Tamoxifen Binding Research Articles

Species differences in the covalent binding of [ 14C]tamoxifen to liver microsomes and the forms of cytochrome P450 involved

Species differences in the NADPH-dependent covalent binding of [ 14C]tamoxifen to liver microsomes have been studied using preparations from humans, female F344 rats and DBA/2 mice. Protein binding has been used as an index of metabolic activation and as a surrogate for DNA binding in order to establish which forms of cytochrome P450 are responsible for genotoxicity. A panel of 12 human liver microsomes has been characterized and immunoquantified for nine cytochrome P450 isoenzymes. Binding of tamoxifen (45 μM) (25 ± 2.5 pmol/15 min/mg protein, mean ± SE) correlated (P < 0.05) with CYP3A4 and CYP2B6 content. Covalent binding of [ 14C]tamoxifen to microsomal preparations from human breast tumour tissue could also be detected but at levels 7-fold lower than in liver. The covalent binding of tamoxifen to mice, rat or human liver microsomal preparations increased with increasing substrate concentration. Covalent binding of [ 14C]tamoxifen (45 μM) in rats was 3.8-fold and mice 17-fold higher than in human liver microsomal preparations. In mice, the apparent K m (9.6 ± 1.9 μM) was very much lower than for rats (119 ± 41 μM). Pretreatment of female rats with phenobarbitone or dexamethasone resulted in a 4- to 5-fold increase in [ 14C]tamoxifen binding, relative to controls, consistent with the involvement of CYP2B1 and CYP3A1 in the metabolic activation. It cannot be distinguished at present if the same reactive metabolites are involved in protein and DNA binding. The greater potential of mouse liver microsomes to activate tamoxifen, relative to rats, does not reflect DNA damage or hepatocarcinogenicity seen following dosing with tamoxifen in vivo. It is concluded that covalent binding of tamoxifen to protein in vitro cannot be directly related to the carcinogenic potential of this compound. However, in the three species investigated, results suggest that the rat is a better model than the mouse for human liver microsomal activation of tamoxifen both with respect to kinetic parameters and the pattern of metabolic products.

Peroxidase activation of tamoxifen and toremifene resulting in DNA damage and covalently bound protein adducts.

When [14C]tamoxifen was incubated with horseradish peroxidase and H2O2, two major metabolites, separated and identified by HPLC, were N-desmethyltamoxifen and tamoxifen N-oxide. Toremifene incubated in a similar system yielded N-desmethyltoremifene and toremifene N-oxide. No 4-hydroxylated metabolites were detected with either drug. When calf thymus DNA was included in peroxidase incubation mixtures, DNA damage, as assessed by 32P-postlabelling, could also be detected. The extent of damage caused by tamoxifen and toremifene was similar. The major adducts formed following incubation of DNA with tamoxifen had similar Rf values to two of the 32P-postlabelled adducts seen following dosing of rats with tamoxifen. Peroxidase was able to activate both drugs to derivatives which covalently bound to bovine serum albumin. The pH optimum for covalent binding and N-demethylation was near to pH 6.0. Results from liquid chromatography-electrospray secondary ion mass spectrometry suggest that tamoxifen and toremifene are metabolized by peroxidase to putative reactive epoxide intermediates responsible for the genotoxic effects. It is proposed that peroxidase oxidizes tamoxifen to a carbon-centred free radical which reacts with oxygen to form peroxy radicals capable of inserting an oxygen atom into tamoxifen. Lactoperoxidase and prostaglandin synthase are also able to catalyse tamoxifen N-demethylation and binding to protein. These data show that peroxidase can activate both tamoxifen and toremifene to an intermediate(s) that can damage DNA and covalently react with protein. Since it is known that women treated with tamoxifen can develop endometrial tumours, it may be relevant to determine whether activation of tamoxifen by peroxidases may contribute to its carcinogenic action at extrahepatic sites.

Relationships between tamoxifen binding proteins in primary breast cancer biopsies

Using high-resolution isoelectric focusing gel electrophoresis (IEF), two tamoxifen binding sites (TBS) with isoelectric point (pI) values of 4.5 and 4.3 were identified, with different affinities for tamoxifen. The form at pI 4.3 (HTBS) displayed high affinity for the ligand (kD ≈ 5 nM), while the protein at pI 4.5 (LTBS) had lower affinity (kD ≈ 50 nM). LTBS was found in the microsomal fraction and HTBS in the cytosol. Of a total of 319 tumours studied, 257 were oestrogen receptor (ER) positive and 106 HTBS positive. In this combined group, thus able to bind tamoxifen either through the presence of ER or HTBS (or both), ER and PR were both negatively correlated with HTBS ( P < 0.0001). The oestrogen-induced protein pS2 was assayed in 92 of the 319 tumours, and was also negatively ( P < 0.0001) correlated with HTBS. The levels of HTBS were similar between infiltrating ductal carcinomas without special features (NOS) and non-NOS forms. However, HTBS concentrations were significantly higher in poorly differentiated grade 3 carcinomas than grade 2 ( P < 0.05) and grade 1 ( P < 0.01) forms. Conversely, ER concentration was lower in grade 3 than grade 1 forms ( P < 0.05). Both the relationship between high affinity TBS and ER and the high concentration of HTBS in ER-poor grade 3 carcinomas may have a bearing on the known variability of tumour response to endocrine therapy and prognosis.

Antiestrogen binding sites in brain and pituitary of ovariectomized rats

In addition to interacting with estrogen binding sites in a number of tissues, antiestrogens have recently been shown to interact with a separate, estrogen-non-competable, antiestrogen-specific binding site (AEBS). In order to better understand possible mechanisms by which the antiestrogens may effect behavioral and physiological changes, we have examined AEBS in several areas of the brain and pituitary in adult, ovariectomized rats. Single point binding assays with 2 nM [ 3H]tamoxifen (TAM) in the presence of saturating amounts (1 μM) E and ± 1μM TAM indicated the existence of specific binding to AEBS throughout the brain and pituitary. In most areas of the brain (cortex, cerebellum, amygdala, area postrema/nucleus of the solitary tract region) as well as pituitary, Scatchard analyses revealed the presence of a single AEBS with a dissociation constant ( K d = 1−4 × 10 −9M ) similar to that previously reported for other tissues. However, in both hypothalamus and preoptic area, an additional, higher affinity site ( K d = 6−9 × 10 −11M ) was found. Competitive inhibition studies revealed that there was little competition by the potent estrogen agonist, diethylstilbesterol, for AEBS binding. Antiestrogens competed in the following order: tamoxifen ≥ nafoxidine ≫ keoxifene. Additional competitive inhibition studies were run using neurotransmitter antagonists. for binding at the AEBS. Other pharmacological substances, including specific antagonists of the D 2 sites, as well as antagonists of the norepinephrine, opiate, histamine, GABA and acetylcholine systems, were ineffective competitors for [ 3H]TAM binding.

Human Albumin Binding of Tamoxifen in the Presence of a Perfluorochemical Erythrocyte Substitute

The binding of tamoxifen (an HSA site IV ligand) to human serum albumin (HSA) in the presence of a perfluorochemical (PFC) erythrocyte substitute has been examined. Standard centrifugation followed by supernatant ultrafiltration was used to study the binding of 0.1 and 0.5 microgram mL-1 tamoxifen at ambient conditions. Tamoxifen was extensively bound (greater than 99%) to the PFC emulsion through an association with the emulsifiers of the droplets. Tamoxifen was also extensively bound (greater than 99%) to HSA. The percent free tamoxifen increased upon HSA dilution. Tamoxifen was extensively bound by various mixtures of HSA and the PFC emulsion and the percent free drug was similar to those obtained with HSA alone. However, the position of drug binding (PFC emulsion vs HSA) varied significantly with changes in the ratio of PFC emulsion to HSA. This could be important in terms of the different distribution of HSA and PFC emulsion in the body. Studies with PFC emulsion components indicated that any displacement of HSA-bound tamoxifen by the PFC emulsion was due to the oleic acid and, to a much smaller degree, Pluronic F-68 components. Other HSA site IV ligands are expected to be similarly displaced.

Involvement of oestrogens in sexual differentiation of gonads as a function of temperature in turtles

Abstract To investigate the possible involvement of oestrogens in the phenomenon of temperature sex-reversal in the turtle Emys orbicularis, the effects of oestrone, oestradiol and an antioestrogen. tamoxifen, on sexual differentiation of gonads were examined at a male-producing temperature of 25° C and at a female-producing temperature of 30° C. When oestrone or oestradiol were injected into eggs incubated at 25° C just before or at the beginning of the thermosensitive period, the gonads differentiated into ovaries instead of testes. Conversely, when tamoxifen was injected, at the same stages, into eggs incubated at 30° C, epithelial cords or tubes, similar to potential seminiferous cords, differentiated in the interior part of the gonads. However, an ovarian-like cortex persisted at their surface. At 25° C, treatment with tamoxifen or with both tamoxifen and oestradiol also resulted in differentiation of ovotestes. These experiments show that tamoxifen binding to oestrogen receptors prevented the inhibitory action of oestrogens on testicular cord development. Maintenance (at 30° C) or development (at 25° C) of ovarian cortex in the presence of tamoxifen can be expected from an agonistic action of this drug, as already described. Preliminary data on steroid content in the gonads indicate that, during the early stages of the thermosensitive period, the level of estrogens is higher at 30° C than at 25° C. It is proposed that in species displaying temperature sensitivity for the sexual differentiation of gonads, temperature acts on the processes regulating the synthesis or the activity of cyto-chrome-P450 aromatase.

Tamoxifen binding sites heterogeneity in breast cancer: a comparative study with steroid hormone receptors

Steroid receptors and tamoxifen binding sites (TBS) were assayed in the soluble fraction of 121 primary breast cancers. Scatchard analysis of TBS in high speed supernatant (100 000 g) showed one population of binding sites; however, biphasic plots were obtained in low speed supernatants (40 000 g). Isoelectric focussing of supernatants preincubated with radioactive tamoxifen identified two classes of TBS (pI 4.1–4.6) which have different binding affinities and bind neither oestradiol nor diethylstilbestrol. Association between TBS and steroid receptors was: TBS positive/progesterone receptor positive 32.6%, TBS positive/glucocorticoid receptor positive 52.7%, TBS positive/oestrogen receptor positive 60% and TBS positive/androgen receptor positive 72.2%. We conclude that heterogeneous TBS are present in low speed fractions and can be easily separated from the oestrogen receptor by isoelectric focussing. The association between TBS and steroid receptor status could be of clinical value in the management of primary breast cancer.

Blood and urinary measures of oxidant damage in healthy human subjects

similarity suggests that loss of protection follows the same time course in each case and indeed the onset of inhibition by tamoxifen of both RNA and protein synthesis has been shown to be simultaneous [3]. These results indicate that the protection of these cellular processes by ascorbate is time dependent and, furthermore, they show that inhibition by tamoxifen becomes irreversible with time. It is possible that cytochrome P-450 enzymes could be the intracellular target site(s) for tamoxifen in yeast [4]. The cytochrome I’-450-dependent lanosterol 14-a-demethylase enzyme on the ergosterol biosynthetic pathway is such an enzyme and is of great importance to yeast growth. Tamoxifen has been shown to inhibit this enzyme, on the analogous cholesterol biosynthetic pathway, in the breast cancer cell line MCF-7 IS]. The binding of tamoxifen to this proposed target site may thus be partially prevented by the simultaneous addition of ascorbate. This time-dependent protective effect of ascorbate could be via the maintenance of essential thiol groups in cytochrome P-450-dependent enzymes such as lanosterol 14-a-demethylase, which are deleteriously affected by the oxyradicals that might be generated by the action of cytochrome P-450 during tamoxifen metabolism 16-91. The award of a Leverhulme Emeritus Fellowship to H.K.V.A. i s gratefully acknowledged. We also thank the Charities Aid Foundation for financial help.

Reversal of acquired resistance to adriamycin in CHO cells by tamoxifen and 4-hydroxy tamoxifen: role of drug interaction with alpha 1 acid glycoprotein

Tamoxifen and 4-OH tamoxifen were used to reverse multidrug resistance (MDR) in CHO cells with acquired resistance to adriamycin (CHO-Adrr). Because alpha 1 acid glycoprotein (AAG) can bind a range of calcium channel blockers that also reverse MDR and rises in malignancy, its interactions with tamoxifen and 4-OH tamoxifen were also studied. Tamoxifen decreased the IC50 of 10 microM adriamycin 4.8-fold in the parent CHO-K1 cell line and 16-fold in CHO-Adrr. Similarly 4-OH tamoxifen decreased the IC50 3-fold in the parent cells, but 13-fold in the resistant cells. Tamoxifen and 4-OH tamoxifen were similarly potent in reversing MDR, although their anti-oestrogen potency differs 100-fold. AAG was added in increasing concentrations to the combination of adriamycin and tamoxifen. As AAG concentrations increased from 0.5 to 2 mg ml-1 (the range found in vivo) the effect of tamoxifen on reversing MDR was gradually decreased. At the highest AAG concentrations, there was complete reversal of the effects of both tamoxifen and 4-OH tamoxifen. AAG was found to bind 3H-tamoxifen in a non-saturable non-specific manner, in contrast to the binding of tamoxifen to albumin. Thus the use of tamoxifen as a reversal agent for MDR in vivo may be impaired by high binding to AAG. However, at the lower range of normal values of AAG, there was still an effect of 10 microM tamoxifen. It may be desirable to select patients for modifier studies based on AAG plasma levels.

High-affinity binding sites for oxygenated sterols in rat liver microsomes: possible identity with antiestrogen binding sites

Oxygenated derivatives of cholesterol are known to exhibit a number of biological activities including the inhibition of cholesterol biosynthesis and of cell proliferation, but their mechanism of action remains unclear. Previous studies have identified a cytosolic protein which binds 25-hydroxycholesterol, as well as several other oxysterols, with high affinity, possibly mediating some of their effects. We now report the existence of a high-affinity oxysterol binding site in rat liver microsomes which is distinct from the cytosolic binding protein. Among the oxygenated sterols examined, 5α-cholestan-3β-ol-7-one (7-katecholestanol) had the highest affinity for this microsomal binding site ( K d = 2.7 nM). Using 7-keto[ 3H]cholestanol as the radioactive ligand, we found that binding of this oxysterol to the microsomal binding site was saturable and reversible and was displaceable by the following oxysterols in descending order of potency: 7-ketocholestanol > 6-ketocholestanol > 7 β-hydroxycholesterol = 7-ketocholesterol > cholesten-3 β,5 α6 β-triol = 7 α-hydroxycholesterol > 4-cholesten-3-one. One other sterols studied, including, notably, 25-hydroxycholesterol, had little or no inhibitory effect on 7-keto[ 3H]cholestanol binding. Additional studies revealed that the microsomal oxysterol binding site was probably identical to the antiestrogen binding site described by other workers. First, saturation analysis and kinetic studies demonstrated that the antiestrogen tamoxifen competed directly with 7-keto[ 3H]cholestanol for the same binding site. Second, tha ability of different oxysterols and antiestrogens to inhibit 7-keto[ 3H]cholestanol binding to the microsomal binding site paralleled their ability to inhibit [ 3H]tamoxifen binding to the antiestrogen binding site. Third, the tissue distribution of binding sites for 7-keto[ 3H]cholestanol was similar to that antiestrogen binding site. We conclude that: (1) in rat liver microsomes there are high-affinity oxysterol binding sites whose ligand specificity is different from that of the cytosolic oxysterol binding protein; and (2) the microsomal oxysterol binding site is probably identical to the antiestrogen binding site. The biological significance of these observations remains to be explored.

Possibility of anti-estrogen action from estriol specific binding sites in rabbit uterus

1. This study was designed to investigate the clomiphene or tamoxifen binding to receptor sites for estradiol-17β (E 2R) and estriol (E 3R) in the rabbit uterus. 2. Those so-called anti-estrogenic compounds tended to inhibit E 2-E 2 R and E 3-E 3R bindings equally. 3. The inhibitor constant of clomiphene for E 2R was approximately 3.8 × 10 −8 M at 4°C and that for E 3R approximately 1.8 × 10 −8 M at 4°C in a given case, determined by charcoal assay. 4. It is suggested that the anti-estrogenic compounds demonstrate their effects after binding either to E 2R or to E 3R. 5. There were some tissue differences of the contents between E 2R and E 3R. For example, the uterus and the cortex contained E 2R, and the pituitary E 3R more than the other.

Cytostatic effect of antiestrogens in lymphoid cells: relationship to high affinity antiestrogen-binding sites and cholesterol

The antiproliferative effect of 10(-6) M antiestrogens in an estrogen receptor-negative lymphoid cell line (K36) was enhanced in lipoprotein-poor growth medium. The enhancement was not due to increased bioavailability because cellular uptake of [3H]tamoxifen was not increased and the lipoprotein fraction of serum had negligible [3H]tamoxifen-binding capacity. Cholesterol and lipoproteins, but not mevalonate, reversed the cytostatic effect of antiestrogens. Reversal by cholesterol was dose-related (10(-7) M to 10(-5) M), while that by lipoproteins could also be demonstrated in medium undepleted of lipoproteins. The cytostatic efficacy of a series of ten compounds correlated well with their relative binding affinities for solubilized antiestrogen-binding sites from K36 cells when log IC50 values (concentration required to reduce [3H]thymidine incorporation by 50%) were plotted against log RBA50 values (concentration required to reduce [3H]tamoxifen binding by 50%) (correlation coefficient 0.94). Transmission electron microscopy of antiestrogen-treated cells showed evidence of disordered cytokinesis which was partially reversed by cholesterol. These observations implicate the antiestrogen-binding protein in the antiproliferative effect of antiestrogens in nonestrogen target cells.

Interactions of sterols with antiestrogen-binding sites: structural requirements for high-affinity binding.

Animal and human tissues contain a microsomal protein that binds nonsteroidal antiestrogens with high affinity and specificity. The functions of these binding sites and the identity of their natural ligands are unknown. Following a report that certain sterols inhibit [3H]tamoxifen binding to this site, we attempted to define the structural requirements for maximal inhibition of [3H]tamoxifen binding to rat liver antiestrogen-binding sites. Our studies identified 5 alpha-cholestan-3 beta-ol-7-one (7-ketocholestanol) as the most potent sterol, having an inhibitory activity that was 12% that of unlabeled tamoxifen and an equilibrium dissociation constant of 6.3 nM. Structural features that appeared important for the inhibitory activity of this sterol include the presence of i) a hydrocarbon side chain at C17; ii) an oxygen function at C7; iii) a hydroxyl group at C3; and iv) the absence of a double-bond between C5 and C6. Saturation analysis and kinetic studies of [3H]tamoxifen binding in the presence of varying concentrations of 7-ketocholestanol clearly indicated that this sterol competed directly with tamoxifen for the antiestrogen-binding site. Unlike tamoxifen, this sterol did not bind to the estrogen receptor. These features make 7-ketocholestanol a potentially valuable tool for studying the properties and functions of this site.

Murine antiestrogen-binding protein: Characterization, solubilization and modulation by lipids

The properties of the antiestrogen-binding protein have been examined in mouse tissues, a species in which nonsteroidal antiestrogens are virtually pure agonists. As in other species studied, this protein wa distributed in all tissues — highest levels being in the liver. Subcellular fractionation of mouse liver showed that 82% of the antiestrogen-binding protein was associated with the rough endoplasmic reticulum where it was confined to the membranous component. The antiestrogen-binding protein was also present in smooth endoplasmic reticulum, nuclei and cytosol. Its concentration in intact nuclei was at least 10-times higher than levels previously reported in intact rat liver nuclei. Binding of [ 3H]tamoxifen to the murine antiestrogen-binding protein was of high affinity ( K d = 1 nM) and was inhibited by unsaturated fatty acids and 7-ketocholesterol. In general, cis-isomers of unsaturated fatty acids were more effective binding inhibitors than trans-isomers. The antiestrogen-binding protein solubilized from rough endoplasmic reticulum membranes by the zwitterionic detergent CHAPS, had a molecular mass of approx. 700 kDa and a sedimentation coefficient of about 19 S. [ 3H]Tamoxifen binding capacity of the solubilized protein was abolished by trypsin and nonspecific proteinases but not by clostripain or Staphylococcus aureus V8 proteinase, suggesting that lysine residue(s) may be involved in [ 3H]tamoxifen binding.

The mechanism of hormone-sensitive breast cancer progression on antiestrogen therapy. Implications for treatment and protocol planning.

Fifteen patients whose tumors progressed while they received tamoxifen citrate therapy were studied by serial determinations of serum levels of estrone (E1), estradiol (E2), and dehydroepiandrosterone (DHEA) obtained during progression after withdrawal from tamoxifen therapy and total endocrine ablation or suppression. Discontinuation of tamoxifen therapy resulted in reductions of DHEA, E1, and E2 levels by 44%, 49%, and 42%, respectively. Ablation or suppression reduced sex steroids to minimal levels and produced responses in all patients. Elevations of DHEA, E1, and E2 could be provoked by readministering tamoxifen to hypophysectomized and oophorectomized, but not adrenalectomized, patients, indicating that the adrenal gland is the source of these sex steroids. We conclude that tamoxifen stimulates adrenal production of DHEA, which is aromatized to E1 and E2. Buildup of E1 and E2 overwhelms the competitive binding of tamoxifen to the estrogen receptor, resulting in tumor progression.

A study on the correlation between estrogen receptor, progesterone receptor and tamoxifen binding sites in human breast cancer tissues.

In order to examine the clinical significance of the tamoxifen binding site, correlations between the concentration of the tamoxifen binding site and either the estrogen receptor (ER) or progesterone receptor (PgR) were studied. A saturable high-affinity tamoxifen binding site was detected in human breast cancer tissues. The concentration of the tamoxifen binding sites in 12,000 g supernatant of ER positive tumors was 122.3 +/- 186.8 (mean +/- SD) fmol/mg protein, and that of ER negative tumors was 68.38 +/- 82.97 fmol/mg protein. The concentration of the tamoxifen binding sites in 12,000 g supernatant of PgR positive tumors was 103.7 +/- 137.3 fmol/mg protein, and that in the 12,000 g supernatant of PgR negative tumors was 90.29 +/- 159.0 fmol/mg protein. There was no significant difference between the concentration of the tamoxifen binding sites in the 12,000 g supernatant of ER positive tumors and ER negative tumors, and there was no significant difference between the concentration of the tamoxifen binding sites in the 12,000 g supernatant of PgR positive tumors and PgR negative tumors. The concentration of the tamoxifen binding sites in the cytosol of PgR positive tumors was 90.55 +/- 103.5 fmol/mg protein, and that in the cytosol of PgR negative tumors was 29.13 +/- 42.22 fmol/mg protein. There was a significant difference between them (p less than 0.05). The concentration of tamoxifen binding sites in 12,000 g supernatant had no correlation with the values of ER and PgR. Only the content of tamoxifen binding sites in cytosol, and the PgR value had a significant correlation.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of unsaturated fatty acids with anti-oestrogen-binding sites.

Specific high-affinity binding sites for non-steroidal anti-oestrogens such as tamoxifen have been identified in many animal and human tissues. The function of these binding sites and the nature of their endogenous ligands are currently unknown. Our laboratory has previously reported that unsaturated fatty acids at micromolar concentrations inhibited [3H]tamoxifen binding to the anti-oestrogen-binding sites in rat liver, raising the possibility that fatty acids might represent endogenous ligands for these sites. These studies have now been extended to examine the mechanism by which fatty acids inhibit [3H]tamoxifen binding to the anti-oestrogen-binding site. Saturation analysis revealed that increasing concentrations of oleic acid progressively decreased the apparent binding affinity of these sites for [3H]tamoxifen without decreasing the total number of binding sites; however, the apparent dissociation constant did not vary linearly with the prevailing oleic acid concentration, suggesting that the inhibition of [3H]tamoxifen binding by fatty acid was not competitive in nature. Kinetic studies of [3H]tamoxifen binding showed that oleic acid did not affect the rate of association, but increased the rate of dissociation of [3H]tamoxifen from the anti-oestrogen-binding site; the latter finding would not be expected if oleic acid acted as a competitive inhibitor. Furthermore, incubation of a rat microsomal fraction with [3H]oleic acid in the absence and presence of excess non-radioactively labelled tamoxifen also failed to demonstrate direct competition between oleic acid and tamoxifen for the same binding site. It is concluded that oleic acid, and presumably other unsaturated fatty acids, do not compete for the anti-oestrogen-binding site and probably reduce its tamoxifen-binding affinity by some other mechanism, such as perturbation of the lipid environment of the binding site. The biological significance of this interaction of unsaturated fatty acids with the anti-oestrogen-binding site remains to be elucidated.

Subcellular and extracellular localization of specific binding sites for triphenylethylene antiestrogens in human breast cancer

MCF-7 human breast cancer cell homogenates and subcellular organelles were submitted to isopycnic centrifugation on Percoll gradients to investigate the subcellular localization of triphenylethylene antiestrogen specific binding sites (AEBS). Electron microscopy revealed that gradient fractions coincident with the migration of [3H]tamoxifen-AEBS complexes were homogeneously represented by rough and smooth endoplasmic reticulum. Eighty percent of AEBS were localized in the endoplasmic reticulum [45,000 ± 4,000 sites/cell, mean ± S.D.), while 20% of these sites were also found in the nuclear fraction (12,000 ± 1,000 sites/cell, mean ± S.D.). A similar subcellular distribution of AEBS was observed in human breast cancer bioptic specimens. No differences in [3H]tamoxifen binding affinity between microsomal and nuclear AEBS were observed in MCF-7 and bioptic breast cancer. No major differences in microsomal AEBS levels were observed in the limited number of estrogen receptor-positive or -negative breast cancer specimens we have studied, whereas estrogen receptor-negative samples had higher levels of nuclear AEBS with respect to estrogen receptor-positive tumors. The presence of AEBS was also detected in the human serum of healthy and tumor-bearing subjects. The affinity and the binding specificity of serum AEBS were similar to those of intracellular AEBS. No differences in the levels of serum AEBS were observed between healthy and tumor-bearing subjects [19 ± 4and22 ± 4 pmoles/ml (mean ± S.D.) respectively. Human serum AEBS did not appear to be associated to lipoproteins, whereas it migrated as a 5.5 S sedimenting molecule.

Subcellular localization of triphenylethylene antiestrogen binding sites (TABS) in rat liver

The subcellular distribution of triphenylethylene anti-estrogen binding sites (TABS) was examined in the rat liver. Nuclear, mitochondrial and microsomal fractions were prepared by differential centrifugation, extracted with 0.5 M KCl and bound [ 3H]tamoxifen was determined by the dextran coated charcoal method. The relative concentration of TABS in each fractions were: nuclear 30.2; mitochondrial, 14.8 and microsomal, 10.2 pmol/g tissues. No TABS were detected in the high speed cytosol. The dissociation constants of nuclear and mitochondrial TABS were similar (1–2 nM); however, a higher number was obtained for microsomal TABS (5–6 mM). The ability of other triphenylethylene drugs to compete for [ 3H]tamoxifen binding to TABS was similar to tamoxifen for mitochondrial and microsomal sites. In contrast, nafoxidine was a more potent inhibitory for nuclear TABS. Exposure of high salt nuclear extracts to charcoal prior to assay did not reveal any evidence for an endogenous ligand of high affinity. We conclude that TABS are present in nuclear, mitochondrial and microsomal fractions of rat liver and that the nuclear fraction contains the highest concentration of these sites.

Tamoxifen binding sites in human mammary cancers.

Tamoxifen binding sites (TBS) were measured using 3H-tamoxifen, the objective being to evaluate the relationships among TBS and hormone receptors and/or clinical and pathological characteristics in malignant tissues from 60 patients with mammary cancer. TBS were detected in most (96.7 per cent) cancers in the breast tissues, and the mean content and affinity were 569 fmol/mg X protein with Kd: 1.98 nM. There was no significant correlation between TBS and the estrogen receptor and/or progesterone receptor, with respect to positivity or content. However, there was a significant correlation between TBS and histological grading, thereby indicating the differentiation and the proliferative activity in this tissue. The content of TBS was significantly higher in the group with a high grade of malignancy. The TBS content significantly increased in parallel with the degree of malignancy, as related to tubule formation, nuclear pleomorphism and mitotic activity. On the other hand, there was no significant correlation between TBS and age, tumor size, lymph node status or clinical stage. These results suggest the possibility that TBS may be associated with differentiation and cell-proliferation in breast cancer tissues.