Studies on type II progesterone receptor in MCF-7 cells

STUDIES ON TYPE II PROGESTERONE RECEPTOR MCFJ CELLS D. E. Saunders, C. Christensen, and S. C. Brooks Department of Biochemistry, Detroit, MI 48201, USA

Wayne

State University

IN

School of Medicine,

Corresponding author: 5. C. Brooks Received Revised

July 17, 1987 February

5, 1988

ABSTRACT These experiments demonstrate for the first time the existence of a Type II progesterone receptor (RpII) in MCF-7 human breast cancer cells. RpII was shown to have a lower affinity for tritiated progesterone (C3HlPg) (Kd > 13 nM) than classical Rp (Kd < 3 nM). RpII was detected Ey cytosolic, nuclear, and whole- cell assays of MCF-7 cells. Scatchard analysis of C3HlPg binding data revealed that classical Rp but not RpII could be recompartmentalized from the cytosolic to the nuclear pool by treating cells 1 h at 37OC with 1 uM Pg. RpII levels were shown to be increased more than two-fold by growing MCF-7 cells for 4 days in 10 nM estradiol (E2) plus 100 nM Pg when compared to either untreated cells or to cells treated with only E2. INTRODUCTION Under in

this

a

variety of growth and treatment regimens carried out

laboratory, very few cases were seen where the cytosolic

compartment

of

progesterone cells

with

compartment grown

in

(Pg) 1

PM

receptor Pg

(1

breast (Rp).

h)

failed

cancer For to

cells was devoid of

example, pulsing MCF-7 deplete

the cytosolic

Furthermore, cells

steroid-free media (e.g., Chemically Defined Media, CDM

observations).

52/3

human

of Rp (unpublished observations).

possessed

[II)

STEROIDS

MCF-7

appreciable This

September

1988

levels

of

Rp

(unpublished

was unexpected, as estradiol (Ep) has been

(249-263)

249

250

Saundersetal:TYPEIIPROCESTERONE

demonstrated

to

be

addition,isolated

for

necessary

nuclei

from

MCF-7

synthesis

cells

of

Rp.

RECEPTOR

In

grown in CDM showed

appreciable C3HlPg binding (unpublished observations). above observations were strikingly similar to the Type II

The

estrogen-binding uterus.

of

a

lower

classical Re

Clark

et --

al

121 had observed in the rat

These investigators demonstrated a form of receptor that

possessed the

that

was

present

Kd (30 nM) and a higher binding capacity than

form

of estrogen receptor (Re).

designated

in

both

Type

the

recompartmentalized

II Re (ReII).

cytosolic

by

E2

and

[2].

This novel class

ReII was shown to be

nuclear

pools but was not

Subsequent studies have shown

an association between ReII and uterine growth [3].

of

The

cytosolic

the

report

extracted

in

by

from

homogenization

and

nuclear compartments are defined in terms

Welshons

the

et al C41. --

nucleus

buffer.

the

nuclei

the

hypotonic

(10

mM

TRIS)

These receptors are subsequently isolated

the 100,000 g supernatant.

in

by

Cytosolic receptors are

Nuclear receptors remain localized homogenization

during

and

subcellular

fractionation. In

view

of

and

the

experiments

were

cells

high-capacity

the similarities between C3HlPg binding in MCF-7

form

observations

on

ReII

carried

to

determine

of

Rp

out

(RpII)

by

exists

Clark

and

et --

al

CZI,

if a low-affinity, to

explore

its

characteristics.

STEROIDS 52/3

September 1988

Saundersetal:TYPEIIPROCESTERONE

RECEPTOR

251

EXPERIMENTAL Cl 2 6 7-3H1Pg (1::) Ci/mmol) was.purchased Purification of the frozm;d Nucieir' (Boston . labeled Pg was performed on ins&t thin layer chromatographic sheets (S. A. Gelman Instrum;;tI)Co., Ann Arbor, MI) with ethyl cyclohexane: acetate as developing solvents. Unlabeled standards were visualized by spraying with a mixture of 20 mL glacial acetic acid, 0.2 mL anisaldehyde, and 0.4 mL heating concentrated sulfuric acid, followed by the chromatographic strip at 110% for 10 min. Cell Culture. MCF-7 cells (a gift of Dr. H. Soule, Michigan were grown in T-75 flasks using Eagle's Cancer Foundation Minimal Essential"')Media (MEM) supplemented with 10% dextran-coated charcoal stripped Donor Calf Serum (Flow Laboratories), 50 pg/mL insulin, L-glutamine, and 0.1 ug/mL gentamycin sulfate (Sigma). Dextran-Coated Charcoal Treatment of Calf Serum. Aliquots (1 mL) of dextran-coated charcoal solution (DCC, 10 parts charcoal to 1 part dextran) were centrifuged 10 min at 1500 g and the Donor Calf Serum (25 mL) was added to the supernatant decanted. pellets and the tubes vortexed vigorously. The tubes were then centrifuged 10 min at 1500 g and the supernatant recovered. This procedure was repeated 2 additional times followed by filtration The stripping procedure was sterilization of the calf serum. carried out at 250C. Assay of Cytosolic Rp. Post-confluent cells were washed twice with 0 9% saline and once with TED buffer (10 nM TRIS, 1.5 mM ethylenediiminetetraacetate CEDTAI, 3 mM dithiothreitol [DTTI and 3 IIM cortisol, pH 7.4 at 4OC). Homogenization of harvested cells was accomplished with 15 strokes in a Dounce homogenizer with a tight-fitting pestle followed by centrifugation of the homogenate at 100,000 g for 1 h. Two hundred microliters of the supernatant (cytosolic fraction) were added to tubes containing [3H]Pg + unlabeled Pg (200-fold excess). The final concentrarions of C3HlPg ranged from 0.5 nM to 30 nM. The mixture was incubated initially for 2 h at 4oC and then for an additional 1 h in the presence of 30% glycerol. Unbound C3HlPg was removed by the addition of 0.4 mL DCC solution (10 mM TRIS, 1.5 mM EDTA, 30% glycerol, 0.5% charcoal, 0.05% dextran, pH 7.4 incubated for 30 min with occasional vortexing, at 4OC), followed by spinning for 15 min at 1500 g. The supernatant (0.25 mL) was counted in 10 mL scintillation cocktail. Binding data were analyzed by the method of Scatchard C61 with application of the Pennock 171 correction technique for multiple classes of

STEROIDS

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September 1988

Saunders et al: TYPE

252

II PROGESTERONE

RECEPTOR

binding. This entailed plotting the data points, then visually both the low-affinity and estimating straight lines for high-affinity components. Radial lines were then drawn beginning at the plot origin and extending through both the estimated component lines and the curvilinear line fit to the data points. The estimated lines for the two binding components were then adjusted until the distance from the origin along each radial to the estimated line equalled the distance from the estimated line to curve fit along the data points. This method yields a mathematical estimate of both the high-affinity (RpI) binding component and the low-affinity (RpII) component and was applied DNA was quantified using to all curvilinear Scatchard plots. Burton's diphenylamine assay C81. Assay of Nuclear Rp. MCF-7 cells were grown and harvested as described above. Detection of nuclear Rp was performed according to the method of Walters et al [9], with minor modifications. Briefly, nuclear fractions fioiii-homogenizedcells were washed 3 times by centrifugation through 0.25 M sucrose buffer (10 mM TRIS, 2.5 mM CaC12, 0.25 M sucrose, pH 7.6 at 4oC). Aliquots (0.4 mL) of the nuclear suspension were incubated overnight in and 3 mM TGC buffer (10 mM TRIS, 30% glycerol, 2.5 mM CaCl DTT, pH 7.4 at 4oC) containing concentrations of f3HlPg (+ a 200-fold excess of unlabeled Pg) ranging from 0.5 to 30 TiM. Maximum [3H]Pg exchange occurred under these conditions ClOl. After incubation, 0.2 mL of a 50% hydroxylapatite suspension in TGC was added, followed by a 10 min incubation and a 10 min centrifugation at 1500 g. The pellets were then washed 3 times Bound [3H]Pg was eluted by centrifuging throuh TGC buffer. overnight at 4oC with ethanol and counted. Whole Cell Assay of Rp. Post-confluent cells were washed 3 times with MEM and harvested in MEM. To minimize cell rupture, cells were syringed loose using a large bore (14 gauge) cannula. Greater than 80% of harvested cells were shown to be intact by A 0.4-mL aliquot of the cell suspension trypan blue exclusion. (approximately lo6 cells) was then incubated with C3HlPg (0.5 to 30 nM) + a 200-fold excess of unlabeled Pg for 2 h at 250C. The cells were then washed 3 times with ice-cold MEM to remove The remaining C3HlPg was eluted into extracellular steroid. 1.5 mL ethanol and the radioactivity monitored. RESULTS Pg MCF-7

Binding cells

to

in

MCF-7 Cells.

The exposure of E2 treated (nM)

concentrations of [3H]Pg ranging from 1 to 12 nM

STEROIDS

52/3

September

1988

Saunders et al: TYPE

yielded

II PROGESTERONE

RECEPTOR

253

a S-shaped saturation curve characteristic of two binding

sites (Figure 1).

I

I

3 nM

I

I

6

9

12

[3HlPg

Figure 1. Saturation curve of [3H]Pg binding in MCF-7 cells. Cell suspensions (lo7 cells per tube) were assayed as described in Materials and Methods. Pg cells

Binding produced

whether

in

Cytosol.

The

cytosol from E2-primed MCF-7

strikingly different Scatchard plots depending on

the culture had or had not been exposed to a l-h

pulse of

1 uM Pg at 370C (Figure 2). These [3HlPg

data

binding

from

the

c71

was

52/3

confirmation

components

and

of

the

applied of

September

to

the

Scatchard

the

two

C3HlPg

1988

high- and low-affinity

dissappearance

cytosol following a Pg pulse.

determinations

STEROIDS

provide

of

the RpI

The technique of Pennock plots

binding

to

give

components.

separate This

Saunderset al: TYPE

254

correction unpulsed dashes)

of

the

cells

non-linear

Scatchard

II PROGESTERONE

plot

RECEPTOR

of the data from

showed a high-affinity component (Figure ZA, long

which possessed a binding capacity of 1.2 fmol/ug DNA and

A. -Pg Scatchard

2

0 s

15-

z

12-

x 8 g z2 =

I

6

4

B. +Pg Scatchard

9630

"@@@-a..._

0

I

2

I

I

4

6

fmol [3HlPs/pg DNA

Figure 2. Detection of RpI and RpII in the cytosolic fraction of A. Scatchard Analysis of Pg binding in the cytosol MCF-7 cells. from Cells which were not administered Pg. Pennock correction indicates two binding sites one (long dashes) of high affinity (RpI) and one (short dashes) with lower affinity (RpII). 6. Scatchard analysis of cytosolic Pg binding following a pulse of Pg (luM, 1 h, 370C); only RpII is evident.

STEROIDS

52/3

September

1988

255

Saundersetal:TYPEIIPROCESTERONERECEPTOR

a

Kd

dashes) of

2.5 nM.

The lower affinity component (Figure 2A, short

possessed

a binding capacity of 2.9 fmol/ug DNA and a Kd

of

14

The curve in Figure 28 represents cytosolic Rp from

nM.

Pg-pulsed

Only the lower affinity RpII is seen.

cells.

components

in

Figures

2A

and

2B

appeared

possessing

similar binding capacities

to

be

The RpII identical,

(2.9 fmol/pg DNA in 2A and

3.1 fmol/pg DNA in 2B) and identical Kd's (14 nM in both). Examination examine in

the

The

nuclei

RpI

suspensions

with

low-affinity the

Scatchard C3HlPg of 4.8 RpII

and RpII in Isolated Nuclei.

To further

from

MCF-7 cells with and without a 1-PM Pg

and RpII levels were measured by incubating nuclear 0.5

Scatchard

unlike

RpI

compartmentalization of RpI and RpII, Rp was assayed

isolated

pulse.

of

plot

RpII

to

30

from

nM

C3HIPg

non-pulsed

component (Figure 3A).

high-affinity

+ unlabeled excess Pg. cells

showed

in

a

This nonlinear plot is

plot expected of RpI and resembles the

previously obtained for nuclear ReII C3l.

binding

only

The graph of

nuclei from cells which received a 1-UM pulse

Pg (Figure 3B) showed both a high-affinity RpI component (Kd = nM

from

replot,

component.

method

of Pennock C71) and a low-affinity

The Pennock replot suggested a Kd of 26 nM and a

binding

capacity

of

7.5

binding

capacity

of

the high-affinity RpI (1.0 fmol/pg DNA) was

strikingly

STEROIDS 52/3

similar

to

fmol/pg DNA for the nuclear RpII.

The

the amount of RpI which migrated from the

September 1988

Saunderset al:TYPE

256

cytosolic

compartment

experiments pulse

(Figure

will

cytosolic

(1.2 3)

effect

to

the

fmol/pg

further

DNA)

support

II PROGESTERONE

(Figure the

recompartmentalization

nuclear

pool

but

will

2).

RECEPTOR

These

premise that a Pg of

not

RpI

from

the

bring about the

nuclear "translocation" of RpII.

A. -Pg Scatchard

fmol [3H]Pg/pg DNA

20-I

B. +Pg Scatchard

0

3

6

9

12

li

fmol [3H]Pg/pg DNA

Nuclear Pg binding before and after a 1-LIMPg pulse. B. Pennock wchard plot analysis of control cells. corrected Scatchard analysis of Pg-pulsed cells.

STEROIDS

52/3

September 1988

Saunderset al: TYPE

II PROGESTERONE

Effects RpI

and

shown

E2

E2

[11,12].

levels, Cells E2

Pg

which

their

binding

in MCF-7

examined plot

48)

levels

fmol/ug cells).

cells was stimulated

after

experiments treated

a

with

better

4 days treatment of

were carried out using calf DCC to reduce its

steroid

indication of E2 and Pg effects.

for

from

total

cellular

RpI binding (Figure 4A). detected.

showed

E2-treated DNA

in

The

RpI

and

RpII.

The

the non-treated cells showed no evidence of

both

The RpI

and

control results

RpII binding (Kd = 13 nM)

plots

and

from

E2-treated

cells

RpII binding as indicated by

respective Kd's (3.1 and 16 nM). in

It has been

4 days in media containing DCC-stripped serum + 1 nM

however,

(Figure

RpII

been

giving

high-affinity was,

Pg Exposure of MCF-7 Cells on

by Whole Cell Assays.

RpII

These

had

grown

Scatchard

and

Experiments were undertaken to determine if

affected

thus

were

Pg

257

3 to 4 days of treatment in media supplemented with

cultures.

serum

E2

Determined

classical

and/or

cell

as

after

nM

Long-Term

RpII

that

5-fold 1

of

RECEPTOR

non-treated

The binding capacities of cells

were

similar

(9.5

cells and 8.4 fmol/pg DNA in E2-exposed show

that

4 days of E2 exposure induced

the binding capacity of RpI but had no net effect on RpII levels. The

effects

of

Ep

and

Pg

were

examined

after 4 days of

feeding

MCF-7 cells MEM plus DCC-stripped serum (control) (Figure

5A)

control

or

(Figure

STEROIDS

52/3

58).

media

supplemented

with

1 nM E2 and 0.1 IIM Pg

Scatchard plots from whole cell assays (Figure 5)

September

1988

Saundersetal:TYPEIIPROCESTERONE

258

indicated fmol/ug

the

DNA

absence of

dramatically control

of RpI in control cells but detected 11.3 in

RpI

E2-

plus

Pg-exposed

cells.

RpII was

elevated

by

E2 plus Pg exposure (36 fmol/ug DNA in

versus

83

fmol/ug

cells 12 -

s 5

RECEPTOR

DNA

in

Ep-

plus Pg-treated

A. Control

9-

J 8 $ z z a

630

I 3

0

I

I

1

6

9

12

fmol [3H]Pglpg DNA

121

B. E2 treared

9-

6\ \ 3-A \_ \

0 0

3

I

I

1

6

9

12

fmol [3H]Pg/pg DNA Effects of a &day exposure of MCF-7 cultures to I nM cellular RpI and RpII. A. Scatchard analysis of RpI in control cells. B. Pennock-corrected Scatchard analysis Of E2-treated cells.

STEROIDS 52/3

September 1988

Saundersetal:TYPEIIPROCESTERONERECEPTOR

cells).

The

Pg-treated

cells

overestimate

higher

apparent

(32.5

caused

by

259

Kd

RpII

for

in

the

E2-plus

versus 17 nM in control cells) may be an the cooperativity of RpII in the nuclear

pool (see Figure 3A).

s

5 -3

24loA. Control

0

10

20

30

40

50

fmol [3H]Pg/pg DNA o^

24-

B.

no

L, 0

30

60

EZ+Pg treated

I 90

I 150

I 120

fmol [3H]Pg/pg DNA Effect of Pg binding of E2 (1 nM) and Pg (0.1 uM) Figure 5. treatment of MCF-7 cells. A. Scatchard analysis of Pg binding in control cells. B. Pennock-corrected Scatchard analysis of E2-plus Pg-treated cells. The adding 2DDU-fold separate

STEROIDS 52/3

specificity

of RpII was examined in whole cell assays by

increasing

concentrations

excess) incubations

of

2-,

dihydrotestosterone

containing

September1988

(l-,

12

nM

20-, (DHT)

[3H]Pg.

200-, and It

E2

and to

required

260

Saunderset al:TYPE II PROGESTERONE

10.&M

DHT

(900-fold to

to

competitively

excess

decrease

Cortisol

[3H]Pg

did

not

block 50% of the binding of C3HIPg A 400-fold excess of Ep was required

DHT) .

of

RECEPTOR

to

binding

interfere

with

50% Pg

(also

binding to

competitively).

RpII since all

assays of cytosolic receptors contained 3vM of this steroid.

DISCUSSION The

data

specific

Pg

affinity

and

by

shown

presented

a

higher

the

this

previously levels

reported

presence

of

a

form

of

Rp

of

[3H]Pg An

(RpI).

conditions receptor

for to

binding

examination

of

the

stimulation of RpI has respond

treatments.

cellular

than that

predictably

For

to

example, its

increased by growing MCF-7 cells for 4 days in media with

for

in

days

nuclear

and

classical

were

increase

capacity

classical

supplemented 4

the

binding (RpII) in MCF-7 cells which displays a lower

compartmentalization shown

document

1 E2

nM E2 (Figure 4). plus

Pg-supplemented

RpI (Figure 5).

in pool

Furthermore, cells grown media

also showed an

RpI moved from the cytosolic to the

when cultures were pulsed 1 h with 1 IJM Pg (Figures

2 and 3). The

responses

of RpII to the above conditions were distinctly

different

from those of RpI.

cytosolic

and

the nuclear pools, a 1-uM Pg pulse failed to cause

recompartmentalization (Figures cells

in

2

Although RpII was found in both the

and

3).

of

RpII

Unlike

E2-supplemented

from

one

pool

into

the other

the RpI response, growth of MCF-7

media failed to increase the levels of

STEROIDS

52/3

September1988

Saundersetal:TYPEIIPROCESTERONE

RpII

(Figure

4).

Pg-supplemented

Although

(Figures

2

and

level of RpII These

observations

to

is,

ligand

rats

[12,131,

plus

in cellular

these

the

have

which

RpII

for

in

of

several

uterine and

ReII

respective

experiments

to

ReII

Ep

days

ReII

with

E2

These

[3].

RpII might serve similar

ligands.

However, unlike ReII

is not inhibited by reducing agents in

Studies have indicated that ReII is related

growth been

response

enhanced

inhibits of

responses

treated

that

their

uterine

the

increase

suggested

for

involvement

RpI

increase

E2-

a l-h E2 treatment recompartmentalized ReI

an

experiments.

findings

dramatic

in

suggest that Pg is capable of increasing the

and

sites,

the

induces

parallel

showed

functions

a

grown

only in E2-primed cultures.

ReII,

injections

these

5)

That

not

binding

E2

results

exposure. but

showed

261

cells

However,

media

RpII.

RECEPTOR

RpII

by

to

Ep

treatment

[3].

These

the discovery of an endogenous

binding of E2 to ReII [14].

The possible

in biologically important events remains to

be demonstrated. The

findings

studies

involving

compartment pool are

was

a

was

RpI.

here The

hold Kd

2.5 nM (Figure 2).

3 nM (Figure 3).

significantly

RpII.

presented

for

several implications for RpI

in

the

cytosolic

The Kd of RpI in the nuclear

Values obtained in

experiments which

higher than these may indicate the presence of

Furthermore residual Rp in the cytosolic compartment after

~-PM

STEROIDS 52/3

Pg

pulse

would

September1988

be

suspected of being RpII.

Likewise,

[3HlPg also

binding

may

indicate [3H]Pg

that

nuclei

by

cytosolic

Pg

that

pulsing it

The

low

and

of

RECEPTOR

non-Pg-treated

the

described Scatchard

cells herein

plots of

(< 6 nM) and high (> 10 nM) ligand

determination RpI

from

experiments

examination

both

allows

and

RpII,

at

isolated

RpII.

careful

binding

examined

in

represent

concentrations and

TYPEIIPROGESTERONE

Saundersetal:

262

of the presence of both RpI

RpII binding can be separated and

cells with 1 uM Pg.

In routine analyses of

is imperative that the concentrations of C3H]Pg

are kept below 6 nM. ACKNOWLEDGMENT This investigation was supported in part by PHS grant number CA-37387 awarded by the National Cancer Institute, DHHS. REFERENCES Studies on the cultivation 1. Higuchi K and Robinson RC (1973). of mammalian cell lines in a serum-free, chemically defined medium. In Vitro 9: 114-121. 2. Clark JH,Har;ain JW, and Upchurch S (1978). Heterogeneity of estrogen binding sites in the cytosol of the rat uterus. i Biol Chem 253: 7630-7634. Two binding sites for 3. MarkavePich BM and Clark JH (1979). estradiol in rat uterine nuclei: Relationship to uterotrophic A::’ ~~~8-~~6rfki J (1984). Nuclear 4. ~~?%~~~' Wm localization of unoccupied estrogen receptors: Cytochalasin enucleation of GH3 cells. Nature 307: 747-749. 5. Soule HD, Vazquez J, LoAlbert S, and Brennan MJ A human cell line from a pleural effusion derived (1973). from a breast carcinoma. --J Nat1 Cancer Inst 51: 1409-1416. 6. Scatchard The attraction of proteins for small G (1949). Ann NY Acad Sci 51: 6601672. molecules and ions.' ---7. Pennock BE (1973). A calculator for finding binding parameters from a Scatchard plot. Anal Biochem 56: 306-309. 8. Burton K (1956). A study of the conditionsand mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. -Biochem J 62: 315-322.

STEROIDS 52/3

September1988

Saunderset al:TYPE

9. 10. 11. 12.

13.

14.

STEROIDS

II PROGESTERONE

RECEPTOR

263

Walters MR, Hunziker W, and Clark JH (1980). Hydroxyapatite prevents nuclear receptor loss during the exchange assay of progesterone receptors. J Steroid Biochem 13: 1129-1132. Masters -Tm Examination of the nuclear Saunders DE. translocation of progesterone receptor in MCF-7 cells. Wayne State University 1983. Brooks SC, Hansen ER, Saunders DE, Battelli MG, and Shafie S.M (1984). Effects of growth on estrogen receptor levels in MCF-7 cells. Cancer Res 44: 3724-3729. Estrogen Horwitz KB, m T and McGuire WL (1978). control of progesterone receptor in human breast cancer: Role of estradiol and antiestrogen. Corombo~nd;;ri n~~~~~ef~3bE17~~11:~~01( Meyers SA, Lozon MM, Christensen C, and Brooks SC (198;). Induction'of porcin: uterine estrogen sulfotransferase activity by progesterone. Biol Reprod 28: 1119-1128. Markaverich BM, Roberts RR, Alejandra MA, and Clark JH. An endogenous inhibitor of 3H-estradiol binding in normal and malignant tissues (1984). -Cancer Res 44: 1515-1519.

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September 1988