Vol. 7, 75-81,
January
Antagonistic G0->G1-->S
Marcella
Actions of Phorbol Cell Cycle Transition’
Faria and Hugo A. Armelin2
Departamonto S#{227}o Paulo,
Cell Growth
1996
do BioquImica, C.P.26077,
do Quimica,
CEP 05599-970,
Abstract We have developed antagonistic
Instituto
S#{227}o Paulo-SP,
de
Brazil
that reveals two of phorbol-12-mynstate-12-acetate
(PMA) on the G0-*G1-*S
transition
of mammalian
cell
cycle. BaIb-3T3 (Clone A31) cells arrested in G0 by serum starvation can be stimulated to traverse the G1 phase and initiate DNA synthesis 12 h later by a 2-h pulse with PMA. In contrast with this early stimulatory effect, PMA has an inhibitory effect when presented to the cells during the last 6 h of G1. PMA is able to inhibit DNA synthesis initiation irrespective of the triggering agent, i.e., serum, fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, or PMA itself (presented as an early pulse) We have established that the critical period for the PMA inhibitory effect is between 6 and 8 h after cell stimulation. This dual effect of PMA is not a peculiarity of BaIb-3T3 (clone A31) cells because it is also observed with other fibroblastic cell lines, namely, SWISS 3T3, NIL 8, and RAT I , and also with the epithelial Y-1 adrenocortical cell line. Treatment with PMA for 0.5 or 2 h activates protein kinase C (PKC) in BaIb-3T3-A31 cells, but is not sufficient to down-regulate the enzyme because a second 30-mm PMA pulse applied between 6 and 6.5 h activates PKC again. On the other hand, a continuous 6.5-h PMA treatment causes PKC down-regulation; therefore, the inhibitory effect of PMA could be mediated by PKC. Growth factor early response proto-oncogenes cmyc, c-fos, and c-jun are induced transiently by both early and late PMA pulses, suggesting that these
genes are not involved in the PMA inhibitory
effect.
Received 8/8/95; revised 10/16/95; accepted 10/31/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1 734 solely to mdicate this fact. 1 This work was supported by FAPESP and CNPq. 2 To whom requests for reprints should be addressed. Phone: 0055-11818.7972;
Fax: 0055-1
1-815.5579;
E-mail:
[email protected].
by peptide
growth
factors
like PDGF3
paper
we show
that
PMA
has a dual
effect
a the (9), the (8). an by
in the
G0-G1-S transition: it is stirnulatory in early G1 (G0-G1 transition) and inhibitory in late G1 (G1-S transition). This novel PMA inhibitory effect was observed in several fibroblastic cell lines, suggesting that it is a general phenomenon in the cell cycle of mammalian cells. PKC isoforms are likely to be mediators of this PMA inhibitory effect, suggesting a link between this enzyme and the pathway that regulates G1-S traversing.
Results The protocols used are shown in Fig. i Fig. iA shows the protocols designed for the DNA synthesis stimulation experiments of Figs. 2-4; Fig. iB refers to PKC activity assays (Fig. .
5) and the induction
of early
genes
(Fig.
6).
G0-arrested
BaIb-3T3 cells initiate DNA synthesis on treatment with a 2-h pulse of FGF or PMA (Fig. 2). However, a second PMA pulse, between 6 and i2 h, blocks G1 traversing, abolishing
DNA
synthesis
initiation
(Fig.
2). The stimula-
tory effects of long (i2 h) treatment with FGF, EGF, or PDGF are also blocked by a late PMA pulse (Fig. 2). In a 12-h PMA treatment, the inhibitory effect prevails (Fig. 2). The
time
of exposure
to PMA
is critical
for
its mitogenic
effect. Thus, a 1 -h pulse is not sufficient but a maximal effect is observed on a 2-h treatment, decreasing upon a 4-h treatment (Fig. 3). The mitogenic effects of both 2- and 4-h PMA pulses are equally abolished on a second PMA treatmont
Introduction Mammalian cell cycle control is exerted mainly at the G0-G1--S transition (i , 2). BaIb-3T3 cells subjected to G0 arrest by serum deprivation can be stimulated to traverse G1
the S phase
and FGF (3). These growth factors are known to induce large set of early response genes among which are c-myc, c-fos and c-jun proto-oncogenes (4-8). The phorbol ester PMA, a well-known PKC activator mimics the mitogenic actions of growth factors, inducing same early response genes and stimulating S phase entry These results have prompted the notion that PKC is essential component in the regulatory routes activated PDGF and/or FGF receptors. In this
75
in Mammalian
and enter Universidade
a protocol
effects
Ester
& Differentiation
applied
between
6 and
i 2 h after
the
initiation
of the
pulse (Fig. 3). This DNA synthesis inhibitory effect of PMA seems to be restricted to a narrow period of the G1 phase between 6 and 8 h: PMA pulses between 6 and 8, 6 and 9, and 6 and i 0 h, respectively, abolish the mitogenic effect of FGF (Fig. 4), whereas later pulses, between 9 and 12 or i 0 and i 2 h, are not effective (Fig. 4). first
The abbreviations used are: PDGF, platelet-derived growth factor; PMA, phorbol-12-myristato-12-acetate; PPP, platelet poor plasma; FGF, fibroblast growth factor; EGF, epidermal growth factor; CDK, cyclin-dependent kinase; GAPDH, glyceraldehyde 3-phosphate dohydrogenase.
3
76
PMA on G0-+G1
Time
Transition
of Mammalian
Cell Cycle
A
(h)
-24
0
2
6xy
I
I
I
II
*
I
12
24
I
I
-R
*
Starvation
by serum depletion
2h pulse with PMA (P2) or FGF (F2)
*-.
-
-
-
Variable PMA pulse (I.e. P6112 describes
-
PBSA washing twice
U
4t 3H Thymldlne ,
Cells
addition
are collected for analysis
a pulse starting
atthe 6th and ending atthe 12th hour) 1 . Schemes of exporimental protocols. A, general procodures followed in the experiments of Figs. 2-4; B, experiments of Figs. 4 and 5, respectively. Fig.
Time
B
(h)
-24 I
00.5
2
3
66.5
9
II
I
I
II
I
* *
4
*
4 4 4
* * *
-j
4 4
*
4-
-
-
*
-
---I
PMA (P) or FGF (F) early pulse
.
Variable PMA (P) late treatment
4, Cells are
Starvation
by serum
PBSA washing
twice
collected
for analysis
depletion
The early response genes are transiently induced during the G0-G1--*S transition; therefore, we monitored c-fos rnRNA levels after 0.5 h and c-myc and c-jun after 3 h stimulation with PMA and/or FGF by Northern hybridizations (Fig. 5). PMA and FGF separately or combined cause identical c-fos, c-myc, and c-jun induction (Fig. 5). It is important to note that a 2-h FGF pulse triggers DNA synthesis stimulation to levels that are higher than that of a 2-h PMA pulse
(Fig. 2). However, the DNA synthesis stimulation due to the combination of FGF plus PMA, in a 2 h pulse, even at nonsaturating concentrations, does not overcome the effect of FGF alone (data not shown), indicating that there is no additive effect between FGF and PMA in this response. Balb-3T3 cells treated with an early 2-h PMA pulse do not display detectable levels of c-fos mRNA at 6.5 h or of c-myc and c-jun mRNA at 9 h (Fig. 5). However, a second PMA
Cell Growth
& Differentiation
U.
U.
3.0
c1
0
0
0
0
U-
Co 0
a-
U-
0
9#{149}E
w
U-
2.5
T
,,J. Fig. 2. Antagonistic actions of PMA. Balb-3T3 cells were serum starved for 24 h in medium containing 3% PPP and stimulated with 10% FCS, 20 ng/ml FGF (F), an early 2-h pulse of either FGF (F2) or PMA (P2), an early 2-h pulse of FGF or PMA, followed by a late 6-h pulse of PMA (F2 P6/12 and P2 P6/12) or with a sustained treatment of FGF (20 ng/ml), EGF(20 ng/ml), and platelet-derived growth factor (PDGF; 1 00 ng/ml), followed, respectively, by a late pulse of PMA (F P6/12, E P6/12, and PDGF P6/12). Mitogenic activity was measured by incorporation of [H]thymidine into DNA between 12 and 24 h; radioactivity was assayed by scintillation counting.
77
a.
x E
2.0
0
0) 0)
I .5 c1
a)
-C
C > C,)
CD
I .0
a. a.
a.
z 0
c,1
CD
CD
0
a.
a. z
CD
a.
a.
0.5
w
c,,1
[In
0.0
T
Treatments
C 0
60-
50U)
40-
Cl)
a, 3. Length of early PMA pulse is critical for stimulation. Serum-starved cells were treated either by a variable early pulse of PMA (U) or an early PMA pulse, followed by a late 6/1 2-h PMA treatment (#{149}). DNA synthesis stimulation triggered by serum and FGF was considered as maximal. Fig.
C
30-
z
20-
E
‘10-
0
0
0
a
2
I
4
Length of Early
treatment at 6.5 therefore,
with ond
initiated h and
at 6 h triggers and
c-myc
following
the
c-fos
c-jun same
induction
the first PMA pulse. The results PMA treatment are the same
mitogenic
factor
used
(Fig. 5). In conclusion, of the induction
G0-G1--S pattern
in the early
a second transition,
of c-los,
first few h, at least at the is blocking G1 transverse initiation.
mRNA
induction
induction
pulse
obtained irrespective
with the secof the first
at 6 h, causes and
c-jun
the
observed
(h)
again
at 9 h (Fig. 5), pattern observed
treatment, i.e. , FGF or PMA PMA treatment in the middle
i.e.,
c-myc,
PMA
We also assaying
modulation
activity
PKC activation
of PKC
activity
by PMA
by
of all kinases dependent on diolin and in both membrane and soluble fractions.
phosphatidylsenine,
is clearly
evidenced
by a 5-fold
increase
in
activity in the membrane fraction of PMA-treated (0.5 h) cells (Fig. 6A). A 2-h PMA pulse leads to similar levels of activation,
whereas
same
pronounced
in the
reduction (Fig. 6A).
mRNA level, even though PMA and abolishing DNA synthesis
measured the
drawal the
cells
a 6.5-h
PMA
treatment
PKC down-regulation, in activity However,
does
in both membrane a 2-h PMA pulse
not cause
resemble
control
is sufficient indicated
a
and soluble fractions followed by PMA with-
down-regulation cells
to cause
by a significant
by two
because criteria:
within (a) low,
6 h rela-
78
PMA
on G0-G1
Transition
of Mammalian
Cell Cycle
mammalian here
CD
I
for
lines,
the
cells,
CD
0
I
I
Results
Balb-3T3
cells
namely
Swiss-3T3
I
cell cycle. RAT
0
transition
is a well-known
cell
the
for
last
that
must
N U-
e
0
a-
a. a.
N
U-
cells.
stages
ofthe
Here
later
in G1
over
its
that
stimulatory
in fact
this
has a dual
effect
at the
and inhibitory effect
the
statement
of PMA
early
when applied
seems
to prevail
effect.
phenomenon
was
to explore
promoting
it is stimulatory
-transition
exten-
as a mitogenic
we show PMA
cells
studied
is known in
because
The late inhibitory
.
early
of Balb-3T3
been
factors
transition:
G0-*G1
has
PMA
growth
be reevaluated G0-G1--*S
that
underlined
by a protocol
the G0-+G1--+S
transition
(Fig.
1A)
in G0-arrested
6 and 8 h of stimulation
(Fig. 4). However,
this
inhibitory
the
0)
effect is not restricted to G0-arrested cells or to the particular experimental conditions used here. In fact, exponentially
U-
U-
and
a.
Co
0
cells),
BaIb-3T3 cells stimulated by serum, growth factors (FGF, PDGF, and EGF), and PMA (Fig. 2). Our protocol has provided evidence that PMA blocks G1 cell traversing between
0
N
fibroblastic
8 (hamster
of the cell cycle
30 years.
mimics
in the
This
1
presented
three
line of functional
response
transition.
designed
N
NIL
for
Discussion
G0-*G1--S
z
cells),
mouse
The G0-G1--S
agent
C,)
to those
found
cells) and, also, for the V-i adrenocortical
a tumorigenic
sively
C >
1 (rat
(mouse
equivalent were
growing Fig. 4. Period covered by the late PMA pulse is critical for inhibition. Balb-3T3 cells were serum starved for 24 h in medium containing 3% PPP and stimulated with serum (10% FCS), early FGF pulse (F2), or early FGF pulse followed by a late PMA pulse covering the indicated times (F2 P6/12, F2P6/1O, F2 P6/9, F2P6/8, F2P1O/12, and F2P9/12). Mitogenic activity was measured 12 h after the end of treatments as indicated in the legend of Fig. 2.
cells
indicating itory
effect
also
between this
Moreover,
other
are
inhibited
by a PMA
that these cells are susceptible
mammalian
G,-blocking
2 and
PMA
4 h before
inhibitory
cell
action
effect
lines
(data
of PMA
pulse
(Fig.
7),
to the PMA inhibentering
was
also
not shown).
described
S phase.
observed
with
Therefore,
here
is likely
the
to be a
common feature of the mammalian cell cycle. Measurements of PKC activity during PMA treatments of Balb-3T3 cells (Fig.
PKC activity in the membrane fraction; and (b) rapid response to a 0.5-h PMA pulse with a 3.5-fold increase in PKC activity in the membrane fraction (Fig. 6A). In conclu-
6) suggest
sion,
proto-oncogenes
tive
treatment
separated with
of BaIb-3T3
by a 4-5
respect
to PKC
cells
with
short
h interval,
yields
activation.
Therefore,
tory and the inhibitory tion may be mediated
PMA
equivalent both
pulses,
responses the stimula-
effects of PMA in G0-G1--S transiby PKC activation. In fact, both effects
display the same dose the PMA concentration
response curve, that coincides range required to activate PKC
with (data
not shown). On the other hand, treatment of Balb-3T3 cells a 2-h FGF pulse does not activate PKC (Fig. 6B). More-
are
that
mediated Induction
both
c-myc,
the activity
resembles
Results
from
several
by us (1 1) and others of c-myc,
stimulation.
(Fig. 6B).
effect
restricted with
checked
the
to the G0-*G1--*S
exponentially
tions. The labeling
G1
whether growing
transition cells,
index results
of exponentially by a PMA pulse 2-4
inhibitory
or could
under
shown
steady
effect
was
state
in Fig. 7 indicate
condi-
that
both G0-arrested cells and exponentially growing cells present a G1 checkpoint at which cells are sensitive to the PMA growth-blocking effect. The dual action of PMA described here is not a peculiarity
fore,
of
the
Balb-3T3
cell
line,
but
may
be characteristic
of the
between These
G1-*S
on
the expression
be observed
growing Balb-3T3 cells is inh before S phase entry. There-
traversing
hibited
PMA
,
Data years
that
of the
the induction
is not cell cycle (i 0). Thus,
0 and results
traversing
is unrelated
by PMA
of PMA treat-
3 h or between that
specific
induction
of the period
suggest
cells the
it is not surprising
and c-jun
c-fos,
irrespective
of these
effects
particularly
indicate
proto-oncogenes
a 2-h FGF treatment renders cells capable of responding to PMA, 4 h later, with a large increase in PKC activity
i.e.
genes,
PMA
is necessary for G0-arrested DNA synthesis (1 0). However,
of these
(Fig. 5) is the same
We have
of growth
induction
that the pattern
inhibitory
and c-jun, by PMA, closely factors like PDGF and FGF.
laboratories
as shown
ment,
and
c-fos,
of these proto-oncogenes to traverse G1 and initiate
with
over,
stimulatory
by PKC activation. of the early response
6 and
the
PMA
9 h of
inhibitory
to its ability to induce
genes.
from a number of laboratories during the last ten have led to the notion that the products of proto-
oncogenes
and
of tumor
suppressor
genes
(anti-oncogenes)
are organized
into two separated circuits that regulate on the G0-G1--*S transition (2, 8). Proto-oncogene
controls
products are
activated
exit
toward
early
This
are concentrated
in the regulatory
growth factor G1. PMA, through
response
mechanism
by
genes
by
the
can explain
pathways
receptors and PKC activation, pathway
of this
the mitogenic
stimulate induces growth
action
the
that G0 the factor.
of PMA.
Cell Growth
& Differentiation
Lt)
0
Lt
LflLflO .
LiQ-U Leftpanel, early gene induction by early PMA treatment. After a 24-h serum starvation in medium containing 3% PPP, Balb-3T3 cells were treated with 10 ng/ml PMA, 20 ng/ml FGF, or the combination of both for 0.5 h (P0.5, FO.5, and F+PO.5) and for 3 h (P3, F3, and F+P3). After the indicated times, RNA was isolated and analyzed for c-fos (A), c-myc (B), and c-jun (C) expression as described in “Materials and Methods.” GAPDH mRNA levels were used as control of the amount of loaded RNA (A, B, and C). Right panel, early gene induction by late PMA treatment. After a 24-h starvation period in medium contaming 3% PPP, Balb-3T3 cells were treated with an early pulse of 10 ng/ml PMA or 20 ng/mI FGF and collected at 6.5 h (P2/6.5 and F2/6.5) or at 9 h (P2/9 and F2/9) or had the early treatmont with PMA or FGF, followed by a late 0.5-h pulse of PMA (P P6/6.5 and F2 P6/ 6.5) or by a late 3-h pulse of PMA (P2 P6/9 and F2 P6/9). After the indicated time penods, RNA was isolated and analyzed for time c-fos, c-myc, c-jun, and GAPDH mRNA levels. Fig.
F
U,
A
GAPDHF
I
0
5.
c-fos
L()
.
U Li
I
,
(OQ.(DO. (s4bsqJ
LLUQ.Q
I
c-fos ,-
GAPDH uQLL
N)
B
c-myc
W
(noQU e LI.LLUQ.Q.
(0 a)O
GAPDH C
-myc
C -jUfl
c-jun
C
GAPDH
GAPDH
On
the
other
blastoma the
circuit
that
CDKs seems be due
ucts, the
with thus,
G1-’S
Materials Cells
and
the
PMA
the
,
and
circuit
products
p1 6) seem the
transition.
response
cycle-specific of a PKC of CDK
and
blocking
Rb phosphorylation
transition
to occur.
genes
event
effect
(retino-
to play a role
G1-S
the early
inhibitory
to activation
and Cell
with
to be a cell
that
nection
anti-oncogene
p21
regulates
in contrast
propose may
the
p53,
activation, tion,
hand,
protein,
Mitogenic
in
CDKs induc-
(2, 12).
We
G1 cell traversing
of
isoform
that
has a con-
anti-oncogenes that
is required
prodfor
Methods
Culture
Conditions
Frozen Stocks of Balb-3T3 Cells. Clone A31 (1 3) was kept in liquid nitrogen. Cultures were grown in DMEM with 1 .2 g/liter sodium bicarbonate plus 1 0% FCS under a 5% C02/air atmosphere. Cultures were grown continuously to near confluence and were subcultuned every 3-4 days.
Activity
Assays
DNA Synthesis Stimulation-Synchronic Populations. Confluent cultures were serum starved for 24 h in medium containing 3% PPP. DNA synthesis of these G0-G1-arrested cells was stimulated by serum, growth factors, or PMA. [3H]thymidine (0.25 MCi/ml;10 M) was incorporated between 12 and 24 h after triggering DNA synthesis stimulation and after incorporation into DNA was measured by scintillation counting as described previously (14). Labeling lndox-Asynchronic Populations. Exponentially growing coverslip cultures were treated with PMA (1 0 ng/ml) for 2 h and then pulse-labeled with [H] thymidine (5 MCi/ml;10 M) for 15 mm every 2 h after the PMA pulse. Coverslips were extracted twice with cold 10% tnichloracetic acid, washed with 95% ethanol, air dried, and covered with Kodak AR-b stripping film. After 10 days of exposure, the film was developed and cells were stained with Giemsa. Labeling index was estimated by scoring the percentage of labeled nuclei under light microscope by counting at least 300 cells/coverslip. The treatment referred to as early PMA pulse consists of: (a) PMA addition
at time
medium
removal;
zero;
(d) washing
(b) incubation for the desired period of time; (C) of cell monolayers twice with PBS; and (e)
addition
of fresh
medium.
Control
dishes
were
subjected
to the
same
79
80
PMA
on G0-#{247}G1 Transition
A
of Mammalian
Cell Cycle
B
PMA
FGF
8
F2 P6/6.5 3SPPP
3sppp
I
C
‘JE
E6 P0.5
x
P2
> .
a
P2
6.5
P2
P6/6.5
EJL1
.5
F2
x2 P6.5
E
iLjL
Fig. 6. PKC activation by PMA treatments. After a 24 h starvation in medium contaming 3% PPP, Balb-3T3 cells were stimulated with 10 ng/ml PMA (A) and 20 ng/ml FGF (B) for the indicated times. PKC activity was measured as described in “Materials and Methods” for the membrane (#{149}) and soluble (LI) fractions. Columns, mean of three independent experiments, each with duplicates. “, statistically significant differences (student t test; P < 0.001) between control and experimental conditions, comparing ities
0
either or
tivities
1
membrane
activ-
membrane:soluble ratios.
ac-
Treatments Membrane Soluble
6045.
40Fig. 7. PMA inhibitory action is exhibited by exponentially growing cultures. Exponentially growing Balb-3T3 cells were treated for 2 h with PMA (#{149}) and then washed twice with PBS. Every 2 h, duplicate coverslips were pulse labeled for 15 mm with H]thymidine and extracted as described in the “Materials and Methods.” The same procedure was adopted for the control population growing in 10% FCS (1) for a 10-h period.
a, 0
35.
z a,
30-
a,
0)
-I
2620-
-U----
Control
-.--
PMA Pulse
150
2
4
washing procedures. PMA removal by PBS washing was monitored by using a radioactively labeled PMA analogue, i.e., [20 N Hlphorbol-12-13dibutyrate. Results shown in Figs. 1-3, and 6 are representative of at least three independent experiments in each case.
RNA
Extraction
and Northern
Hybridization
Analysis
Confluent cultures in 1 50-mm diameter dishes were serum starved for 24 h in medium containing 3% PPP. Treatment with FGF (20 ng/ml), PMA(10 ng/ml), or a combination of both was performed for different periods of time as indicated in “Results.” Cell monolayers were lysed in 4 M guanidine isothiocyanate (1 5), layered on top of a 5.7 M CsCI-25 mi sodium acetate (pH 5.0) solution, and centrifuged to equilibrium in a SW5O.1 rotor. Total RNA recovered from the CsCI pellet was dissolved in water, quantified by UV absorption, and used for Northern hybridization analysis as follows: 10
8
6
Time After Treatment
10
(h)
ng total RNA in 4-morpholinepropanesulfonic acid-formaldehyde was heated for 1 5 mm at 65”C, fractionated in 1 % agarose gels, and transferred to nitrocellulose filters in 1 5x SSC. The DNA probes used for hybridization were: pFBJ-2fos for c-fos (16), LK588-c-jun4 for c-jun, pBCOOlmyc for c-myc (17), and prGAPDH1 for GAPDH (18). All of the plasmids were labeled with [a-32P]dCTP by random primer extension (19).
Protein
Kinase
C Activity
Determination
Confluent cultures in 1 50-mm diameter dishes were serum starved for 24 h in medium containing 3% PPP. PMA, FGF, or a combination of both
4
R. Bravo,
unpublished
data.
Cell Growth
were added for different periods of time as indicated in “Resuits.” Cuitures were washed twice with a physiological saline buffer without calcium; cells were collected in 1 .5 ml buffer A [20 mi Tris-HCI (pH 7.5), 2 mti EDTA, 0.5 M EGTA, 2 mM phenylmothylsulfonyl fluoride, 25 ng/mI loupeptin, and 0.33 M Sucrose] and homogenized with 30 strokes of a Teflon glass homogenizer. The homogenates were centrifuged at 100,000 x g for 45 mm at 4#{176}C, and the supornatants were collected and kept at 4”C to assay for PKC activity associated with the soluble fraction. The pellets were washed in 1 .5 ml buffer B (buffer A without the sucrose) and homogenized with 20 strokes, with NP4O added to a 1 % final concentration. The pellet homogenates were mixed for 40 mm at 4”C and centrifuged at 100,000 x g for 45 mm at 4”C. The supematants were collected to assay for PKC activity associated with the membrane fraction. Soluble and membrane samples were loaded onto a DEAE-cellulose column (300-pJ bed volume), oquilibrated in 20 mM Tris-HCI (pH 7.5), and kept under agitation for 40 mm at 4”C. Columns were washed with 2 ml buffer B for 40 mm and eluted with 0.5 ml buffer B containing 0.15 M NaCI. PKC activity was assayed in so-M1 aliquots containing 20 m Tris-HCI, 25 ng histone Hi , 0.75 m CaCI2, 10 rnM magnesium acetate, 50 mg/mI leupoptin, 100 m [y-32P]ATP (120 cpm/pmol), and with or without lipids as described (20).
Gmwth
Factors,
Drugs,
and Isotopes
C. S. Arrnelin
for critical
reading
of the manuscript.
References 1 . Pardee, (Washington 2. Scherr, 1994.
A. B. Gi events and DC), 246: 603-608, C. J. Gi phase
regulation 1989.
progression:
cycling
of cell proliferation.
5. Kelly, regulation 603-610,
ofpeptide growth Genes and Growth Inc., 1987.
B. H., Reffel, A. C. and Stiles, C. 0. Molecular cloning regulated by PDGF. Cell, 33: 939-947, 1983. K., Cochran, ofthe c-myc 1983.
Science
on CUE. Cell, 79: 551-554,
3. Armelin, H. A., and Armelin, M. C. S. The interactions factors and oncogenos. In: G. Guroff (ed), Oncogenes, Factors, pp. 331-373. New York: John Wiley & Sons, 4. Cochran, sequences
8. Herschman, H. R. Primary and tumor promoters. Annu. 9. Nishizuka, duction
response genes induced by growth Rev. Biochom., 60: 281-319, 1991.
Y. The role of protein
and tumor
promotion.
Nature
kinase
C in cell surface
(Lond.),
308:
693-698,
factors
signal
trans-
1984.
10. Angel, P., and Karin, M. The role of Jun. Fos and the AP-1 complex in cell-proliferation and transformation. Biochim. Biophys. Acta, 1072: 129-157, 1991. 1 1 . Kimura, Induction of ester but not Endocrinol.,
E., Sonobe, M. H., Armolin, M. C. S., and Armelin, H. A. FOS and JUN proteins by adrenocorticotropin and phorbol by 3,5-cyclic adenosine monophosphate derivatives. Mol. 7: 1463-1471 , 1993.
12. Moreno, phase ofthe 1994.
S., and Nurse, P. Regulation cell cycle by the sumi +Gene.
of progression through the Gi Nature (Lond.), 367: 236-239,
14. Armelin, M. C. S., Gambarini A., and Armelin H. A. On the regulation of DNA synthesis in a line of adronocortical cells. J. Cell Physiol., 93: 1-10, 1977. 15. Chirgwin, J., Przybyla, A. E., McDonald, R. J., and Ruttor, W. J. Isolation of biologically active RNA from sources enriched in ribomilease.
Acknowledgments Dr. Man
7. Almendral, J. M., Sommor, 0., Macdonald-Bravo, H., Burckhardt, J., Perera, J. and Bravo, R. Complexity of the early response to growth factors in mouse fibroblasts. Mol. Cell. Biol., 8: 2140-2148, 1988.
13. Aaronson, S. A., and Todaro, J. G. Basis for the acquisition of malignant potential by mouse cells cultivated in vitro. Science (Washington DC), 162: 1024-1026, 1968.
FGF and EGF from Boehringer Mannheim; PMA from Sigma; and [H]mothylthymidine (83 Ci/mmol), [‘y2P]ATP (3000 Ci/mmol), and [a32P]dCTP (3000 Ci/mmol) were from Amersham.
We thank
& Differentiation
of gene
B. A., Stiles, C. 0. and Leder, P. Cell specific gene by lymphocyte mitogens and PDGF. Cell, 35:
6. Lau, L F., and Nathans, during GO-Gi transition 3151, 1985.
D. Identification of a set of genes expressed of cultured mouse cells. EMBO J., 4: 3145-
Biochemistry,
18: 5294-5299,
1979.
16. Curran, FBJ murine
T., Peters, G. Van Boveren. C., Teich, N. M. and Vorma, I. M. osteosarcorna virus: identification and molecular cloning of
biologically
active
proviral
DNA.
J. Virol.,
44: 674-682,
1972.
1 7. Stewart, T., Pattengali, P. K., and Leder, P. Spontaneous adenocarcinoma in transgonic mice that carry and express fusion genes. Cell, 38: 627-637, 1984.
mammary MMTV/myc
18. Piechaczyc, M., Blanchard, J. M., Marty, L, Dany, C., Panabieres, F., El Sabouty, S., Fort, P. and Jeanteur, P. Post-transcriptional regulation of glyceraldehyde-3-phosphate-dihydrogenase gene expression in rat tissues. Nucleic Acids Ros., 12: 6951-6963, 1984. 19. Feinberg, A. P., and Vogelstein, B. A technique for radiolabelling restriction endonuclease fragments to high specific activity. Biochem., 132: 6-13, 1983.
DNA Anal.
20. Thomas, T. P., Gopalakrishna, R., and Anderson, W. R. Hormone and tumor promoter-induced activation or membrane association of protein kinase C. Methods Enzymol., 141: 399-408, 1990.
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