Hydroxylapatite extraction index and heterogeneity of oestrogen receptors
Data 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 < 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 > 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.
Figure 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.
Figure 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).
Figure 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.
Figure 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).
Figure 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.
Figure 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.
Table 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
Table 2  Comparison of EIs of adsorbed ER onto hydroxylapatite matrix in the absence and presence of protease inhibitors  hER, human full-length ER.
Table 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]. IHC, immunohistochemistry; IS, immunostaining score.