PMC:13922 / 21444-34197 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/13922","sourcedb":"PMC","sourceid":"13922","source_url":"http://www.ncbi.nlm.nih.gov/pmc/13922","text":"Results\n\nAdsorption of oestrogen isoforms onto hydroxylapatite at low ionic strength\nPrevious studies from our laboratory [12,36] revealed that ER preparations (human ER expressed in yeast, cytosols from MCF-7 breast cancer cells and uterus) adsorbed onto hydroxylapaatite display a lower [3H]oestradiol-binding capacity than those provided by the conventional DCC assay; interference of the phosphocalcic matrix of the hydroxylapatite with ligand binding was advocated to explain this discrepancy. Assessment of cytosols from human primary breast tumours revealed that this property also holds for their receptors.\nIn 97 cases out of a series of 102 cytosols, assessment of the binding parameters of [3H]oestradiol (multipoint Scatchard plot analysis) gave Kd values of the same order of magnitude for both assays (median Kd values of 0.30 for DCC and 0.40 nmol/l for hydroxylapatite; Table 1). Binding capacities established by these two methods were also significantly correlated (r = 0.79; P \u003c 0.001), with systematically higher values for the DCC assay (slope of the regression line= 1.47; Fig 1). The remaining five outlayers bound [3H]oestradiol with a relatively lower affinity (Kd about ten times higher; Table 1). These outliers were also characterized by a weak [3H]oestradiol-binding capacity by the DCC assay and a wide range of values by the hydroxylapatite procedure (Fig. 1; inset). They did not differ from the others with regard to their protein contents, thus refuting the hypothesis of false-negative or false-positive cases associated with a low or high protein level [37]. The nature of these low affinity-binding sites remains unknown.\nUsing [125I]TAZ instead of [3H]oestradiol as the labelling agent in another series of 36 cytosols, we established a similar correlation (r = 0.83; P \u003c 0.001; Fig. 2). The slope of the regression line was similar to that established with [3H]oestradiol (1.40), indicating a similar interference of the phosphocalcic matrix of the hydroxylapatite in the binding of [125I]TAZ. Hence, at low ionic strength, ligand-binding capacities of cytosolic ER from breast tumours are significantly correlated when assessed by DCC and hydroxylapatite assays.\n\nRelease of cleaved oestrogen receptor isoforms from hydroxylapatite with KCl\nData reported above suggest that the ER domains that are required for hydroxylapatite adsorption (ABC domains) and ligand-binding (E domain) are present in most tumour cytosols. In view of the high frequency of cleaved ER isoforms in breast cancer [11], such domains should be dissociated at high ionic strength, as demonstrated here using [125I]TAZ as a labelling agent.\nER isoforms labelled with radiolabelled TAZ were easily detected by successive immunoprecipitation and SDS-PAGE (full-length 67 kDa and cleavage products of 50 and 37-28 kDa) [19,38,39]. Using this approach we observed that a pool of breast cancer cytosols labelled with [125I]TAZ gave different electrophoretic patterns, depending on the nature of the anti-ER monoclonal antibody used in the immunoprecipitation step preceding electrophoresis (H222, H226, ER1D5; Fig. 3a, left). The carboxyl-terminal-specific antibody H222 precipitated all ER isoforms, whereas the amino-terminal-specific antibodies H226 and ER1D5 precipitated only the full-length and a partially truncated 50 kDa isoform, indicating a lack of corresponding antigenic sites in the 37-28 kDa isoforms. Of note, the 50 kDa band was more intensely labelled with H226. The [125I]TAZ-labelling intensity of all of these bands was suppressed with a 200-fold excess of unlabelled oestradiol, establishing their specificity.\nPart of this [125I]TAZ-labelled cytosol pool was adsorbed onto hydroxylapatite and subsequently subjected to KCl extraction (Fig. 3a; right). Elutes were then immunoprecipitated before being subjected to SDS-PAGE. Absence of 67 and 50 kDa isoforms under all immunoprecipitation conditions confirmed their adherence onto hydroxylapatite due to their strong interaction (ABC domains) with the matrix. On the contrary, and as expected, ER isoforms with a molecular weight of between 37 and 28 kDa were detected in the elutes when the immunoprecipitation was carried out by H222; their absence after exposure to H226 or ER1D5 confirmed the cleavage of these isoforms at a site(s) downstream of ABC domains. Hence, hydroxylapatite extraction assay easily identifies ER isoforms that lack amino-termini. Figure 3b illustrates the presumed structure of these various ER isoforms, as well as their sizes as determined by SDS-PAGE.\n\nAssessment of oestrogen receptor mRNA size in breast cancer\nTotal RMA from 46 breast tumours was qualitatively and quantitatively analyzed by hybridization with an ER-α full-length probe (Northern blotting). All tumours expressed a full-length 6.6-kb ER mRNA (small-sized species were not recorded). Moreover, a good correlation was obtained when the amount of 6.6-kb ER mRNA estimated by densitometry was compared with corresponding [3H]oestradiol-binding capacities (DCC values; Fig. 4). Hence, ER isoforms of low molecular weight did not appear to be encoded by truncated ER mRNAs, suggesting that they were generated by proteolysis. Whether such a phenomenon is an intracellular process is analyzed below.\n\nOrigin of cleaved oestrogen receptor forms\nCytosol samples of a series of breast tumours were labelled with [125I]TAZ in the presence of a cocktail of compounds that are known to inhibit the action of a wide range of proteolytic activities (final concentrations: 1 mmol/l for AEBSF, antipain and chymostatin; 5 mmol/l for PMSF; 0.1 mmol/l for leupeptin; and 0.1 mg/ml for calpastatin); this cocktail was added before or shortly after the homogenization of the samples. These inhibitors failed to maintain the native 67 kDa nature of the receptor as demonstrated by SDS-PAGE and autoradiography (Fig. 5); the ER electrophoretic pattern was not significantly modified, still showing bands of low molecular weight.\nIn order to determine the molecular weight of intracellular ER molecules, we applied an in situ labelling approach along with an extraction procedure to minimize ER proteolysis [31,32]; tumour slices were incubated with [125I]TAZ at 37°C before homogenization and lysis at 100°C in the presence of SDS. Labelled proteins extracted with phenolic solution were precipitated by acetone and finally identified by SDS-PAGE. ER electrophoretic patterns remained almost identical to those usually found with cytosols, still showing bands of truncated receptors (mainly 50 and 37-28 kDa; Fig. 6). Hence, the high proportion of the low-molecular-weight isoforms appeared already to be present within tumour samples.\nFinally, breast cancer cytosols were heated at 37°C for 2 min in the absence or presence of a cocktail of protease inhibitors in order to determine whether they possess proteolytic activities that are able to cleave native ER. Samples were subsequently adsorbed onto hydroxylapatite and successively subjected to KCl and ethanol extraction. No significant increase in EI was recorded (Table 2); inhibitors also failed to induce any drastic change in EI values, refuting the hypothesis that isoforms devoid of ABC domains may emerge during assay procedures. Of note, treatment at 37°C of control preparations of native ER (human recombinant ER, MCF-7 cells) resulted in similar behaviour. Hence, major proteolysis did not occur at the time of manipulation. Taken together, these data suggest that dominant ER proteolytic cleavage is an intracellular process.\n\nHydroxylapatite extraction index and heterogeneity of oestrogen receptors\nData reported here clearly show that low-molecular-weight ER isoforms extracted from hydroxylapatite matrix with KCl were not recognized by the ER1D5 monoclonal antibody. Because this antibody is often used in immunohistochemical assessment of ERs, we assessed whether immunohistochemical data are related to hydroxylapatite ER adsorption characteristics measured in cytosolic preparations from the corresponding tumours. For this purpose, cytosols from a set of 15 ER-positive tumours (by DCC assay), for which nuclear ERs had been detected by immunohistochemistry (IS cutoff ≥ 5), were labelled with [3H]oestradiol and were then subjected to hydroxylapatite assay (Table 3). A significant correlation between the two sets of measurement was recorded (IS versus total number of binding sites assayed by hydroxylapatite, r = 0.71; P \u003c 0.001; Table 3). Sequential extraction of bound [3H]oestradiol from hydroxylapatite with KCl and ethanol revealed an EI of over 30% in the large majority of these cytosols (11/15), indicating a high frequency of cleaved ER. Of note, no significant correlation between IS and EI data (r = 0.2; P \u003e 0.05) was detected, clearly establishing that identification of ABC domains within the cell (indicated by IS) does not imply the presence of (native) full-length ER in the corresponding cytosol.\nFigure 1 Comparison of [3H]oestradiol (E2)-binding capacities of a series of human breast cancer cytosols simultaneously measured by DCC and hydroxylapatite (HAP) assays (ethanolic extraction). The ordinate corresponds to values established by DCC, and the abscissa to those by hydroxylapatite (data established by Scatchard plot analysis). A significant correlation was obtained between the two assays in 97 out of 102 (95%) samples; five outlayers are represented by closed symbols in the insert.\nFigure 2 Comparison of [125I]TAZ-binding capacities (5 nmol/l) of a series of human breast cancer cytosols simultaneously measured by DCC and hydroxylapatite (HAP) assays (phosphate extraction).\nFigure 3 (A) Molecular weight and monoclonals anti-ER recognition of [125I]TAZ-labelled ER isoforms extracted from hydroxylapatite (HAP) with KCl. (left) Part of human breast cancer cytosol pool, after labelling with 1 nmol/l [125I]TAZ for 1 h at 0°C in the presence or absence of a 200-fold excess of radioinert oestradiol, was immunoprecipitated with H222, H226 or ER1D5 anti-ER monoclonal antibodies, and then analyzed by SDS-PAGE and autoradiography. (right) Another part of this cytosol pool was adsorbed onto HAP, labelled with 1 nmol/l [125I]TAZ, extracted with 0.5 mol/l KCl, and immunoprecipitated before being subjected to electrophoresis. (B) Presumed structure of ER isoforms extracted from HAP with KCl. Potential sites of covalent attachment of TAZ [40,41] are indicated by open circles; antigenic sites for anti-ER monoclonal antibodies are shown above ER structure. The predicted ER isoforms extracted from HAP as well as their sizes determined by SDS-PAGE are shown below.\nFigure 4 Correlation between ER mRNA and ER (by DCC assay). Breast tumours mRNA abundance (intensity of the 6.6-kb band) was expressed relatively to mRNA levels in MCF-7 cells (MCF-7 = 1).\nFigure 5 Effect of heat treatment on the relative expression of ER isoforms. Human breast cancer cytosols were heated at 37°C for 2 min in the presence or absence of protease inhibitors. They were then labelled with [125I]TAZ in the presence or absence of a 200-fold excess of radioinert oestradiol, immunoprecipitated with H-222 anti-ER monoclonal antibodies, and then subjected to SDS-PAGE. Lane 1, unheated control; lane 2, plus an excess of oestradiol; lane 3, 2 min heating in the absence of protease inhibitors; lane 4, 2 min heating in the presence of protease inhibitors.\nFigure 6 In situ labelling of ER with [125I]TAZ. Breast tissues slices (samples 1-4, ER positive; sample 5, ER-negative) were incubated with 1 nmol/l [125I]TAZ for 1 h at 0°C and the unbound ligand was removed. Then, all tissues were mixed with Krebs-Ringer phosphate buffer containing 1% SDS, 1.6 mmol/l EDTA and 2% β-mercaptoethanol, and briefly homogenized. After lysis at 100°C, proteins were extracted with phenol, precipitated by acetone and were finally analyzed by SDS-PAGE. The figure shows the electrophoretic patterns of these tissue ERs.\nTable 1 Comparison of Kd values estimated by DCC and hydroxylapatite (HAP) assays in the whole series of human breast cancer cytosols(102 cases) as well as in the five cases not included in the correlation\nTable 2 Comparison of EIs of adsorbed ER onto hydroxylapatite matrix in the absence and presence of protease inhibitors hER, human full-length ER.\nTable 3 Comparative analysis of ER by immunohistochemistry (ER1D5), DCC ([3H]oestradiol-binding) and hydroxylapatite ([3H]oestradiol-binding and EI) *Hydoxylapatite corresponds specifically to [3H]oestradiol extracted successively from hydroxylapatite with KCl and ethanol (ie 27 = 20 + 7). †EI = ([3H] oestradiol) [KCl] × 100/([3H]oestradiol) [KCl] + ([3H]oestradiol) [EtOH]. 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