of proliferation cells involves diminished efficacy of prostaglandin E1-mediated inhibition. Effector function for RAS oncogene in interleukin-3-dependent myeloid.
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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
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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
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
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
From bloodjournal.hematologylibrary.org by guest on July 10, 2011. For personal use only.
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
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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
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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-
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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,
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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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