of proliferation cells involves diminished efficacy of prostaglandin E1 ...

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1989 74: 1942-1951

Effector function for RAS oncogene in interleukin-3-dependent myeloid cells involves diminished efficacy of prostaglandin E1-mediated inhibition of proliferation HG Derigs, D Klingberg, GJ Tricot and HS Boswell

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Effector Myeloid Cells

Function Involves

By Hans cell

growth

in by

growth

Guenter

the

and

The

Derigs,

Oncogene in Interleukin-3-Dependent Efficacy of Prostaglandin of Proliferation

Debra

Klingberg,

Guido

J. Tricot,

and

H. Scott

E1-Mediated

Boswell

tion.

respectively.

These

differences

cell

products.

including

tion

by adenylate

cyclase

agonists

the

inhibitory

regulator

tant cells inhibition)

marrow

stromal

factors

for RAS Diminished Inhibition

microenvironment

of

stromal to

be

lular

(lower were

cAMP

and

is

than

cells

in

cells

been ade-

intoxicated activation

by

were

ever,

similar

in

(IL-3)-

tion

by

ii%. +

i%,

4

mM

6 ±

myeloid-ceII G protein,

cellular

response

the

p21

RAS-containing

and

appears

This

IL-3

where

be an

the

to

or

important

with

pertussis

cyclase

inhibition

cyclase

of

agonists

with

effector

partoxin-

adenylate

diminished

compared

& Stratton,

activa-

inhibitory

compared of

adenylate

cells

compared

of direct

diminished

by

observed

are minimal. These studcyclase activity in mutant

is linked

to

to

forskolin.

How-

was

conditions

membranes

Gi.

of membranes

pertussis-

nucleotide.

activity

independent

activity

of

membranes

under

of

Mg2-dependent

guanine

influences adenylate

membranes

cells activity

that on

RAS-containing

G-protein diminished

genic p2l RAS. @1989 by Grune

than

cyclase

and

pertussis-

RAS-transfected

lower

and/or

Mn2

(greater Intracel-

of

cyclase

membranes

membranes

RAS-bearing sensitive

times

inhibi-

in RAS-transfectant

adenylate

adenylate

p2l

parental

macti-

two

hormone

oncogenic

parent cells (P < .0005).

pertussis-intoxicated

to

for

RAS-transfec-

stimulation

lower

The

parent-cell

with

ent-cell

±

1 .5

very

1 MM.

prolifera-

±

was

stimulatory ies showed

Hi

i9

from

PGE1

1 50%

cells.

Cells

mode

on

was

parent

membranes have

inhibition) versus highly significant

all

formed

intoxicated

second

in capacity between

of

parental

function

cells of once-

Inc.

combina-

MAY

ARISE

as

possess

the action elements signals interest

of types.

The to interact

in majority with cellular

that

pepeither

intrinsic

tyrosine

kinase

nuclear

gene

c-myc

product.5’

phospholipid-dependent School

of

Adminis-

IndianApril

dent

on initiation

pase

C.89

Research

Hematology/

first

amine,

muscarinic,

The

Medical IN by page

1942

regulation

guanine

teins),

myeloid

Inc.

of phospholipid

marked to

that

at

of the

least

recognized

metabolism

prostaglandin

C activation proteins between

is

(G

pro-

catechol-

cell-surface

receptors

proposed aspect myeloid

that of the cells,

guanine nucleotide-binding of IL-3-dependent control

of

cell growth.’4 association

but

cellular peptide receptors, guaproteins, and phospholipase C at the has been studied in detail in several model between

nine nucleotide-binding plasma membrane systems neutrophil,

depen-

by phospholi-

of phospholipase

as intermediaries and

calcium-

C is normally

nucleotide-binding

an observation that implicates proteins as possible mediators

ofthe

Walther

solely

by

kinase

and adenylate cyclase.’#{176}” It was recently activation of protein kinase C is an integral interleukin-3 (IL-3) signal in IL-3---dependent

USPublic

hereby

or

Activation

protein

In turn,

mediated

14.

activity

propagate tyrosine phosphorylations on cytosolic substrates.’4 However, an early observation showed that induction of a state of cellular competence (for cell-cycle traverse and, hence, proliferation) may also be associated with phosphorylation on cellular substrates by the serine/threoninespecific protein kinase C and subsequent expression of the

not in IL-3-dependent myeloid cells. In the such studies have demonstrated that the receptor

for the chemotactic

peptide

nylalanine protein,

(FMLP) Gi (named

cyclase),

which

serves

Blood,

N-formyl-methionyb-leucyl-phe-

is physically coupled to the 4l-Kd for its inhibitory effect on adenylate as the substrate

Vol 74. No 6 (November

for the bacterial

1), 1989:

toxin,

pp 1942-1951

G


RAS

ONCOGENE

pertussis also

2l

toxin.

activates

of

FUNCTION

In

the

which

neutrophil

locks

modification, responses.’5” may also

receptor-mediated because of a reliance protein-mediated

study

to

protein,

Gi, and

Our role

was

the

previous of

by covalent and

functional

C activation,

role the

of

studies

clarify

in the

in myeloid

cells,

role ofa

adenylate

to

proteins the

pertussis

mutant

alterna-

the control with

role

of

of

IL-

particular

toxin-sensitive

p21

of myeboid

cyclase

or,

reversal of the tonic cyclase in these celbs.”24

performed

proliferation

attention

and

Pertussis

proliferation in certain cells, either of their proliferative response upon Gi

nucleotide-binding

3-dependent

yield

suggest that pertussis toxin of growth-factor peptide-

phospholipase

present

guanine

to

response.

conformation

tively, by pertussis toxin-mediated inhibitory effects ofGi on adenylate The

breakdown

C activation

biochemical

These observations be a useful probe

protein

(PIP2)

migratory

these

Gi

generates

kinase

Gi in its inactive

ablates

this

bisphosphate

to protein

1943

CELLS

conformation,

4,5

leading of

MYELOID

C, which

inositol

diacylglycerol, toxin,

its active

phospholipase

phosphatidyl

induction

IN

cell

RAS

lines

activation

0

protein. showed

by

that

the

prostaglandin

E,

(PGE,) is on cyclic AMP (cAMP)-mediated inhibition of IL-3-dependent proliferation.25 With these data in mind, we studied the effect of pertussis toxin and POE, treatment on a naive

11-3-dependent

laboratory, Neo

Ras.F3,

lipid

studies

revealed

metabolism

the

by the

inactivation

of a 0

the

generated

of

the

codon

naive

line,

IL-3.2’

line phospho-

The

observed

cellular proliferation by pertuscould therefore not be explained by

protein

that

functions

solely

to activate

C. The loss of effectiveness of pertussis of IL-3-dependent proliferation in the

transfectant mechanistically

derivative and

Hi

12 (T24)

parent-cell

of stimulated factor

in this

cell

H-RAS

absence

growth

of IL-3-dependent on this cell line

phospholipase as an inhibitor

recently

on a derivative

with

Primary 1 Hi

inhibition sis toxin

line and

transfected

oncogene.26 NFS/N

cell

NFS/Nl.Hi,

cell related

toxin RAS-

line Hi Neo Ras.F3 was studied to the alternative signaling sys-

tem relevant to IL-3-dependent proliferation-namely, the adenylate cyclase system. The present study shows that the RAS oncogene product affects IL-3-dependent proliferation, in part, as a result of its inhibition of POE, (hormone)dependent the

adenylate

importance

cyclase

of p21

leukemic cells may prostaglandin-mediated the

hematopoietic

Murine

myeloid

oncogene

be its abrogation inhibitory

and

suggests

expression

that in early

of the transmission of signals generated within

microenvironment. MATERIALS

line

activation,

RAS

cell

lines.

AND The

METHODS fastidious

IL-3---dependent

cell

NFS/NI Hi was developed from bone marrow cells of the NFS/N mouse, as described elsewhere.2’ This cell line was generated as a model of the fastidious growth requirements of immortal myeloid clones that could develop in the absence of ecotropic type C murine leukemia viruses and in the presence of a paucity of xenotropic and mink cell focus-forming (MCF)-endogenous leukemia viruses, which, if present, might sequentially activate growthrelated genes by viral insertional mutagenesis,2’ The NFS/N mousedonor strain has severely limited copies of these endogenous murine leukemia virus sequences. 11-3 concentrations of 250 to 500 U/mI were required to attain maximal proliferation of the cell line (data

not shown). When washed free of 11-3, these cells also manifested a proliferative response to interleukin-4 (11-4), as assessed by incorporation of 3H-thymidine in the presence of, but not in the absence of, lL4.26 The 11-4 response in these cells was not dependent on cellular protein kinase C, as assessed by the failure ofcellular protein kinase C depletion to diminish the proliferative response.’ The RAScontaining clones, Hi NeoRas.F3 and H7NeoRas.E5, were generated by electroporation of the NFS/N 1 .H7 cell line with the vector pAL8, which contains a mutant (codon i 2) human H-RAS genomic clone (6.6-kb Bam-HI insert) derived from the T24 bladder carcinoma and driven by an SV4O promoter.2’ After electroporation, cells were selected for neomycin resistance (also encoded by the vector) in G4l8, I mg/mL, and subcloned by limiting dilution. Studies of Hi Neo Ras.F3 showed that it contains the 6.6-kb human T24 oncogene and displays constitutive p2 1 RAS activity in overproduction of 3H-diacylglycerol as compared with parent NFS/Ni.Hi cells.’ The RAS-transfected cell lines were shown to express the mutant HRAS (Val I 2) protein by flow cytometric immunofluorescence analysis with a Val 12 RAS-specific monoclonal antibody after fixation of cells with methanol to expose the inner cellular membranes, as described elsewhere’ (data not shown). H7 Nec Ras.F3 and Hi NeoRas.E5 cells were maintained in McCoy’s 10% fetal calf serum (FCS) and 10% WEHI-3 CM with G4i8, I mg/mL. These cells were strictly 11-3 dependent, as assessed by their failure to proliferate in the absence of 11-3. Control clones were formed by transfection of NFS/NI Hi cells with a neomycin-resistance geneexpression vector pNeo.3, as described elsewhere.’ Proliferation assay. Cells were washed free of factor and then plated into assay in triplicate microwells of 96-well flat-bottomed plates at 5 x l0 cells per well in a final volume of 100 ML and incubated overnight at 37#{176}C in 5% CO2. On the following day, each well was pulsed with 0.5 uCi 3H-thymidine, harvested after 6 hours’ incubation, and counted by liquid scintillation, as described elsewhere.2#{176} The incubation media consisted of McCoy’s 10% FCS with or without (a) 8-bromo-cAMP (8 Br cAMP) (Sigma, St Louis, MO), 0.5 mmol/L (b) POE, (Sigma) 10’ mol/L (1 Mmol/L), or (c) pertussin toxin, 0.1 to 1,000 ng/mL (List Biologics, Campbell, CA). To these media, purified hematopoietic growth factors were added in a range of concentrations. Purified murine IL-3 and purified recombinant 11-4 were obtained from Genzyme (Boston, MA). Homogeneously purified 11-3 was a gift of Dr James N. IhIe, St Jude’s Children’s Research Hospital, Memphis, TN. Membranepreparation. Cells were incubated at a concentration of I x 106 cells/mL in McCoy’s 10% FCS with or without pertussis toxin 0.1 to 100 ng/mL for 4 to 16 hours at 5% CO2. 37#{176}C. Viability, as assessed by trypan-blue exclusion, was not affected by this treatment (greater than 90%). Cells were washed twice in serumfree medium and homogenized in a buffered solution of 15 mmol/L Tris,

pH

7.4,

1 mmol/L

EDTA,

i mmol/L

EGTA,

and

1 mmol/L

phenyl methylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 400 g for 10 minutes to remove nuclei, and membranes were isolated by ultracentrifugation (200,000 g for 20 minutes) of the supernatant. Adenylate

cyclase

activity

assays.

Adenylate

cyclase

reactions

were initiated by the addition of 20 ML membrane suspension containing 40 g protein to a final volume of 100 ML containing 40 mmol/L Tris, 1 mmol/L NaATP, 0.5 mmol/L cAMP, 2.5 U/mI adenosine deaminase, 0.4 mg/mL bovine serum albumin (BSA), 80 mmol/L

NaCI,

10

mmol/L

MgCl2,

and

an

ATP-regenerating

system consisting of 5 mmol/L creatine phosphate and 5 U/mI creatine phosphokinase.2#{176} Reactions were performed to assess generation of 32P-cAMP from 2 iCi a 32P-ATP at 30#{176}C for iO minutes and were terminated by the addition of 0.8 ml of a solution of 0.25% sodium dodecyl sulfate (SDS), 5 mmol/L adenosine triphosphate (AlP), and 0.175 mmol/L cAMP (pH 7.5). The nonhydrolyzable


1944

DERIGS

GTP was

analogue used

guanosine

as the

5’ (fi, ‘y-imido)

stimulus

in some

triphosphate

reactions

5,000

Gpp(NH)p

in concentrations

Mmol/L GTP and 10.0 mmob/L MgC12. In other reactions, MnC12 4 mmol/L with or without forskolin i04 mol/L was substituted for MgCl2 to assay adenylate cyclase activity by direct activation of the catalyst (bypassing Gs).#{176}32 After termination of each reaction, #{176}H-cAMP(l0 cpm) was added to judge recovery of cAMP after chromatographic isolation by the method of Salomon and colleagues.#{176}3 Each data point represents the mean ± SEM of duplicate reactions. ADP ribosylation for SDS polyacrylamide-gel electrophoresis (PAGE) assay of Gi. The [#{176}2PJ ADP-ribosylation reactions were performed by a modification of the procedure described previously.#{176}’#{176}2 The major substrate of pertussis toxin-catalyzed ADP ribosylation has been previously demonstrated to be Gi, the adenylate cyclase inhibitory GTP-binding protein (4i-Kd subunit).TM Determination of intracellular 3H-cAMP generation. Determination of intracellular 3H-cAMP was performed according to the methodology of Kienast et al with modification, as described previously.25’35 Following preincubation in culture medium with pertussis toxin

100

ng/mL

or

control

medium

for

4

hours,

cells

Specific

inhibition

a specific proliferation

studied

(Fig

by

pertussis

toxin

of

the

cell

was

not inhibited

lower

levels

of thymidine

it was

of interest

of IL-3 and proliferation

250 was

10,000

effect

line

stimulated

by

by pertussis

toxin,

incorporation that

IL-4 in combination of NFS/Nl.Hi

(Fig

was

I

like

for

50

to

2

A

25 lL-3,

0

lrA

200

00

50

(Uil)

U/mi

lL-4,

U/ni

(lOCU/mO

‘I

flk1.

250

B

00

IL-3.

50

1000

500 L-4.

U/n4

250

(10uml)

U/ni

(50(1/mI)

iOI.i/mI)

IL-4 (500U/mO(2

Fig 1 . Inhibitory effect of pertussis toxin on proliferation of NFS/Nl .Hi cells at different doses of IL-3 and IL-4. alone or in combination. In successive experiments (A versus B) 5 x 1 O’ cells per replicate microwell were plated in medium with different doses of IL-3 orIL-4. without (0) and with (0) pertusais toxin 1 .000 ng/mL. Cells were incubated for 18 hours before addition of 0.5 MCi 3H-thymidine and then harvested 6 hours later. Background proliferation in the absence of factor was less than 500 cpm. (A) lL-3, 10 to 50 U/mL; 11-4. 50 to 200 U/mL; and suboptimal dose combinations. (B) IL-3. 50 to 250 U/mL; IL-4. 250 to 1 .000 U/mI; and suboptimal dose combinations. Data from A and B are representative results from different experiments and reveal minor variations in absolute amounts of proliferation stimulated by the individual growth factors.

of suboptimal

toxin,

dependent

relate

to distinguish the

the

functional

to the

stimulation

inhibition

specific

by

of

biochemical

11-3-

action

of

pertussis toxin, dose-response curves for pertussis toxin inhibition of IL-3-dependent proliferation and for ADP ribosylation of cellular 0 protein were performed (Fig 2). With the toxin

of

maximal

effective

varying

held

the

for

ng/mL

incorporation proliferation cells

were

doses,

subjected

to

pertussis

toxin

in

vitro

activated

stepwise assay

pertussis (Fig

toxin 2A).

then ADP

overnight ribosybation

in vitro

with

of

the

half-

inhibition

(Reduction

to be of

3H-

15,000 cpm, with In parallel, NFS/

with

membranes

dilution

revealed

of 30,000 to of 500 cpm).

incubated and

constant,

proliferation

dose 10

thymidine background N I .Hi

iL-3

in

approximately

of

serve

proliferation

pertussis

doses

of proliferation

pertussis

these cytokines. In order to

concentration

or

hematopoietic postreceptor

by

500

1). In addition,

resulted in supraadditive 1). The observation

inhibition

5.000

pro-

at comparable

effect) between the IL-4 implies different which,

specific because

IL-4,

(Fig

addition

to was

1B), cellular 50% inhib-

.Hi

a

by The mac-

10 to 50

U/mL (Fig approximately

of NFS/Ni

to observe

mechanisms,

concentrations,

1 ). This

(Fig

synergism (supraadditive growth factors IL-3 and signaling

to

of IL-3

proliferation

liferation

proliferation

by RAS oncogene. ADP-ribosylates and

nucleotide-binding protein parent-cell line NFS/Ni.Hi,

a range

IA) or 50 incorporation

IL-3-dependent U/mL,

JL-3-dependent

guanine ofthe

i). Over

U/mL (Fig 3H-thymidine ited

of

toxin or PGE, is reversed of pertussis toxin, which

tivates inhibit

-I

were

RESULTS

cotro +Pertussis

at iO’ cells/mL in serum-free medium with 3H-adenine for 30 minutes at 20#{176}C. Cells were centrifuged and in medium at the same cell density. Cell aliquots of 450

ML were added to 50 iLL medium containing 3.isobutyl-i-methylxanthine (MIX) (a phosphodiesterase inhibitor; final concentration, I mmol/L), with or without POE,, and incubated at 37#{176}C for 20 minutes. Reactions were stopped by addition of 50 ML 3 mol/L trichloracetic acid containing ‘4C-cAMP (ICN Biochemicals, Costa Mesa, CA), 0.2 iCi/mL, vortexed, and then centrifuged at 10,000 g for 5 minutes. cAMP was isolated from the supernatants by the method of Salomon Ct al.#{176}3 Results were expressed as the percentage of incorporated 3H-adenine converted to 3H-cAMP.

pertussis capacity

0

from

iO-9 to i0 mol/L to assess stimulatory regulatory GTP-binding protein (Gs)-mediated adenylate cyclase activity specifIcally. Forskobin l0 mol/L was used as the stimulus in other reactions performed with various concentrations of the divalent cations Mg2 or Mn2 in the presence ofguanine nucleotides to evaluate inhibitory influences (Gi) on direct activation of the adenylate cyclase catalyst.#{176}’ Stimulation of adenylate cyclase by POE, in concentrations between l0 and l0 mob/I was assessed in the presence of 10

suspended 15 sCi/mL resuspended

ET AL

pertussis

were

prepared

with

‘2P-NAD

dithiothreitol.

toxin

at and and

These


RAS

ONCOGENE

2l

FUNCTION

IN

MYELOID

CELLS

1945

40,000

a) 30,000

.

C

.“

E >,

-C I-

I

20,000

C,,

l0,0001 0

0.049

0.097

0.195

0.39

A

0.78

Pertussis

l.56

3.l2

6.25

12.5

25

50

00

ng/n

toxin,

kD

NFS/N1.H7

CONTROL

NFS/N1.H7+

P.tox

O.lngAnl

P.tox

Ingftnl

ii

Si

ii

ii

IOngAnl

+P’NAD

NFS/N1.H7+P.tox+32P-NAD IS

SI

B Fig 2. 1 . Dose-dependent antiproliterative effect of pertussis toxin (P. tox) in IL-3-treated NFS/N1 .Hi cells. (A) 5 x iO cells per replicate microwell were plated in medium with IL-3. 5 U/mI. with stepwise twofold dilutions of pertussis toxin, beginning with 100 ng I ml. Cells were incubated for 1 8 hours before addition of 0.5 MCi 3H-thymidine and harvested 8 hours later. Background H-thymidine incorporation less than 200 cpm. (B) Pertussis toxin-mediated in vivo ADP ribosylation of cellular 41 -Kd G protein in NFS/Ni .Hi cells blocks subsequent in vitro ADP ribosylation with P-NAD. Isolated membranes of NFS/Nl .Hi cells, which were incubated overnight without or with different doses of pertussis toxin (0.1 ng/mI. 1 ng/mI. 10 ng/mI). were subjected to in vitro ADP ribosylation with P-NAD and pertussis toxin. The membrane protein was subsequently analyzed by SDS-PAGE and autoradiography. NFS/N1 Hi control lane involves incubation of membranes with P-NAD but without pertussis toxin. Densitometry of the resulting bands revealed stepwise reduction of integral density (OD x width units [U]. 1 D Analyst. Biorad) by increasing pertussis toxin concentration that paralleled the slope of decrease in proliferation: control lane. 10.38 U; pertussis toxin. 0.1 ng/mI. 8.043 U; 1 ng/mI. i.895 U; 10 ng/mI, 3.0i5 U.

membranes

were

diography cated at

2B).

overnight

41

then

(Fig Kd,

substrate

analyzed

demonstrated

putative

Gi

(Fig

of ADP-ribosyl

and

membranes a single 2).

transfer

no ‘2P-ADP was transferred in the absence of the toxin doses

by SDS-PAGE

NFS/Nl.Hi

intense

This

band

autora-

intracellular dependent

proliferative

substrate

band

hypothesis

to hold

true,

adenylate

cyclase

agonist

was

by pertussis

a specific

toxin,

because

proliferation

in the presence of ‘2P-NAD but (control lane, Fig 2B). Increasing

of

pertussis toxin in the overnight intoxication of increasingly derivatized the 4l-Kd substrate, preventing “P-ADP-ribosylation in the in vitro reaction 2). At 10 ng/mL pertussis toxin overnight, only a small

residual

capacity 2).

to

directly

41-Kd

(Fig

One

activity

ribosybation

with

was

two dose-response

toxin-induced

in vivo

still

curves

ADP

present appeared

ribosylation

of

inhibition

of DNA

toxin-induced

the

consequence

substrate

of the

by pertussis

of adenylate

cyclase,

ribosybation

toxin

was

the

so that

small

increases

IL-3 concentration We also observed

that

that

the

3).

IL-3For

the capacity

to inhibit

(Fig

this of the

IL-3-dependent

Indeed,

inhibition

and amount ofproliferation in the RAS-transfectant

Neo Ras.F3, the resistant to POE,

N 1 .H7

3). In order

cell line,

cent in

PGE,

cell line Hi proliferation (Fig

greater

resistance

compared

with

proliferation of experiments was

of the

we reasoned

be high

affect

of NFS/Nl.Hi.

of

IL-3-dependent proliferation in NFS/Nl.Hi by 1 MM POE, was quite significant and was inversely related in degree to

to greater

of a 4l-Kd

alteration

significantly

capacity

should

observed

2).

possible

G-protein basal

these

relate

protein

synthesis

for ‘2P-ADP

Therefore,

(Fig

would

intoxi-

NFS/NI.H7

thus (Fig

cAMP

not

inhibition

to POE, NFS/N

by

POE,

trations

from

the

possibility

of the 1 .Hi,

Hi

that Neo

the

Ras.F3

was simply

due

at a given IL-3 concentration, a series performed to individually examine per-

of proliferation

Ras.F3

amount of IL-3-dependent was greater than in NFS/

to discount parent

(Fig 3). derivative

1 itmob/L

10 U/mL

of NFS/Nl.Hi at

a large

to 100 U/mL

range

(Table

or Hi of 11-3

Neo

concen-

1). In fact,

the


DERIGS

Ras.F3

but led to almost

(Table

1).

analogue

On

the

8 BrcAMP

of inhibition

cell

experiments,

intracellular

cAMP

cell

and

line

to reverse

was

p21

RAS,

in Hi

of

0 proteins The failure

an

inhibitory

effector

a decreased

Neo

cyclase

Opp

may

0

Hi

-

be

by

PGE,

Hi

agonist

on

cyclase

Ras.F3

(Fig

of

of

(Fig

4).

of

v

top

Differences the p21

NFS/N

perNeo

I Hi

adenylate decrease

or

alter

OppNHp

79.0

±

1 1 . 1 % (1 2)

51.2

±

6.9%

(12)

in adenybate

Hi

SO U/mL, plus H7

(Fig 4, top). We cyclase activity from

compared Neo.Ras.F3

NeoRas.E5

activity

H7 on

by hormone

and/or

Neo

(data

of membranes

Ras.F3

versus

Mg2-dependent

guanine

nucleotide

assess

parent

activation

coupling whether

ruled

of

out

to Os in Hi diminished

activity might be a result of quantitative of catalyst in the membrane, forskobinby PGE1,

Pertussis

Toxin,

and 8 BrcAMP

Pertussis

eight;

inhibitor(s),

Neo

Ras.F3

Toxin

+ PGE,

5 1 .2

±

8.6%

(1 3)

92.0

±

2.0%

(8)

5.7

±

2.0%

(9)

58.5

±

8.8%

(8)

.0003

106

versus

H7Neo difference

in membranes

cyclase

membranes

.00041 to

the

TKNeo.O9, from H7

clone

Pertussis Toxin (1 00- 1 .000 ng/mL)

in lL-3

and

not

cells, H7 membranes

RAS-containing

Cells

PGE,

30

of

activity

SEM (No.

±

(1

six;

by

cyclase

cells

RAS-transfectant

cyclase in amount

RAS-Transfectant %

by the

of the catalyst

adenylate

a simple defect of hormone-receptor Neo Ras.F3 (Fig 4). To directly and Hi

stimulated

shown).

the catalyst

in

1 .H7, from

NFS/Hi.H7 did

hormone

from

intoxica-

augmentation

versus NFS/N of membranes

when

both

bottom),

stimulated

line of

the

was always lower than NFS/Nl.H7, by POE, or OppNHp (Fig 4, bottom). In intoxication, which mildly increased adeactivity

similar

H7 in

To localize

Mn2.

Adenylate

and RAS-transfectant twofold greater

not

from

observed by

Ras.F3

between parent also observed

or the

this

catalyst

membranes

or pertussis-toxin

control neomycin-resistant with p21 RAS-containing Pertussis

to

for

cyclase

or on stimulation

Os

4,

by

cells possibly

responsible

corrected

Neo

POE,

NHp-activated

nybate

through

protein,

NFS/N1.H7

of POE,

Hi Neo Ras.F3 whether stimulated addition, pertussis

.Hi x Neo

act

complex cyclase cata-

adenylate

activity with

and

NFS/Nl.Hi

that

Ras.F3.

defects

hormonal

in

that inhibit (Oi) or of pertussis intoxica-

of cAMP generation in HiNeoRas.F3 we measured adenybate cyclase activity IL-3

1).

greater

of RAS-transfectant to another

Me

the putative

bevels (Table

were

to agents

pointed

compared

presence

in 11-3

resistant

resistance

adenylate

NeoRas.F3

cAMP

E,-generated

transmembrane-signabing receptor, the adenybate

or simply

Lower

to IL-3

prostaglandin

POE, as

resistance, activity

the

by

soluble

comparable

NFS/Nl.Hi cells than in H7 Neo also pertussis-intoxicated (Table 2). these data suggested that the p21 RAS-

lyst, as well as the regulatory stimulate (Os) the catalyst. inhibition

the

and

grown

AL

ofNFS/Nl.H7

of

in response

in cells

the adenybate cyclase consisting of the POE,

tion

use

lines

basal

pertussis-intoxicated Ras.F3 that were Taken together, containing

inhibition

hand,

bed to significant

of both

In other

complete

other

ET

75

to

100

multiplied by the stated

U/mL, by

100.

agent.

three.

Percent

inhibition

equals

cpm


RAS

ONCOGENE

Table

2.

2 1 FUNCTiON

Excess

Above

IN MYELOID

Intracellular

Hi

Neo

cAMP

Ras.F3

Pertussis

Stimulus

0.1

PGE,

Mean

SEM

±

of two

Corrected

for

intoxicated

for

ADP-ribosylated

.Hi

Fold Stimulation

Generated Nec Ras.F3

of

CAMP Over Basal by PGE, in NFS/N 1 .H7

:

Q.

a -

139

±

18%

161

±

25%

15

±

2

146

±

18%

24

±

2

>.

> I-

experiments.

incorporation

4

in NFS/Ni Intoxication

SEM

±

imoI/LPGE,

1 jzmol/L

1947

Cell Types

Percent CAMP NFS/N 1 .H7/H7

Basal

Generation

After

of Both

CELLS

hours

of

at

1 00

all detectable

3H-adenine. ng/mL

Cells

of

were

toxin,

a

pertussis-

maneuver

that

30

00

0,3

G,.

w

2o-

stimulated range

reactions

were

of Mn2

concentrations

Mn’

independently

Because an allosteric

site

0 proteins range, 0.5

and

reactions

added

OppNHp

(Fig

mmol/L, significantly

differentially

were 5).

forskolin-stimulated greater in Ras.F3,

and

cyclase

as the Mn2 4 mmol/L, activity

0.5 to 4 mmol/L

presence

5).

adenylate

cyclase

at

uncouples

regulatory

Mn2

the

(Fig

concentration the difference

5). These observations pertussis-intoxicated

(Fig

5, top).

and

NFS/N

below

B.

>.

ade-

a

40

I-

> I--..

However,

00i

was diminished

made and

l0

50

2

v right).

I .H7

were also NFS/Nl.H7

.(

activity was than in

augmented

left

0

with

was increased above 2 mmol/L to between the adenylate cyclase

Ras.F3

(Fig from

or

concentrations

5, bottom,

Lu

ade-

without

significantly

z

concentration

adenylate cyclase parent NFS/NI.H7

OppNHp

activity

of H7 Neo

performed

At

of a (Fig

from the cyclase catalyst in the to 4 mM,”” forskolin/Mn2-stimubated

cyclase

nylate

in the

from

activates

also

nylate

HiNeo

performed

1 >.

for membranes H7 Neo Ras.F3

(1)

.c-.. -‘.5 0E 2oLu 1 >.

z

I-

Lu

0 .(

DISCUSSION The

present

of myeloid exert

study

cell

known

effects

by hormones IL-3-dependent

and and

H-RAS

studies

cell

line

oncogene,

the

most

tumors,

performed

give

frequent

including rise

to the

RAS

myeloid RAS

inhibitors

POE,,

which

signaling

transfected

Neo

Hi to gain

gene

protein’s

into

the

cell

in mammalian

involve

guanine

the

role of

hematopoietic have demonstrated

mutations

leukemias,

with

Ras.F3.slas.2s

insight

an RAS oncogene in a model of neoplastic proliferation. Studies in other systems that

and

for their action in inhibiting of a naive parent-cell line,

a derivative

were

of two

toxin

of transmembrane

growth factors, proliferation

NFS/Nl.Hi, mutant

the activity pertussis

on elements

T24 These

examined

proliferation,

codons

that

nucleotide-binding

effector

functions

of p2 1 RAS

at the

effector systems

(DAO), through

the primary activator of protein kinase C, perhaps prolonged phospholipase-C activation at the memThe

kinase

C

requirements that function

resulting activation

constitutive would

relieve

p21

membrane.

The first in model

brane.

function of oncogenic was in the overproduction

plasma

RAS elucidated of diacylglycerol

DAO-mediated certain

for transcription of nuclear in initiation of DNA synthesis

growth

Fig 4. Adenylate cyclase activity assays in membranes from NFS/Nl .Hi and Hi Neo Ras.F3 cells. Membranes were isolated from NFS/Ni Hi (left) or Hi Neo Ras.F3 (right), either untreated (bottom), or incubated overnight with pertussis toxin (iOO ng/mL) (top). Adenylate cyclase reactions were performed in the presence of 10 mmol/L Mg2 (basal) and iO smoI/L GTP without (U) or with (0) 100 MmoI/L PGE, or i to 100 MmoI/L GppNHp (). Mean ± SEM of duplicate reaction. Data are plotted as adenylate cyclase activity (pmol/min/mg cAMP ± SEM) on the ordinate, compared with negative logarithm of molar concentration of reaction constituents on the abcissa (eg. i04 mol/L corresponds to - log mol/I 4). Representative of four separate determinations.

core, like the T24 codon I 2 mutation of this study.”9 As a result of these mutations, the intrinsic OTP hydrolytic capacity of p21 RAS is severely impaired, which maintains the

-Iog(M)

protein factor

protooncogenes and mitosis.#{176}

More systems

recently, transformed

it has

been reported

by RAS

capacity of hormone-stimulated ate intracellular cAMP.4””

that

oncogenes

These

model display

tumor-cell diminished

adenylate cyclase to generobservations were of inter-

est to us because previous studies from this laboratory had demonstrated that one relevant signal-transduction pathway affecting myeboid-cell proliferation ating intracellular cAMP.25 Those

is that involved in generstudies demonstrated that

a sustained antiprobiferative

cAMP IL-3-dependent

increase in intracellular signal for both

was

a strong and IL-


1948

DERIGS

300

FORSKOLIN.

k

ET AL

1 00MM

+GppNHp

250

FORSKOLIN, No GppNHp

100MM

:I:II::::

#{149}P4FS/P11.H7 P#{149}rtussisIntoxicated #{163} Hi

NeoRas.F3

P#{149}rtusalsIntoxIcated

Pertusais

,M

FORSKOLIN.100

+GPPNHP

Fig cyclase and Hi from

0 NFS/N

OPIFS/N1.H7 OH?

OH?

1 .H7

NioRas.F3

NeoRaa.F3

concentrations

‘

[ mMJ

Mn2

the

[mM

rele-

at for cAMP-

p21 C activation

IL-3RAS

inhibition of intoxicaOi on the inhibitory

of the parent 3-stimulated

as comby Oi

by

of

SEM

of two

were isolated NFS/Nl.Hi

0.5 to 4 mmol/I. reactions. Representa-

Mn2

from

of duplicate

consecutive

NFS/N activation

determinations.

I .H7 cell line of phospholipase

be on signaling

systems

influence growth factor-stimulated in the present study we were the

adenylate

other

failed to reveal ILC,26 which caused of the

than

4i-Kd Oi mitogenesis

phospholipase

C,

ent-cell proliferation the growth factor

mitogcnesis.#{176} Therefore, particularly receptive to data cyclase

responses to pertussis toxin. Our studies demonstrated

system

inhibition

by pertussis IL-3 and that

in

functional

of NFS/N

toxin that was strictly

1 Hi

par-

was specific for related to the

ADP ribosylation of a single 4i-Kd substrate, putative Oi (Figs I and 2). The failure of pertussis toxin to inhibit the IL-4 proliferative response in these parent cells may be related to the intrinsic resistance of this growth factorproliferative

cyclase

agonists,

8 BrcAMP Pertussis RAS

on

response

including

(data toxin

to inhibition

POE,,

and

not shown). was also a useful

IL-3-dependent

proliferation

loss

toxin’s

3-dependent ing Hi Neo POE,

in

of

in

RAS-

of

signal

concept

to the

signal-transduction RAS itself might

that

pertussis

proliferation Ras.F3 cells

the

of has

or

such as the adenylate cyclase system. Reports from other laboratories have suggested that p21 RAS may also interact with signaling systems other than phospholipase C to

absolute intoxication the parent

cells. Membranes untreated (0)

us to postulate that the role of inactivation by pertussis toxin in inhibiting 11-3-dependent

mediated

RAS

±

tive

implicating the

of mutant

Mean

I

might

p21 of

NeoRas.F3 NFS/Nl.Hi

adenylate NFS/Nl Hi

from

treated overnight with pertussis toxin 100 ng/mI ( . ). or from Hi NeoRas.F3 untreated (0) or treated with pertussis toxin 100 ng/mI overnight (A). Adenylate cyclase reactions were performed in the presence of 100 MM forskolin. with (right) or without (left) 1 or 10 lLmol/L GppNHp and graded

0 ‘5 . Mn2

5. Forskolin/Mn2-dependent activities in membranes

inhibiting these

observed paralleled such

two

agents

probe

of the in

pathway (Table be an adenylate

pertussis

toxin).

adenybate analogue

effect

of p21

study. effect

The on

IL-

in mutant RAS-containthe diminished efficacy their

supporting effects

1). The cyclase

strengthened by the failure of pertussis reverse resistance of Hi Neo Ras.F3 to POE, plus

our

inhibitory

proliferation, exert

by

to the cAMP

on the possibility inhibitor

the same that was

intoxication to (Table I ; POE,


RAS

ONCOGENE

Our divided

21

FUNCTION

studies of membrane into those in which

on OTP-dependent in which activation

forskolin

and

RAS

to inhibit assays under

observed presence

cyclase

would

in the

or without

presence

conditions

occur

independent

a capacity

for

and

might

OppNHp

(Fig

cyclase guanine

4).

direct

in

guanine

activation

of inhibitory

Pertussis

of

Neo Ras.F3 cells toxin. We therefore parental

cells

We

did

not

inactivation adenylate

membranes

Ras.F3 it is known

indirect

dependent Such

of the adenylate

of 3H-diacylglycerol nist) from the mutant compared directly

gisticably the

augmented

presence

uncouples high Mn2 the

by guanine

RAS

Oi-mediated concentrations

forskolin

except

nucleotide Mn2,

con-

forskoldirectly

In the

present

cyclase

of Hi

Neo

by guanine

mmol/L)

(Fig

OppNHp between of Hi

study,

(through

ty.

treatment Indeed,

5,

(Fig 5, adenylate Neo

Ras.F3

pertussis-intoxicated

left)

Mn2

and

concentrations

was

augmented

was

still (Fig

observed 5,

forskobin (data very reproducible observe

of parent

and

mutant

RAS-containing

for membrane inhibitors.

preparation

(Fig

particular

activi-

NFS/Nl.Hi but from

RAS

RAS nature

for

inhibition

is subject

of adenybate

of mutant paracrine

method

of phosphatase

basis

by p21

the we

phosphorybaby our

absence

mechanistic

importance

purely

is supported in the

activity

therefore, that cyclase activity

is not

conclusion

exact

function of the

cyclase

of

to debate, inhibition

as

in myeboid cells may be a of leukemic cell growth It has been

release

not

advantage

of cyclase

of growth

factors

but

initiating

to 2

from

only

on

(0.5

of

microenvironment.

exerts an inhibitory influence proliferation.5354 Recent evidence transformation,

activation

may but to an

not

be the

rather

already

may

advantage tors

clone of

would locally

over

other

of PGE,

transformed

which

progenitor-cell that RAS oncoevent

confer

dependent and stromal cell-associated crine growth model, RAS oncogene kemic influence

also

myeloid suggests

observed

in beukemic

a further but

growth

clone.55 activation

growth factorIn a parain the leu-

thus block the growth-regulatory produced PGE, allowing for growth normal

or dysplastic

myeboid

progeni-

(progression).

with

Mn2 concentrations of 4 mmol/L is consistent with the interpretation that p21 RAS directly mediates adenylate cyclase inhibition, which is GTP-dependent and is uncoupled by Mn2

the cyclase

gene

cells

We conclude, in adenylate cells

This

that

observation

not shown). decrease

in RAS-transfected

Although

cyclase

of parent

at of

the difference .Hi and that

adenylate

on adenylate

treatment

cells compared with control memreproducible between experiments and, not corrected by 4 mmob/L Mn2 and

tion-dependent.

ade-

by addition

The

kinase C agoH7 NeoRas.F3

cells labeled with 3H-gbycerol,26 we effect of phorbol (16 to 160 nmob/L ester

phorbol ester-treated branes was poorly when present, was

than

in membranes

top).

of forskolin-stimulated

a protein clone

On

recovery

that growth factor-dependent beukemic cells constitutiveby produce interleukin-1 (IL-i).52 The stimulation of bone marrow microenvironmental cells by IL-i results in the

Os is

higher

left v right). In addition, cyclase activity of NFS/N1 cells

near-equalization activities

at lower

itself.5’

in the hematopoietic

of Gs

membrane was

catalyst

in

that

uncoupling

of NFS/Nl.Hi

previously

of augmented

of NFS/N1.H7

phorbob

an effector consequence

Gs)

nucleotide-activated

forskolin-stimulated

activity Ras.F3

the

observed is syner-

inhibitory influences.M However, (eg, 4 mmol/L), augmentation

response

nylate

when

a concentration

diminished because of the Mn2-dependent from adenylate cyclase.”

cyclase

observation

(like phorbol, RAS-containing

parent the

was

reduced adenylate cyclase activity in some experiments, the reduced adenybate cyclase activity of membranes

the

from p21 4). Such a adenybate

It has also been cycbase activity

of 0.5 to 2 mmol/L

with assessed

components.

systems

correct

p21

proteins,

of our

cyclase

cells C-

treated with phorbol ester and from protein kinase C-mediated

previous

the

G

is high.#{176}’7 adenybate

erythrocytes to result

of diminished

RAS-containing protein kinase

signaling

phosphorylation basis

development

of adenylate

between

the

adenylate

regulatory

phosphorylation

observed in frog was demonstrated

adenylate

cyclase

for

activity in mutant p21 RAS-stimubated

“crosstalk”

activate adenylate cyclase but that their activation is subject to modulation by the inhibitory (Oi) or stimubatory (Gs) Mn2 concentration that forskolin-stimulated

mechanism

TPA)

of Gi by pertussis cyclase activity of versus

An

adenybate cyclase might be through

whether GTP ana-

in the presence of Mn2 and that both forskolin and Mn2

Neo

in the Hi

or to the existence of another inhibitory influence in Hi

left after compared

NFS//Nb.H7

taming Hi in. In fact,

toxin

the

influences.

cyclase activity of membranes cells to the parental level (Fig due either to greater inherent

activity in parent nucbeotide-dependent

p21

not be observ-

cells compared to parent NFS/Nl.Hi, by hormone or by nonhydrobyzable

lower adenylate RAS-containing result could be

to by

be demonstrable

but of

auxiliary catalyst

of Mg2

hormone

ver-

significantly bower adenylate cyclase activity of Mg2 from membranes of RAS-transfectant

NeoRas.F3 stimulated bogue,

cation-dependent)

that

certain

that

1949

cyclase activity were absolutely dependent

reasoned

adenylate

with

catalyst

We

performed

nucleotide

(Mg2

CELLS

GTP-dependent signals were of the adenybate cyclase

Mn2.

those

adenybate activity was

signaling

sus those primary

able

IN MVELOID

5).

ACKNOWLEDGMENT The authors thank Drs John W. Fleming, Henry August M. Watanabe, whose prior investigations cyclase system allowed us to pursue these studies.

R. Besch, Jr. and of the adenylate

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by

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ResCommun 37. Jakobs involved

Cyclic

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38.

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