Binding of [3H] methyltrienolone (R 1881) in rat prostate and human benign prostatic hypertrophy (BPH)

449

BINDING

OF

C3H1 HUMAN

JACQUES

ASSELIN,

METHYLTRIENOLONE BENIGN PROSTATIC

(R 1881) IN HYPERTROPHY

YVES

FERNAND LABRIE, and JEAN-PIERRE

RAT PROSTATE (BPH)

GOURDEAU,

CLAUDE

AND

BONNE

RAYNAUD,

Medical Research Council Group in Molecular Endocrinology, Le Centre Hospitalier de l'universite Laval, L'HGpital de l'Enfant-J@sus, Quebec, GlV 462, Canada, and Centre de Recherches Roussel-UCLAF, 93230 Qu&bec, Romainville, France Received:

6/22/75

ABSTRACT Methyltrienolone (R 1881 - 17B-hydroxy-17a-methyl-estra4, 9, 11-trien-3-one) binding to rat ventral prostate cytosol has a specificity typical of an androgen receptor. In human benign prostatic hypertrophy (BPH) tissue, the specificity of C3H1 R 1881 binding is different from that measured progesterone and R 5020 (17, 21-dimethyl-19in rat prostate: nor-4,9-pregnadiene-3, 20-dione) being more potent while 19nortestosterone is less potent competitor. Moreover, the synthetic progestin C3HI R 5020 binds to BPH tissue with a similar specificity. These data suggest the presence of progestin binding components or of an atypical androgen receptor in human BPH cytosol. INTRODUCTION The teins the

absence

greatly

of

facilitated

specific

androgen

gen-dependent

tissues

contamination

of

(SBP)

or

significant

SBP-like

interference

the

use

receptor

in

in

tissues protein

the by

rat

of

DHT

prostate (l-6).

a plasma

which

C3Hl

binds

sex

by

plasma

for

and

pro-

studies

other

andro-

In man,

however,

binding

protein

both

DHT

and

of

T with

Abbreviations and trivial names: R 1881 = methyltrienolone = 17B-hydroxy-17a-methyl-estra-4,9,ll-trien-3-one; DHT q androstanolone q 5a-dihydrotestosterone q 17B-hydroxy-5a-androstan3-one; T = testosterone = 17B-hydroxy-4-androsten-3-one; epitestosterone = 17a-hydroxy-4-androsten-3-one; androstenedioq 4-androstene-3, 17-dione; progesterone =4-pregnene-3, FE-dione ; cyproterone acetate - 6-chloro-17a-acetoxy-1, 2amethylene-4,6-pregnadiene-3, 20-dione; estradiol q 1,3,5 (lo)-estratriene-3, 17B-diol; 19-nortestosterone q 17@-hydroxy-4-estren-3-one; androstanediol = 5a-androstane-3a, 178R 5020 = 17,21-dimethyl-19-nor-4,9-pregnadiene-3,2O-dione, diol; VoZwne 28, Number 4

S

TIIBOXD6

October,

2976

450

TTESIIBLOSDI

s

high

affinity

drogen

(5,

7)

receptors.

specific

androgen

be

observed

(8-17)

(12,

cytosol

the

8S

has

area

components We steroid during

4S

the

has of

and

rat

components

recent 8s

similar

found

that

does

bind

to

be

used

not can

androgen

The

present

report

the

characteristics

human

BPH

to

the

could

were

indicate

while

an-

presence

also

that

components: SBP

of

the

4s

binding

androgen

BPH

in

binding

prostate.

recently

the

of

the

results

(11)

binding

characteristics

have

of

in

negative data

DHT

studies

limitations,

some

properties

incubation,

studies

although More

complicated

these

binding

l3).

contains

component

greatly

Despite

of

reported

has

that

of

SBP

receptor

extends C3Hl

our

C3Hl

R 1881,

and

as

an in

is not

metabolized

advantageous rat

previous

R 1881

a synthetic

prostate studies

binding

in

tracer (14, and

human

for

15).

describes BPH

tis-

sue. MATERIALS Human

AND

METHODS

tissues

Human prostatic samples from patients suffering from benign prostatic hypertrophy were obtained at surgery at the Hbpital de l'Enfant-Jesus. Immediately after removal, the tissue was kept in ice-cold 0.9% NaCl until its transport (l2 hours) to the laboratory. The tissue was then transferred in Buffer A (25 mM Tris-HCl, 1.5 mM EDTA, 70 mM a-monothiopH 7.4) and kept at 0-4oC until glycerol and 10% glycerol, Human uteri were obtained from normal women the assay. treated for 5 days with a daily dose of 5 mg of conjugated estrogens (Premarin) before hysterectomy performed 24 hours The tissue was treated after the last estrogen treatment. as described for BPH. Rat

material

Ventral prostates (obtained from Canadian

were obtained from Sprague-Dawley rats Breeding Laboratories, St.Constant,

S

451

-EDPBOXD_

Quebec) weighing 175-250 g and castrated 19-20 hours before Rat uteri were also obtained from Sprague-Dawley sacrifice. The female rats were castrated animals weighing 200-250 g. and injected with 17B-estradiol (10 ug/day, S.C. for 10 days). The uteri were removed 24 hours after the last steroid injection. Steroids C6,7-3H1 17a-hydroxy-17a-meth 1-estra-4,9,11-trien-3-one, C6,7- r HI 17, 21-dimethyl-19-norC3HI R 1881,(58.2 Ci/mmole), 20-dione,i3H1 R 5020,(51.4 Ci/mmole) and pregna-4,9-diene-3, unlabelled steroids were synthesized at the Roussel Research The labelled compounds were stored at -200C in toCenter. luene-methanol (9:l) and their purity was checked by thinAfter evaporation of the layer chromatography before use. solvent under a stream of nitrogen, the labelled steroids were dissolved in buffer B (10 mM Tris-HCl, 1.5 mM EDTA and Stock solutions of the 10 mM a-monothioglycerol, pH 7.4). unlabelled steroids were kept at 2-4oC at a concentration of The steroid solutions were prepared 4 x lo-4M in ethanol. by appropriate dilution with buffer B. 13-ethyl-17-ethinyl178-hydroxy-4-gonen-3-one (Norgestrel) was also used. The steroids were added to the incubation in a 10 ~1 volume and the final ethanol concentration never exceeded 0.2%. Tissue

preparation

After rinsing in ice-cold buffer A, the tissue was minced with scissors and homogenized in 3 (BPH) or 9 (human and rat uterus) volumes (v/w) of the same buffer using a Polytron PT-10 homogenizer at a setting of 4 for three periods of 10 seconds. All operations were performed at 0-4oC. The homogenate was centrifuged at 40,000 xg for 20 minutes and the resulting supernatant was centrifuged at 105,000 xg for 90 minutes to obtain the cytosol fraction. All binding assays were performed with fresh cytosol. Protein concentration was measured (16) using bovine serum albumin as standard. Steroid sorption

binding

assays

by

dextran

coated

charcoal

(DCC)

ad-

For measurement of C3H1 R 1881 binding, 0.20 ml aliquots of cytosol were incubated for 15-17 hours at 0-4oC with 0.050 ml of 3 x 10-8 C3Hl R 1881 in the presence or absence of increasing concentrations of unlabelled steroids added in a 0.010 ml volume. C3HI R 5020 binding was measured as described (17) in a total volume of 0.4 ml. Unbound steroid was then removed by incubation for 10 min at 0-4OC with 0.3 ml of 0.5% charcoal-0.05% dextran T-70 in buffer B and centrifugation at 2000 xg for 10 min at the same temperature. 0.3 ml aliquots of the supernatant were then removed and after

452

‘EBEOXDI

m

the addition of 10 ml of Aquasol, in a Beckman liquid scintillation efficiency of 35%.

radioactivity spectrometer

was measured at a counting

RESULTS Since most te

specificity

exclusively (l-6),

ficity

we

of Table

ty

of

and

1 and

in

are

in

Blondeau

rat

with

al

receptor

C3H1

in

other

initial in

good

some

SPECIFICITY

OF

C3H1

R 1881

PROSTATE

Steroid

R 1881 19-nortestosterone 5a-dihydrotestosterone Testosterone 17a-methyl-testosterone D-Noraestrel Cyproterone acetate R 2956 Androstanediol Progesterone R 5020 Estradiol 19-hydroxytestosterone Epitestosterone Androstenedione 5-androstene-3,17-dione Androsterone

ventral of

potency

compete

with

TABLE LIGAND

the

al-

prosta-

the

Shain

of

for

These

cytosol.

although

rat

been

speci-

tissue.

to

agreement

has

studies

this

steroids

prostate

(5)

DHT

1 illustrate

ventral

fairly

androgen

our

binding

Figure and

et

the

extended

R 1881

androgens

binding data

studied

have

C3H1

of

a varie-

['HI

R 1881

competition

and

difference

Boesel are

(18)

apparent.

1 BINDING

IN CASTRATED

RAT

CYTOSOL

Displacing

ability

165 125 100 60

4.5 2 1
(%)

At

variance

deau

et

al

tosterone R 1881

with (5) has

from

Liao

and

our

about

its

et

al

(6)

and

previous

the

same

in

data

agreement

(15),we

potency

as

DHT

with

find to

that

Blontes-

displace

C3H1

receptor.

RI981 P~ogestereee R5020 Cypraterone Acetate

o-o +-a 4-a

“7

,’

’

25~10-9

10-B

STEAOIO

m--o ci +-% X--I

3x1043

19-No~testesterunc 9-Nefgestrel Testosterone Dih~drotestosterene

10-7

CONCENTRATION

3x0-7

10-6

(M)

Effect of increasing concentrations of unlaFigure 1: belled R 1881, R 5020, progesterone, dihydrotestosterone, D-Norgestrel, testosterone, 19"nortestosterone and cyproterone acetate on the binding of 6 nM C3HJ R 1881 to castrated rat ventral prostate cytosol. After incubation at 0-4oC for 16 hours, the bound fraction was separated by dextran-coated charcoal. 10,000 CPM were bound per tube in the control group. It should titors

to

be

displace

mentioned C3H1

that

R 1881

the

potency

is markedly

of

weak

influenced

compeby

the

time

of

incubation.

efficient long

to

displace

incubation

binding

studies

androstanediol, diol

have

R 1881 after

while

by

the

data

with

seen gen

to

As

receptor

the

selected artefacts

steroids

R 5020

and

progesterone

reduced

prostate of potency

are

in

R 1881 BPH

the

present

O°C

show

that

Blondeau

of

twice

related

the that

human

to

C3Hl

(except

Norgestrel)

much

while binding

cytosol.

be

better

the

it can

seen

that

competitors

19-nortestosterone,

Identical

be

andro-

R 1881 In

contaminating

in rat

andro-

unpu-

cytosol.

to

can

R 1881

prostate

binding

It

that

DHT.

of

BPH

incu-

C3H]

15),

of

be

appear

Labrie,

rat

(5)

performed

of

(14,

al

steroids

it would

competition in

et

short

the

and

C3Hl

could

studies

(Drouin

estra-

displace

incubations

for

and

finding

From

R 1881

to

to

to

why

during

closely

SBP.

cytosol

C3H1

This

previously

used

for

by

culture,

more

the

due

affinity

competitor

in

steroids

low

rat

O'C.

approximately

having

than

of

DHT

found

compounds

2 illustrates

un labelled

of

more

compared

at

transformation

reported

were

androstenedione

long-term

affinity is

hours

conditions

cells

are

short

explain

15-17

incubations.

with

of

the

to min imize

would

were at

studied

after

ability

some

cytosol

data).

bindin g by

the

values

pituitary

obtained

that

1%

long-term

Figure

the

or

activity

blished

for

incubation

prostate

genie

performed

non-equilibrium

during

anterior

R 1881 This

than

a 2-hour

periods

the

C3H1

competitors

periods.

higher

bation

in

weak

epitestosterone,

less

explained

All

tissue, results

order SBP,

were

those

Norgestrel, in

BPH

a strong shows were

much ob-

S tained

in

ten

different

Since

such

with

those

of

dies

the

samples

binding

of

of

BPH

tissue.

characteristics

a progestin

specificity

455

'EBBOXDILI)

receptor C3H1

have

(17,

R 1881

binding

M 0-o b--d o-u m-m

STEROID

19,

much 20), in

in we

human

common next

stu-

myome-

R1991 Progesterone A5020 Cyproterone Acetate 19-Nortestosterone Cl-Norgestrel

CONCENTRATION

(M)

Figure 2: Effect of increasing concentrations of unlabelled R 1881, R 5020, progesterone, D-Norgestrel, 19nortestosterone and cyproterone acetate on binding of 6 nM CsHl R 1881 to human BPH cytosol. After incubation at 0-4OC for 16 hours, the bound fraction was separated by dextran-coated charcoal. 2500 CPM were usually bound per tube in the control group. trium

after

re

C3H1

3,

priming R 1881

with binds

estrogens. with

high

As

illustrated

affinity

in

the

in human

Figumyome-

trium

and

terone study

R 5020

for the

C3H1

a potent

to

with

19,

20).

R 1881

specificity

tissue, bind

competesmore

Using

binding. of

synthetic

high

affinity

the

efficiently

same

It was

binding

of

progestin to final

the

than then

['HI

19-nortestos-

of

R 5020

found

to

in

BPH

human

in other

progesterone

concentration c--o o-0 H n-a

interest

systems

receptor of

(17,

6 x lo-'M,

R1881 Progesterone R5020 Cyproterone Acetate 19-Nortestosterone

STEROIDCONCENTRATION(M) Figure 3: Effect of increasing concentrations of unlabelled R 1881, R 5020, progesterone, 19-nortestosterone and cyproterone acetate on the binding of 15 nM C3HI After incubation at OR 1881 to human uterus cytosol. the bound fraction was separated by 4oC for 16 hours, 12,000 CPM were usually bound dextran-coated charcoal. per tube in the control group. it was

found

that

0.7

and

1.0

pmole

was

bound

per

g of

wet

S tissue

with

C3H1

R 5020

though

the

binding

of

that

of

C3H1

progesterone than

by

C3H1

R 5020

4s an

the

analysis, region

and

excess

of

C3H1 R 5020

in

being

more

(data

not

binding

only

R 1881,respectively. human

specificity

19-nortestosterone

gradient the

and

and

the

R 1881,

457

TIlEOXDI

50%

unlabelled

the

was

Using

R 5020

binding

lower

than

similar:

competitors

shown). C3H1

was

binding

efficient

of

of

of

BPH

Al-

sucrose

was

can

be

found

in

displaced

R 5020.

DISCUSSION The previous C3H1

data

presented

observations

R 1881

similar

to

Coupled higher

with

to

ing

to

the

usefulness

we

have

of

this

conditions

to

human

the

specificity

BPH

cytosol

the

same

BPH

present

tracer

in

of

is markedly

widely

used after

the

of

as model 16

hours

in of of

cytosol

15)

of

is

very

describing

high

of

or

with

different

our

specificity

affinity data

SBP-like

labelled that

rat

ventral

androgen incubation

recep-

androgens,

C3HI

R 1881

data

show

steroid

from

bind-

components.

endogenous

present

human under

prostate

receptor O'C,

bind-

that

to

measured

at

its

indicate

androgen

of

the

receptor,

specificity

The

conditions

of

SBP

the

the

androgen

presence

(15).

binding

the

studies

exchange

cytosol

(14,

absence

with

1 extend

DHT.

for

the

previously

incubation

In fact,

and

of

that

data

R 1881

Figure

prostatic

E3H1

contaminated

observed

show

previous

C3H1

1 and

ventral

with

proteins,

ing

a tissue

of

tissues

Using

our

and

rat

degradation

plasma

in

to

obtained

affinity

resistance

tors

(15)

binding that

in Table

gland,

(l-6). the

spe-

S

458

cificity

of

found

estrogen-primed

in

C3H1

high

concentration

both

tissues,

progestin, ing

is

used

model

tin

binding

in

city

of

binding

that

of

C3H1

however

It

hours likely

The of from

and

findings

The

found

in

specific

only

minimal

data

in

in rat

or from

due

an SBP

to

and

BPH

of the

same

but

BPH

with gra-

incubation

could

be

also

the

is markedly

prostate

human

detec-

be men-

(15-17

endogenous

that

to

sucrose

conditions

clearly

of

specifi-

to

should

with

proges-

similar

R 5020

It

exchange

ventral

atypical

C3H1

the

the

the

si-

widely

measured

of

using

incubation

human

characteristic be

of

reverse

finding

R 5020

that

region

that

indicate

binding

measured

4s

the

being

than

in

bind-

of

tissue,

C3H1

cytosol.

present

present

could

the

binding

prostate

by

progestin of

The

presence

this

lower

be mentioned

the

in

binding 30%

occurred.

receptor

Separation

is

with

R 1881 that

R 5020

R 1881

a tissue

supported

labelled

have

properties

tin

R 1881.

O'C),

to

of

the

ventral

that at

of

potent.

The

a

synthetic

C3H1

prostate,

that

Moreover,

a potent of

receptor.

to

containing

receptors.

less

ventral

similar

a tissue

R 5020,

is

further

binding

no

in rat

tioned

is

is

competitors

androgen

should

conditions, ted

and

approximately

R 1881

dient.

rat

BPH

BPH

progesterone

potent

in

of

affinity

C3H1

most

in

myometrium,

of

found

high

is

binding

19-nortestosterone

tuation as

R 1881

progesterone

are

while

?FDEOXDI

DHT

is

specificity different rather

shows

a progestin

receptor.

These

presence

a specific

proges-

androgen

specificity

of

receptor measurements

in BPH

tissue.

under

con-

S ditions

of complete

rization show

exchange

of the binding

also

clearly

that

should

R 1881

and progestin

receptors

should

be taken

using

both

permit

molecule(s)

drogen

when

459

1c13IlOXDSD

binds and

further

characte-

in this

tissue.

equally

well

to the an-

that appropriate

this marker

Our data

in tissues

care containing

receptors,

REFERENCES :: 3. 4. 5. 6. 7. 8. 9. 10.

17. 12. 13,

16. 17. 18.

Mainwaring, W.I.P. J. ENDOCRINOL. 45, 531 (1969). Unhjem, O., Tveter, K.J. and Aakvaag, A. ACTA ENDOCRINONOL. 62_, 153 (1969). Liao, S. and Fang, S. VITAMINS AND HORMONES 27, 17 (1969). Baulieu, E.E. and Jung, I. BIOCHEM. BIOPHYS. RES. COMMUN. 38, 599 (1970). Blondeau, J.P., Corpechot, C., Le Goascogne, C., Baufieu, E.E. and Robel, P. VITAMINS & HORMONES 33, 319 (1975). Liao, S., Liang, T., Fang, S., Castaneda, E. and Shao, T.C. J. BIOL. CHEM. 248, 6154 (1973). Westphal, U. IN: STEmD-PROTEIN INTERACTIONS, SpringerVerlag, Berlin (1971). Davies, P. and Griffiths, K. MOL. CELL. ENDOCRINOL. 3_, 143 (1975). Mainwaring, W.I.P. and Milroy, E.J.G. J. ENDOCRINOL. 57, 371 (1973). Fraser, H.M., Mitchell, A.J.H., Anderson, C-K, and Oakey, R.E. ACTA ENDOCRINOL. 76_, 773 (1974). Rosen, V., Jung, I., Baulieu, E.E. and Robel, P. J. CLIN. ~NDOCRINOL (Kbhf 4l_, 761 (1975). Steins, P., Krieg, M., Hollman, H.J. and Voigt, K.D. AcTA END~CRINOL. (KBH! fi, 773 (1974). ~~~~~~ J.K. and Giorgi, E.P. J. STEROID BIOCHEM. 3_, 471 . Bonne; C. and Raynaud, J.P. STEROIDS a, 227 (1976). Bonne, C. and Raynaud, J.P. STEROIDS 27_, 497 (1976). Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. J. BIOL. CHEM. 193, 265 (1951). Asselin, J., Labrie, F., Kelly, P.A., Phifibert, D. and Raynaud, J.P. STEROIDS 27_, 395 (1975). Shain, S.A. and Boesel, R.W. J. STEROID BIOCHEM. 5, 43, (1975). Philibert,

Philibert, (1974).

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J.P. J.P.

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