Comparison of dexamethasone binding and resistance to inhibition of induced differentiation in subclones of murine erythroleukemia cells

BIOCHEMICAL

Vol. 101, No. 3,198l August

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages 823-829

14, 1981

COMPARISON OF DEXAMETHASONE BINDING AND RESISTANCE TO INHIBITION OF INDUCED DIFFERENTIATION IN SUBCLONES OF MURINE ERYTHROLEUKEMIA CELLS. H.Beverley OSBORNE Laboratoire de Biologie Moleculaire et Cellulaire, (E.B. 199 du C.N.R.S.) Departement de Recherche Fondamentale, Centre d'Etudes NuclGaires de Grenoble, 85X, F 38041 GRENOBLE Received

May 19,

1981

SUMMARY: Subclones of the murine erythroleukemia cell line DS 19 with different sensitivities towards inhibition by dexamethasone (DEX) of the hexamethylene bisacetamide induced differentiation have been obtained. Scatchard analysis of DEX binding to whole cells and the time course of DEX binding to nuclei show that resistance to inhibition of erythrodifferentiation by DEX is neither associated with a reduction in specific binding of the hormone nor in a lack of activation or translocation to the nuclei of hormone receptor complexes. Hence resistance in these subclones must result from a defect at an event after binding of hormone-receptor complexes to chromatin if inhibition of differentiation is mediated by these complexes. INTRODUCTION: tion

Glucocorticoid

of murine

erythroleukemia

(DMS0)(1,2,3),

Specifically

I-synthase(5)

and ornithine and commitment

contain

affinity

(5,s)

that

cell mutant

where

or variant

with

defects

(for

receptor

complexes clones

(for

see 10).

icoids

on these

receptors(7,8)

a defect resistant

and dimethyl-

inhibit

exhibit

has been

which This

to the nucleus. in some later comparison

and sensitive

823

are

suggested induced

In many other

resistant

glucocorticoid

have

clones

associated

of the steroid-

However the

to the effect

is often

ability

event

between

mRNA

on gene expression,

resistance

or in the

on the

receptors.

an effect

see 9).

globin MEL cells

and it

by these

the

urophyrinogen-

of glucocorticoids

mediated

of receptors

Potentially

later

sulfoxide

shown to inhibit

They also

have been obtained

to migrate with

by dimethyl

differentiation(6).

effect

is

a review

in the number

resistant ref.

inhibitory

clones

erythrodifferen-

dehydratase,

decarboxylase(4).

glucocorticoids

of glucocorticoids

induced

have been

to terminal

of MEL cells

systems

the

bisacetamide(HMBA),hypoxanthine

glucocorticoid

the observed

inhibit

of b-aminolevulinate

synthesis(5)

differentiation

cells

glucocorticoids

in enzyme activity

high

hormones

(MEL)

hexamethylene

formamide(4). increase

steroid

also

effect should

been

reported

of glucocortyield

informa-

0006-291X/81/150823-07$01.00/0 Copyright 0 1981 by Academic Ress, Inc. All rights of reproduction in any form reserved.

BIOCHEMICAL

Vol. 101, No. 3,198l

tion

on the genes

complexes.

whose

In addition

and consequential induced icoid

expression it

should

events.

to one such MEL cell

AND

is

differentiation

is

where not

modified

be possible

We report

clone

BIOPHYSICAL

here the

RESEARCH COMMUNICATIONS

by the hormone-receptor to distinguish

between

the characterisation

resistance

correlated

with

casual

of DEX binding

to the effect a deficiency

of DEX on the in the glucocort-

receptors.

MATERIALS AND METHODS: DS 19 clone derived from was obtained from Dr.J.Yu. Stock cultures were diluting to 1x10' cells /ml in Eagles modified mented with penicillin (lOOU/ml), streptomycin serum(KC Biologicals Inc.,lot no. 300070). Cells media by limiting dilution (12).

the 745 MEL cell line (11) passaged twice weekly by essential medium(MRM) supple(lOOpg/ml) and 15% fetal calf were cloned in conditioned

Differentiation was induced by seeding exponentially growing cells into MEM supplemented with 5% fetal calf serum and 4mM HMBA. When required was added to yield the DEX(Sigma) from a stock solution (500uM) in ethanol desired final concentration of DEX. The final concentration of ethanol was always less than 0.2% and no effect of this concentration of ethanol on cell The proportion of hemoglobin containgrowth or differentiation was observed. ing cells was determined by benzidine staining(l2). [3H] DEX(Nuclear New England) binding and Scatchard analysis of binding to whole cells was measured as described by Neifeld et al(13) with the following modifications. We determined .that equilibrium binding to MEL cells was attained after a 25 min incubation at 37'C, hence all incubations were performed for 30 min at 37°C. At the end of this period 2 ml of ice cold HEPES 15mM,pH 7.4; NaCl 135mM was added to the cell suspension. The cells ation at 4'C(15Oxg,5 min) and washed twice with the were collected by centifu same buffer. To measure [8 H]DEX binding to nuclei in intact cells, 8~10~ cells previously incubated with [3~1~~~ and washed as above were lysed in 1OOpl of lysis solution(TRIS lOmM,pH7.4; MgC12 4mM; CaCla 2mM; Sucrose. 0.6M; Triton X-100 0.1% and the protease inhibitor Aprotinin(Sigma), 0.1 trypsin inhibitor units/ml). After a 2 min incubation on ice the nuclei were collected by centrifugation( 4"C, 5OOxg, 10 min). They were washed once with 1OOpl of lysis solution and then with 500~1 of lysis solution without Triton X-100. Under phase contrast microscopy the nuclei appeared intact, nonaggregated and devoid of attached cytoplasm. The final pellet of whole cells or of purified nuclei was suspended in 7501~1 of HEPES 15mM, pH 7.4; NaCl. 0.2M; sodium dodecyl sulfate 0.5%, and sonicated briefly. Duplicate 50~1 and 200~1 aliquots were taken for determination of protein(14) and of radioactivity by liquid scintillation spectrometry respectively. The free [3H]DEX was calculated from the radioactivity remaining in the first supernatent obtained at the end of the incubation. Specific binding was taken as the difference between the amount of [3~]~~~ bound in the presence and absence of a large (500,-1000x) excess of non-radioactif DEX (15).

RESULTS: We have previously tion their for

of MEL cells HMBA induced publication).

and testing

observed

that

are heterogeneous

with

differentiation( We have

the various

extended

subclones

the individual respect

for

observation the

824

ability

of a popula-

to the inhibition

H.B.Osborne,A.C.Bakke this

cells

and J.Yu by subcloning

of DEX to inhibit

by DEX of submitted DS 19 cells their

BIOCHEMICAL

Vol. 101, No. 3,198l

AND

16’”

BIOPHYSICAL

IO' Dexamethosone

IO"

RESEARCH

16'

COMMUNICATIONS

10'

CM)

Fig.1 : Effect of dexamethasone on the induced differentiation of subclones DI (A) and B6 (0) of DS 19 cells. Exponentially growing cells were seeded into MEM with 5% fetal calf serum, 4mM HMBA and various concentrations of dexamethasone. The cell concentration was maintained between 2~1%~ and 2~10~ cells/ml. The proportion of hemoglobin containing cells (benzidine positive) was determined after 72h in culture. In the absence of dexanethasone 759: of the B6 cells and 90X of the Dl cells stained benzidine positive at this time.

HMBA induced analysis.

differentiation.

Two of these

One clone(B6)

and the

other

clone

The differentiation trations

of Dexamethasone

receptors, binding

data

three

for from

separate

binding

the

per

of bind

cell

(n)

000 sites/cell

binding

the

further

of DEX (see

Fig]).

even at DEX concen-

site, would

amount

dissociation calculated

but

B6 cells

for

the

Dl cells.

number inhibited

the

glucocorticoid

analysis given

bound of the

in F'ig.2.

(Kd)

per !?-llDEX

From

and the number

and Kd= 14+1 nM and Hence at DEX concentrations

more DEX due to the greater

at concentration

be less

is

DEX

between

of DEX specifically

constants

the

bind

measuring

to be : Kd= 5+1 nM and

for B6 cells

with

differences of their

experiment

were

B6 and Dl cells.

associated

The Scatchard

the

more DEX due to the greater

differentiation

total

one representative

1nM the sensitive

their

problems

due to eventual

two subclones.

n= 34 OOOrt-5 000 sites/cell n= 54 00055

40% inhibited

or ease of extraction

experiments

sites

below

possible

we have measured cell

for

of differentiation effect

in undifferentiated

fractions,

of the stability

an intact

was only

binding

to avoid

to sub-cellular

subclones

to this

chosen

as 1uM.

In order binding

were

to DEX inhibition

was semi-resistant

of Dl cells

as high

Comparison

was sensitive (Dl)

subclones

above about of binding in

825

sites

the presence

affinity

1OnM the Dl cells even

though

of HMBA. It

their appears

of

BIOCHEMICAL

Vol. 101, No. 3,198l

2

6

4 (@m&/cell)

Bound

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

8

03 Fig.2 : Scatchard analysis of r3H] dexamethasone binding to intact undifferentiated DI (A) and B6 (0) cells. 4~10~ cells were incubated 37’C for 30 min in MEM containing TRICINE IOmM, pH 7.4 and increasing concentrations of [sH]dexamethasone from 0.5nM to 30nM. The amount of specific binding was determined as described in Materials and Methods.

at

Fig.3 : Time course of specific [slildexamethasone binding to nuclei of Dl(A) and B6 (0) cells. 6x10s cells were incubated for various periods of time at 37’C in MBM containing TRICINE IOmM, pH 7.4 and 10nM[3H]dexamethasone. For comparison the amount of non-specific binding (+), determined by incubating duplicate samples with 10nM[3H]dexamethasone and 5nM unlabeled dexamethasone is also shown. After the desired time the cells were washed and the nuclei prepared as described in Materials and Methods.

therefore

that

of DEX being In

this

order

to

asses

B6 and Dl cells

was compared.

various

with

periods duplicate

In these these the

conditions

two subclones amount

subclones almost

to

of

DEX

nuclei

of

lo6

nuclei

in

the

Aliquots

samples

with

of these

1OnM

the

total

amount

is

almost

identical. to

CdCUlate

presence

of

where

10 nM

to

anything

[3~]

826

DEX.

the

was

nuclei

of

for

determined

intact

by

DEX cells

(15). of

3 no difference of

these

binding

in two

was

the binding

experiments).

fraction

no aggregation 12 fmole

cells

incubated

was

of this

faster(2

that

to

fraction

course

in the nuclear

in conditions us to

bound

If

Dl

5 uM unlabeled

nuclear

time

slightly

concentration

and

FHIDEX

the

the

the

were

binding

As shown in Fig

Dl

was

cells

[3H]DEX

of

bound

In addition

of

bound

Non-specific

the

of nuclei/ml,

amount

of the hormone-

specifically

the B6 and the Dl cells.

ration

due to a smaller

translocation

BH]DEX

E3~l~~x.

cells

not

semi-resistance

for

of the protein allowed

of

specifically

ation

observed,

the

or nuclear

10 nM

was observed. identical

the

whether

the amount

is

as a whole.

in activation

complex,

incubating

in the Dl cells

to the cell

due to a defect receptor

resistance

bound

of DEX

Normalis-

to the concentof the nuclei

of r3H] DEX was bound

was per

Vol. 101, No. 3,198l

BIOCHEMICAL

TABLE

1: Effect

of

AND

prolonged

culture

nuclear

Time

BIOPHYSICAL

in

13HlDEX

in

HMBA and

COMMUNICATIONS

[3~]~~~

on

binding.

[3HlDEX/A26a

CP~

culture

RESEARCH

(h) Dl

0.5 48 72

cells

B6 cells

469 426 2lD

450 463 187

DI and B6 cells were maintained between 2~10~ cells/ml and 2~10~ cells/ml in MEM with 5Z fetal calf serum,4mM HMBA and IOnM [‘HI DEX. After 0.5, 48 and 72h aliquots of 4~10~ cells were taken and the amount of nuclear bound [‘HI DEX determined as described in Materials and Methods.

Comparison

of

Dexamethasone

hexamethylene

of DEX bound

lower

than

observed both

subclones

bound

cultured

72h.

After

of nuclear

in

with

to asses

of

that

the

total

subclones

was

DEX measured.

condensation between

DEX is

proportional have

performed

ific

binding

this

data

given

time

about

to

further

after

an

the ratio

in Table

or

(given

827

48 and

and the amount

1. Several

points

of MEL cells of protein Therefore

the absorbance

differentiated

cells

0.5;

more of the Dl cells

cells.

In addition

to the

present, for

taken

Upon differentiation

by using

fraction

were

experiments

and hence

undifferentiated

to the nuclear

is

and undifferentiated here

in

committed

was continually

In these

to the DNA concentration. on

are

not decrease

of the culture

This data.

occurs(l6)

normalised

Dl cells

from

nuclei

of HMBA on the DEX binding

the hormone

the B6 cells.

differentiated

differentiated

of 4mM HMBA and 1OnM [3~1~~~

bound

we

decreased

HMBA.

aliquots

than

which

of 4mM HMBA. At this

of DEX did

when

intervals about

In addition

binding

fmol/106

90% of the

the effect

these

are differentiated

of bound

the presence

and to its cells.

the presence

than

the presence

stressed

to 2.5

binding

of B6 and D1 cells,

were

nuclear

in

we observed

in the nuclear

48h in

and more

The nuclear

In order

report(7)

to undifferentiated nuclei

24h in culture

be

D1 cells

and

DS 19 cells

reduction

after

the B6 cells

additional

vary

to differentiated

the amount

differentiate.

must

B6

a previous

undifferentiated

the

nuclei

with

a concomitant

12fmo1/106 75% of

to

bisacetamide.

In agreement amount

binding

to DNA may the amount

at 260 nm which

in all

is

the experiments

MEL cells

by the radioactivity

we

the non-specbound

in the

BIOCHEMICAL

Vol. 101, No. 3,1981

presence

of excess

specifically close

estimate

cultured

cells

the presence occurs

(58% benzidine

tion

in the

occurs

amount

about

In this

cells).

For both

DEX, observed

in the presence

correlated

with

inhibition

curve

DEX( about

40%) is

inhibition

curve

(Kd)

for

(Kd=

14nM)

defect

than

in the

However for

for

the

bound

the

for

conditions

and varies

the nuclear presence

are

to the nuclear similarly

fraction

occurs

and HMBA also

by DEX. Hence commitment

of the

to differentiation

Several

other

studied

also

the glucocorticoid activation,

semi-resistant present

similar

receptors

receptors translocation subclones

total

is

with

of DEX

almost in the

of DEX bound in the

differentiation

is

necessary

to

inhib-

for

this

to occur. of this subclone, is not due to a respect

of DS 19 cells characteristics.

828

is time

to the nucleus

DEX binding

effect

two subclones.

induced

is not

inhibition

of

A similar

are

These results imply that the semi-resistance by DEX of its HMBA induced differentiation,

DEX binding,

of DEX specifically

in the amount

when this

of glucocorticoid

a

so that

80%, the amount

when MEL cells occurs

that

the resistance.

subclones

in the

for

for

of these

reduction

modification

number

almost of both

reduction

constant Dl cells

of 4mM HMBA and ~OI-&~[~HIDEX,

inhibited

the

to the

the B6 cells,

amount

fraction as a function

words

, which

of DMSO(7)

presence

by

seem to imply

than

not

of DEX. The

higherforthe

would

total

by

inhibited

relative

to the B6 cells.

in the

absence.

of DS 19 cells,is

was responsible 1OnM the

the reducof HMBA,

the dissociation

also

5nM) this

than

cultured

of HMBA. In other

Since is

Dl

to inhibition

is

of DEX to the nuclei

the B6 cells

bound

identical

greater

its

resistance

more receptors

about

binding

cells

where

presence

is

specific

specifically

ited

above

in

binding

cells

(Kd=

possess

subclones

differentiation

B6 cells.

by

of bound

the presence

DEX concentrations

receptors

also

to the Dl cells

In addition

sensitive

glucocorticoid

DEX concentrations

seen for

to higher

the B6 cells

the DI cells

the

inhibited

semi-resistant

or the nuclear

whose

of DEX to intact

for

that

total

Dl cells

displaced

in

of a subclone

in the

those

the binding

shown

in the

of these

1 is a

the B6 cells is

1OnM DEX than

differentiation,

a reduction for

of

10% of the

retention

retained

bound

DEX of the HMBA induced

for

no additional

to that

we have

than

in Table

the differentiation

positive

study

less

the data

We see that

cells),

relative

Hence

of nuclear

24h later

DISCUSSION:

3).

DEX binding.

positive

RESEARCH COMMUNICATIONS

was always

Fig

of HMBA, where

13% benzidine

the nucleus

BIOPHYSICAL

competitor(l5))

radioactivity(see

of the specific

in

DEX( only DEX in

unlabeled

bound

AND

that

to their or nuclear we have However

to

affinity binding. partially Mirendorf

BIOCHEMICAL

Vol. 101, No. 3,1981

and Mueller(8)

have

to DEX inhibition number is

characteristic particularity availability

that

for

of the DMSO induced

of receptor

required

reported

AND

per

cell.

to determine

Hence

whether

BIOPHYSICAL

T3 Cl2 and GM 86 cells differentiation

further the data

of MEL cells,semi-resistant of DS 19 cells. of these

of the modifications

different induced

More

RESEARCH COMMUNICATIONS

is

analysis that

subclones in MEL cells

effect

though should

is

allow

sensitivity

correlated

of other

we report

to the important

the

with

MEL cell

here

is

fact

a

that

us to define

by HMBA are directly

lines

a general

of DEX,or the

the

the which

inhibited

by DEX.

ACKNOWLEDGEMENTS: This work was supported in part Delegation G&&ale 1 la Recherche Scientifique.

by grants

from

the

REFERENCES: Scher,W., Tsuei,D., Sassa,S., Price,P.,Gabelman,N., and Friend,C. (1978) I.' Proc.Natl.Acad.Sci.tiSA 75,3851-3855. 2. Lo,S-C.,Aft,R.,Ross,J. and Mueller,G.C. (1978) Cell 15, 447-453. 3. Santoro,M.G.,Benedetto,A. and Jaffe,B.M. (1978) Biochem. Biophys. Res. Comm. 85, 1510-1517. 4. Gazitt,Y. and Friend,C. (1980) Cancer Res. 40, 1727-1732. 5. Scher,W.,Tsuei,D. and Friend,C. (1980) Leuk.Res. 4, 217-229. 6. Tsiftsoglou,A.S., Gusella,.J.F.,Volloch,V. and Housman,D.E. (1979) Cancer Res. 39, 3849-3855. 7. Golde,D.W.,Bersch,N.,Lippman,M.E. and Friend,C. (1979) Proc.Natl. Acad. Sci.USA 76, 3515-3517. 8. Mierendorf,R.C. and Mueller,G.C. (1979) Molec. Cell. Endocrinol. 13, 301-316; 9. Harris, A.W. and Baxter,J.D. (1979) Monographs on Endocrinology,Vol 12 pp 423-448, Springer-Verlag, Berlin, Heidelberg. 10. Cohen,L. and Sachs,L. (1981) Proc. Natl. Acad. Sci. USA 78, 353-357. 11. Ohta,Y.,Tanaka,M.,Terada,M.,Miller,O.J.,Bank,A.,Marks,P.A. and Rifkind,R.A. (1976) Proc.Natl.Acad.Sci.USA, 73, 1232-1236. 12. Orkin,S.H.,Haros,P.I. and Leder,P. (1975) Proc.Natl.Acad.Sci.USA 72, 98-102. 14. Neifeld,J.P.,Lippman,M.E. and Tormey,D.C. (1977) .T.Biol.Chem. 252, 2972-2977. 15. Baxter,J.D.,Higgins,S.J. and Rousseau,G.G. (1975) Methods in Enzymology Vo1.36,pp 240-248, Academic Press, New York. 16. Friend,C.,Scher,W.,Holland,J.G. and Sato,T. (1971) Proc.Natl.Acad.Sci. USA, 68, 378-382.

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