Characterization of unoccupied (R) and occupied (RA) androgen binding components of the hyperplastic human prostate

561

2263 CHARA~~RI~ATION OF UNOCCUPIED (R) AND OCCUPIED (RA) ANDROGEN BINDING COMPONENTS OF THE HYPERPLASTIC HUMAN PROSTATE Sydney A. Shain, 1l2 p4 Robert W. Boesef,’ Donald L. Lamm,*p3 and Howard M. Radwin3 lDepartment of Cellular and Mofecular Biology, Southwest Foundation for Research and Education, 2Audie Murphy Memorial Veterans Administration Hospital, and 3Department of Surgery, University of Texas Health Science Center, San Antonio, Texas Received

12-27-77 ABSTRACT

Saturation protocols were developed for measurement of unoccupied (R) and steroid~ccupied (RA) androgen binding components of human hyperplastic prostate. The concentration of unoccupied cytoplasmic binding sites (2 hr incubation at 2°C) for the synthetic androgen RI 881 (17&hydroxy-17~-methyf~stra~,9,11 -trienS-onef and the synthetic progestin R5020 (17a,21 -dimethyl-lQ-norpregna~,gdiene-3,2Odione) respectively was 10.7 + 1.4 and 14.3 + 3.2 fmofes per mg cytosof protein and the apparent steroid affinity respectively was 9.6 f 0.8 and f .6 f 0.4 x IO* M-l. Steroid specificity of the unoccupied cytopfasmic R1881 and R.5020 binding sites was similar. When RI881 and R5020 were employed as probes of total, R plus RA, cytopfasmic binding components (20-24 hr incubation at 15°C) saturable binding of R5020 was not detectable. Total cytopfasmic R1881 binding site concentration and apparent affinity for R1881 were 51.7 + 3.3 fmoles per mg cytosol protein and 2.7 + 0.6 x f07 M-l. R5020 was a poor inhibitor of R1881 binding to total cytopfasmic R1887 binding components. Incubation

(4

hr

at

37°C)

of

human prostate mince with Se-RHT a single class of KCI extractable nuclear binding component(s) which sedimented at 4-55 on linear sucrose gradients confining 0.6 M KCI. Binding site concentration and apparent affinity for SEU-DHT were IS fmofes per 100 &g DNA and 1.0 x lOa M‘“. The crude nuclear pellet obtained from human prostate homogenates contained binding components which were detectable by incubation of the clarified nuclear KC1 extract with R1881 at 15°C for 20-24 hr. Binding site concentration and apparent affinity for R’f881 were 29 t 8 fmoles per 100 pg DNA and 3.1 f 0.4 x IOa M-‘. Steroid specificity of the nuclear Se-DHT and RI881 binding sites was comparable. Unoccupied KCf-extractabfe RI881 binding sites were not detected subsequent to 2 hr incubation at 2°C. The data were consistent with identification of the nuclear binding components and the R1881 cytopfasmic binding component measured subsequent to incubation at 15°C as human prostate androgen receptors.

(17~-hydroxy-Sff-androstan-3~ne~ demonstrated

4Address reprint requests to Dr. Sydney A. Shain, Southwest and Education, P. 0. Box 28147, San Antonio, TX 78284.

Foundat~n

for Research

INTRODUCTION The

notable

a predictive gators

to

success of estrogen

index

of

examine

receptors

in

response

androgen

human

globulin-like

limited-capacity

binding

column

(lo-12),

provide

only

semiquantitative

binding

component

Following Raynaud

the

and

unoccupied

ventral

human

plasma

SHBG

R1881

binding

to

components

was

specificity (17).

of

(151,

These observations,

human

benign

have also

been

investigators

In this suggest consist

that

stroma

report

plasmic

and

the properties

nuclear

same

binding

that

sulfate

sex

high-affinity,

of

Sephadex

Se-DHT

to

from

SHBG.

a prostatic

specific

activity

R1881

by

(8,ll)

human These

have

prostate

procedures

cytoplasmic

5a-DHT

also

components

et al. (18)

and

failed

probe

of

to

bind

cytoplasmic

reported

steroid

by

demonstrated

was similar

the unoccupied

R1881

a potent

prostate

they

principally

was

subsequently

hyperplastic

Moreover,

radiolabeled

receptors

investigators

cytosol

precipitation

binding

androgen

benign

indicate

Dubk

we will

that

binding

that binding

the

to that

to

steroid

of

RI881

component(s)

of

may be progesterone-receptor-like,

and Grant

and colleagues

the progesterone-receptor-like

present

steroid

of androgen-receptor-like demonstrate

plasma

(19).

component

The

is present

latter only

(19).

unoccupied

we will

high

cytoplasmic

prostate

have suggested

in the prostatic

to

by

of

R5020.

which

reported

androgen

(S-8),

to unoccupied

demonstrated

human

by

hyperplastic

centrifugation

investi-

specificity.

cytoplasmic

These

binding

of

demonstrate

binding

of SHBG

steroid

they

prostate

inhibited

R5020

Se-DHT

synthesis

unoccupied

of

presence

“receptor”

Se-DHT

from

defined

(16).

the

which

gradient of

many

Characterization

by

and protamine

separation

colleagues

rat

Sucrose

(13,14),

successful

prostate.

breast tissue as

7u-acetoxy-ltu,2ff-methylenepregnalt,6diene-3,20-dione)

receptors

of poorly

(l-4)

complicated

to distinguish

androgen

has prompted

inhibition

(6-chloro-l

cytoplasmic

therapy

macromolecules

Se-DHT.

to attempt

in neoplastic

human

been

(SHBG)

agar gel electrophoresis

been employed

by

has

(g-11),

acetate

determinations

endocrine

binding

of

chromatography

cyproterone

to

prostate

hormone-binding

receptor

that binding

of androgen

data

which

binding

sites

confirm of

the

human

benign

and progesterone-receptor-like

R1881

is a highly

components receptors.

selective

of human

observations

hyperplastic

components.

probe

benign

preceding

of total

hyperplastic

and

prostate In addition,

(R plus RA) prostate

that

cytohave

S

TPIEOXDI

MATERIALS

.AND METHODS

Tissue specimens: All benign hyperplastic prostate tissue specimens were obtained from consenting donors at the time of transurethrai resection or open prostatic enucleation for relief of obstruction. The tissue was immediately placed in ice-cold Earle’s medium (Gibco) and transported on ice to the laboratory for processing within 30 min of completion of the surgery. Representative samples of each specimen were processed for histologic examination as previously described (20). 5a-[ 1 ,2-jH2 JDihydrotestosterone, specific activity 5.5 Cijmmole Chemicals: (Amersham~Searie Corp.), was purified by paper chromatography (21) shortly before use. [l 7-C3 H3 JR5020, specific activity 86 Ci/mmole, was obtained from New England Nuclear Corp. [6,7-3 H2 ]R1881, specific activity 58.2 Ci/mmole; [6,7-3 Hz ]R2858 (1 I&methoxy17e-ethynyl-1,3,5( lO)~stratriene-3,17-dial), specific activity 52 Ci/mmole, radioinert R1881, R5020, and R2858 were generously provided by Dr. Jean-Pierre Raynaud (Centre de Recherches Roussel-Uclaf, France) and were used without further purification. 7e-Methyf-l7~-hydroxy-4,14~stradien-3~ne (A I4 -7cu-methyl-l9-nortestosterone) was generously provided by Dr. Albert Segaloff, Alton Ochsner Foundation, New Orleans. Sa-Androstane-3a,l_lrucliol was obtained from the steroid reference collection of the Medical Research Council of Great Britain. Other radioinert steroids were obtained from Steraioids, Inc. and purity was established as previously described (22). Human r-globulin (Fraction II) was from Calbiochem and Dextran T70 was from Pharmacia Fine Chemicals Inc. All other chemicals were the best reagent grade available. Aqueous solutions were prepared with water which was distilled, deionized, and redistilled from glass. The preparation and storage of aqueous solutions of radiolabeled steroids has previously been described by us (21). Aqueous solutions of radiolabeled R.5020 additionally contained 0.1% bovine serum albumin (Sigma). Saturation analysis and steroid specificity of cytoplasmic steroid binding components: Unless otherwise stated all procedures were performed at 2°C. Prostate tissue was thoroughly minced with fine surgical scissors and homogenized in 3-5 volumes (w/v) of buffer TES (50 mM TrisCl, 0.1 mM EDTA, 380 mM sucrose, 0.5 mM &mercaptoethanol, pH 7.4) in a Dual1 glass/glass homogenizer maintained in an ice slush. Cytosol was prepared by centrifugation for 60 min at 100,000 x g. Endogenous steroids were removed by mixing cytosol with a charcoal pellet obtained by centrifugation, 1000 x g x 5 min, of one-tenth volume of DGCC (5% Norit, 0.5% dextran T70, 1% human r-globulin in buffer TE: 50 mM TrisCI, 0.1 mM EDTA, 0.5 mM &mercaptoethanol, pH 7.4). After 10 min incubation, charcoal was removed by centrifugation at lO,O~ x g (2 x 5 min) (22) and 100 ~1 aliquots were incubated with steroid in a final volume of 220 ~1 of buffer TES. Saturation analysis for quantitation of unoccupied cytoplasmic binding sites employed 2 hr incubation at 2°C as previously described (21). Saturation analysis for quantitation of total (R plus RA) cytoplasmic binding sites employed incubation for 20-24 hr at 15°C with 10 nM radiolabeled steroid and sufficient radioinert steroid to achieve the following final nM concentrations of ligand: 10, 15, 20, 30,40, 50, 80, and 110 (22). Nonspecific ligand binding corrections were determined as previously described

S

fJfBEOXD8

(21,22).

Determination

of cytoplasmic

labeled

steroid

and

terminated

probe

by treatment

Saturation Two

quantitation ization

of nuclear

of

canine

obtained

tissue homogenates

extracted

mM

M KCI),

extracted extracts

were

100 ~1 aliquots (triplicate

concentration binding, DGCC

with

in the

treatment

Other described

(21).

either

RI881

of steroid preceding

the exceptions

protocol

the

employed

crude

binding

nuclear

(w/v)

Washed

EM

of buffer

pellets

EMK

After

centrifugation

were

(buffer

each

.i’ S. x! ‘I C 7 i j

sequerl,r;:!ll,

EM containir+:

incubation

at 50,000

analyses were performed

the

exception

that

EMK.

of nonspecific

of incubations

.The

multiple

for 20-24

in a final volume of 220 ~1 buffer

and termination

interval.

x g at 2°C.

by incubating

was 0.5 to 20 nM. Determination with

TES,

x :: at

(10 mM Tris-Cl,

nuclear

5 and 5 min.

RI881

section

Tissue

buffer

5 min at 5000

the pellet

components.

volumes

at 2°C for 2 hr (single points) or at 15°C

binding

that in the

steroid

were performed

ethanol

hi Thi

was omitted

as and

was for 20 min at 2°C.

methods:

the methodology

7.4).

15,

of steroid binding

the pellet was washed three times by rcpc

by 5 min

radiolabeled

range for

specificity

described

pH

and saturation

of extract

points)

of

in 5 ml per g tissue buffer

collected

combined

second

tubes, and centrifuged

supernatant,

binding procedure

by us for character-

with

in 5-10

at 2°C with three aliquots

were

The

extraction

described,

centrifuge

1 ml per g tissue, for

proteins

mM.

KCI

first

the

and the sucrose concentration

as the source of nuclear

p-mercaptoethanol,

by incubation

100

as previously

and centrifugation

0.5

to

repeated

steroid

In

described (21)

were

(21,22).

and determination

components

10 nM radio-

incubations

nuclear

analyses.

previously

mg tissue mince

by

removal of the resultant

EDTA,

binding

All

described of

these

components

employed

inhibitor.

specificity for

protocols

was reduced

recovered

to polycarbonate

suspension

binding

500

steroid

as previously

used

nuclear

homogenized,

transferred

0.6

buffer

from was

After

steroid

binding specificity

steroid

were

the tissue mince prostate

fraction

mince

and

flasks contained

homogenization

steroid radioinert

DGCC/ethanol

procedures

employed

incubation protein

with

analysis

components: specificity

1 PM

Sample

preparation

for quantitation Sucrose

binding components

for the quantitation

of radioactivity,

density

gradient

was performed

protein

sedimentation

as previously

of protein

and DNA analysis

described

and DNA

have previously of

nuclear

and been

androgen

(21).

RESULTS Quantitation steroid

binding

radiolabeled

and

determination

components:

probe occurred

4 hr incubation.

Saturation

of

RI881

and

cytosol

preparations

both

prostate and

R5020

binding

R5020

of

steroid

Preliminary during

specificity

examination

2 hr incubation

analyses demonstrated to cytoplasmic

unoccupied

indicated

maximal

cytoplasmic binding

at 2°C and was maintained limited

components

(Fig. 1). Simultaneous

site concentration

of

and apparent

capacity,

high affinity

in human

determinations steroid

benign

through binding

hyperplastic

of unoccupied

association

of

constant

RI881 in the

S same cytosol binding

preparations

TBEOSDrn

indicated

both

steroids

sites (Fig. 1 and Table 1). However,

was only

17% of that for R1881.

not detectable.

Limited

not detectable

in human

the apparent

Saturable,

capacity,

were

high affinity

high affinity

binding

binding

to a similar

number

binding site affinity cytoplasmic

of R1881,

R2858

R2858

of

for R5020 binding was

or R5020

was

male plasma.

0.02

004

0.06

Radioligand BoundfnM)

Figure

1: Saturation

unoccupied with

binding

R1881

analyses

for

sites. Aliquots

(o-o)

or R5020

quantitation of cytosol

(e-e)

of

(1.15

cytoplasmic

mg protein

for 2 hr at 2°C

R1881

and

R5020

per tube) were incubated

and specific

binding

of probe was

determined.

R1881

binding

either from

The

prepared

from

electrocautery

absence of detectable, transurethral

resection

concentration

and

in cytoplasmic

by open prostatic saturable

resection

and 9.6 f

different

patient

prostatic

enucleation M-l.

prepared from

specimens,

apparent

protein

lo8

comparable

steroid

specimens

binding

1 .O x 10” specimens.

M-r

enucleation

binding

affinity

(mean

were 12.0

binding

specimens.

cutting

10.5

prepared

or by transurethral

in cytoplasmic associated

current

f

1.0

with

data for prostate

extensive

always

detectable

and their

per mg cytosol

performed

obtained

per mg cytosol

extracts

was used to obtain

fmoles

for determinations

+ 2.0 fmoles

sites were

binding

extracts

sites were always detectable

were

f SEM)

The comparable

patients

R1881

R1881

enucleation

was always

damage of the tissue. When principally

transurethral

x

were

prostate specimens obtained

resection.

1.2

characteristics

from

upon 11 three open

protein

in cytoplasmic

and 9.4 + extracts

S

546

Table

1: Site Concentration

the Human

Benign

Rl881

~_”

TCBEOIDI and Apparent

Hyperplastic

and R5020

Steroid

Prostate

Binding

Affinity

of

Cytoptasmic

Componentsa

.II.__

&and --

Incubation

Apparent

Conditions

(lo+

M-‘)

Affinity

Site Concentration (fmoles

per mg protein)

R188lb

2 hr, 2OC

9.6

+0.8

10.7&l

R5020C

2 hr, 2’C

1.6

+0.4

14.3k3.2

R1881d

20-24

hr, 15°C

R5020=

20-24 _

hr, 15°C -__-_..__

5 1.7k3.3

0.27kO.06 ~_~ aData are the mean 2 SEM; bN = 14; CN = 3; dN = 7.

Comparative RI881

binding

only

studies

sites was similar

moderately

progesterone

effective

and some

and cortisol

indicated

natural

and

of

inhibitors

determination

of

performed

receptor since

during

of rodent

controlled

course

incubation activity.

indicated occurred

at 15°C. No

incubation

during Incubation

hr

at higher

binding

of

and

and testosterone

R5020

were highly

specificity

binding,

effective,

were whereas

and estradiol

not

radiolabeled

maintained resulted was

to

androgen

human

Preliminary

RI881

R5020

for

tissues

binding

cytoplasmic

through

cyto-

assay, which

total

be performed

of

temperatures

(R plus RA)

cytoplasmic

feasible.

was

totar

of this binding

of

not

binding

and

of

validation

could

were

saturable

18-20

or

quantitation

(22),

experiments

SC&H-F

R5020

at

binding

least

in loss of RI881 detectable

time-

28

hr

binding

subsequent

to

at 15°C. analysis,

class of

binding

steroid

for

prostate

maximal

saturable

Saturation single

and canine

unoccupied

2).

The rigorous

development

orchiectomy

studies

components

its

-. ---._____

-

of

RI881

androgens

(Table

none detectable

specificity

either

or synthetic

plasmic steroid binding components: we

steroid

(Table 2). Significantly,

inhibitors

were ineffective

Quantitation

the

.4

limited

parameters

cytoplasmic

extracts

enucleation

specimens.

at

15”C,

capacity for

cytoplasmic

these

prepared The

employing

R1881 from

data

radiolabeled total

binding

suitable

demonstrate

androgen sites

number

as probe

binding

(Table I)

transurethral the

RI881

resection of

sites

were and

cytopiasmic

revealed

a

(Fig. 2).

The

comparable

in

open total

prostatic Rl881

S

547

%?DEOXD~

Table 2: Steroid Specificity of the Human Benign Hyperplastic Prostate Cytoptasmic R1887 and R5020 Binding Componentsa -

-

DPM Specifically Bound (% of control) Total’

Uno~~upiedb Inhibitor

-. None RI881 So-~ihydrotestosterone 19Nortestosterone ‘I7p-Testosterone 17a-Testosterone A’4-7~-Methyl-19”nortestosterone Sa-AndrostaneSo,l?&diol So-Androstane-3~,~ 7e-dial Sa-Androstane-3@,17fldiol 4-Androste*e-3,17~io~e 17~-Estradio~ Cortisol Progesterone R5020

Probe:

R5020

R1881

R1881

100 13rt: 3 42+ 7 22* 7

100 0 57% 9 9* 2 48+ 3 12t 5 114+10 96410 71* 6 1o.V 7 20+ 7 14+ 6

100 0 2453 4rt4 27rt2 85+2 4rt2 7226 1O&3 @I+2 9126 881t5 114ir9 6428 51+4

?.d 30*13 77&15 79411 75*9 92& 4 13+ 7 0

--

aData are the mean f SEM for three to seven determinations. bln~ubation at 2°C. CIncubation at 15°C. dDetermination not performed.

binding sites to be fivefold greater than the number of cytoplasmic unoccupied Rl881 binding sites, This

result

suggests that

the majority of these sites are occupied by

endogenous steroid prior to exchange (15°C)

incubation. Examination of the steroid

specificity of cytoplasmic total androgen binding sites demonstrated the effectiveness of 17jWestosterone and Scu-DHT as inhibitors of R1881 binding to be markedly increased, whereas the effectiveness of progestins was markedly decreased (Table 2, compare results at 2°C and 15°C). Greater than 90% of the radioactive material recovered subsequent to 20-24 hr incubation at 15°C was isopofar with R1881 as determined by silica gel thin layer chromatography in the solvent system btnzene:ethyl acetate (7 : 1, v/v).

Q~a~titation

components: by a 100-fold

and

determination

of

steroid

specificity

of

nuclear steroid binding

Preliminary experiments demonstrated that maximal specific (inhibitab~e molar excess of radioinert Sa-DHT)

nuclear accumulation of Sol-DHT

occurred between 2 and 4 hr incubation and was maintained for up to 12 hr incubation

S

548

TIlEOIDS

0.06 -

1

5

I

-a,

I

0.02

[L

0.06

&]

Figure

2:

R1881

total

Double

incubated

for

at

All

37°C.

remaining Thin

inhibitable

nuclear

that

with

Sucrose fraction

having for

synthetic

90%

gradient that

as determined

and 1 .O x lOa M-l. were

Sol-DHT

or R1881

in nuclear

homogenates

c/:o~,ia~~

per tube)

by buffer

recovered

c.

here

bound

EMK.

by ethanol

ether,

2O:l

radiosteroid

of the DGCC-treated,

coefficient

(Table

of uterine

the

centrifugation

a sedimentation

component

When

of the specifically

ot

was determined.

was extracted

was quantitatively

radioactivity

androgens

quantitation

quantitation

(1 .lO mg protein

of RI881

radiosteroid

the

binding

for

binding

bound

for

on silica gel [dichloromethane:ethyl

%DHT,

per 100 pg DNA and

data

of cytosol

The

extraction.

(v/v)]

in the

KCI

demonextract

Sol-DHT.

density

affinity

saturation

aliquots

radiosteroid

than

demonstrated

molecules

of

and specific

nuclear

bound

greater

was isopolar

plot

24 hr at 15°C

0.08

(ll”‘-’

sites. Triplicate

layer chromatography

strated

and

reciprocal

binding

J

I

1

0.04

clarified

nuclear KCI

Steroid

using the

estrogen

extract

was used as the source

to

of

of nuclear

in association

analysis studies

nuclear

with

macro-

site concentration

(Fig. 4), were revealed

15 fmoles

that

only

natural nuclear

of

Sa-DHT

binding

to

this

measure

specific

binding

of

radiolabeled

procedures

receptor crude

only

(Fig. 3). Binding

specificity

inhibitors

Attempts

pellets

of 4-5s

by saturation

effective

3).

was found

KCI-extracted

described

by Clark

et al. (23)

were unsuccessful.

nuclear binding

pellets activity,

obtained

from

maximal

specific

prostatic binding

S

TPIlEOXDI

549

Gradient Effluent (ml)

Figure

Sucrose density gradient sedimentation

3:

DGCC-treated associated

KCI

extract

with 260 pg DNA,

radiolabeled 5~DHT.

nuclear So-DHT

Arrow

obtained

in the

indicates

aliquots,

absence

from (m-0)

I

a

nuclear binding component.

to

nuclear

human prostate

position of r-globulin

T

of Sa-DHT

equivalent or

presence

(o-o)

sedimentation

I

binding

mince incubated of

1 .O PM

component with

10 nM

radioinert

standard.

I

2 *.o-

$

1.6-

P 8

1.2-

% 0.6P a ", 0.4-& Li

Figure 4:

Mince

saturation

A 0

1.0

2.0

analysis of nuclear Sa-DHT

Data are the mean _+ SEM for four independent

3.0

binding

determinations.

sites in the KCI extract.

S

550

Table 3: Steroid

TDEOXDI

Specificity

of the Human

Prostate Nuclear

Benign Hyperplastic

Binding Componenta

DPM Specifically Inhibitor

Bound (% of control)

Sa-DHTb

Probe:

R1881C 100 0 23+ 5 26+ 7 31+ 9 94214 26+ 7 87k13 lOI? 4 58+ 1 56+ 5 105* 3 73+ 3 82+ 8

100 0 0 0

None R1881 Sa-Dihydrotestosterone 19-Nortestosterone 17p-Testosterone 17a-Testosterone A’ 4 -7a-Methyl-19-nortestosterone Sa-Androstane-3a,l7p-diol Sa-Androstane-3ct,l7adiol Sa-Androstane-3fl,17pdiol 17p-Estradiol Cortisol Progesterone R5020

_d _ _

79+10 93k 6 78+ 8 _

bMince aData are the mean k SEM ‘for three to seven determinations. protocol. ‘Nuclear KCI extract protocol. dDetermination not performed.

I

1

I

Figure (0.22

5:

Saturation

mg protein

for 20-24

hr (04)

analysis of R1881

Bound (nM)

binding sites in the KCI nuclear extract.

per tube) were incubated and specific binding

I 0.04

0.02 RI881

incubation

with

R1881

was determined.

at 2°C for 2 hr (o-o)

Aliquots

or at 15°C

of

R1881

was demonstrable

analysis

revealed

a single

(Fig. 5).

Binding

per 100

pg DNA

class of

and

demonstrated

R1881

binding

3.1

+ 0.4

that

only

binding

20-24

hr

capacity,

and apparent x 1Oa M-r natural

to these nuclear

R1881

to

limited

site concentration

studies

unoccupied

subsequent

components

affinity

for

f

Saturation

binding

androgens

specificity

effectively

inhibited

KCI-extractable

subsequent

sites

were 29 ?r 8 fmole

N = 3). Steroid

3). Saturable

sites were not detectable

15°C.

R1881

R1881

SEM,

synthetic (Table

at

high affinity

(mean

and

incubation

to incubation

nuclear

at 2°C for

2 hr (Fig. 5). DISCUSSION Assay plasmic

procedures

and

nuclear

Unoccupied, R1881

estrogen

for the R1881 of R5020 R1881

for

and R5020 unoccupied

(17,18)

The

preceding

1) R1881

and

different

unoccupied

binding

cant simultaneous R1881

and

do

affinity,

sites with binding

R5020

binding

steroid

component

as a

component

as an androgen

to cytoplasmic 50% of the total

binding

R1881

androgen

incubation

reports

(Table 1)

whereas the affinity

was significantly

the

at 2°C.

binding sites for the

were comparable,

and extend

permit

distinction

from

R1881

and R5020

steroid

R1881

sites. The

less than that of the laboratories

particular

receptor

receptor.

or

the

However,

the maximal

androgen-receptor-like measured at 2°C.

and 3) that

specificity

identification upon

amount binding

binding

of

the

of R1881

site(s)

is signifi-

unoccupied

of the cytoplasmic of the R5020

of

that:

cytoplasmic

there

cytoplasmic

the identification

based

possibilities

bind to different

to distinct

steroid

the

unoccupied

specificity,

or R5020

sites does not permit

binding

between

to the same cytoplasmic

2) that

or testosterone,

unoccupied

sites for the synthetic following

cyto-

prostate.

(Table 2) and site concentration

components

comparable

progesterone

by Sa-DHT

not

of either

unoccupied

binding

binding

and total

hyperplastic

to detect similar cytoplasmic

component(s)

are binding

apparent

benign

were detectable

binding

confirm

human

unoccupied

(19).

data

R5020

R5020

of

binding

specificity

data

and Grant

in

Steroid

steroid

(Table 1). These

Raynaud

sites

analyses failed

R2858.

quantitation

cytoplasmic

progestin

preliminary

for

binding

high affinity

and the synthetic

synthetic

developed

steroid

saturable,

Comparable

with

were

the

R1881

inhibition

of

binding binding R1881

binding attributable

components

is approximately

Subsequent binding

to incubation

components

progesterone

and at

determined

at 2°C.

prostate

manner and

(21).

rodent

which

binding

(24)

x IO’ 15°C

potency

prostate

at 15°C)

(22).

These properties

with the identification Human

prostate

binding

for

prostate,

3.0

androgen

receptor

by

x lo*

incubation

Steroid

canine

(21)

M-l

f21),

the

prostate

KCI

comparable

to that of the rodent

component

or

rat

human

These

are consistent

prostate

receptors

protocols

prostate

nuclear

(25) of

with its identification

x lOa

human

(21)

prostate

the nuclear

nuclear of

as determined

also is comparable.

component

binding

only in

The affinity

3.1 x IO8 M-l,

prostate

receptor.

hlV1 (25),

Additionally,

androgen

are consistent

of

protocol.

preparations

receptors.

RI881

to that of the canine

(Table 3) is identical

and canine the

3.7

binding

for

the

measured at

androgen

affinity

by us

Finally,

were demonstrable

incubation

nuclear

synthetic

for canine (6.5

androphile

The

for R1881,

nuclear

androgen

properties

prostate,

of crude

and

of R1881

androgen

preparations.

mince

to that

relationship

andro~hile

as a cytoplasmic

ventral

by the

incubation

nuclear

natural

1 .O x IO’” M”’ , was comparable

extract

of

receptor.

cytopiasmic

component(s)

nuclear

determined

coefficient

androgen

binding

is comparable

androgen receptors.

prostate cytoplasmic

crude

of the human

the

cytoplasmic prostate,

dependent

has also been described

to the binding affinity

androgen of

by the mince or KCI extract or

androphile

nuclear binding component

with

specificity

M-l)

Sa-DHT,

when

prostate

human

studied

that

binding to canine

an apparent

of the human prostate cytoplasmic

nuclear fraction

the human

prostate

the

of

during

binding to

temperature

(21,22)

Both

less than

R1881

of RI881

absence of

of the binding component

the salt-extractable component

affinity

is comparable

and rat (4.1 x 10’

receptors

The

cytoplasmic

(27,22)

inhibition

binding

to threefold

in an identical

androgen

relative

two-

cytoplasmic

(Table 3).

RI881

that progestins inhibit

receptors

prostate.

of this heat-labile,

M-’

M-I)

and

and canine

affinity

x 107

human

capacity

for androgens

cytopiasmic

being

reported

cytoplasmic

for

for the human

for rodent

of

inhibition

androgen

to limited

high specificity

the degree of progestin

total

report

bound

inhibitors

We have previously

In addition,

biologic

poor

percentage

cytoplasmic

now

androgens

were

the

prostate

we

between

(2.7

15°C;

RI881

demonstrated

R5020

incubation

canine

which

at lS”C,

as measured either to that of rat (26) the

sedimentation

component, KU-extractable

nuclear

as a nuclear androgen

androgen receptor.

4-5S,

is

nuclear binding

Human

prostate

cytosol

protein.

concentration

is 11

mg

activity

prostate

with

mg

represent

binding

of

components.

These

species

the

or

rat

ventral

of human

prostate

or

the

as

of

content

our

canine

(22)

this

latter

less than

binding androgenhuman

macromolecules.

This

prostate

determinations and

site

10% of

of human

direct

per

binding

unoccupied

steroid-free

or canine

in human

prostate

2) that

than

that

the

unoccupied

this

rodent

agrees well

of

unoccupied

prostate

unoccupied

from

(Shain

and

other

a variable

with

of androgen

was

decrease

in

had

androgen

from

100

that

about

in both

that:

of canine

the

to that

of

receptor,

10% of that

of androgen

and rodent

the possibility

of the

Although

androgen

is only

of canine

and

is comparable

the distribution

is less than

content

pg DNA.

cytoplasmic

receptor

that

between

receptor

receptor

prostate

and ranges from

age-dependent

per

androgen

receptor

receptor

prostate.

The

1) mean androgen or rodent

containing

cells in human

a

describing

preparation,

in human

progestins

cytosol

of rat ventral

prostate

prostate

is less

prostate.

receptors.

which

prostate

is age-invariant

concentration

modestly

data suggest that

choice

receptor

androgen

fmoles

rodent

nuclear

The

Nuclear

230

different

receptors

androgen

tissue

The

human

manuscript

and

performed

prostate

for

(22).

is only

cytoplasmic

androgen

age and breed dependent

(27).

(25)

values

or rodent

androgen

R5020

lower

number

of canine

While

prostate

cell

is both

protein

an unequivocal per

protein

is about

is markedly

content

and

receptor

prostate.

do not permit

cytoplasmic

mg cytosol

prostate

of human

of rodent

receptor

per

concentration

the concentration

noted

of

prostate

per mg cytosol

mean

concentration

tion

and

50%

that

is 52 fmoles

R1881

cytoplasmic

suggest

exist

(7),

Only

to

receptor

content

androgen

180 fmoles (22)

were

al.

of canine

72 fmoles

cytoplasmic

canine

that

et

unoccupied

protein.

androgen

receptors

receptor

concentration

prostate

data

cytosol

concentration

in preparation).

averages

canine

cytoplasmic

cytoplasmic

Rosen

androgen

The

25 to

of

receptor

considerations

androgen

estimate

cytoplasmic

and

fmoles

androgen

prostate

of the androgen-free

the

Boesel,

Human

cytoplasmic

estimate

total

per

could

receptor-like

cytoplasmic

failed

Noteworthy

preparations been

in R5020

stored binding

prostate to

report

appeared

inhibit

is the fact which

(28).

R1881 that

In

to cytoplasmic

These authors binding

to

the binding

had been frozen

at -70°C.

measurement

at -20°C

preliminary unoccupied

of

reported

cytoplasmic

determinations after

experiments binding

preparawe had

sites when

S

554

cytosol

TDEOXDI

was prepared

from

frozen

also decreased about

25%,

we did not pursue our observation.

and that

of others

component

androgen

is unstable

androgen

that

It is worthwhile of heat degradation

5 nM

R1881.

remained.

or 37°C.

From

incubation

of

equilibrium, that

the

Time

human

prostate

determination

of

receptor

the data

with

et

transfer

al.

(28)

from

0°C

their

incubated

at 0” with

for incubation

R1881

binding

at 15, 23,

calculate

would

be

to 15°C

more

a

that

produce,

These considerations

may

at

determina-

35% of the initial

5 nM

here

incubation

one may readily

receptor.

cytoplasmic

presented

during

previously

report,

of androgen

after

binding

also suggests that

performed

approximately

at 15°C

prostate

is stable

et al. (28)

in our current

Snochowski

sites were

cytoplasmic

experience

4 hr were not reported

cytosol

of equilibrium

R5020

however,

preparations

at 15”C,

15% saturation

Our

at 37°C;

with

points beyond

at

suggest

measure

of

than a determination

of

degradation.

5o!-Androstane-3a,l7adiol, be in

reported

to

Canine

prostate

Vitro

terone

Sa-androstane-3fl,17fldiol

stimulators

cytoplasmic

5a-androstane-3a,l7a-diol

human

37°C.

androgen

of receptor

the data presented

reestablishment receptor

cytoplasmic

binding

In view of our experience

suggest that human

or

is unstable

4 hr incubation

less that

(28)

23

R1881

freezing.

to note that Snochowski

tions

After

15,

since total

the heat-labile

by repeated

at

receptor

demonstrate

activity

that

these same investigators

receptor

cytoplasmic

15°C.

we conclude

also may be inactivated

In addition,

clearly

(17-19)

tissue. However,

of canine and

nuclear

and canine

has been reported a previous report inhibitor current

of

prostate

to be a product (21),

Sa-DHT

report

(33)

has been mince

of human

we demonstrated binding

demonstrate

Se-androstane-3p,l7fldiol

are It

to

(30,31). reported

preparations.

which

that

prostate

inhibitors

of

R1881

therefore

appears

that

if

receptors.

3a,17adiol,

and Sa-androstane-3fl,17pdiol

are regulators

mode of action does not involve interaction

specifically

bind

of ePit=toSincubation

also (32).

The

data

of the

5a-androstane-3a,l7a-diol binding

In

was an ineffective

receptors.

to

epitestosterone, of prostatic

with the cytoplasmic

androgens.

of

Se-Androstane-3p,l7adiol

androgen

epitestosterone,

androgen

(29).

in vitro

for

been

polymerase

The formation

that 5a-androstane-3a,l7adiol

canine

prostatic

RNA

have

prostate in vitro mince incubations

poor

which bind the more common

Se-DHT

nuclear

androphiles

also have been described

(17a-hydroxy-4-androsten-3-one) (32)

prostate

and

human

and prostate

Sa-androstanemetabolism,

their

and nuclear androphiles

In summary, quantitation androgen

of

we have described highly sensitive and reproducible unoccupied

receptors.

The protocols

tions of total cytoplasmic trations

of

ligand,

prepared from useful diagnostic

and occupied

human

cytoplasmic

are simple and rapid. Moreover,

performed

with

300 mg of tissue. These saturation for a test of the hypothesis

cytosol

that androgen

for the nuclear

determinatwo concen-

and KCI nuclear

analysis protocols

prognostic value in the selection of therapy for treatment

and

triplicate

and nuclear androgen receptor content, employing

may be readily

tool

prostate

protocols

should

extract

provide

a

receptors may be of

of prostatic carcinoma.

ACKNOWLEDGEMENTS This study was supported in part by funds from the National Cancer Institute, NOI-CP-33379, and by Biomedical Research Support Funds from the National Institutes of Health. The support and assistance of the surgical staffs of the Urology Services of the Audie Murphy Memorial Veterans Administration Hospital and The University of Texas Health Science Center is gratefully acknowledged. REFERENCES 1. McGuire, W. t., Horwitz, K. B., Pearson, 0. H., and Segaloff, A., CANCER 39, 2934 (1977). 2. Heuson, J. C., Longeval, E., Mattheiem, W. H., Deboel, M. C., Sylvester, R. J., and Leclercq, G., CANCER 39, 1971 (1977). 3. Nomura, Y., Kobayashi, S., Takatani, O., Sugano, H., Matsumoto, K., and McGuire, W, L., CANCER RES. 37, 106 (1977). 4, McGuire, W. L., Carbone, P. P., and Vollmer, E. P. (Eds.), Raven Press, New York, 284 pp., 1975. 5. Mainwaring, W.I.P. and Milroy, E.J.G., J. ENDOCRINOL. 57, 371 (1973). 6. Davies, P. and Griffiths, K., MOL. CELL. ENDOCRINOL. 3, 143 (1975). 7. Rosen, V., jung, I., Baulieu, E. E., and Robel, P., J. CLIN. ENDOCRINOL. METAB. 41, 761 (1975). 8. Menon, M., Tananis, C. E., McLaughlin, M. G., Lippman, M. E., and Walsh, P. C., j. UROL. 117, 309 (1977). 9. Hansson, V., Tveter, K. J., Attramadal, A., and Torgersen, O., ACTA ENDOCRINOL. (Kbh.) 68, 79 (1971). 10. Geller, J., Cantor, f., and Albert, J., J. CLIN. ENDOCRINOL. M&TAB. 41, 854 (1975). 11. Mobbs, B. G., Johnson, I. E., and Connolly, J. G., PROC. AM. ASSOC. CANCER RES. 17, 9(35), (1976). 12. Mobbs, B. G., Johnson, I. E., Connolly, J. G., and Clark, A. F., J. STEROID BIOCHEM. 8,943 (1977). 13. Krieg, M., Bartsch, W., Herzer, S., Becker, H., and Voigt, K. D., ACTA ENDOCRINOL. (Kbh.), 86, 200 (1977).

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1,3,5(10)-

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19-nortestosterone: 17o-testosterone, 17p-hydroxy-4-

J*