Specific binding of 3H-estradiol to rat prostate nuclear matrix

Vol.

128,

No.

May

16,

1985

3, t 985

BIOCHEMICAL

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

SPECIFIC

BINDING OF 3H-ESTRADIOL George

April

1381-l

387

TO RAT PROSTATE NUCLEAR MATRIX

E. Swaneck*

*The Rockefeller 'Washington University Received

AND

and Juan M. Alvarez+

University, New York, N.Y.10021 School of Medicine, St. Louis, MO 63110

9, 1985

SUMMARY: Specific estradiol binding activities can be demonstrated in Some of nuclear matrix preparations obtained from intact rat prostate nuclei. the characteristics of these --in vitro binding activities to intranuclear components are presented and compared to those exhibited by purified nuclear fractions. Examination of the effects of exposure to castration and testosterone on the number of nuclear matrix binding sites revealed that the quantity and quality (Type) of receptors was modified. Furthermore, these changes are prevented when protein synthesis was inhibited. @ 1985 Academic Press, Inc. It tors

has been reported

in rat

class

uterus

are

receptors

opposition

salt-resistant

point

differences

ties

and association

to DNA pellets

During

few years

resistant protein

estradiol

specific existence

binding

for

binding

structural Our recent

matrix

last

studies sites

of specific and how the

number

for

growth

have considered

that

of

(2-4).

In

methodolog-

as solubilization

has been demonstrated are

associated

that

estrogens

with

cells

proper-

(10).

MATERIALS

the

prostate

The present

sites

sites

that

most of the

nuclear

matrix,

nucleus

there

salta

(7-9).

in the rat

binding

of these

and another class

uterine

characteristics

of eukaryotic

estradiol

treatment

recep-

(5,6).

it

indicate for

salt

of estrogen

salt-resistant

essential

those

sites

framework

These

some authors

ical

one class

by low

(l-4).

of view,

may account

that

extracted

have been considered

to this

the

authors

can be partially

of receptors

estrogen

by several

in the

is modified

study rat

documents

prostate

are the

nuclear

by the hormonal

milieu.

AND METHODS

Preparation of Nuclear Matrix from Purified Nuclei of Rat Prostate. Nuclei were isolated as described previously (10). Purified nuclei were processed for the isolation of nuclear matrix as described by Barrack and Coffey (8) and Ciejek et al. (ll), at 4oC, in the presence of the protease inhibitor Phenylmethylsulphonyl fluoride (PMSF) in all buffers. Pancreatic DNase I, electrophoreti0006-291X/M 1381

All

Copyright 0 1985 rights of reproduction

$1.50

by Academic Press, Inc. in any form reserved.

Vol. 128.No.3,

1985

BIOCHEMICALAND

8lOPHYSlCALRESEARCHCOMMUNlCATlONS

tally purified, from Worthington (Waltham, MA), was used at '250 ug/ml in the presence of 0.25 M Sucrose, 10 mM Tris-HCL, 25mMKC1, 5 mM MgC12 buffer (0.25 STKM), pH 7.4, 10% glycerol, at 22OC for 30 min. Nuclear spheres were pelleted, and resuspended in 1% Triton X-100, 0.25 M STKM at 4oC for 30 min., sedimented and resuspended in 0.2 MgC12, 10 mM Tris-HCL buffer, pH 7.4 (Low salt buffer = Spheres were extracted in the presence of 1 M NaCl, LMB at 4oC LMB) on ice. for 30 min., pelleted and redigested with DNase I as described above. Nuclear spheres were extracted again in the presence of 2 M NaCl, LMB, 4oC for 30 min. and sedimented. All centrifugations were at 2200 x g x 20 min. 3H-estradiol Exchange Assay. Specific estradiol binding sites were measured by an jH-estradiol LjH-E?] exchange assay at 370C x 30 min. or at 4 oC x 16 hours as described previous y (10). Triplicate 250 ul aliquots of nuclear matrices suspensions in 0.25 M Sucrose, 10 mM Tris-HCl, 25 mM KCl, 5 mM MgCl buffer (STKM), pH 7.4, 10% glycerol, were incubated with a wide range of 3i-E (137 Ci/mmol, NEN, Boston, MA) concentrations (0.2 - 80 mM) in the absence PTotal binding) and presence (non-specific binding) of 300-fold molar excess unlabeled estradiol (E2). Incubations were terminated by precipitating nuclear matrices with protamine sulfate (1 mg/ml 0.25 STKM, 20% glycerol, 0.02% Triton buffer (0.25 M SGT) and applied to glass-fiber filters (Whatman, GC-A) presoaked in buffer (12). Filters were washed with three 5 ml portions of 0.25 M SGT buffer with gentle vacuum in the cold room. Filters were placed in vials and digested with 1 ml TS-1 (RPI, Mt. Prospect, IL), and radioactivity was measured in 1 ml of Biofluor. Assays were done in triplicate and the specific binding of # HIestradiol to nuclear matrices was calculated by subtracting the radioactivity of non-spe ific binding (300-fold E2) from the total radioactivity that corresponds to [ 5 H]-est.radiol in filters. Parallel incubations done without nuclear matrices revealed no specific binding of [3H]-steroids to borosilicate tubes or filters. The specific binding of r3H]-estradiol molecules per mg of DNA or per mg of protein was calculated from the specific activity of the radiolabeled steroid and DNA or protein determinations done in nuclear matrices pellet extracts by the diphenylamine method of Burton (13) using calf thymus DNA as a standard, or Lowry's method for protein (14). Chemicals. Unlabeled steroids, Triton X-100, DES, and Pronase were from Sigma (St.s, MO). RNase-free sucrose was from Bio-Rad (Richmond, CA); DTT and PMSF from Calbiochem (LaJolla, CA). All other reagents were of highest available grade. RESULTS A. Nuclear and chemical nuclear

Matrix

analysis

matrices,

subcellular Barrack

was free

and Coffey that

nuclear

the nuclear

intact

envelope sphere

The chemical 21% of initial

I)

the

spherical

nuclear

Triton-,

rat

microscopy

composed

as originally

of the

4% of the 1382

1) of

with defined

nucleus

where

structure NM is

principally

contaminated

in a continuous

network

(Figure

other by

and 2 M NaCl-resistant

prostate,

retained

of prostatic protein,

(NM),

integrity

adult

and a granular

fraction

matrix

a DNase-,

have been

analysis

final

by electron

and was slightly

The nuclear

the

from

As judged

of nuclei

represents

retains

shows NM obtained

tain

(Table

components.

structure

of the

Isolation.

fills

summarized

(9).

Figure

the residual

elements

structure the

1

surrounding

interior.

in Table

DNA and 36% of the

I.

They con-

RNA of the

Vol. 128,No.

3, 1985

Fi gure

BIOCHEMICALAND

Electron

1.

matrix,

micrograph after

nucleus.

Since

our

estradiol

binding

objective

sites,

in the presence

of high

Dithiothreitol

(DTT),

of such treatments

TABLE I.

rat prostate

I treatment

and

COMMUNICATIONS

nuclear

2 M NaCl

extraction.

17,400X.

was to examine

additional

digestions

concentrations and DNase I,

are

of mature

DNase

Magnification:

BIOPHYSICALRESEARCH

the

intranuclear

of NM spheres

of DNase I plus

were

RNase,

RNase and DTT at 4OC for

shown in Table

distribution

of

performed

DNase I plus

30 min.

The results

I.

EFFECT OF DIFFERENT TREATMENTS ON THE REMOVAL OF NUCLEAR COMPONENTS % Recovery

Treatment*

None (Nuclei

Protein

DNA

RNA

)

100

100

100

*DNase, 2 M NaCl (matrices)

21

4

36

DNase

+ RNase

15

2

8

DNase

+ DTT

8

1

10

5

0

1

DNase + RNase + DTT

* Concentrations OTT = 1 IIt'!.

of

enzymes

were:

DNase

I (250

1383

us/ml),

RNase

A (10

ug/ml)

;

Vol. 128, No. 3, 1985

BIOCHEMICAL

B. Intranuclear

Estrogen

following

extraction

activity

corresponding

the

radioactivity binding

by heat

denaturation

are

that

to high

capacity

soluble

(0.3

remained than

to hefine

(see

Methods)

mM MgC12).

specifically

10% of the

mately

with

tested

(10). with

Pronase,

These

in prostate

most of the

sites

bound

results nucleus

are

not

in a

residual

DNA (see Table were

treated

remained

This

differential

sensitivity

(5,

10,

None (initial)

with

low salt

of sites

that

contained

less

DNase I and RNase A, approxi-

only

the

15% of the

of Type II

These

recovery

sites

at different results

of Type

II

initially

bound

to RNase treatconcentrations

suggested

to us that

RELATIVE RETENTION OF SPECIFIC BOUND3H-E2 IN RAT PROSTATE NUCLEI Specific

Treatment

pellet

while

was effective

50 and 100 ug/ml).

TABLE II.

with

present,

and consituted

to Type I sites

3 and 40 mM) in an

ONase i and washed

nuclear

binding

II).

less

respect

(at

of estrogen

56% and 84% of each class

to the

nuclei

localization

with

was significantly

enzyme

bound

digestion

present

radio-

and 72% of

5 minutes.

3H-estradiol

Approximately

initial

3H-estradiol.

the

activities

78% of the

intranuclear

remained

or 980C for

intranuclear

were

54% of Type I sites

ment with

sites

and that

the

incubated

When radiolabeled

sites

binding

remained

by proteolytic

1 hour

proteins

nuclei

assay

buffers

sites

that

phase.

purified

exchange

affinity

we have reported

0.1 to 0.4 M KCl,

was destroyed

estradiol

In an attempt sites,

Previously

containing

at 68OC for

the

Sites.

to low affinity

temperative-sensitive

nuclear

Binding

buffers

associated

Estradiol

indicated

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Binding

Type 1 13.8 * 0.8 (100%)

(fmole/mg DNA initial) Type II 1203 * 68 (100%)

Recovery DNA Protein (%I 100

100

DNase I

7.7 * 0.7

(56%)

1006 f 11

(84%)

9

29

DNase I + RNase A

7.5 * 0.5

(54%)

182 f 11

(15%)

6

19

* Mean values for 6 experiments f S.E.M. BH-Estradiol done in triplicate at 37OC for 30 min (10). 1384

exchange assays were

of

Vol.

128,

most

of the

could

specific

correspond

of estrogen

to NM.

sites

To further exchange estradiol

than

with

found

activity

in the

of intact

incubated

with

of the matrix

our

adult activity

initial

by Testosterone

(T)

the distribu-

II),

nuclear

findings

rats

when we compare RNase treatment.

contain

of these fraction.

3H-estradiol,

we have performed

Type I and Type II

sites

was 3 times

Nhen NM were

digested

60% of Type II estradiol

was lost.

TABLE III.

COMPARISONOF SPECIFIC 3H-ESTRADIOL BINDING BY ISOLATED PROSTATIC NUCLEI AND NUCLEAR MATRICES

Hormonal Status

Specific

(fmol/mg

Fraction

Type

Intact

Binding

Recovery

DNA Initial)

ONA Protein

II

Type

1

(%I

Nuclei

60.(1.0)

890.(1.0)

N. Matrix

39.(0.65)

704.(0.8)

6

21

ID +RNase

29.(0.48)

227.(0.25)

2

15

100

100

Castrated

Nuclei

260.(1-O)

3430.(1.0)

100

100

+Tx40H

N. Matrix

108.(0.4)

2048.(0.6)

4

20

40.(0.15)

186.(0.05)

2

18

100

100

ID +RNase Castrated

Nuclei

257.(1.0)

+ T + CHX

N. Matrix

159.(0.6)

68.(0.5)

6

12

ID +RNase

168.iD.6)

44.(0.3)

3

8

x 40 H

Testosterone was given 100 ug/lOO G.B.W./day, ethanol. Daily release

in in of

in DNA

in the number

to examine

NM and NM after

The specific

involved

increase

induced

(Table

summarizes

of nuclei,

prostates

NM is

that

growth.

findings

III

that

we decided

prostatic

structures

a 4-fold

growth (17)

previous

NM. Table

sites.

RNase and then

binding

our

from

that

in insoluble

has been proposed

castration

activities

binding

were

prostatic

in NM during

binding

estradiol

it

after

with

NM isolated

Because

during

validate

assays

estradiol

sites

immediately

of these

for

and because we had observed

binding

treatment

higher

sites

(9,15,i6)

synthesis

tion

8lOCHEMlCALAND8tOPHYSlCALRESEARCHCOMMUNlCATlONS

No. 3, 1985

136.(1.0)

subcutaneous Silastic subcutaneous injections T from implants: =60

Values are averaged of two experiments. Incubations were carried for 16 hours at measured as indicated in Reference 10.

1385

implants (T); Cycloheximide, (CNX) in saline, 0.2% ug, determined by dry weight.

Assays were 4oC. Type

I

done in triplicate. and Type II sites

were

Vol. 128, No. 3, 1985 Within observed diol

the

BIOCHEMICAL

first

40 hours

an increase

binding

prostate

over

was studied,

the

(Type

of initial

matrix

of the

estrogen

trated

rats

I and II) values.

associated

rats

abolition while

newly internal

to one half

This

finding

suggests

observed

of sites

was

and one tenth,

respectively,

that

an important from

proportion

T-treated

cas-

(RNP) components.

received

g.b.w.,

in T-treated

twice

T implants,

increase These

proteins;

T-treated

binding

in prostates

and T-induced

present

When NM from

estra-

of NM, estradiol

(100 ug/lOO

was not modified.

we have nuclear

in number

RNase digestion

reduced

or processed

network

rats.

increase

had simultaneously

activity

in prostatic

in intact

to ribonucleoprotein

that

synthesized

to 4-fold

was administered

I increase

II binding

found

RESEARCH COMMUNICATIONS

and T treatment,

were

of the castrationType

Type

that

activities

When cycloheximide to castrated

3.5

Following

binding

is

castration

same proportional

III).

(Table

activities

following

of approximately

activities

demonstrated

AND BIOPHYSICAL

there

suggest

prostates

that

is:

and 2) associated

was an

Type II

of nuclear

data

in 40 hours)

sites,

estradiol

1) related with

to

the RNP

of NM.

DISCUSSION Several

investigators

NM of mammalian

cells

have studied in direct

ing have

been described

prostate

(8,19).

for

We have

exchange

estrogen

lobes

prostatic

classes

of binding

are present:

for

E2, DTT resistant;

another

and RNase-sensitive. both

type

of sites

Type II sites are

possibly

processing. this

component

in the

3H-estradiol

and Dorsolateral

During

with

after

dependent Type

on newly

II sites

are

may be associated

binding results

affinity

and high

and T treatment 40 hours

RNase-sensitive

proteins

and this

to the RNP network

1386

a 3-fold

combined two

for

E2, DTT-

increase

of

The increase that

and/or

T in rat

that

capacity

suggests

bind-

and low capacity

of treatment.

and this

synthesized

indicate

with

affinity

and for

to NM from

high

affinity

is cycloheximide-sensitive

and high

(8,9,18)

liver

one with low

of sex steroids

--in vitro

and our

castration

is detectable

interactions

assays

studied

Ventral

sites

the

these

in

sites

on protein-

finding

of the matrix.

suggests

that

Because

Vol.

128,

No. 3. 1985

castration

per -se increases

binding

sites

effect

observed

understand It ticles

in the

the

role

prostate

of these

has been shown from

target have

may involve

nuclear

regulation

accounts

castrated

animals,

further

estradiol

binding

androgen

that

binding

interact

poly

(A)

gene regulation

with functions

sites the

for study

with

containing

Our findings

that

30 to 50% of the is

necessary

nuclear

to

RNP par-

RNA (21).

by androgen-receptor

associated

idea

COMMUNICATIONS

sites.

receptors

and with

RESEARCH

Type I and II estrogen

increase

RNP and DNA associations.

are consistent of prostatic

BIOPHYSICAL

of nuclear

and this

(20)

suggested

of Type II estrogen

T treatment

that

tissues

AND

the number

in T-treated

observations

tion

BIOCHEMICAL

with

estradiol

complexes

of a great

NM-RNP network may play

These

a role

proporduring in the

and growth. ACKNOWLEDGEMENTS

We thank Dr. Gerald Sufrin for helpful discussions and support. This research was funded by the American Cancer Society[IN-261 and the National Institutes of Health CAM-206021 grants.

REFERENCES 1. 2.

7.

a. 9. 10. Il. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21.

Mester, J. and Baulieu, E.E. (1975) Biochem. J. 146, 617-623. Baudendistel, L.J. and Ruh, T.S. (1976)o??isx, 223-237. Clark, J.H. and Peck, E.J., Jr. (1976) Nature 260, 635-637. Ruh, T.S. and Baudenistel, L.J. (1977) Endocrinology 100, 420-426. Juliano, J.V. and Stancel, G.M. (7976) Biochemistr 15, 916-920. Muller, R.E., Traish, A. and Wotiz, H.H.d.%-oc. 36, 911 ~Barrack, E.R., Hawkins, E.F., Allen, S.L., Hicks, L.L. and Coffey,'D.S. (1977) Biochem. Biophys. -Res. Commun. 79, 829-836. Barrack, E.R. and Coffey, D.S.(1980) J. --Biol. Chem. 3, 7265-7276. Barrack, E.R. and Coffey, D.S. (1982) Recent Prog. Horm. Res. 38, 133-189. Swaneck, G.E., Alvarez, J.M. and Sufrin,(l982) BhechBiophys. Res. __ Comnun. 3, 1441-1447. Ciejek, E.M., Nordstrom, J.L., Tsai, M.J. and O'Malley, B.W. (1981) J. Cell Biol. 91, 132a. iTel= K.L., Miller, A.L. and Peck, E.J., Jr. (1980) J. Receptor --Res 1, 215-237. Burton, K. (1956) Biochem. J., 62, 315-322. . Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. --Biol. Chem. 193, 265-278. Fardoll, D.M., Vogelstein,,B. and Coffey, D.S. (1980) Cell l$l-, 527-536. Vogelstein, B., Pardoll, D.M. and Coffey, D.S. (1980) ma, 79-85. Swaneck, G.E. andAlvarez,J.M.(1985) Endocrinology submmd. Simmen, R.C.M., Dunbar, B.S., Guerriero, V., Chafouleas, J.G., Clark, J.H. and Means, A.R. (1984) J. -Cell. -Biol. 99:588. Barrack, E.R. Bujnovsky, P. and Walsh, P. (1983) -Cancer -.Res. 43, 1107. Liao, S., Liang, T. and Tymoczko, J.L. (1973) Nature mew Biol.) 241, 2 11-2 13 -Lin, S.Y. and Ohno, S. (1982) __Eur. J. Biochem. 124, 283-287.

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