Inhibition of dimethylsulfoxide-induced differentiation in friend erythroleukemic cells by diacylglycerols and phospholipase C

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 14, 1984

Pages 491-499

INHIBITION OF DmDE-INDDCED DIFFERENTIATION INFEIEND EFYIBROLEDEEMICCFLISBYDIAcYLGLYcERoLs ANDPmSPI-I0L1PASE c S.M. Pincus, B.S. Beckmanaud W.J. George* DepartmentofPharmacology n&meCniversity School of Medicine 1430 Tulane Averr~e,Ne~ Orleans, LA 70112 Received October 29, 1984 SU$iARy: The effects of diacylglycerols and phospholipase C on dimethylsulfoxide (cMso)-induced differentiation were investigated in Frierxd erythroleukemic cells (FEIC). Greater than 80%of cells becomebenzidinepositive when incubated with 1.5% DMSO. The tumor pranoter, 12-O-tetrar-phorbol-l3-acetate (TPA), inhibits DMSO-induceddifferentiation in Diacylglycerols wre four-d to inhibit CMSO-induceddifferentiation dose ~ivelywiththeorderofpotencybei~lI-oleayl-2-acetylglycerol(OAG) > dicaprylin > dilaurin > diolein. Phospholipase C Which releases endogenaus diacylglycerols fran membrauephospholipids also inhibited t.MSO-induced differentiation dose responsively. Theseresultssupportthehypothesisthat diacylglycerols can have effects similar to tumor promoters ard suggest that protein kinase C my be a ccammnmechanismfor tumor pramtion. ~1984 Academic Press.

Inc.

Friend erythroleukemic

cells

(FEIC) have been widely utilizeo

for studying the processes of proliferation cells. state,

These cells

normally proliferate

but can be induced by certain

differentiate

into cells

and differentiation rapidly

in erythroid

in a poorly differentiated

drugs such as dimethylsulfoxide

resembling orthochromic normoblasts (1).

accumulate hemoglobin, lose the capacity specific

as a Mel

membraneantigens,

to divide,

and decrease in size (2).

chemically induced differentiation

correlate

in FELC (3).

This property

(TPA) inhibit of TPA and

has beenextensivelysMiedinFELCandisknownt0

highly with the tumor promoting ability

ester studied.

These cells

develop erythrocyte-

Tumor promoters such as 12-O-tetradecamylphorbol-13-acetate

relatedphorbolesters

(DMSO)to

of the particular

phorbol

TheFEU:systemcanthusbeusedasamodelforstudyingthe

mechenismof action of tumor promoters. * To whomall corresporxlence should be addressed. DMSO,dimethylsulfoxide; FELC, Frienderythroleukemic cells; Abbreviations: OAG, l-oleoyl-2-acetylglycerol; TPA, 12Utetr adecamylphorbol-13-acetate. 0006-291X/84 $1.50 491

Copyright 0 1984 by Academic Press, Inc. All righrs o] -vroduction in any form reserved.

Vol. 125, No. 2, 1984 It

BIOCHEMICAL

has been recently

affinity

binding site

demonstrated specific

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that TPA interacts

established

in other tissues (4).

Yamasaki, et al.,

co-purified

high

have

binding sites for tumor pranoting phorbol esters in

When this binding site was isolated

FEM: (5).

with a specific

with a calcium-phospholipid

protein kinase C (6).

from mousebrain,

it

dependent protein kinase termed

Nishimka and coworkers have demonstratedthatphorbol

ester tumor promoters can activate

protein

kinase C in vitro

(7).

studies and others have led to the suggestion that the receptor

Those for TPA is a

site on the protein kinase C molecule, and binding of TPA to protein

kinase C

causes enzyme activation.

studies

in the platelet serotonin,

This hypothesis is supportedbyextensive

which show that WA treatment can cause secretion of

platelet

aggregation and phosphorylation

to be phcsphorylated by protein Diacylglycerols initiation activate its

receptor

activators

(8).

platelet

kinase C in vitro

(10).

aggregation.

for

have been proposed as endogenous

kinase C. of diacyl-

as well as the enzyme, phospholipase C, which can release membrane

bound diacylglycerols,

on DEO-induced differentiation

demonstrate that diacylglycerols tiation

can also

(9) and ccmpete with TPA competitively

Thus, diacylglycerols

for protein

Diacylglycerols

The purpose of the present study was to examine the effect glycerols

proteins kncwn

are phospholipid metabolites which are released following

of thrombin-irduced protein

kinase C -in vitro

of specific

ti

in FELC. The results

phospholipase C can inhibit

differen-

in FEW induced by EMSO. MiWERIALSANDMETHODS

Chemicals: TFA was purchased from Consolidated Midland Corp., Brewster, NY. l-Cleoyl-2-acetylglycerol (OAG) was a gift of Dr. Y. Takai at Xobs University, Japan. Dicaprylin, dilaurin, diolein, distearin, phospholipase C (fran C. perfrim) and diaminobenzidine were obtained from Sigma Chemical Co., St. Louis, MO. DISC was supplied by Fisher Scientific Corp. Cells and Culture: FELC clone DSlS-10s was kindly provided by Dr. Shigeru Sassa, NewYork University, NY. Cells were cultured in Minimal Essential Medium (MEM) containing Earle's salts supplemented with heatinactivated fetal bovine serum (lo%), penicillin (100 U/ml), streptomycin (100 pg/ml), amphotericin B (0.25 pg/ml), nonessential amino acids (3X), and MEMvitamins (3X). Cultures were maintained at 37' C with 5% CC2 in humidified air. Cultures were diluted twice weekly to maintain the cells in 492

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

logarithmic phase. All culture supplies were obtained from GIHCO, Grand Island, NY. The fetal bovine serum consisted of a single lot of serum that had been tested for the ability to support growth and DMSO-induced differentiation in FELC. Cell doubling time was approximately 11 hr. Culture Experiments: Cells growing in kg phase were collected and washed twice by centrifugation at 1000 x g for 10 min. The cells were resuspended in the MEMculture mediumti added at a final concentration of 104 cells/ml in a total volume of 10 ml to 25 cm2 culture flasks containing MEMculture medium. EWSO(1.5%) was added to selected cultures to induce differentiation. Either PA, dicaprylin, dilaurin, diolein, distearin, OAG, or phospholipase C was added to the cultures at zero time to test their effects on cellular differentiation and proliferation. Culture flaskswerethenincubated for 4 days with 0.2 ml aliquots of cells being removed daily for cell countiq. On day 4, aliquots of cells were stained for hemeusixq benzidine. Due to limited solubility, TPA and the diacylglycerols were dissolved in a small volume of DMSOwhich accounted for a maximumof 0.05% lBi!SOin the cultures. Henxidine Staining: The benzidine stain was prepared fresh just prior to use Three mg of 3~,3'-disminobenzidine was added to a mixture of 10 as follows: ml of 50 mMTris-HCl buffer (pH 7.5) and 2 ml of 3% hydrogen peroxide. The stain was allowed to stand for 10 min and was then added to the aliquots of cells (1:l) in a test tube. At least 30 min was allowed for staining to be completed before the cells were counted. The number of cells which stained brawn per 100 total cells was termed the % of benzidine positive cells. HESULTSANDDISCUSSION E'EW clone DSlQ-10s spontaneously differentiate

at a rate such that the

percentage of benzidine stained cells is less than 1% of the total. of EELCwith DMSOresults

in a dose responsive increase in the percentage of

cells which differentiate.

In the present studies it was fourXl that 1.5% DMSO

produces a maximal stimulation cells

differentiating

proliferation.

of differentiation

with approximately

while having minimal inhibitory

The tumor promoter, PA,

inhibits

effects

after

4 days.

a 40-50% inhibition

80%of

on

DMSO-induced differentiation

also in a dose-dependent manner. At a concentration approximately

Treatment

of 160 nM there was

of the differentiation

induced by 1.5% tB!SO

This response to TPA was used as a positive

control

for other

treatments. Table 1 shows the effect

of dicaprylin

EELC. Increasing concentrations in the inhibition

of dicaprylin

of differentiation

cultures

from 29 PM to 870 PM resulted

significantly

of FFJE by approximately

treated with DMSOalone.

in

of FELC in a dose-dependent manner. At

the highest dose (870 UM), dicaprylin DMSC-induced differentiation

on DMSO-induceddifferentiation

Dicaprylin 493

(p<.O5) inhibited 20%when canpared to

also inhibited

cell

Vol. 125, No. 2, 1984 TABLE

1:

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EFFEXS OF TPA AND DICAPFWLIN AND DIFFERENTIATION IN FRIEND

ON DMSO-INDUCED EFLYI?m-c

PFEOLIFERRTION CELLS

INHIBITION

TREAmEm

INHIBITION OF DI-TION

OF PROLIFZRATION Doubling (h-j

maso (1.5%) Imo (1.5%) + TF'A (160 nM) mSO‘(1.5%)-+ DICAF'RYLIN (29 PM) Ix60 (1.5%) + DICAF'RYLIN (87 !JM) m60 (1.5%) + DI-IN (290 PM) r&so (1.5%) + DIUPFZYLIN (870 PM) cells

were

cultured

for

Doubling Tim (% increase)

Time

(% -=a=)

12.4 11.8

--- 4.0

0 f 4.7 54.4f6.0

12.1

- 2.4

4.9

i6.9

14.0

12.9

6.6

+ 4.8

14.7

18.5

15.0

f 5.7

16.8

35.5

20.3

f3.1

4 days.

Doublingtimewas

(6) (6)* (3) (3) (6) (6):

calculatedbetweendaya

and 3 whenall the cultures were growing logarithmically. Inhibition of proliferation is given ae percent increase in doubling time of FELC calculated as percent differentiation benzidine

increase

frauWSOtreatmntakme. presented aa mean

is

Percent inhibition of and is besed on percent

f SEM(n)

2

of

positive cells seen with dicaprylin or TPA treatment with IXSO as canpxedtotreaQnentwithDMSOalone. *SQnificantly different from IMSOtreatment group (~0.05)

proliferation

in a dose-responsive manner (Table 1).

Dicaprylinincreased

doubling time frcm 12.4 to 16.8 hrs at 870 PM. When dicaprylin the cultures at a concentration Figure 1 shows the effect

of 2.9 m&l, lethal of dilaurin

effects

on differentiation

in FELC irduced

used, dilaurin

inhibited

of FEW approximately

number of differentiated trations

of dilaurin

diacylglycerols, effect

had essentially

diolein

and dicaprylin with

their

the

19%as the

on differentiation.

Tvm other

were faurd to have no significant of FEIC (not shown).

and distearin

is that the limited

One possible

and the small effect

solubility

of the diacylglycerols

rapid rate of metabolism in the cell

for a significant

accmnilatkm

of

of

the

(11,12)

diacylglycsrols

cell.

Due to the limited asynthetic

of diolein

apparently

would not have allmed within

no effect

and distearin,

reason for lack of effect

coupled

(660 vM) significantly

cells was reduced from 83% to 68%. Lower concen-

on DMSO-induced differentiation

dilaurin

was added to

were observed.

by LBISO. At the highest concentration DMSO-induced differentiation

the

cmpour&

solubility

of the naturally

occurring diacylglycerols,

l-oleoyl-2-acetylglycerol (OAG) wastested. 494

The

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0 CONTROL

LMSO (1,5X)

DNSO + TPA (160 Nfl)

DNSO +

DNSO +

DlLAURlN

Q,2

DRSO +

DILMIRIN

(22 rfl)

dl)

DMSO +

DILAURIN

DILAURIN

(220 NM)

(660 dl)

Figure 1: Effects of dilaurin ard TPAon IXBo-itiuced differentiation in FEZ. Cells were treated for 4 days with 1.5%IZSO, anl either TF'Aor different concentrations of dilaurin as shmn. The percentage of benzidine positive cells per 100 total cells on the ordinate represents the percent of differentiated cells. N=6except for E&So alonegroup,v&reN=9. *Irmiicatee significant difference from DSCI treatment alone (pCO.05). substitution

of an acetyl

group for a loqer

chain fatty

acid in the 2

position makes OAGmore soluble in aqueous medium than the other diacylglycerols.

The effect

of OAGon proliferation

of I&SO-treated

FEK is

presented in Figure 2.

EMS0plus OAGat 7.5 PM or 25 ~.IMconcentrations

inhibited

of FFLC only slightly

proliferation

to DMSOalone.

When 75 I.~MOAGwas included in the incubations,

inhibition

of proliferation

paralleled

that seen with TPA treatment.

lethal

during the first

exposed

there was an

tm days of incubation which

OAGat a concentration

of 250 PM was

to the cells.

The effect in Figure 3. positive

when canpared to cells

of OAGon DISO-induced differentiation OAG (75 LJM)significantly

reduced the percentage of benzidine

cells on day 4 from 85% to 60%of total

in magnitude (30% inhibition)

in FELC is demonstrated

FEZC. This effect

and occurs with a concentration

l/10 that required for significant

effects

with dicaprylin

is greater

of OAGabout

ard dilaurin.

When

canpared to the response seen with TPA, OAGgives a less dramatic response ard 495

Vol. 125, No. 2, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

V Control. v .

n X A

1 Incubation

Figure

!mso (1.5%). EMSO + OAG (7.5 uM), CMY) + OAG (25uM). CWO+TPA(~~OEM). DMSO + OAG (75 uM),

3

2 time

4

5

(days)

2: Effect of OAG ard TPA on proliferation in lBSO-treated cells. z&ycally gruvirg cells were seeded at 10 /ml and incubated . Daily aliquots were removed for determination of cell cancentration. Eachpoint is the average of the determinations fran three sewate cultures from a single representative experiment.

20 ,o 0 Ii-

* CONTROL

DMSO (1.5%)

DMSO (1,52) TPA (160

496

NM) OAG (;.51iMJ

OAG i5

IIN)

OAG (75

ufl)

Vol. 125, No. 2, 1984

is less potent. of TPA results diacylglycerols,

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

This was expected for two reasons: the more rigid in a greater binding affinity

structure

than that seen with

and the rate of metabolism of TPA is very slow canpared to

that of diacylglycerols

(13), resulting

in a more prolonged exposuretoTPA

during the incubation. None of the cell.

compounds

tested are naturally

In order to study the effect

addedtotheincubationmedium.

Phospholipase C is a potent toxin purified on proliferation

in the mammalian

of release of endogenous diacylglycerols

inFE?LC. theenzyme phospholipaseCwas

effect

occurring

from Clostridium perfriwens.

in FEW is shown in Figure 4.

Its

Therewas adose-

P

/? k

106

: Z F! ; : : 0 =

105

:

v Control, v

Dmo

(1.5%),

xEt+EO+TPA( . m+PLX

160 (0.01 (0.05 (0.25

0 DMSC+PL-C A rSO+PL-C

104,

I 1

Time

Figure

4: Effects zgitcally

Figure

3: Effects of OAG ar-d TPA Cells were treated for different concentrations benzidine positive calls represents the precent significant difference

IN), u/ml),

u/ml),

u/ml).

1

after

2 treatment

3

4 (davs)

of phcspholipase C (PLC) and TPA on proliferaticn in -treated growing cells y+3re seeded at 104/ml and incubated . Dally aliqucts were removed for determination of cell concentration. Each point is the average of the determinations frun three separate cultures of a single representative experiment.

on CMSO-induced differentiation in FFU. four days with 1.5% LMSO ti either TPA or of OAG as shcwn. The percentage of per 100 total cells on the ordinate of differentiated cells. N=6, * irdicatff frcm DMSO treatment alone (pcO.05).

497

cells.

Vol. 125, No. 2, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DMSO

CONTROL

iL52) DNSO (1,5X) MS0(105%)DKSO + TPA (160

+ Nfl)

PLc(.OlO/ML)

Um5X) +

PL’3,050/M~)

DRSO (105%) + PLc(.25

O/ML)

Figure 5: Effect of phoqholipase C and TPAon IMSO-induceddifferentiation in FEE. Cells were treated for four days with 1.5%CMSO and either TPAor different concentrations of phcsphlipase C (PLC), as shorn. The percentage of benzidhe positive cells per 100 total cells, on the ordinate, represents the percent of differentiated cells. N=6, l indicates significant difference from DMSO treatment alone (PCO.05).

depe&ent inhibition

of the rate of proliferation

phaspholipaseCincreased. trations

As shown in figure

of 0.01, 0.05 and 0.25 U/ml inhibited

30 and 31%, respectively. as an added control. proliferation

5, phospholipase C at concendifferentiation

Phospholipase A2 had no significant

of FEU: by 7,

effect

at 4 days of incubation.

acids and not diacylglycerols

these results suggest that diacylglycerols differentiation

of

Phospholipase A2 was used at the sameconcentration

or differentiation

A2 releases fatty

as the ccncentration

a& proliferation

on either

Since phospholipase

from membranephcspholipids,

are involved

in the regulation

of FELC.

Recent reports provide strorg evidence that TPA works via activation protein

kinase C (4,6,7,8).

receptor ccnnpetitively

Diacylglycerols

and can also activate

in FELC on proliferation

protein kinase C in vitro

and differentiation 498

of

can displace TPA from its

The present experiments suggest that TPA and diacylglycerols effects

of

(10).

cause similar

at concentrations

that

Vol. 125, No. 2, 1984

correspond with -in vitro TPA and diacylglycerols

1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13.

BIOCHEMICAL

potencies.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Our findings supportthehypothesisthat

work via a commonmechanisminvolving

protein

kinase C.

Friend, C., Scher, W., Holland J.G., and Sate, T. (1971) Proc. Natl. Acad. Sci. USA 68, 378-382. Reuben, R.C., Rifkird, R.A., and mks, P.A. (1980) Biochim. Biophys. Acta 605, 325-346. Yamasaki, Ii., Fibach, E., Nudel, U., Weinstein, I.B., Rifkind, R.A., and Marks, P.A. (1977) Prcc. Natl. Acad. Sci. USA 74, 3451-3455. Driedger, P.E., ad Blumberg, P.M. (1980) Proc. Natl. Acad. Sci. USA 77, 567-571. Yamasaki, H., Drevon, C. and t&rtel, N. (1982) Carcinogenesis 3, 905-910. Niedel, J.E., Kuhn, L.J., and Vandenbark, G.R. (1983) Proc. Natl. Acad. Sci. USA 80, 36-40. Castagna, M., Takai, Y., Kaibuchi, K., Sam, K., Kikkawa, U., ard Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851. Kikkawa, U., Kaibuchi, K., Castagna, M., Yamanishi, J., Sane, K., Tanaka, Y Takai, Y. and Nishizuka, Y., (1984) In pdvances in C&EyZe%de and Protein Phosphorylation Research, Vol. 17 (Greengard, P. et al., eds), pp. 437-442, Raven Press, NY. Takai, Y., Kishimoto, A., Kikkawa, U., Mori, T., ard Nishizub, Y. (1979) Biochem Biophys. Res. Cumun. 91, 1218-1224. Sharkey, N.A., Leach, K.L., and Blumberg, P.M. (1984) Proc. Natl. Acad. Sci. USA 81, 607-610. Michell, R.H. (1975) Biochim. Biophys. Acta 415, 81-147. Bell, R.L., Kennerly, D.A., Stanford, N., and Majerus, P.W. (1979) Proc. Natl. Acad. Sci. USA 76, 3238-3241. Yamasaki, H., Mufson, R.A., and Weinstein, I.B. (1979) Biochem. BioFhys. Res. camMun. 89, 1018-1025.