ORG-2058 as a ligand in the assay of progesterone receptor in breast cancer

ORG-2058 AS A LIGAND RECEPTOR IN BREAST

IN THE ASSAY CANCER

OF PROGESTERONE

Yoav Sharoni, Bianca Feldman, Noga Karny, and Joseph Levy Endocrinology Laboratory, Clinical Biochemistry Unit, Soroka University Hospital and Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel Received February 1 1986 Revised July 31, 1966 ABSTRACT Tritiated [(15a-ethyl-21-hydroxy-l~-n~r-pregn-4-en~-3,2O-di~ne)-~~-~Hl (3RG-2059) and 1’~,21-dimethyl-19-nor-pregna-4,~-diene-3,2O-di~ne (R5020) were compared as ligands in the assay of progesterone receptor in human We found that ORG-2058 is a better Ligand beand rat breast tumors. Most of the nonspecific binding cause of its low nonspecific binding. of the other ligand R5020, is to proteins which bind corticosteroids. In cancerous tissue ORC-2058 binds to progesterone receptor linearly in a range of protein concentrations which are normally used in the receptor On the other hand, R5020 exhibits binding linearity over a assay. narrower protein concentration in many tumor biopsies, which may cause severe limitation in the assay procedure or frequent underestimation of receptor content. INTRODUCTION The

assay

routine has

of

procedure

been used

rone

ence

in

found

progesterone

in

when

some the

cancer.

has

Some with

of

assay of rat

(1,2)

tissues been

these in

R5020

demonstrate

studies

other

in the

in

laboratories.

assays

receptors

replacing

receptors

many

these

ORC-2053, was

RS020

in

in

receptors

ligand,

is

progesterone

and

many

for

ligands

the

progesterone

these

advantages

in

same

were

is

now a

use

receptors have

progestesynthetic No differ-

purpose. in

the

Nevertheless

In the

of

another

compared

(5-7).

laboratories.

of

cancer

measurement

recently

the

cancer

advantages

mammary tissue

for

More

suggested

breast

breast

The synthetic progestin R5020

(3,4).

two

human

this of

in

previously

assay

CRG-2058

communication

ORG-2058 human

and

been

of

rather rat

we than

mammary

suggested

in

(3.9).

EXPERIMENTAL ORG-2058 [(16a-ethyl-21-hydroxy-l~-nor-pregn-4-ene-~,2O-dione~6,‘~-3Hl @5Ci/rmnoL) hamshire,

and radioinert ORG-2058 were obtained from Amersham, England. R5020 1(17,21-dimethyl-19-nor-pregna-4,9-diene-3,20-

STEROIDS 48 / 5-6

November-December

1986 (4 19-426)

Bucking-

419

Sharoni et a/

420

dione), Nuclear,

17,21-dimethyl-3H Boston, MA, and

(80-90 Ci/mmol) was obtained from New England progesterone from Ikapharm, Ramat Gan, Israel.

Tissues Human breast tumor tissue was transferred from the operating room as soon as possible (<30 min), examined macroscopically, and immediately placed at -7O’C until further evaluation. 7,12-Dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats (10) and rat uterine tissue (11) were obtained as described previously. Preparation of cytosol for receptor assay Frozen tumor or uterine tissue was minced and homogenized in five volumes (w/v) of TED buffer (10 mM Tris-HCl, 1.5 mM EDTA, 0.5 mM dithiothreitol pH 7.4), using a Polytron PT-10 homogenizer (Setting 4) for two periods of 10 set with intervals of 20 set for cooling. The homogenates were centrifuged at 800 x g for 10 min, and the resulting supernatants were centrifuged at 105,000 x g for 60 min to obtain cytosols (supernatant fraction). All binding assays were done with fresh cytosol. Prior to receptor assay the cytosol was treated for 20 min with a pellet of 1 vol of Dextran-coated charcoal (DCC, 250 mg Norit A, 2.5 mg Dextran in 100 ml, 10 mM Tris-HCl buffer pH 8.0). The supernatant obtained after centrifugation at 2000 x g was used for progesterone receptor assays. Cytosolic progesterone receptors Progesterone receptors in cytosol were measured by the Dextran-coated charcoal technique (12). Glycerol (40%) in TED buffer was added to 0.15 mL of the cytosol to obtain a final concentration of 10% glycerol in TED buffer. L3HlR5020 or [3H10RG-2058 (0.1 - 12.0 nM) was incubated with the cytosol at 4’C for 18 h. Parallel incubations with an excess of 100fold unlabeled progestin were also pet-formed. The specific binding at each concentration was determined, and the results were calculated by Scatchard analysis. Results are expressed as fmol progestin bound per mg cytosolic protein. Statistical analysis The statistical significance tal values was assessed by use

of difference between various of paired Student’s t-test.

experimen-

RESULTS Cytosolic

progesterone

DMBA-induced and

normal

normal using

rat

rat rat

obtained

using

ficantly

more

were

mammary tumors,

uterine

uterine

ORG-2058

receptors

as

tissue

tissue the

R5020

(Table

progesterone

(as

the

ligand

33

biopsies

control).

calculated were 1).

quantitated

not

For

significantly when

binding

16 biopsies

both

tumor

tissues

constants different

ORG-2058 sites

from

human mammary tumors

dissociation

However

receptor

from

in

was

(50-70%

and

obtained from

used, on

those signi-

average)

ORG-2058

detected

were

tained

in

using

R5020.

three-fold. values

were Two of

2a). Table

1:

and

rat

human

In

some

However,

in

found

the

the

in rat

of

25%

the

of

two

tumor

cancer

the

the

human

biopsies

were

those was

similar

procedures

that

to

difference

biopsies,

experimental

R5020

2.

tumors

compared

as

ob-

almost

receptor

(see

estimated

RELATIVE NUMBER OF BINDING SITES (5)

n

Uterus

as

42 I

also

Fig.

progesterone

sites and affinities of R5020 and Comparison of binding ORC-2058.in different tissues.to progesterone receptor. Number of binding sites was calculated from the full Scatchard plot Binding sites were between 0 and 200 fmol/mg with each ligand. cytosolic protein. 'The results are Mean 2 S.E.M. N.S. - not zignificantly different from binding sites measured with R5020. PCO.05.

TISSUES

1.

tissue

AS LIGAND

H5020

ORG-2058 ---

ORG-2058

9

100

122 + 13N.S.

1.1

+ 0.2

0.8

_+ 0.2

DMBA-induced mammary tumor (rat)

16

100

1 47 +

0.9

z

0.1

0.5

_+ 0.1

Breast cancer (human)

33

100

176 -+ 24

0.4

+ 0.2

1.0

-+ 0.3

3.

(rat)

Kd (M x 109)

receptor-negative assayed The

(borderline

with

ligand

receptors

R5020 used

did

detected

in

Because

of

the

cytosolic

with tor

in

tumor

the

with

in

the

lower

the

protein

not

rat

protein of

(Fig.

cytosolic tested range

seem uterine

and

7 fmol/mg

when

affect

the

the

linearity

concentration.we

The

protein)

tested

number

with of

The

binding off

specific

measured

protein specific

of

when

ORG-2058.

progesterone

ligand

binding

of

at

of

0.5

R5020

was

linear

1.5

3.0

mg

-

rccep-

the

same

[3H10RG-2058

between of

binding

progesterone

concentrations

concentrations

leveled

cytosolic

tissues.

of

protein

cytosols.

**

positive

to

different

lb).

-

were

ligand

importance

presence

cytosols

linear all

as

value

9"

and

was 6.0

only of

rat

me;/m.L in

cytosolic

the

422

Sharoni

protein

per

tumor

cytosols-

was

observed

results fic

R5020.

protein such

Similar

mL.

(Fig. with

may explain

binding

with

et al

obtained

rat

2b).

R5020

uterine

were

However, up to

why in with

The binding

concentration as

results

6.0

some

the

tested

in

tissue

in

the

mg/mL

as

two

(Fig.

about

remaining

ligands with

not

30% of

cytosols

example,

but ligand

tissue

in

(see

cytosols,

ORG-2058 of

obtained

in

Fig.

is

similar

higher

2a). the

than

These speci-

obtained

throughout

specific

human

linearity

others,

was higher

the

the

binding,

la).

3

0

6

protein-mg/mL Fig.

1.

Nonspecific

Effect of protein concentration on specific ligand binding in and tumor (b) cytosols. Uterine and DMBArat uterine (a) induced mammary tumor cytosol was prepared ds described in the Methods section. Specific binding was calculated from the full saturating curve with either ligand in cytosol aliquots, suitably diluted. Results shown are from one of several experiments performed with different tissue cytosols as explained in the Results section.

binding

was significantly

higher

in

the

presence

of

Hi020

ORG-2058 AS LIGAND

than with r)R!G2!?!33 (results earity

in the

CFlE.

l),

t3t31

Specific

its

tias generally

and binding the higher

A5320 at. high often

hand,

the total

binding

of

of

lin-

concentrations

progesterone

binding

binding bindint:

In cytosols

capacities.

absence

more than 50% of

the nonspecific

IO-15% of

nonspecific

the

protein

reached

50 fnsl

containini:

in the range of

conccntrati?ns level,

of

On the other

Becaus,e of

shown).

binding

in cytos9ls

per irlg protein.

tor

bio$in&

nonspecific

binl.inC

not

423

with

of

the

sites

ORG-2058

a% a21 protein a lower

recep-

R5020 nay be quantitatively

raven more important

-b

c ytoso1ic Fig.

2.

Effect of protein concentration on specific ligand binding in Fig. 1. two human tumor cytosols. For det,ails, see legend. Case a represents 20% of the tumor cytosol tested. Case b repre:s?nts the rest of the biopsies assayed.

Competition

experiments

between steroids

centrations

of

radioinert

specificity

of

binding

tumors

protein-mglmt

(Fig.

3).

All

of

trit

ligands

highlighted

[3HlORC-2058

progestins

ated

tested

and

and increasing

the

difference

[3HIR5020

displaced

the

con-

in

rat

in

the

mammary

two tritiated

li-

Sharoni et al

424

gands

with

terone

was

A

a similar the

lowest

IOO-fold

excess

[ 3H lR5020

binding.

affect binds tors

of

of

and

the

three

Tne

ssme

binding,

receptor

binding

for

sites

efficiency

previously

of

proges-

described

(13).

approximately

concentration suggesting

Molor excess of

as

displaced

non-progesterone proteins

displacement

tested

corticosterone

LjHlORG-2058 to

the

efficiency;

of

corticostcroid

that which

502

the

former

most

of

the

did

not

ligand

probably

also

are

recep-

corticosteroids.

nonradlooctwe

competitor

( Fold 1 Fi .g.

3.

Effect of various competitors on the binding of [3H10RC-2059 and with to tumor cytosols. Competition experiments [ jH 1 R5020 3H-ligands (4 nM) and an excess of nonradioactive steboth The roids were performed as described in the Methods section. nonspecific binding was subtracted from all values, and the specific binding is depicted as percent of controls in the absence The data shown are from three different of competing steroids. experiments. Pg - progesterone; Cort - corticosterone; Dex dexamethasone.

DISCUSSION ORG-2058 is terone specifically

a better

receptor

in to

the

ligand

human receptor

and

than rat for

R5020

for

breast progesterone

the

cancer. in

estimation ORG-2058 these

tissues

of

proges-

bound

more

because

ORG-2058 AS LIGAND

it

bound less this

over,

problem

well

ligand

of

high

human breast

to

manifested

tumors

the nonspecific

of

total

tant

finding,

receptors

(Figs.

1,2).

tested

with

range

of

seriously biopsy

of test

rat

explain

large

of

the

the

the

is

reason the

assays.

small

but

higher

range

not

with

1) that

other is

binding

with

rat

protein over

its

impor-

to proges-

tumor cytosols over

a narrow

linearity

may

reasons:

1)

The

repetition

of

the

receptors

in many

concentration which

in the

R5020 binding

were reported

uterine

tissue

is

of

did

not

receptor

low protein

however, attendant

there

find

previously Cd),

ORG-2058 than with

(5-T)

At such concentrations,

this

tumors.

with

use

of

the

the progesterone

groups their

range

progesterone of

as

concentrations

only

several

the two ligands

when tested

two ligands

nonspecific

of

the tested

between

The most

protein

permit

2 and 6 mg/mL, a range of

results.

narrow

to

In

ORG-2058 binding

for

The

such

content.

was linear

assay

More-

general.

tissues

the human and rat

binding

preferred

in

3).

which are more than 50%

cytosolic

This

too

(Table

that

the of

of

low content

mammary tissue our finding

between their

usually

differences

tumor tissue

the

receptor

in the majority

Similar

haps

is

is between

not linear

with

the

biopsies,

tube

range

concentrations.

2) Because

these

distort

in most of

R5020 as ligand

handicap

assay.

a wider

in

receptor values

wxs the linearity

In contrast,

received

can reach

and thereby

over

protein

important

is

(Fig.

binding

progesterone

binding

however,

terone

low

proteins

nonspecific

binding

with

binding

binding

less

nonspecific

tissue the

corticosteroid

425

and may

content R5020.

distortions

Per-

any difference

concentrations is

in

a likelihood of

in of

receptor

426

Sharoni et al

concentrations.

Unfortunately the data about protein concentration and

nonspecific binding are missing in their reports.

ACKNOWLEDGMENTS This work was supported in part by the Beatty Malkin Grant for cancer research and grants from the Chief Scientist's Office, Israeli Ministry of flealth.and the Israel Cancer Association. The authors wish to thank Mrs. M. Duman for her excellent editorial assistance. REFERENCES 1.

2.

3. 4. 5. 6. 7. a. 9. 10. 11. 12. 1' 3.

Clark, G.M., McGuire, W. L., Hubay, C.A., Pearson, O.H.. and Marshall, J.S., NEW ENGL J MED ?og, 1343 (lY33). and Levy, J. and Glick, S.M., in: Progesterone Receptors in Normal -Neoplastic Tissue (McCuire, W.L., Raynaud, J.P.. and Baulieu, E.E., Editors), Raven Press, New York (1977), p 211. SC1 g, 970 MED Levy, J., Liel, Y.. and Glick, S.M., ISR J (1981). Sharoni, Y., Feldman, b., Teuerstein, I..and Levy, J., ENDCCRINQLQGY 2, 1918 (1984). J CANCER 3, 1181 (19,/Y). Duffy, M.J. and Duffy, G.J., EUR Koendcrs, A.J.M., Geurts-Moespot, J., Beex, L.V.A.M.. and Benraad, 15P (1981). Th.J., PROC SOC ENDOCRINOL J CANCER 5, '78s (lY7Y). Keightley, D.D., EUR ENDOCRINOLOGY 111, Quirk, S.J., Gannell, J.E.. and Funder, J.W., 1383 (1982). Quirk, S.J., Gannell, J.E.. and Funder, J.W., J STEROID BIOCHEM 20, 803 (1934). Levy, J., Teuerstein, I., Marbach, M., Radian, S.. and Sharoni, y., 3IOCHEM BIOPHYS RES COMMUN 123, 1127 (1984). Sharoni, Y., Teuerstein, I., Shirman, A., Feldman, B.. and Levy, J., ENDOCRINOLOGY 115, 229'7 (1984). LieI, Y., Marbach, M., Bear-man, J.E., Feldman, B., Glick, F5.M..2nd J CLIN ONCOL 4, 1389 (1932). Levy, J., EUR 500 BIC'PHYS ACT4 E, Fleischmann, G. and Beato, M., BIOCHIM (19'78).