Estrogen-like effects of combined dexamethasone and tamoxifen in the chick oviduct

Vol. 124, No. 1, 1984 October 15, 1984

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 57-62

BIOCHEMICAL

ESTROGEN-LIKEEFFECTSOF COMBINEDDEXAMETRASGNE AND TAMOXIFRNIN THE CHICK OVIDUCT Achille

Gravania,

Nadine Binart, Paul Robel, Etienne-Emile and Maria-Grazia Catelli

INSERM U33 and CNRSER 125, Lab Hormones, 94270 Bicttre,

Baulieu France

Received August 2, 1984 SunHART : The effects of dexamethasone alone on withdrawn chick oviduct weight, DNA, protein content and progesterone receptor concentration were barely detectable, whereas ovalbumin and conalbumin synthesis were increased. When dexamethasone and tamoxifen were combined, a marked increase of total DNA and wet weight occurred. proteins, including egg white proteins, result was the Progesterone receptor also was increased. The most striking stimulation of DNA polymerase-o activity by combined dexamethasone and tamoxifen, whereas either compound was completely ineffective., e~a4 Acadaic Press,

Inc.

The nonsteroidal

triphenylethylene

antiestrogen

influence

the growth parameters , the level

synthesis

of egg white proteins

reported

that

estrogen

primed,

egg white protein Same effects

conditions

increase oviduct

of this

or progesterone

enzyme activity

(6) and rat uterus

synthesis

are

(4).

In appropriate are

In the present

activity

Several reports during

in

additive

(5).

chick oviducts

of antiestragen-glucocartico-

have not been described.

growth potential.

(2, 3).

resemble those

on the growth of withdravn

used the measurement of DNA-polymerase-a the oviduct's

properties

on

of the responses obtained

with estrogen.

and the results

to

of progesterone

of dexamethasane and of progesterone

have not yet been reported,

or the

and progesterone

and conalbumin

and kinetics

act synergistically

combinations

receptor,

on the chick oviduct

with estrogen

of glucacorticasteroids

does not

We had previously

the effects

They induce avalbumin

to those obtained

the effects

The effects

of tamaxifen

of glucocorticosteroids

Glucacorticosteraida

(1).

and produced growth promoting

chicks and the magnitude

comparable

steroid

in the chick oviduct

chicks potentiated

synthesis

produced by progesterone. withdrawn

of progesterone

the combined administration withdrawn

tamoxifen

estrogen

as

a

work, we have

sensitive

had previously stimulated

parameter described

of the

growth of chick

(7). 0006-291X/84

57

$1.50

Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 124, No. 1, 1984 MATERIALS

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

AND METHODS

Experimental animals : Newborn chicks were primed with estradiol benzoate and withdrawn for 4-6 weeks. After this period, they were reinjected in the upper leg muscle with the appropriate test compounds, as indicated in the Results section. Animals were killed 18 h after the last injection. The oviducts were removed, fragments of each oviduct magnum were pooled, and the relative rates of ovalbumin and conalbumin synthesis were measured as previously described (2. ._ 8)._ Progesterone receptor measurement : The technique utilized allowed measurement of total (filled and unfilled) PR sites in the cytosol, as reported in detail by Bayard et al. (9), with modifications. The cytosol was stripped of endogenous steroids by dextran-charcoal adsorptiog, then samples were incubated at O'C for 15 h followed by 25'C for 2 h with H-progesterone (range 0.5-10 nM) in presence of 0.5 nM cortisol. Unbound ligand was separated by dextran-coated charcoal adsorption and the equilibrium binding constants were determined by Scatchard analysis, using the Rosenthal correction for non saturable binding (10). The results were expressed in sites/cells, assuming a DNA concentration of 2.5 pg per cell of chick oviduct

(6).

DNA-Polymerase-c : Oviducts were homogenized in 5 vol of chilled buffer containing 20 mM Tris HCl pH 7.5, 0.5 M KCl, 2 mM dithioerythritol, and 0.5 % Triton X iO0. The homogenates were sonicated for 20 set using a Branson I-22 sonicator and kept at O'C for 60 min. The 105,000 g supernatant was used for DNA Polymerase-a assay according to Bertazzoni et al. (11). The results were expressed in pmol thymidine triphosphate incorporated per min per mg protein. and DNA were measured in samples of Measurement of DNA and proteins : Protein homogenates and/or cytosol using the techniques of Bradford (12) and Burton respectively. (13), RESULTS Progesterone withdrawn

oviducts

dexamethasone for

(Figure

600 sites/cell).

; however

control)

Results

although

were

benzoate

the

statistically

Estradiol

benzoate

was given

in % of

control

PR concentration control

+

than

4

and

Dexamethasone

a slight

increase

(14,800 more

value

benzoate.

tamoxifen

significant

in with

of estradiol with

measured

stimulation

PR 20 % below

combined

PR were

secondary

expressed

increased

effect

when

90 h of

or combined.

to decrease

completely

ineffective

alone

1).

tended

of cytosol

and after

Estradiol

Tamoxifen

counteracted

(control)

and tamoxifen,

comparison

fold.

: The concentrations

receptor

was

(20 % above

of cytosol

PR occurred

(p < 0.5). Weight, alone Estradiol

DNA and protein or combined benzoate

oviduct

growth

minimal

effects

with

content tamoxifen,

was given

parameters

: The effects

for

(data

on wet weight,

were

determined

comparison. not

of dexamethasone.

shown)

DNA and protein 58

after

Tamoxifen (3).

18, alone

Dexamethasone content

(Figure

injected 24 or 90 h. had no effect alone 2).

had Tamoxifen

on

Vol. 124, No. 1, 1984

E:

BIOCHEMICAL

AND BIOPHYSICAL

lml T

E+T

RESEARCH COMMUNICATIONS

0’2

Oex Dex+T

I

I

18

24

'%

HOlMS

Figure

1 : Effects

of

estradiol,

dexamethasone,

and/or

tamoxifen

on

cytosol

PR. Withdrawn chicks received daily injections of estradiol benzoate (E, tamoxifen (TAM, 10 mg/kd/d), or dexamethasone (DEX, 2 mg/kg/d) 1 mg/kd/d), for 4 d and were killed 18 h after the last injection. E and DEX have also been combined with TAM. Results are expressed in % of control (C) PR concentration (14,800 + 600 sites/cell, mean +- sd, n = 3). Figure weight,

2 : Effects of estradiol, dexamethasone and/or tamoxifen on oviduct DNA, and proteins. Withdrawn chicks were treated as reported in the legend of Fig. 1, and sacrificed 18, 24, or 90 h after the first injection. Open symbols correspond to DEX alone (2 mg/kg) and closed symbols to DEX + TAM (10 mg/kg). Results are expressed in % of control. l : Protein (5.5 + 0.2 mg per oviduct ; A: DNA (1.26 + 0.1 mg per oviduct ; n : wet weight (76 + 4 mg per oviduct). Insert : Estradiol benzoate (1 mg/kg) has been injected instead of DEX. The same symbols are used.

completely On the

counteracted contrary,

content

of

after

the

combined

chick

oviduct

effects

of estradiol

dexamethasone more

than

benzoate

and tamoxifen

7 fold,

(Figure

increased

and DNA content

2, Insert). the

more

protein

than

2 fold

a 90 h treatment. The stimulatory

ovalbumin tamoxifen. detailed

effects

and conalbumin A representative report

will

of dexamethasone

synthesis

were

experiment

be published

also is

elsewhere

59

on the greatly

reported (5).

relative potentiated on Table

rates

of

by 1. A more

Vol. 124, No. 1, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE TREATMENT

OS'ALBDMIN 0.2

0.8

Tamoxifen

0.2

1.6

Dexamethasone

2.8

4.4

7.2

7.8

Relative

rates

DNA

total

extracts

18,

tamoxifen, DNA under

protein

alone

polymerase tamoxifen

dexamethasone

and

daily

alone

or

conalbumin

synthesis

injections

of

combined

for

in

chick

(10

rag)

Results

are

expressed

measured

in

tamoxifen

90 h.

oviduct. and in

%

synthesis.

: The 24

ovalbumin

received (2 mg)

Polymerase-a

+

of

chicken

dexamethasone of

CONALBUMIN

Control

Tamoxifen Dexamethasone

Withdrawn

1

and or activity (Figure

increased

90

enzyme h

activity

after

the

combined.

has onset

of

Estradiol

was

unchanged

3).

Nevertheless,

DNA

polymerase-a

been

treatment

benaoate by

was

given

dexamethasone combined

I

I

18

24

and tamoxifen

activity

I

with

almost

chick

oviduct

dexamethasone for

and

comparison.

tended

to

decrease

and 2 fold,

The

‘ti

HOWS

Figure benzoate

3 : Effects of dexamethasone or/and tamoxifen, and of estradiol on DNA-polymerase-a activity. Withdrawn chicks were treated as reported in the legend to Fig. 1, and sacrificed 18. 24, or 90 h after the first injection. Open symbols correspond to control (CJ), DEX (2 mg/kg) (0) or estradiol benzoate (1 mg/kg) (A). Closed symbols correspond to treatments with tamoxifen alone (10 mg/kg), (m) or combined with dexamethasone (0) or with estradiol benaoate (A). Results are expressed in % of control DNA polymerase-o activity : 14.7 + 0.4 pmol TTP/min/mg protein.

60

Vol. 124, No. 1, 1984 time-coursesof

BIOCHEMICAL

DNA polymerase-a

dexamethasone

+ tamoxifen

AND BIOPHYSICAL

stimulation

were

similar

RESEARCH COMMUNICATIONS

by estradiol

with

benxoate

or by

a maximum at 24 h.

DISCUSSION The effects withdrawn work

chick

utilized

oviduct

--in vivo

withdrawal,

were

a single

interval

have

magnum explant

dexamethasone

(1-8

mediate

of dexamethasone

h)

(4).

of

relationships

progesterone.

Using

chick

the

oviduct's

tamoxifen. protein

growth

control

The effects content,

of proteins,

slightly

but

tamoxifen inhibitory.

Similar

compound

synergistic

effective

than

on the

progesterone

contrary,

it

antagonistic

action

or the

of

benzoate

estradiol

has been growth

with

of

with

that

dexamethasone synthesis.

to compare

weight,

of weight,

reported

that

of quail

oviduct

was

was the

dexamethasone

alone

after

a marked

receptor

result

ineffective

dexamethasone

61

even

combined,

if

had been previously

(16).

DNA or

Progesterone striking

the with

detectable, were

by combined

the

injected

on oviduct barely

or

as concerns

or combined

occurred.

increases

can and

dexamethasone

and tamoxifen

The most

time

alone,

were

d of

estrogen

undertaken

alone

10-12

kinetics

protocols,

was completely

although

3 or

of

glucocorticosteroids

observed

essentially

in

the published

The effects

and conalbumin

results

of

and a short

we confirm

activity

and progesterone,

On the

h),

increased.

either

after

dose

receptor

tamoxifen

(3).

the

DNA and wet weight

of DNA polymerase-a whereas

al.

experimental

of dexamethasone

significantly

et

to those

When dexamethasone

increase

15).

by dexamethasone

and progesterone

4 d of stimulation.

stimulation

ias

14,

proteins

of ovalbumin

work

Most

(4,

that

egg white

(18-90

present

reported.

concluded

(4 to 6 weeks)

rates

and mRNA synthesis

of dexamethasone,

different

interval

protein

by Hager

similar

relative

However,

cultures

authors

rather

time

the

organ

the major

of withdrawal

increases

previously

reported

These

dose-response

(2 mg) and the

been

5 or 1 mg dose

an induction

duration

on egg white

and not

reported

with

is much less DNA, and proteins

dexamethasone stimulated

exerts by high

an doses

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 1, 1984 In mammalian both the

estrogenic

and antiestrogenic

withdrawn

chick,

combinations

of

oviductal

response

estradiol

(18).

related

to organ

underlying tamoxifen

tamoxifen,

species,

activities

contrary

tamoxifen

to

with

, which

molecular

are

effects

with

which

in this

tamoxifen

dexamethasone

are

alone

of those

respect

the

by itself

reasons

or dexamethasone

are

DNA polymerased can explain

displays

undefined

reminiscent

importance in particular

of (3)

qualitatively

mechanisms

when injected

the

--in vivo

for

progesterone

Of particular growth,

administered

the

activity.

(17).

(l),

the

produce produced

by

parameters The

estrogen-like

completely

In

effect

of

unknown.

ACKNOWLEDGEMENTS We thank

M. Rossillon,

and J.C.

Lambert

for

the

preparation

of

the

manuscript. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Sutherland, R.L., Mester, J. and Baulieu, E.E. (1977) Nature 267, 434-435. Catelli, M.G., Binart, N., Elkik, F. and Baulieu, E.E. (1980) Eur. J. Biochem. 107, 165-172. Binart, N., Mester, J.. Baulieu, E.E., Catelli, M.G. (1982) Endocrinology l&, 7-16. Hager, L.J., M&night, G-S., Palmiter, R.D. (1980) J. Biol. Chem. 255, 7796-7800. Le Bout, Y., Groyer, A., Robel, P. and Baulieu, E.E. Submitted for publication. Sutherland, R.L., Lebeau, M.C.. Schmelck, P.H. and Baulieu, E.E. (1977) Febs Letters 2, 253-257. Harris, J.N. and Gorski, J. (1978) Mol. Cell. Endocr. lo, 293-305. Palmiter, R.D., Oka, T. and Schimke. R.T. (1971) J. Biol. Chem. 246, 724-737. Bayard, F., Damilano, S., Robel, P. and Baulieu, E.E. (1978) J. Clin. Endocr. Metab. s, 635-640. Rosenthal, H.E. (1967) Anal. Biochem. 20, 526-567. Bertazzoni, V., Scovassi, A. and Brun, G. (1977) Eur. J. Biochem. 81, 237-240. Bradford, M.M. (1976) Anal. Biochem. 11, 248-254. K. (1956) Biochem. J. 62, 315-322. Burton, M&night, G.S. (1978) Cell 14, 403-413. E.R. (1978) Science 201, 356-358. Palmiter, R.D., Mulvihill, Boisvieux-Ulrich, E., Laugier, C. and Sandoz, D.1982) Biol. Cellg, 175-188. Jordan, V.C. and Dix, C.J. (1979) J. Ster. Biochem. 2, 285-291. O'Malley, B.W., McGuire, W.L., Kohler, P.O. and Korenman, S.G. (1969) Rec. Progr. Harm. Res. -25, 105-160.

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