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Androgen receptor binding and androgenicity of methylated 4-ene-3-ketosteroids having no 17-hydroxy group
ANDROGEN RECEPTOR BINDING AND ANDROGENICITY OF ~ETHYLATED 4-ENE-3-KETOSTEROIDS HAVING NO 17-HYDROXY GROUP KENNETHM. B. CHAN, STEPHANIE SMYTHEand SHUTSUNGLIAO
The Ben May Laboratory for Cancer Research and the Department of Biochemistry, University of Chicago, Chicago, Illinois 60637, U.S.A. (Received 12 January 1979) SUMMARY
and steroids that have no hydroxy group on ring D can compete with 5a-[3H3-~hy~otestosterone.(DHT) for binding to the androgen receptor protein of rat ventral prostate. The relative receptor binding affinities and corn~tition indices (in p~enthe~) for these steroids are in decreasing order: 7a-methyl-19-nortestos~rone (2.65) > DHT (1.00) > 7a-methyl-19-norCertain 7u-methyl-19-norandrostenes
androst-4-ene-3-17-dione (0.16) > 7a,l7,17-trimethylgona-4,13-dien-3-one (TMGD) (0.08) r 4-androstenedione (0.03) B 17-deoxy-5a-dihydrotestosterone (0.00). The results confirm our earlier finding that the 7u-methyl-19-nor-4-ene-3-keto structure can contribute substantially to the high receptor binding activity of androsta(e)nes. Thus, TMGD, which does not have an oxygen function on ring D has a surprisingly high receptor binding affinity. TMGD aiso competed with C3H]-DHT for nuclear binding if the steroids were incubated with the prostate homogenate; such competition was not observed when the incubation was carried out with minced prostate. The results suggest that, in the intact cell, TMGD does not readily penetrate into the intracellular site to interact with the receptor protein. It is apparently for this reason that TMGD was found to be neither androgenic nor antiandrogenic at doses from 5 to 30 pg per 50 g rat.
We have suggested [l, 21 that the androgen-receptor protein of the rat ventral prostate interacts with androgen as if the hormone were recognized from all sides (Fig. 1). Such a mechanism is in distinct contrast to that for many steroid-metabolizing enzymes or blood steroid-binding proteins, which generally recognize only a portion of the steroid molecule [3,4]. This suggestion is supported by our finding that the removal of the 1Pmethyl group and 7a-methylation of 17j-hydroxy-4-androstenes can enhance both the receptor binding activity and the androgenicity of the
steroid [2]. The increase in these activities can be attributed to (a) the flattening of the steroid molecule which is less hindered in receptor binding, (b) the interaction of the ‘la-methyl group with a receptor site to enhance the affinity, and/or (c) the protection of the A,B-ring structure from enzymatic alteration 12,51. In addition to the gross geometric structure of the steroid, the l7j-hydroxy group also contributes greatly to the receptor binding affinity and androgenicity of androgens [2,6]. It is not clear, however, whether the hydroxy group is needed for high-affinity
ANDROCEN-RECEPTOR
I
COflPLEX
II
III
Fig. 1. Hypothetical diagram of the interaction of androgens with the androgen-receptor protein. Steroids appear to be recognized from all sides by the receptor. For Sa-dihydrotestosterone (I) the M site on the receptor does not contribute to the binding affinity; for certain synthetic methyIat~ steroids, however, the 7a-methyl group may interact with the M site and significantly increase the binding affinity, as has been observed for 7a-17u-dimethyl-19-nortestosterone (II). Thus, the synthetic steroids, such as 7a,17,17-trimethylgona-4,13-dien-3-one (Ill) which has the 7a-methyl group but no oxygenated group at the C-17 position, may bind tightly to the receptor. Studies of receptor binding and of the biological activity of these steroids may provide a better understanding of the role of the 17/?-hydroxy group in androgen action. 1183
1194
KENNETHM. B. CHAN, STEPHANIE SMYTHE and SHUTSUNG
receptor binding or for the trigger action of the androgen-receptor complex or both. As an initial effort to investigate this, we have studied the receptor binding capacity of several 7umethyl-19-norandrostenes. Some of these methylated steroids may bind tightly to the androgen receptor even if they lack a hydroxy group on ring D; if such a steroid is androgenic, the need for the 17/I-hydroxy group in the triggering process may be questioned.
LIAO
radioactive androgen for binding to the receptor protein, we used our earlier method 123. In this procedure, if a function Y is defined as
Y=
3 S receptor-bound
c.p.m. in the presence of a competitor
3 S receptor-bound
c.p.m. in the absence of a competitor
’
then
EXPERIMENTAL
PROCEDURE
Animals. Male Sprague-Dawley the entire study. The radioactive
rats were used in androgen 5x-[1,2-
3H]-dihydrotestosterone (44 Ci/mmol) was obtained from New England Nuclear Corp. Nonradioactive steroids were generously supplied by Drs. John C. Babcock and by Allan J. Campbell of the Upjohn co. Receptor binding assay. Male rats (body weight: 300-4OOg) were castrated 18 h before being killed. The ventral prostates were dissected free of their capsules, minced, and homogenized in 3 volumes of Medium A (0.32 M sucrose containing 3 mM MgC12, 5% glycerol, and 20mM Tris-HCl buffer, pH 7.0) in an all-glass Potter-Elvehjem homogenizer at O-2°C. The homogenate was centrifuged at 12,000 g for 10min. (Most of the microsomal materials sediment during this centrifugation [7].) The supernatant (5 ml) was mixed with 2.5 &i of the radioactive steroid in the presence or absence of the nonradioactive steroid. The mixture was incubated at 0°C for 1 h and then centrifuged at 100,000 g for 60 min for removal of the microsomal materials. Granular (NH&SO, (enzyme grade, 243 mg per ml of cytosol) was then added to the cytosol fraction slowly over 10min. The mixture was allowed to stand at 0” for 20min and was then centrifuged at 10,000g for 10min. The protein precipitate was dissolved in 0.5 ml of Medium B (1.5 mM EDTA and 20 mM Tris-HCI buffer, pH 7.5) containing 0.4 M KCl. Two-tenths milliter of the protein solution that contained androgen-receptor complex was analyzed by gradient centrifugation with a linear sucrose gradient (5-20x) medium containing 0.4 M KCl, 1.5 mM EDTA, 20 mM Tris-HCI buffer, pH 7.5, and 10% glycerol. Centrifugation was performed with a Spinco SW 60 rotor at 56,000 rev./min for 22 h. Fractions (0.2 ml each) were collected and numbered starting from the bottom of the tube, and the radioactivity was measured in a scintillation counter [2]. The sedimentation coefficient of the radioactive androgenreceptor complex ranged from 3.0 to 3.5. The above procedure was modified from that which we described previously [2]. In the modified method, we added the steroids at an early stage of the preparation of the cytosol to save time, and also to prevent the denaturation of the unbound receptor protein. Relative competition index. For comparing the capacity of various steroids to compete with the
Y=
[C3H]-DHT] [C3H]-DHT]
+ a [competitor]
’
where [C3H]-DHTl and [competitor] are, respectively, the concentrations of 5a C3H]-dihydrotestosterone and the competitor in the assay system and a is a factor characteristic of a competing steroid. By plotting l/Y as a function of [competitor]/[[3H]-DHT], one can obtain a from the slope. Theoretically, a is 1 for Sa-dihydrotestosterone; however, the presence of endogenous androgen and other factors in the receptor preparation may affect this value. Therefore, a for Sa-dihydrotestosterone (aDHT)and for the competitor (a,,& are measured experimentally. The relative competition index (RCI) for a competitor is defined as
RCI,,,,
=p,acomp aDHT
An RCI value larger than 1.00 suggests that this competitor has a receptor binding affinity higher than that of Sa-dihydrotestosterone. Nuclear retention assay. The retention of androgen was studied by the method previously described in detail [2]. Minced prostate (1 g) was incubated with 5 &i of radioactive Sa-dihydrotestosterone in the absence or presence of different concentrations of nonradioactive steroids in 3 ml of Medium A at 37°C for 45 min. The minced prostate was washed and homogenized, and the cell nuclei were isolated by centrifugation in Medium C (2.2 M sucrose containing 1 mM MgC12 and 20mM Tris-HCl buffer, pH 7.0). The androgen-receptor complex retained by the isolated nuclei was extracted with 2 volumes of 0.6 M KC1 containing 1.5 mM EDTA and 20 mM Tris-HCl buffer, pH 7.5. For comparison, the radioactivity associated with the extract and with the whole nuclei was measured. In some experiments, the prostate was homogenized, and the whole homogenate was then incubated with the radioactive androgen and the competitors in the way described above. Other methods. Determination of protein and DNA as well as thin-layer chromatography of steroids were carried out according to methods described elsewhere [2,8]. The androgenic potencies of the steroids were determined as reported previously [2] using normal and castrated male rats (S&55 g) in groups of eight each. Each rat was given a daily injection of 0.1 ml of sesame oil containing 0 to 30 pg of the test steroids.
Steroid binding to androgen receptor
1195
RESULTS AND DISCUSSION
In the studies illustrated in Figs 2 and 3, we used gradient centrifugation to compare the capacity of several 7a-methyl and D-ring deoxylated steroids to compete with [“HI-Sa-dihydrotestosterone for binding to the prostate cytosol receptor protein. As shown in Fig. 2, the formation of the 3.5 S radioactive androgen-receptor complex was reduced very slightly in the presence of nonradioactive 4-androstenedione at ten times the concentration of the radioactive ligand. The 7a-methyl-19-nor analogue was far more competitive than 4-androstenedione in binding to the receptor protein. The RCIs, determined at different concentrations of these compounds (Fig. 4) indicate that 7a-methyl-1Pnorandrostenedione had an affinity comparable to that of testosterone or 7a-methyltestosterone [2,6]. Thin-layer chromatographic analysis showed that there was no metabolism of the methyl derivative during the receptor binding assay. The binding activity, therefore, was not due to the transformation of this steroid into a 17fl-hydroxy compound. The receptor-binding capacity of tritiated Sa-dihydrotestosterone was not competed by high concentrations of 17-deoxy-5a-dihydrotestosterone (Fig. 2). However, 7a,l7,17-trimethylgona-4,13-dien-3-one (TMGD), which has no oxygen at the D-ring, is fairly active in competing with Sa-dihydrotestosterone for binding to the receptor protein. According to the thin-layer chromatographic analysis, TMGD was not metabolized during the binding assay. The binding
n 6
I’HID
H
T
r.7
0
-4 x
z1
0
2
x 10
L
5
10
15
20
FRACTION
Fig. 2. Receptor binding of 5@H]-dihydrotestosterone in the absence and presence of 4-androstenedione and ‘la-methyl-19-norandrostenedione. The receptor binding assay was carried out as described under Experimental Procedure. The concentrations of radioactive androgen (C3H]-DHT) and of the competitor,, incubated with the receptor preparation were 11 and 110 nM, respectively.
5
IO FRACTION
15
20
NUMBER
Fig. 3. Receptor binding of Sa-dihydrotestosterone in the absence and presence of 17-deoxy-5a-dihydrotestosterone, 7a,17,17-trimethylgona-4,13-dien-3-one, 7a-methyl-19-norandrost-4-ene-3,17-dione, and 7a-methyl-19-nortestosterone. The receptor binding assay was carried out as described under Experimental Procedure. The concentrations of radioactive androgen and of the competitor incubated with the receptor preparation were 11 and llOnM, respectively.
activity, therefore, was due to TMGD and not to its metabolite. We also investigated whether TMGD could compete with tritiated Sa-dihydrotestosterone for nuclear retention. We found that, when the radioactive androgen and different concentrations of TMGD were incubated with whole-prostate homogenate, TMGD reduced the nuclear retention of androgen in a concentration dependent manner. If both the radioactive androgen and the competitor were incubated with minced prostate, however, there was no reduction in the amount of radioactive Sa-dihydrotestosterone retained by the prostate cell nuclei (Fig. 5). We also compared the capacity of TMGD and Sa-dihydrotestosterone to stimulate the growth of rat ventral prostate. The daily dose of Sa-dihydrotestosterone needed to maintain the weight of the ventral prostate at twice that of the control castrates was about 0.8 pg per 50 g castrated rat; at 5-30 pg per rat, however, TMGD did not affect the growth of the prostate in either castrates or normal rats. Whether TMGD is androgenic or antiandrogenic at higher concentrations was not tested because only a limited quantity of TMGD was available to us. Such a test is desirable since the receptor binding affinity of TMGD is comparable to that of certain androgenic steroids such as 7a-methyl-19-nor-testosterone [2], and of antiandrogens such as hydroxytlutamide or cyproterone [8,9].
1196
KENNETH
M. B.
CHAN. STEPHANIE SMYTHE and SHUTSUN~ L~AO
100
-I
80
0 CL c
60
sz Q u
(0.08) 0
m
‘\
\
/ I I
--
t
PROSTATEMINCE, T M G D \
\
'Q \ I \\ 1,HOMOGENATE,T M G D I LI a\l \
40
\
'H,DHT
. .
H 20
\
. .
9.
M,DHT
t 10
i 20
30
40
NONRADIOACTIVE COMPETITOR/I"HIDHT /'
I 10
20
30
iCORPETITORI/IC3HlDHTI
Fig. 4. Relative ~m~tition of various methyl steroids and steroids having no 17&hydroxy group. The receptor binding assay was carried out as described under Experimentai Procedure, in the presence of 11 nM .5a-[3H]-dihydrotestosterone and various concentrations of the nonradioactive competitors, -la-methyl-lPnortestosterone, 5a-dihydrotestosterone, 7a-methyl-l9-norandrost~-ene-3,17-dione. 7a.17, 17-trimethylgona~,i3-dien-3-one, 4-androstenedione, and 17-deoxy-5a-dihydrotestosterone. The amount of the radioactive androgen-receptor complex was estimated from the 3 S protein-bound radioactivity seen in the gradient centrifugation patterns. As described under Experimental Procedure, l/Y is the ratio of the 3 S protein-bound radioactivity in the absence of a competitor to the 3 S proteinbound radioactivity in the presence of a competitor whose concentration is shown on the abscissa. The numbers in parentheses below the steroid structures are the relative competition indices which are the ratios of the slopes relation to the slope of Sa-dihydrotestosterone.
The above studies suggest that TMGD may be biologically inactive, for it does not interact with the receptor and does not bind to nuclei in the intact cells. Since TMGD did compete with the nuclear retention of S@H]-dihydrotestosterone if the incubation was carried out with prostate homogenate, it is possible that TMGD does not readily penetrate through the cell membrane or translocate to the intracellular site, where it can interact with the receptor protein. Without an oxygen function on ring D, TMGD may behave like a monopolar lipid molecule and easily intercalate with other lipid materials of the ceflular membranes or other celfular orgenelles.
Fig. 5. Nuclear retention of 5a-[3H]-dihydrotestosterone in the absence and presence of nonradioactive Sa-dihydrotestosterone (DHT) or 7a,l7,l?-~rimethylgona~,i3-dien3-one (TMGD). The radioactive androgen (37nM) and the competitor (at different concentrations as shown on abscissa) were incubated with minced prostate (-) or prostate homogenate (---) in 3 ml of Medium A at 37°C for 45 min. The cell nuclei were isolated and then extracted with 0.6 M KC1 and the radioactivities in the KC1 extracts were compared. The control value in the absence of a nonradioactive competitor was 7700 d.p.m. Other results were expressed in y/, of this control value.
REFERENCES 1. Liao S., Liang T and Tymoczko J. L.: Structural recognitions in the interactions of androgens and teceptor proteins and in their association with nuclear acceptor components J. steroid Biochem. 3 (1972) 401-408. 2. Liao S.. Liane T.. Fanrr S.. Castaiieda E. and Shao T.-C: Steroid Structure and .androgenic activity: specificities involved in the receptor binding and nuclear retention of various androgens. f. bioi. Chem. 248 (1973) 6154-6162. 3. Liao S.: Cellular receptors and mechanisms of action of steroid hormones. Inc. Rev. Cytology 41 (1975) 87-172. 4. Castaiieda E. and Liao S.: The use of anti-steroid antibodies in the characterization of steroid receptors. J. b&l. Chem. 250 (1975) 883-888. 5. Liao S.: Molecular actions of androgens In Biochemical Actions ~~~~~~~es Vol. 4 (Edited by G. Litwack). Academic Press. N.Y. 11977) 351-406. 6. Liao S. and Fang S.: Recepior proteins for androgens and the mode of action of androgens on gene transcription in ventral prostate. Warn. Harm. 27 (1969) 17-90. 7. Liao S. and Williams-Ashman H. G. An effect of testosterone on amino acid ~ncorvoration by Drostatic ribonucleoprotein particles. &oc. narn. -A&d. Sci. U.S.A. 48 (1962) 1956-1964.
research was supported by Research Grant AM-09461 from the United States National Institutes of Health and by Grant BC-151 from the American Cancer Society. Acknowledgements-This
8. Fang S. anh L&o S.: Androgen receptors: steroid- and tissue-specific retention of a 17/I-hydroxy-5a-androstan-3-one-protein complex by cell nuclei of ventral prostate. J. biol. Chem. 246 (1971)16-24.
The Ben May Laboratory for Cancer Research and the Department of Biochemistry, University of Chicago, Chicago, Illinois 60637, U.S.A. (Received 12 January 1979) SUMMARY
and steroids that have no hydroxy group on ring D can compete with 5a-[3H3-~hy~otestosterone.(DHT) for binding to the androgen receptor protein of rat ventral prostate. The relative receptor binding affinities and corn~tition indices (in p~enthe~) for these steroids are in decreasing order: 7a-methyl-19-nortestos~rone (2.65) > DHT (1.00) > 7a-methyl-19-norCertain 7u-methyl-19-norandrostenes
androst-4-ene-3-17-dione (0.16) > 7a,l7,17-trimethylgona-4,13-dien-3-one (TMGD) (0.08) r 4-androstenedione (0.03) B 17-deoxy-5a-dihydrotestosterone (0.00). The results confirm our earlier finding that the 7u-methyl-19-nor-4-ene-3-keto structure can contribute substantially to the high receptor binding activity of androsta(e)nes. Thus, TMGD, which does not have an oxygen function on ring D has a surprisingly high receptor binding affinity. TMGD aiso competed with C3H]-DHT for nuclear binding if the steroids were incubated with the prostate homogenate; such competition was not observed when the incubation was carried out with minced prostate. The results suggest that, in the intact cell, TMGD does not readily penetrate into the intracellular site to interact with the receptor protein. It is apparently for this reason that TMGD was found to be neither androgenic nor antiandrogenic at doses from 5 to 30 pg per 50 g rat.
We have suggested [l, 21 that the androgen-receptor protein of the rat ventral prostate interacts with androgen as if the hormone were recognized from all sides (Fig. 1). Such a mechanism is in distinct contrast to that for many steroid-metabolizing enzymes or blood steroid-binding proteins, which generally recognize only a portion of the steroid molecule [3,4]. This suggestion is supported by our finding that the removal of the 1Pmethyl group and 7a-methylation of 17j-hydroxy-4-androstenes can enhance both the receptor binding activity and the androgenicity of the
steroid [2]. The increase in these activities can be attributed to (a) the flattening of the steroid molecule which is less hindered in receptor binding, (b) the interaction of the ‘la-methyl group with a receptor site to enhance the affinity, and/or (c) the protection of the A,B-ring structure from enzymatic alteration 12,51. In addition to the gross geometric structure of the steroid, the l7j-hydroxy group also contributes greatly to the receptor binding affinity and androgenicity of androgens [2,6]. It is not clear, however, whether the hydroxy group is needed for high-affinity
ANDROCEN-RECEPTOR
I
COflPLEX
II
III
Fig. 1. Hypothetical diagram of the interaction of androgens with the androgen-receptor protein. Steroids appear to be recognized from all sides by the receptor. For Sa-dihydrotestosterone (I) the M site on the receptor does not contribute to the binding affinity; for certain synthetic methyIat~ steroids, however, the 7a-methyl group may interact with the M site and significantly increase the binding affinity, as has been observed for 7a-17u-dimethyl-19-nortestosterone (II). Thus, the synthetic steroids, such as 7a,17,17-trimethylgona-4,13-dien-3-one (Ill) which has the 7a-methyl group but no oxygenated group at the C-17 position, may bind tightly to the receptor. Studies of receptor binding and of the biological activity of these steroids may provide a better understanding of the role of the 17/?-hydroxy group in androgen action. 1183
1194
KENNETHM. B. CHAN, STEPHANIE SMYTHE and SHUTSUNG
receptor binding or for the trigger action of the androgen-receptor complex or both. As an initial effort to investigate this, we have studied the receptor binding capacity of several 7umethyl-19-norandrostenes. Some of these methylated steroids may bind tightly to the androgen receptor even if they lack a hydroxy group on ring D; if such a steroid is androgenic, the need for the 17/I-hydroxy group in the triggering process may be questioned.
LIAO
radioactive androgen for binding to the receptor protein, we used our earlier method 123. In this procedure, if a function Y is defined as
Y=
3 S receptor-bound
c.p.m. in the presence of a competitor
3 S receptor-bound
c.p.m. in the absence of a competitor
’
then
EXPERIMENTAL
PROCEDURE
Animals. Male Sprague-Dawley the entire study. The radioactive
rats were used in androgen 5x-[1,2-
3H]-dihydrotestosterone (44 Ci/mmol) was obtained from New England Nuclear Corp. Nonradioactive steroids were generously supplied by Drs. John C. Babcock and by Allan J. Campbell of the Upjohn co. Receptor binding assay. Male rats (body weight: 300-4OOg) were castrated 18 h before being killed. The ventral prostates were dissected free of their capsules, minced, and homogenized in 3 volumes of Medium A (0.32 M sucrose containing 3 mM MgC12, 5% glycerol, and 20mM Tris-HCl buffer, pH 7.0) in an all-glass Potter-Elvehjem homogenizer at O-2°C. The homogenate was centrifuged at 12,000 g for 10min. (Most of the microsomal materials sediment during this centrifugation [7].) The supernatant (5 ml) was mixed with 2.5 &i of the radioactive steroid in the presence or absence of the nonradioactive steroid. The mixture was incubated at 0°C for 1 h and then centrifuged at 100,000 g for 60 min for removal of the microsomal materials. Granular (NH&SO, (enzyme grade, 243 mg per ml of cytosol) was then added to the cytosol fraction slowly over 10min. The mixture was allowed to stand at 0” for 20min and was then centrifuged at 10,000g for 10min. The protein precipitate was dissolved in 0.5 ml of Medium B (1.5 mM EDTA and 20 mM Tris-HCI buffer, pH 7.5) containing 0.4 M KCl. Two-tenths milliter of the protein solution that contained androgen-receptor complex was analyzed by gradient centrifugation with a linear sucrose gradient (5-20x) medium containing 0.4 M KCl, 1.5 mM EDTA, 20 mM Tris-HCI buffer, pH 7.5, and 10% glycerol. Centrifugation was performed with a Spinco SW 60 rotor at 56,000 rev./min for 22 h. Fractions (0.2 ml each) were collected and numbered starting from the bottom of the tube, and the radioactivity was measured in a scintillation counter [2]. The sedimentation coefficient of the radioactive androgenreceptor complex ranged from 3.0 to 3.5. The above procedure was modified from that which we described previously [2]. In the modified method, we added the steroids at an early stage of the preparation of the cytosol to save time, and also to prevent the denaturation of the unbound receptor protein. Relative competition index. For comparing the capacity of various steroids to compete with the
Y=
[C3H]-DHT] [C3H]-DHT]
+ a [competitor]
’
where [C3H]-DHTl and [competitor] are, respectively, the concentrations of 5a C3H]-dihydrotestosterone and the competitor in the assay system and a is a factor characteristic of a competing steroid. By plotting l/Y as a function of [competitor]/[[3H]-DHT], one can obtain a from the slope. Theoretically, a is 1 for Sa-dihydrotestosterone; however, the presence of endogenous androgen and other factors in the receptor preparation may affect this value. Therefore, a for Sa-dihydrotestosterone (aDHT)and for the competitor (a,,& are measured experimentally. The relative competition index (RCI) for a competitor is defined as
RCI,,,,
=p,acomp aDHT
An RCI value larger than 1.00 suggests that this competitor has a receptor binding affinity higher than that of Sa-dihydrotestosterone. Nuclear retention assay. The retention of androgen was studied by the method previously described in detail [2]. Minced prostate (1 g) was incubated with 5 &i of radioactive Sa-dihydrotestosterone in the absence or presence of different concentrations of nonradioactive steroids in 3 ml of Medium A at 37°C for 45 min. The minced prostate was washed and homogenized, and the cell nuclei were isolated by centrifugation in Medium C (2.2 M sucrose containing 1 mM MgC12 and 20mM Tris-HCl buffer, pH 7.0). The androgen-receptor complex retained by the isolated nuclei was extracted with 2 volumes of 0.6 M KC1 containing 1.5 mM EDTA and 20 mM Tris-HCl buffer, pH 7.5. For comparison, the radioactivity associated with the extract and with the whole nuclei was measured. In some experiments, the prostate was homogenized, and the whole homogenate was then incubated with the radioactive androgen and the competitors in the way described above. Other methods. Determination of protein and DNA as well as thin-layer chromatography of steroids were carried out according to methods described elsewhere [2,8]. The androgenic potencies of the steroids were determined as reported previously [2] using normal and castrated male rats (S&55 g) in groups of eight each. Each rat was given a daily injection of 0.1 ml of sesame oil containing 0 to 30 pg of the test steroids.
Steroid binding to androgen receptor
1195
RESULTS AND DISCUSSION
In the studies illustrated in Figs 2 and 3, we used gradient centrifugation to compare the capacity of several 7a-methyl and D-ring deoxylated steroids to compete with [“HI-Sa-dihydrotestosterone for binding to the prostate cytosol receptor protein. As shown in Fig. 2, the formation of the 3.5 S radioactive androgen-receptor complex was reduced very slightly in the presence of nonradioactive 4-androstenedione at ten times the concentration of the radioactive ligand. The 7a-methyl-19-nor analogue was far more competitive than 4-androstenedione in binding to the receptor protein. The RCIs, determined at different concentrations of these compounds (Fig. 4) indicate that 7a-methyl-1Pnorandrostenedione had an affinity comparable to that of testosterone or 7a-methyltestosterone [2,6]. Thin-layer chromatographic analysis showed that there was no metabolism of the methyl derivative during the receptor binding assay. The binding activity, therefore, was not due to the transformation of this steroid into a 17fl-hydroxy compound. The receptor-binding capacity of tritiated Sa-dihydrotestosterone was not competed by high concentrations of 17-deoxy-5a-dihydrotestosterone (Fig. 2). However, 7a,l7,17-trimethylgona-4,13-dien-3-one (TMGD), which has no oxygen at the D-ring, is fairly active in competing with Sa-dihydrotestosterone for binding to the receptor protein. According to the thin-layer chromatographic analysis, TMGD was not metabolized during the binding assay. The binding
n 6
I’HID
H
T
r.7
0
-4 x
z1
0
2
x 10
L
5
10
15
20
FRACTION
Fig. 2. Receptor binding of 5@H]-dihydrotestosterone in the absence and presence of 4-androstenedione and ‘la-methyl-19-norandrostenedione. The receptor binding assay was carried out as described under Experimental Procedure. The concentrations of radioactive androgen (C3H]-DHT) and of the competitor,, incubated with the receptor preparation were 11 and 110 nM, respectively.
5
IO FRACTION
15
20
NUMBER
Fig. 3. Receptor binding of Sa-dihydrotestosterone in the absence and presence of 17-deoxy-5a-dihydrotestosterone, 7a,17,17-trimethylgona-4,13-dien-3-one, 7a-methyl-19-norandrost-4-ene-3,17-dione, and 7a-methyl-19-nortestosterone. The receptor binding assay was carried out as described under Experimental Procedure. The concentrations of radioactive androgen and of the competitor incubated with the receptor preparation were 11 and llOnM, respectively.
activity, therefore, was due to TMGD and not to its metabolite. We also investigated whether TMGD could compete with tritiated Sa-dihydrotestosterone for nuclear retention. We found that, when the radioactive androgen and different concentrations of TMGD were incubated with whole-prostate homogenate, TMGD reduced the nuclear retention of androgen in a concentration dependent manner. If both the radioactive androgen and the competitor were incubated with minced prostate, however, there was no reduction in the amount of radioactive Sa-dihydrotestosterone retained by the prostate cell nuclei (Fig. 5). We also compared the capacity of TMGD and Sa-dihydrotestosterone to stimulate the growth of rat ventral prostate. The daily dose of Sa-dihydrotestosterone needed to maintain the weight of the ventral prostate at twice that of the control castrates was about 0.8 pg per 50 g castrated rat; at 5-30 pg per rat, however, TMGD did not affect the growth of the prostate in either castrates or normal rats. Whether TMGD is androgenic or antiandrogenic at higher concentrations was not tested because only a limited quantity of TMGD was available to us. Such a test is desirable since the receptor binding affinity of TMGD is comparable to that of certain androgenic steroids such as 7a-methyl-19-nor-testosterone [2], and of antiandrogens such as hydroxytlutamide or cyproterone [8,9].
1196
KENNETH
M. B.
CHAN. STEPHANIE SMYTHE and SHUTSUN~ L~AO
100
-I
80
0 CL c
60
sz Q u
(0.08) 0
m
‘\
\
/ I I
--
t
PROSTATEMINCE, T M G D \
\
'Q \ I \\ 1,HOMOGENATE,T M G D I LI a\l \
40
\
'H,DHT
. .
H 20
\
. .
9.
M,DHT
t 10
i 20
30
40
NONRADIOACTIVE COMPETITOR/I"HIDHT /'
I 10
20
30
iCORPETITORI/IC3HlDHTI
Fig. 4. Relative ~m~tition of various methyl steroids and steroids having no 17&hydroxy group. The receptor binding assay was carried out as described under Experimentai Procedure, in the presence of 11 nM .5a-[3H]-dihydrotestosterone and various concentrations of the nonradioactive competitors, -la-methyl-lPnortestosterone, 5a-dihydrotestosterone, 7a-methyl-l9-norandrost~-ene-3,17-dione. 7a.17, 17-trimethylgona~,i3-dien-3-one, 4-androstenedione, and 17-deoxy-5a-dihydrotestosterone. The amount of the radioactive androgen-receptor complex was estimated from the 3 S protein-bound radioactivity seen in the gradient centrifugation patterns. As described under Experimental Procedure, l/Y is the ratio of the 3 S protein-bound radioactivity in the absence of a competitor to the 3 S proteinbound radioactivity in the presence of a competitor whose concentration is shown on the abscissa. The numbers in parentheses below the steroid structures are the relative competition indices which are the ratios of the slopes relation to the slope of Sa-dihydrotestosterone.
The above studies suggest that TMGD may be biologically inactive, for it does not interact with the receptor and does not bind to nuclei in the intact cells. Since TMGD did compete with the nuclear retention of S@H]-dihydrotestosterone if the incubation was carried out with prostate homogenate, it is possible that TMGD does not readily penetrate through the cell membrane or translocate to the intracellular site, where it can interact with the receptor protein. Without an oxygen function on ring D, TMGD may behave like a monopolar lipid molecule and easily intercalate with other lipid materials of the ceflular membranes or other celfular orgenelles.
Fig. 5. Nuclear retention of 5a-[3H]-dihydrotestosterone in the absence and presence of nonradioactive Sa-dihydrotestosterone (DHT) or 7a,l7,l?-~rimethylgona~,i3-dien3-one (TMGD). The radioactive androgen (37nM) and the competitor (at different concentrations as shown on abscissa) were incubated with minced prostate (-) or prostate homogenate (---) in 3 ml of Medium A at 37°C for 45 min. The cell nuclei were isolated and then extracted with 0.6 M KC1 and the radioactivities in the KC1 extracts were compared. The control value in the absence of a nonradioactive competitor was 7700 d.p.m. Other results were expressed in y/, of this control value.
REFERENCES 1. Liao S., Liang T and Tymoczko J. L.: Structural recognitions in the interactions of androgens and teceptor proteins and in their association with nuclear acceptor components J. steroid Biochem. 3 (1972) 401-408. 2. Liao S.. Liane T.. Fanrr S.. Castaiieda E. and Shao T.-C: Steroid Structure and .androgenic activity: specificities involved in the receptor binding and nuclear retention of various androgens. f. bioi. Chem. 248 (1973) 6154-6162. 3. Liao S.: Cellular receptors and mechanisms of action of steroid hormones. Inc. Rev. Cytology 41 (1975) 87-172. 4. Castaiieda E. and Liao S.: The use of anti-steroid antibodies in the characterization of steroid receptors. J. b&l. Chem. 250 (1975) 883-888. 5. Liao S.: Molecular actions of androgens In Biochemical Actions ~~~~~~~es Vol. 4 (Edited by G. Litwack). Academic Press. N.Y. 11977) 351-406. 6. Liao S. and Fang S.: Recepior proteins for androgens and the mode of action of androgens on gene transcription in ventral prostate. Warn. Harm. 27 (1969) 17-90. 7. Liao S. and Williams-Ashman H. G. An effect of testosterone on amino acid ~ncorvoration by Drostatic ribonucleoprotein particles. &oc. narn. -A&d. Sci. U.S.A. 48 (1962) 1956-1964.
research was supported by Research Grant AM-09461 from the United States National Institutes of Health and by Grant BC-151 from the American Cancer Society. Acknowledgements-This
8. Fang S. anh L&o S.: Androgen receptors: steroid- and tissue-specific retention of a 17/I-hydroxy-5a-androstan-3-one-protein complex by cell nuclei of ventral prostate. J. biol. Chem. 246 (1971)16-24.