Mab 18. The discrepancies observed in these assays are best explained by assuming the existence of antigenic variants of NCA which have not been described previously. These variants appear to exist in various proportions in the different sera. The variants may represent antigenically complete and incomplete molecules. Alternatively, most of the NCA molecules may be incomplete, lacking one or another of the several NCA-specific epitopes. Sequential immunoprecipitation experiments were in favor of the second hypothesis, showing that most of the NCA molecules were incomplete, lacking either epitope A or B.

Presence of alpha-lactalbumin, epidermal growth factor, epithelial membrane antigen, and gross cystic disease fluid protein (15,000 daltons) in breast cyst fluid.

The frequency of gross cystic breast disease in premenopausal women and its possible association with increased breast cancer risk emphasize the importance of investigations relating to breast cyst fluid composition. In order to contribute to a better analysis of this medium, we have measured four proteins the presence of which in human milk was well documented. Breast cyst fluid specimens from 266 breast cyst disease patients were assayed and compared as to concentrations of alpha-lactalbumin, gross cystic disease fluid protein (GCDFP-15), epidermal growth factor (EGF), and epithelial membrane antigen (EMA). All the analyzed cyst fluids contained GCDFP-15, EMA, and EGF whereas alpha-lactalbumin was detected in only 14.2% of fluids assayed. Positive correlations were observed between GCDFP-15 and EMA concentrations (P less than 0.005), as well as GCDFP-15 and EGF concentrations (P less than 0.0005). The cyst fluid GCDFP-15 and EGF levels were higher when alpha-lactalbumin concentrations were below detection limits. This association was statistically significant for GCDFP-15 (P less than 0.03) and for EGF (P less than 0.001). These results suggest that GCDFP-15 and EMA could be the biochemical expression of apocrine metaplasia and epithelial hyperplasia, respectively, two histopathological features which characterize breast cystic disease. On the other hand, the occasional presence of alpha-lactalbumin in the cyst fluid would reflect the persistence of differentiated cells in the epithelium surrounding the cyst and would be inversely proportional to the degree of cellular proliferation. The omnipresence of EGF in the cyst fluid argues for the hypothesis of its production by the mammary gland. The highly significant relationship between GCDFP-15 and EGF levels in the medium remains to be elucidated but may be related to an androgen sensitivity in the breast epithelium surrounding the cyst.

Breast cyst fluid proteins and breast cancer.

A specific breast cyst fluid protein was purified by the following steps: ultracentrifugation, gel filtration, DEAE and Con A chromatography, and gel filtration with guanidine, 6 M. The protein was pure, having a molecular weight of 17,800 daltons on SDS-PAGE and 68,000 daltons on gel filtration. The GCDFP 17,800 is immunologically distinct from other breast cyst fluid components and known milk and plasma proteins. A specific radioimmunoassay was developed and used to determine GCDFP 17,800 in 158 samples of breast cancer cytosol. The GCDFP 17,800 levels were significantly different between grade I tumors (mean of 813 ng protein per mg +/- 430 SEM) and grade III tumors (mean 184 ng protein per mg +/- 59 SEM) and were correlated with progesterone receptor values in postmenopausal women (Spearman's correlation, p = 0.03) but not in premenopausal women. The value of GCDFP 17,800 did not differ between the pre- and the postmenopausal women. By immunocytochemistry the intracellular localization of the GCDFP 17,800 was also found in relation to tumor grading and in correlation with PR values. GCDFP 17,800 appears as a hormone-induced protein of the breast cells. Its intracellular detection by means of radiolabeling allows a more sensitive and precise evaluation of the hormone-dependence of the breast cancer cells and emphasizes the heterogeneity of the tumor cell population.