0021-972X/78/4606-0998$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society

Vol. 46, No. 6 Printed in U.S.A.

YOICHI MURAYAMA, TOMOKO SAKUMA, HIDEO UDAGAWA, JOJI UTSUNOMIYA, RYOHEI OKAMOTO, AND KENICHI ASANO Second Department of Surgery, and Department of Endocrinology, Tokyo Medical and Dental University, School of Medicine, Tokyo, Japan ABSTRACT. The binding capacities of SHBG (sex hormone-binding globulin) and ER (estrogen receptor) were measured by agar gel electrophoresis with dextrancoated charcoal treatment and also by sucrose density gradient ultracentrifugation in the sera from 19 women with breast cancer to assay the correlation between SHBG and ER in breast cancer. SHBG levels were also measured by agar gel electrophoresis with dextrancoated charcoal treatment in control sera from 20 normal women. Two kinds of 17/?-estradiol-binding substances were recognized in the cytosol of breast cancer cells and they were confirmed to be ER and HSBG. Out of 13 postmenopausal patients with breast cancer, the SHBG levels in 6 ER-positive cases (66 ± 13 pmol/ml plasma, mean ± SD) were significantly higher than the levels of 10 controls (35 ± 7.6 pmol/ml; P
0.05). The findings suggest that correlation between SHBG and ER in postmenopausal breast cancer is possible, but no significant difference was noted between the SHBG levels in ER-positive premenopausal breast cancer patients and premenopausal normal women. (J Clin Endocrinol Metab 46: 998, 1978)

breast cancer by means of clarifying the interrelationship between SHBG and estrogen receptor. Materials and Methods Subjects Nineteen women with primary breast cancer, consisting of 6 with premenopausal breast cancer and 13 with postmenopausal breast cancer, ages 33-71 (averaging 54 yr in age), were selected as the subjects. Liver function and thyroid function tests remained within normal ranges in all of the patients. They had no endocrine diseases nor any liver diseases. None of them were pregnant or on any continuous medication such as thyroid hormones, estrogens, androgens, cortisols, sedatives, or any drugs for hypertension which can change SHBG titers. The controls consisted of 10 ovulating normal women and 10 postmenopausal women of ages 30-70 yr, averaging 38 and 60, respectively. None had any endocrine diseases, nor were they pregnant or on any medication. Liver function and thyroid function tests were within normal ranges in all of the controls.

998

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Sex Hormone-Binding Globulin and Estrogen Receptor in Breast Cancer: Technique and Preliminary Clinical Results*

COMMENTS

999

and Jungblut (5). Frozen tissue (500 mg) was ground with an auTen milliliters of dextran charcoal (0.01 M Tristopulverizer (Thermovac) in liquid nitrogen and HC1 buffer plus 0.05% dextran T70 Pharmacia and was homogenized with a Teflon glass homogenizer 0.5% carbon Norit A) were centrifuged at 2000 rpm in ice-cold 0.01 M Tris-HCl buffer (pH 7.4) into a x to obtain fa> g carbon as the sediment, to which 3 volume four times that of the tissue. The homogeml Tris-HCl buffer was added to prepare the dexnate was centrifuged in a cooled centrifugal butytran-charcoal solution for use. Serum (100 fd) and late tube at 130,580 X g at 4 C for 30 min to obtain 3 1.5 nmol 170-[2,4,6,7(n)- H]estradiol (SA, 95Ci/ cytosol fractions to be used as the supernatant. 17/8mmol; radiochemical purity, 99%; Amersham) were Estradiol-receptor complex was prepared as deincubated at 0-2 C for 2 h to obtain equilibrium scribed above. All available receptor-binding sites between SHBG-bound and free 17/?-estradiol. were completely saturated under these conditions. Then, 3 ml dextran-charcoal solution was added Thirty microliters of each charcoal-extracted suand incubated at 0-2 C for 1 h. The formed SHBGpernatant was assayed by agar gel electrophoresis. 17/?-estradiol complex was separated from charcoalThey were allowed to run at 120 V and 130 mA at bound free-17/?-estradiol by centrifugation at 3000 4 C (temperature at the center of agar) for 2 h. rpm for 10 min at 0-2 C and was used as the After the agar layer was fractionated into 3-mm supernatant. On the other hand, 60 fil cytosol and widths, the radioactivity of each fraction was mea0.4 nmol tritiated 17/?-estradiol were incubated at sured by a liquid scintillation counter. 0-2 C for 2 h to form an estrogen receptor (ER)radioactive 17/?-estradiol complex, which was proc- ER assay by sucrose density gradient ultracentrifessed in the same manner as stated above to sepa- ugation (6) rate free radioactive 17/?-estradiol. Up to this step, Cytosol fraction (supernatant) was separated by 99.8% of the added radioactivity was recovered as the same procedure as stated above. One-half milthe complex and the free fraction. liliter of 0.01 M Tris-HCl buffer (pH 7.4) and 0.0005 M dithiothreitol were measured into one of two Assay of SHBG cooled small test tubes and 0.1 ml CN-55, 945-27 SHBG was determined by the method of Wagner 10~6 M, an antiestrogen agent, was poured into the (4) and by dextran-coated charcoal treatment. other tube. Supernatant from one tumor (0.3 ml) A mixture of 60 ml warm 1% agar solution (noble was measured into each of the two test tubes and agar; Difco) in sodium diethylbarbiturate-acetate the contents were stirred. buffer (pH 8.2, fi = 0.05) was poured into a perspex After the mixtures were incubated at 0 C for 10 mould (110 X 100 X 7 mm) to form a 5-mm thick min, 0.1 ml 2.5 X 10"9 M 17/?-[2,4,6,7(n)-3H]estradiol layer of gel. The gel was cooled overnight at 0-4 C. was added to each tube and the contents were stirred. Nine sample wells, each 3.0-mm in diameter, were Sucrose density gradient solutions (5-20%), 4.8 ml filled with 30 /il charcoal-extracted preparation. in total, dissolved in 0.01 M Tris-HCl buffer (pH Another sample well was filled with a control mix- 7.4) containing 0.001 M EDTA and 0.01 M KC1 were ture consisting of 30 /xl undiluted serum as mea- prepared in a pair of centrifugal tubes. A portion sured by a densitometer. Electrophoresis was then (0.2 ml) of each of the aforementioned mixtures performed on each sample in a closed chamber at prepared in the two small test tubes was gently 4 C. Connections to the buffer trays, which were superimposed on each of the sucrose density grafilled with sodium diethylbarbiturate-acetate buffer dient solutions, which was then centrifuged at (pH 8.2, /i = 0.05), were established with 1% agar 130,580 X g at 4 C for 15 h. After the ultracentrifsolution in the same buffer. The samples were run ugation, each tube was placed in a fractionator to at 130 mA and 120 V for 120 min. After the electro- collect three drops each from the pore provided at phoresis, fractions were collected by slicing the gels the bottom to form a fraction, and the radioactivity lengthwise. These strips were then cut into 3-mm was measured by a liquid scintillation counter. sections with a special gel slicer. The radioactivity of each fraction was measured in a scintillation fluid Determination of dissociation constant (Kd) and (80 g napthalene, 5 g PPO, 50 mg POPOP-1000 ml binding capacity xylol-dioxane, 1:2) by a liquid scintillation counter Amounts of either receptor- 17/?-estradiol comwith a counting efficiency of 30%. plex or SHBG-17/8-estradiol complex were determined at various amounts of added tritiated 17/?Assay ofER by agar gel electrophoresis estradiol, according to the methods aforemenER was determined by the method of Wagner tioned.

Dextran-coated charcoal treatment (DCC)

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JCE&M • 1978 V0U6 • No 6

COMMENTS

1000

Results

cpm

Presence of two kinds of 17/3-estradiol-binding substances in cytosol of breast cancer cells Results obtained by the addition of 10" n mol 17/?-estradiol to 30 jul cytosol are shown in Fig. 1. As noted in Fig. 1A, three peaks were recognized by the addition. Of these three

Agargel Electrophoresis

10000

ER-Ej Complex free

5000

28

26

24

22

20

18

B FIG. 1. Electrophoresis pattern without charcoal treatment. A, Electrophoresis of cytosol fraction incubated with tritiated 17/?estradiol. Three radioactive peaks (fractions 5-10, 12, and 17) are distinctly separated. B, Two peaks (fractions 12 and 17) disappeared by the addition of CN-55, 945-27 to the cytosol fraction, before preparation of receptor-17/?estradiol complex. C, Electrophoresis pattern of buffer control and free radioactive 17/?-estradiol. Sodium diethylbarbiturate-acetate buffer (30 jul) was incubated for 2 h at 0-2 C with 10"" mol tritiated 17/?-estradiol. E2, 17/?-Estradiol.

16

12

14

t

0

10

8 6 4 21 Fraction Number

Start

cpm 15000

0

Agargel Electrophoresis

10000

5000

28

26

24

22 20 18

e

16 14

12 ' 10

8 6 4 21 Fraction Number ©

Start

Agargel Electrophoresis

cpm

free estrogen

50000

25000

5000 28 ©

26

24

22

20

18

16 f

14

12 10

Start

8 6 4 21 Fraction Number

©

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A Scatchard-type plot (15) of the ratio of concentrations of specifically bound 17/?-estradiol to free hormone (B:U) vs. bound hormone concentration (B) was prepared. Where a single binding component was present, the equilibrium constant of dissociation, Kd, was equal to the reciprocal of the slope of this curve, and the extrapolated intercept on the abscissa was equal to the concentration of the binder-steroid complex (17).

COMMENTS

Agargel Electrophoresis

600

ER-E 2 Complex

400

SHBG-E 2 Complex

200

BG 28

26 24

22

20 18 16

14 12

10

4 Start

8

6

4

21

Fraction number

B X10" 4 10

Kd=2.5±0.2XKTnM(0~2'c: 0.12X" M \ 0.12X10~ 8 M

0.52X

M 0.2IX M I 0.8XI0'9M

1*1 5

10 20 (B) f mole/mg of cytosol protein

X10

Kd=1.4X10

100

200

(B)

M(0~2C)

300

f

0

FIG. 2. Electrophoresis of incubated cytosol fraction after charcoal treatment, 30 jul charcoal-extracted supernatants of cytosol were used for measurement. A, Only two binding peaks are observed; the peak of free radioactive 17/?-estradiol has disappeared. BG, Background. B, Scatchard plot of binding peak of fraction 15. Dissociation constants (K

Sex hormone-binding globulin and estrogen receptor in breast cancer: technique and preliminary clinical results.

0021-972X/78/4606-0998$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society Vol. 46, No. 6 Printed in U...
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