Like sugar for poison: glucose as a substitute for benzene.

The concentration of oestrogen receptors (ER) in a breast cancer is now well established as an index for predicting the subsequent response of the disease to endocrine therapy (Hawkins et al., 1980). Oestrogen receptor concentration is influenced by menstrual status, tumour cellularity and tumour grade (Steele et al., 1982), and also rises with increasing age (e. influence of tissue and plasma levels of oestrogen on ER concentration, however, is as yet less clearly understood: whilst high levels of circulating oestrogen have been found to be associated with low/absent tumour ER in premenopausal women, less is known about this relationship in postmenopausal women. In the present study, we have examined the quantitative relationship between plasma oestrogens and ER in the tumours from 50 postmenopausal women. Plasma for the assay of oestrone and oestradiol-17/3 and tumour for the assay of ER were obtained from 50 postmenopausal women with primary cancer of the breast. Plasma samples were obtained betweem 9 a.m. and 11 a.m. in the week prior to operation, and at operation, the tumour was placed on ice for immediate transfer to the laboratory. Oestrogen receptor activity was determined by by saturation analysis (Hawkins et al., 1975) with minor modifications. A portion of tumour (300mg) was homogenised (1:10 w/v) in Tris buffer (10mM Tris, 0.25 M sucrose, 1 mm ethylene diamine tetrace-tate, containing 10% v/v glycerol and 1% monothioglycerol) and centrifuged at 2040g for 20min. Aliquots of the resulting cytosol were in-cubated overnight at 40C with 0. fractions were separated by the addition of dextran-coated charcoal suspension, adsorption and centri-fugation, and the radioactivity in the bound fraction was determined by liquid scintillation counting. The concentration of receptor was calculated by 6-point Scatchard analysis (Scatchard, 1949). Cytosolic protein concentration was determined by the method of Bradford (1976) and receptor levels in excess of 5 fmol mg-1 tumour cytosol protein were regarded as positive. Radioimmunoassay of plasma oestrone Plasma samples (1 ml), diluted 1: 1 (v/v) with water plus 7.4fmol tracer [2,4,6,7-3H], oestrone, were extracted with 10ml of Analar ether. The extract was backwashed with 0.2 ml of 8% (w/v) sodium bicarbonate solution, evaporated to dryness and dissolved in Tris buffer. A portion (1/3) was removed and counted to assess manipulative losses and the remainder was used for radioimmunoassay. After overnight incubation at 4°C with 0.1 ml of antioestrone antibody (1:62,750 v/v in buffer), 0.1 ml of [2,4,6,7-3H] oestrone solution (10,000cpm was added). Bound …

The concentration of oestrogen receptors (ER) in a breast cancer is now well established as an index for predicting the subsequent response of the disease to endocrine therapy (Hawkins et al., 1980). Oestrogen receptor concentration is influenced by menstrual status, tumour cellularity and tumour grade (Steele et al., 1982), and also rises with increasing age (e.g., Clark & McGuire, 1983;Mercer et al., 1983;Daxenbichler et al., 1982). The influence of tissue and plasma levels of oestrogen on ER concentration, however, is as yet less clearly understood: whilst high levels of circulating oestrogen have been found to be associated with low/absent tumour ER in premenopausal women (Maass et al., 1972;Nagai et al., 1979;Hawkins et al., 1979), less is known about this relationship in postmenopausal women.
In the present study, we have examined the quantitative relationship between plasma oestrogens and ER in the tumours from 50 postmenopausal women.
Plasma for the assay of oestrone and oestradiol-17/3 and tumour for the assay of ER were obtained from 50 postmenopausal women with primary cancer of the breast. Plasma samples were obtained betweem 9 a.m. and 11 a.m. in the week prior to operation, and at operation, the tumour was placed on ice for immediate transfer to the laboratory.
Oestrogen receptor activity was determined by by saturation analysis (Hawkins et al., 1975) with minor modifications. A portion of tumour (300mg) was homogenised (1:10 w/v) in Tris buffer (10mM Tris, 0.25 M sucrose, 1 mm ethylene diamine tetracetate, containing 10% v/v glycerol and 1% monothioglycerol) and centrifuged at 2040g for 20min. Aliquots of the resulting cytosol were incubated overnight at 40C with 0.031 nM [2,3,6,7-3H] oestradiol and varying amounts of competing non-radioactive oestradiol (0, 0.03, 0.09, 0.15, 0.21, 0.28, 3.06 and 61.2 nM). Free and bound steroid fractions were separated by the addition of dextrancoated charcoal suspension, adsorption and centrifugation, and the radioactivity in the bound fraction was determined by liquid scintillation counting. The concentration of receptor was calculated by 6-point Scatchard analysis (Scatchard, 1949). Cytosolic protein concentration was determined by the method of Bradford (1976) and receptor levels in excess of 5 fmol mg-1 tumour cytosol protein were regarded as positive.
Radioimmunoassay of plasma oestrone Plasma samples (1 ml), diluted 1: 1 (v/v) with water plus 7.4fmol tracer [2,4,6,7-3H], oestrone, were extracted with 10ml of Analar ether. The extract was backwashed with 0.2 ml of 8% (w/v) sodium bicarbonate solution, evaporated to dryness and dissolved in Tris buffer. A portion (1/3) was removed and counted to assess manipulative losses and the remainder was used for radioimmunoassay. After overnight incubation at 4°C with 0.1 ml of antioestrone antibody (1:62,750 v/v in buffer), 0.1 ml of [2,4,6,7-3H] oestrone solution ( 10,000cpm was added). Bound hormone was separated from free by the addition of dextran-coated charcoal and adsorption: after centrifugation, the radioactivity in the bound fraction was measured by liquid scintillation counting. The amount of oestrone present was determined by reference to a standard curve derived from known amounts of radioinert oestrone (0.003-0.617pmolml-') and was corrected for manipulative losses. Values were expressed as nmol oestronel-1 plasma.
Radioimmunoassay of oestradiol-17# Similarly, 2ml samples of plasma, after the addition of 0.2ml of 0.3M sodium hydroxide solution and 7.4fmol [3H] oestradiol-17/3 tracer, were extracted with ether. The extract was backwashed with 0.2ml of water and the ether extract was evaporated to dryness. After removal of a portion (1/3) for counting to monitor losses, the remainder was subjected to radioimmunoassay using an antibody against oestradiol-17/ (1:83,000 v/v) and [3H] oestradiol as radioligand. The mass of oestradiol-17/ present was read from a standard curve prepared using radioinert oestradiol-17/ and after For the 36 ER-positive tissues, the quantitative relationships between the concentration of ER in the tumour and of those oestrone or oestradiol in the plasma are shown in Figures 2 and 3. A significant positive correlation was found between the level of ER in the tumour and the level of oestrone in the plasma (r=0.50, P<0.01, Spearman's rank correlation test). There was also a tendency for the levels of tumour ER to rise with increasing plasma oestradiol concentration but this did not attain significance (r=0.26, P>0.10, Spearman's rank correlation test).
These results demonstrate that in postmenopausal women, in contrast to our earlier findings in premenopausal subjects (Hawkins et al., 1979), the level of oestrogen receptor activity detected in a breast cancer, which has prognostic and predictive value, is correlated with the concentration of the major circulating, unconjugated oestrogen, oestrone. The plasma levels of oestradiol-17# were much lower, and showed a similar, but nonsignificant relationship with tumour ER activity. Our findings are in agreement with those of Saez and her colleagues (1978)  ER levels were significantly correlated with plasma oestrogen level in a group of postmenopausal patients with tumours positive for both oestrogen and progestogen receptors. Drafta et al. (1983), on the other hand, found that the plasma oestradiol concentration was higher in patients with receptorpositive tumours than in those with receptornegative tumours. The present study implies that the levels of circulating oestrogens, ( -0.02-0.08 nmol I1for oestradiol, -0.04-0.20 nmol 1 ' for oestrone) are sufficient to half-saturate the oestrogen receptor (-~0.03 nmol -' for oestradiol, -0.09 nmol l-1 for oestrone at 4°C) and may thus influence the rate of its synthesis or degradation in the tumour. However, it seems likely that it is the level of . . oestrogen in the tissue, rather than that in the plasma which ultimately influences ER levels. In tumour tissue, the endogenous oestrogens derive not only from the plasma, but also from biosynthesis within the tumour (Miller & Forrest, 1974;Miller et al., 1981;Mason et al., 1981) and hydrolysis of oestrone sulphate (Santner et al., 1984); oestradiol rather than oestrone appears to be the major free oestrogen (Poortman et al., 1983). In general, in tumour tissue, oestrogen concentrations are likely to be higher when ER are present than when they are absent (Fishman et al., 1977;Edery et al., 1981;Thorson et al., 1982;Drafta et al., 1983), and in one study (Edery et al., 1981), tumour oestradiol content rose with increasing ER level.
Oestrogens can affect receptor levels in at least two ways: (i) they fill empty receptor sites and (ii) they may influence the rate of synthesis or degradation of receptor (see e.g. Hawkins et al., 1977;Bayard et al., 1978). This study suggests that whilst the former effect may predominate in premenopausal women, where progesterone levels may determine oestrogen receptor synthesis (Saez et al., 1978), the latter effect may occur, to some extent, in postmenopausal subjects.