(Received for publication, October 31, 1995; and in revised form, February 5, 1996)
From the
Prostaglandin A (PGA
) reversibly blocked
the cell cycle progression of NIH 3T3 cells at G
and
G
/M phase. When it was applied to cells synchronized in
G
or S phase, cells were blocked at G
and
G
/M, respectively. The G
/M blockage was
transient. Microinjected oncogenic leucine 61 Ras protein could not
override the PGA
induced G
blockage, nor could
previous transformation with the v-raf oncogene. The
serum-induced activation of mitogen-activated protein kinase was not
inhibited by PGA
treatment. These data suggest that
PGA
blocks cell cycle progression without interfering with
the cytosolic proliferative signaling pathway. Combined microinjection
of E2F-1 and DP-1 proteins or microinjected adenovirus E1A protein,
however, could induce S phase in cells arrested in G
by
PGA
, indicating that PGA
does not directly
inhibit the process of DNA synthesis. In quiescent cells, PGA
blocked the normal hyperphosphorylation of the retinoblastoma
susceptible gene product and the activation of cyclin-dependent kinase
(CDK) 2 and CDK4, in response to serum stimulation. PGA
treatment elevated the p21
protein
expression level. These data indicate that PGA
may arrest
the cell cycle in G
by interfering with the activation of
G
phase CDKs.
For cell cycle transition from G/G
to S
phase, at least three types of molecular systems are involved in a
concerted manner. These include: 1) the signal transduction pathway
which receives extracellular signals and transmits these into the cell,
2) cyclins with associated kinases and modulators which may regulate
passage through the G
restriction point and other cell
cycle check points, and 3) the metabolic processes required for
doubling the essential cellular components including DNA.
While a
variety of signaling systems work together to either induce or block
proliferation, one of the best characterized, and perhaps the most
universally required signaling systems for proliferation, involves
proto-oncogenes including cellular Ras proteins. When growth factors
bind to their tyrosine kinase receptors, a series of phosphorylations
and resulting intermolecular interactions induce the activation of
cellular Ras proteins(1) . Active Ras in turn binds to cellular
Raf kinases resulting in their activation and ultimately the activation
of mitogen-activated protein kinases (MAP ()kinases)(2) . The activated MAP kinases enter the
nucleus and presumably stimulate the activity of genes required for
proliferation (3, 4) .
Even though cellular proliferation in most cell types requires the activity of the above signal transduction system, the orderly transit of the growth factor-stimulated cell through the cell cycle depends upon the action of the second class of nuclear proteins, including cyclins, CDKs, and proteins which modulate the activity of the complexes they form. It is believed that cyclins and associated CDKs control progress through cell cycle phases. In so doing they would regulate the activity of the third group of molecules required for cell proliferation, those which catalyze the metabolism required to duplicate DNA and other critical cellular components necessary for cell division. For example, active cyclin D/CDK4 is known to phosphorylate the pRb(5, 6) . Hyperphosphorylation of the pRb results in the release of the transcription factor complex E2F/DP which is bound to and inactivated by hypophosphorylated pRb(7) . Active E2F/DP is known to induce the transcription of molecules required for DNA synthesis, such as dihydrofolate reductase(8) .
A full
understanding of the control of cell cycle progression from G to S phase will require not only an understanding of these three
separate processes essential for cell cycle progression (signaling
molecules, cyclins and associated proteins, and enzymes required for
DNA synthesis), but an understanding of how these classes of proteins
interact with each other. As described above and elsewhere (9) , the molecular mechanism connecting the activity of
cyclins and their associated proteins to DNA synthesis is well
characterized. Of particular interest in this study is the interaction
between proto-oncogene signaling molecules and cyclin-associated
proteins. Direct evidence for such an interaction was obtained in a
recent microinjection study. The proliferation of most normal cells is
blocked following the microinjection of a neutralizing anti-Ras
antibody(10) . When cells which have received such injections
receive a subsequent injection of purified adenoviral E1A protein which
is able to release E2F/DP from pRb(7, 11) , the cells
are able to rapidly enter S phase with high efficiency(12) .
Thus, the activity of E2F/DP, a normal consequence of cyclin-associated
protein action, is able to compensate for blockage of the action of
proto-oncogene signaling molecules. It therefore appears that Ras
activity in the cell is required for entry into S phase to a certain
extent based upon its ability to ultimately lead to the stimulation of
cyclin/CDK complexes.
In addition to the injection experiments
described above, other types of studies, such as cyclin D1 elevation by ras(13) or cyclin D1 promoter activation by ras(14) , make it clear that proto-oncogene signaling
molecules must be involved in controlling cyclin-related proteins,
although how this occurs is not well understood yet. This study has
identified a reagent which is likely to provide important information
in the search for this connection. The evidence presented here
indicates that PGA, a member of the growth-inhibitory
cyclopentenone prostaglandin family(15) , does not interfere
with the action of any of the proto-oncogene signaling molecules listed
above, whereas it does block the activation of G
phase
cyclin/CDK complexes and the hyperphosphorylation of pRb. The fact that
its inhibition can be overcome by E2F-1/DP-1 injection or by adenoviral
E1A protein injection clearly indicates that it does not block any
enzymatic function required for DNA synthesis. These are the
characteristics predicted for an inhibitor whose target is closely
related to the connection point between the action of signaling
molecules and cell cycle regulatory proteins.
To determine an inhibitory range of PGA, rapidly
growing cultures of NIH 3T3 cells were treated with various
concentrations for 24 h, labeled with
[
H]thymidine for 4 h at the end of the PGA
treatment, fixed, and autoradiographed. Parallel cultures were
treated similarly except that, after 24 h, the PGA
was
removed and replaced with normal medium containing
[
H]thymidine for an additional 24 h, to determine
reversibility. Concentrations of PGA
higher than 20
µM efficiently blocked thymidine incorporation, while
concentrations of 25 µM or less were found to be
reversible (Fig. 1A). Reversible inhibition was
observed even after 48 h of PGA
treatment, although
continued inhibition required addition of fresh PGA
every
24 h (data not shown). Concentrations above 35 µM were
cytopathic.
Figure 1:
Reversible inhibition of
NIH 3T3 and v-raf transformed cells by PGA. A, rapidly growing NIH 3T3 cells (circles) or
v-raf transformed NIH 3T3 cells (triangles) were
treated with various concentration of PGA
for 24 h. For the
last 4 h, the cells were labeled with
[
H]thymidine (open symbols). In parallel
plates, after 24 h of PGA
treatment, the cells were washed
3 times with fresh medium containing no PGA
and cultured
for another 24 h in the presence of [
H]thymidine (closed symbols). After autoradiography, the labeling index
was determined by counting more than 200 cells. The error bars indicate standard error (n = 2). B,
rapidly growing NIH 3T3 cells were treated either with 1 mM hydroxyurea (squares) or 25 µM PGA
(open circles), or ethanol, the solvent used for
PGA
treatment (closed circles). The cells were
pulsed with [
H]thymidine for 1 h at the indicated
times after addition of chemicals. After autoradiography, the labeling
index was determined by counting more than 200 cells. Similar results
were obtained in two separate experiments.
To further analyze the point at
which PGA inhibits cell cycle progression, asynchronously
growing cultures were treated with PGA
for 24 h, and their
DNA content was analyzed by flow cytometry. In accordance with
previously reported data, it was apparent that blockage at multiple
points takes place(28) . Such cultures exhibited an increased
proportion of the cells in both G
and G
/M
phases, while the number of the cells in S phase was reduced (Fig. 2, A and B).
Figure 2:
PGA arrested the cell cycle
at G
and G
/M. The DNA content of the cell was
analyzed by flow cytometry monitoring the fluorescence intensity of the
propidium iodide-stained cells. A-G, NIH 3T3 cells; H and I, v-raf-transformed NIH 3T3 cells. A, rapidly growing cells; B, cycling cells treated
with 25 µM PGA
for 24 h; C,
serum-starved quiescent cells (G
arrested population); D, serum-starved cells were stimulated with 10% serum for 15 h
(synchronous S-phase population); E, quiescent cells were
treated with 25 µM PGA
in the presence of 10%
serum for 15 h; F, serum-starved cells were stimulated with
10% serum for 24 h; G, PGA
was added to
synchronous S-phase cells for 9 h; H, rapidly growing
v-raf-transformed NIH 3T3 cells; G,
v-raf-transformed NIH 3T3 cells treated with 25 µM PGA
for 24 h.
In order to confirm that
cell cycle blockage can take place in either G or
G
/M phase, synchronized cultures were treated with
PGA
. When the culture was synchronized in G
by
serum deprivation (Fig. 2C) prior to treatment with
PGA
and stimulation with serum, essentially all the cells
were blocked in G
as indicated by their DNA content (Fig. 2E). Control cultures stimulated with serum in
the absence of PGA
progressed into S phase (Fig. 2D). Cultures treated with PGA
at 15
h after serum addition, when most cells were in S phase (Fig. 2D), appeared in G
/M phase after 24 h (Fig. 2G). The fact that most of the S phase cells
progressed into G
/M phase in the presence of PGA
confirms the previous results indicating that PGA
does not block the progress of an ongoing S phase. The fact that
few of these cells had progressed through mitosis into G
,
as would have been the case in the absence of PGA
(Fig. 2F), indicated the presence of a
G
/M phase block by the inhibitor. There was, however, a
clear distinction between G
and G
/M blockages.
While the G
arrest could be maintained for 48 h by
replenishing the PGA
-containing medium every 24 h, the
majority of G
/M arrested cells, in the presence of
PGA
, eventually went through the mitosis and were blocked
in G
at 20 h after the addition of PGA
to
synchronous S phase cells (data not shown). This indicates that
PGA
-induced G
/M blockage is transient or that
PGA
treatment results in the elongation of time required
for progression through G
/M phases. The blockage at two
different cell cycle points may indicate that PGA
targets a
molecule(s) important for the regulation of cell cycle progression at
multiple points.
Finally, the point in G at which
PGA
exerts its inhibition was determined. For this
experiment, NIH 3T3 cultures were synchronized in G
by
serum deprivation for 48 h. Serum and
[
H]thymidine were then added to the cultures. In
one set of such cultures, PGA
was added to separate dishes
at various times after serum addition. These cultures were all fixed at
24 h after the initial addition of serum. If the cells failed to become
labeled, it would indicate that the added PGA
had blocked
entry into S phase. As an indication that PGA
functions
late in G
, it was found that even when it was administered
6 h after serum addition, PGA
was efficiently able to block
DNA synthesis. The efficiency of inhibition was reduced when PGA
was added later than 6 h after serum addition, and, at 10 h,
little inhibition was observed (Fig. 3). To determine the normal
time of entry into S phase, a parallel set of quiescent cultures
received serum and thymidine, but no PGA
. These cultures
were fixed at various times after the addition of serum. In these
cultures, the cells became labeled with thymidine beginning 10 h after
serum addition until reaching a maximum at 15 h. This indicates that S
phase began between 10 and 15 h after the addition of serum to these
cultures. It is not known how long it takes PGA
to exert
its inhibitory effects after being added to a culture, but these data
indicate that it is inhibitory as long as it is added approximately 3 h
prior to the onset of DNA synthesis. This result is consistent with its
execution point at or near the restriction point late in G
at which a cell becomes committed to enter S phase and complete
another cell cycle.
Figure 3:
PGA-induced G
arrest is localized to late G
phase. NIH 3T3 cells were
made quiescent by serum starvation. To determine the time course of
entry into S-phase after serum restimulation, the cells received 10%
serum and [
H]thymidine together and were fixed at
various times there after (closed circles). To locate the
PGA
blockage point, cultures received serum and
[
H]thymidine simultaneously but were treated with
25 µM PGA
at various times after the serum
addition (open circles). These cultures were fixed at 24 h
after the serum addition. In this case, labeled cells indicate the
failure of the PGA
treatment to block the cells in the
G
cell cycle phase. Labeling index was determined by
counting more than 200 cells after autoradiography (n =
3).
Figure 4:
Microinjection studies. NIH 3T3 cells on
coverslips were made quiescent by serum starvation for 48 h. Quiescent
cells then received medium containing 10% calf serum and 25 µM PGA and were cultured for 12 h. All the cells inside
the circle, drawn on the back of the coverslip (approximately
100-200 cells), were injected with the protein(s) indicated (solid bars in PGA
section). Injected proteins
include Leu-61 Ras , E2F-1, DP-1, E2F-1/DP-1, and E1A. As positive
controls, serum-starved (SS) quiescent NIH 3T3 cells were
injected (solid bars in SS section). The noninjected cells
outside the circle served as controls for each injection (shaded
bars). After microinjection, cells were labeled with
[
H]thymidine for 20-24 h, and the labeling
index was determined by autoradiography. For the noninjected cells,
more than 200 cells were counted, and for injected cells, the mean
value of counted cells was 125 with the minimum counting of 80 cells.
The number of experiments for each injection is indicated by numbers (n =) in the graph.
To confirm the observation that PGA interferes with proliferative signaling at a point subsequent to
the action of cellular Ras, the action of two well characterized
downstream targets of Ras activity were analyzed. The effect of
PGA
upon v-raf-transformed cells was first
examined. A number of biological and biochemical observations confirm
that biologically active cellular Ras proteins induce the activation of
cellular Raf proteins. When PGA
was added to NIH 3T3 cells
transformed by oncogenic raf, thymidine incorporation was
blocked in a dose-dependent and reversible manner (Fig. 1A). In addition, when raf-transformed
cells were treated with PGA
and subjected to FACS analysis
24 h later, the cells were seen to be inhibited in G
and
G
/M (Fig. 2, H and I), as was
observed with nontransformed NIH 3T3 cells. The fact that raf-transformed cells were effectively inhibited in cell cycle
progression by PGA
is consistent with the idea that
PGA
inhibits a molecule whose function is required
subsequently to the action of cellular Raf protein.
The biological
studies were extended with biochemical analysis of MAP kinase, a
molecule whose activity is stimulated by both Ras and Raf activity, and
which therefore functions downstream of both in proliferative
signaling. The activity of MAP kinase was assessed by
immunoprecipitation with specific anti-ERK2 antibodies followed by
incubation with a substrate, myelin basic protein, in the presence of
[-
P]ATP. The amount of the myelin basic
protein phosphorylation indicates the level of MAP kinase activity in
the cell at the time of immunoprecipitation. Addition of serum to
serum-deprived cells results in a rapid, more than 10- to 20-fold,
increase in the activity of MAP kinase compared to quiescent cells (Fig. 5, lanes 2 and 3). When PGA
was added together with serum or when the cells were pretreated
with PGA
for 2 h prior to the addition of medium containing
serum and PGA
, the activation of MAP kinase was not altered (Fig. 5, lanes 4 and 5). PGA
itself has little effect on the basal MAP kinase activity (Fig. 5, lanes 1 and 2). The fact that neither
injected Leu-61 Ras nor transformation with oncogenic raf is
able to override PGA
inhibition, together with the fact
that serum-induced activation of MAP kinase is not altered by PGA
treatment, clearly indicates that it blocks an activity which is
essential to proliferation but which is not apparently involved in the
action of several well characterized proto-oncogene components of the
proliferative signal transduction pathway.
Figure 5:
Serum-induced MAP kinase activation was
not inhibited by PGA treatment. Serum-starved quiescent NIH
3T3 cells were harvested after treatment with 25 µM PGA
for 2 h (lane 1), nothing (lane
2), 10% serum and solvent for 5 min (lane 3), 10% serum
and 25 µM PGA
for 5 min (lane 4), or
25 µM PGA
pretreatment for 2 h followed by 10%
serum and 25 µM PGA
for 5 min (lane
5). Cell lysates were made and MAP kinase was immunoprecipitated
using anti-ERK2 antibody. The kinase activity in the immunoprecipitate
was determined using myelin basic protein as a substrate. Similar
results were obtained in all three such experiments
performed.
Although E2F-1 microinjection induced S phase in serum-starved
cells, it failed to efficiently override the PGA G
arrest (Fig. 4). Since DP-1 is another transcription
factor which associates with E2F-1 and potentiates the E2F
transcription activity (7) , we tested the S phase inducing
activity of DP-1 protein. While microinjection of DP-1 alone could
induce S phase in serum-starved cells, it also failed to override
PGA
blockage. Combined microinjection of E2F-1 and DP-1,
however, was able to override the PGA
growth arrest
efficiently. As a biological marker of proliferative control which is
somewhat upstream of E2F/DP-1 activity, we next microinjected E1A
protein. When purified E1A protein was microinjected into NIH 3T3 cells
treated with PGA
, the cells were efficiently induced to
incorporate labeled thymidine (Fig. 4). Because E2F-1/DP-1 and
the modulation of the activity of the molecules targeted by E1A were
able to overcome PGA
inhibition, this inhibition must
involve molecules required for the control of proliferation rather than
a metabolic process required for DNA synthesis. Furthermore, it is
clear that PGA
inhibits a step involved in cell cycle
control which functions prior to the action of the targets of E1A and
the release of E2F/DP-1 transcription factor.
Figure 6:
The hyperphosphorylation of pRb was
inhibited by PGA treatment. Serum-starved NIH 3T3 cells (lane 1) or serum-starved cells stimulated with serum and
solvent (lane 2) or with serum and 25 µM PGA
(lane 3) were analyzed. After 12 h of
culture (at early S phase; see Fig. 4.), cells were lysed and
pRb was immunoprecipitated. The electrophoretic mobility of pRb was
monitored by Western blotting. Fast migrating hypophosphorylated pRb (Hypo-P-pRb) and slowly migrating hyperphosphorylated pRb (Hyper-P-pRb) were detected as well as IgG heavy chain (IgG HC) and light chain (IgG LC) which were used for
immunoprecipitation. Similar data were obtained in two separate
experiments.
There is extensive evidence that pRb phosphorylation requires the
activation of cyclins and their associated kinases. Cyclin D/CDK4
becomes an active kinase in mid to late G and has the
ability to phosphorylate pRb directly(32) . Later in
G
, apparently near the restriction point, cyclin E/CDK2
becomes active. The effect of PGA
upon these two kinases
was, therefore, analyzed. NIH 3T3 cells were deprived of serum for 48 h
prior to the addition of serum alone, or serum and PGA
together. These cells were cultured for an additional 10 h and
lysates were prepared. From these lysates, immunoprecipitates were made
with antibodies against CDK2, CDK4, cyclin D, or cyclin E. The
immunoprecipitates were then incubated with the appropriate substrate
to detect kinase activity; recombinant pRb in the case of CDK4 and
cyclin D or histone H1 in the case of CDK2 and cyclin E. The
phosphorylated substrates were then separated by SDS-PAGE and
autoradiographed. The activity of both kinases was low in quiescent
cells. The added serum stimulated the ability of the cyclin D/CDK4
complex to phosphorylate pRb as indicated either in the cyclin D or in
the CDK4 immunoprecipitate. In the presence of PGA
,
however, the kinase activity in each immunoprecipitate was equal to or
even less than that seen in quiescent cells (Fig. 7A).
In addition, the activity of the cyclin E/CDK2 complex was inhibited by
added PGA
. As above, the added serum greatly stimulated the
activity of this complex as assessed in immunoprecipitates with either
cyclin E or CDK2, while no stimulation was seen in cells treated with
serum and PGA
together (Fig. 7B).
Figure 7:
Activation of G CDKs were
abolished by PGA
. Control cells were serum-starved for 48 h
without any treatment (lanes 1 and 4). Serum-starved
NIH 3T3 cells were either stimulated with serum and solvent (lanes
2 and 5) or serum and 25 µM PGA
(lanes 3 and 6). After 10 h of culture (late
G
phase, see Fig. 4), cells were lysed and CDK4 (A, lanes 1-3) or cyclin D1 (A, lanes 4-6), CDK2 (B, lanes 1-3),
or cyclin E (B, lanes 4-6) were
immunoprecipitated using respective antibodies. The kinase activity
associating with the precipitate was determined using recombinant pRb
(for CDK4 and cyclin D1) or histone H1 (for CDK2 and cyclin E). The
result was reproducible in 2 (for CDK4, cyclin D1, and cyclin E
immunoprecipitation) or in 4 (for CDK2 immunoprecipitation) separate
experiments.
Figure 8:
Induction of p21
protein by PGA2. A, rapidly growing NIH 3T3 cells were treated
with solvent ethanol as control (lane 1), 25 µM PGA
(lane 2), O gray of mock irradiation (lane 3), or 5 gray of
-irradiation (lane 4).
Cells were harvested after 6 h of PGA
treatment or 6 h
after the irradiation. B, serum-starved NIH 3T3 cells received
serum and solvent (lanes 5 and 7) or serum and 25
µM PGA
(lanes 6 and 8).
Cells were harvested 6 h (lanes 5 and 6) or 9 h (lanes 7 and 8) after the serum addition. Each cell
lysate containing 50 µg of protein was analyzed by Western blot for
p21
protein. The result was reproducible in 2
separate experiments.
Although its mechanism of action is unknown, pharmacological
studies suggest that PGA is taken up by a carrier system at
the plasma membrane. After internalization, PGA
binds to
cytosolic proteins and moves to the nucleus(35) . Cell cycle
blockage in mid to late G
has been well
documented(36) . In this study, the blockage by PGA
in NIH 3T3 cells was confirmed to be in late G
,
within 3 h of the initiation of DNA synthesis. This observation
suggests that PGA
might be interacting with a molecule
(molecules) critical for passage through the restriction point just
prior to S phase. In addition, a blockage in G
/M is also
observed as reported previously(28) . To confirm these results,
cells were synchronized in either G
or S phase at the time
of PGA
addition. In such cultures, cells became blocked
either in G
or in G
/M, respectively, although
blockage in G
/M was transient. No evidence for blockage in
S phase was obtained. It, therefore, appears likely that PGA
targets a molecule(s) required for cell cycle progression both in
late G
and in G
/M phase.
Analyses were
performed to localize PGA inhibition in relationship to
well characterized molecules involved in cell proliferation. The fact
that microinjected oncogenic Ras was unable to overcome the inhibitory
effects of PGA
treatment indicates that PGA
directly targets Ras, a molecule required downstream of Ras
action, or a molecule involved in an entirely separate pathway required
for cell cycle progression from G
to S phase. This result
is the opposite of that observed with TGF-
. In mink lung
epithelial cells, the proliferation inhibitory action of TGF-
was
completely overcome by injection of oncogenic Ras(30) . These
results were extended by demonstrating that NIH 3T3 cells transformed
by oncogenic raf were also inhibited by PGA
treatment. This observation indicates PGA
apparently
acts downstream of Ras and even downstream of Raf. When E2F-1 and DP-1
or adenoviral E1A protein was injected into PGA
-treated
cells, however, efficient entry into S phase was observed. The results
reported here indicate that the target(s) of PGA
action
functions between these two markers of proliferative induction, Ras/Raf
and the activity of cellular molecules triggered by E1A or E2F-1/DP-1.
A number of treatments are known to block cell cycle progression in
mid to late G. In Balb/c 3T3 cells, treatment with sodium
butyrate or a combination of the ion channel blockers amyloride and
bumetanide efficiently block cell cycle progression in mid-G
at a point which is prior to the requirement of Ras in late
G
(31) . Interestingly, the effects of these
inhibitors were not overcome by injection of either oncogenic Ras or
E1A. We interpret this observation to indicate that these two
inhibitors block metabolic processes essential during preparation for
cell cycle transit, processes such as the duplication of important
cellular components. PGA
, on the other hand, apparently
interferes with signaling or cell cycle control molecules which
directly regulate transition between different phases of the cell
cycle, leaving all preparatory metabolic requirements unaffected as
indicated by the fact that E2F-1/DP-1 or E1A could overcome PGA
inhibition.
A number of biochemical markers of proliferative
signaling and cell cycle control were analyzed next. MAP kinase, whose
activity is stimulated by the Ras-Raf-MEK pathway, was shown to be
unaffected by PGA treatment, while the kinase activity of
both cyclin D/CDK4 and cyclin E/CDK2 were completely inhibited, as was
the hyperphosphorylation of pRb. Although it is p130, not pRb, which is
primarily associated with E2F in G
phase of mouse cells (37) , there are several reasons to believe that pRb functions
as one of the regulators of E2F in these cells and can serve as an
indicator of the activity of the class of E2F regulating proteins. It
has recently been shown that dihydrofolate reductase, an enzyme whose
expression is regulated by E2F, is expressed at higher levels in pRb
negative murine cells than normal cells(38) . In addition, over
expression of cyclin D in murine cells, which presumably induces
hyperphosphorylation of pRb (6) , can induce the activity of a
reporter gene controlled by an E2F promoter(39) . The facts
that PGA
blocks the hyperphosphorylation of pRb, and that
E2F-1/DP-1 or E1A microinjection overcomes PGA
-induced
growth arrest, are taken to indicate that PGA
ultimately
blocks the normal release of E2F/DP-1.
A possible explanation for
the inhibition of G phase cyclin-CDK kinase activity by
PGA2 is the induction of p21
protein, since
this protein has been shown to inhibit both CDK4 and CDK2
activity(33) , and its binding to CDK2 prevents the activating
phosphorylation by CDK activating kinase(40) . In this study we
have shown that PGA
-induced G
arrest was not
overridden by oncogenic Ras. On the other hand, although TGF-
also
induces CDK inhibitory proteins expression levels including
p21
(41) , Ras can override
TGF-
-induced G
arrest(30) . A possible
interpretation of these observations is that interruption of mitogenic
signaling at any site along the pathway, upstream of Ras in the case of
TGF-
and downstream of Ras in the case of PGA
, may
result in the up-regulation of CDK inhibitory protein(s). If so,
p21
induction might be a consequence rather
than a cause of inhibition of the proliferative process. On the other
hand, the possibility that PGA
directly induces
p21
synthesis is supported by the fact that
other genes are reported to be induced by PGA
treatment,
including gadd153, heat shock proteins, and hemoxylase (42, 43, 44) .
We conclude that PGA is targeting downstream of most elements of the Ras-Raf pathway
and elements prior to or at the site of the activation of most cyclins
and associated kinases. Consequently, PGA
apparently
interrupts an activity which might be closely related to the point at
which these two critical cell proliferation control mechanisms
(proliferative signaling and cell cycle regulation systems) interact.
Interestingly, however, microinjection of either E2F-1 or DP-1 alone
could not efficiently override PGA
cell cycle arrest while
each was able to induce S phase efficiently in serum-starved, quiescent
cells. This may indicate the possibility that PGA
blocks
the cell cycle not only by disrupting the connection between the two
proliferation control systems described above, but also by affecting
the interaction of the cell cycle controlling system and the DNA
synthesis process, where pRb, pRb-related proteins, and E2F/DP
transcription factors are involved(9) . Another possible
explanation is that by suppressing the cyclin E-CDK2 activity,
PGA
can shut off the positive feed back loop of cyclin E,
pRb, and E2F proposed by DeGregori et al.(45) , which
may result in the insufficient release of free E2F/DP transcription
factor. On the other hand, since the E2F-1/DP-1 complex has more potent
transactivation activity than each protein
alone(7, 46) , E2F-1/DP1 combined microinjection may
readily be able to induce the molecule required for S-phase without
this cyclin E-pRb-E2F amplifying loop.
In this study we have not
tested the possible involvement of PGA in several other
pathways with potential to modulate the activity of the cell
proliferation controlling molecules discussed above. For instance,
cyclic AMP is inhibitory in many cell types, and it has been suggested
that PGA
might function by elevating the intracellular
concentration of cyclic AMP. Since antiproliferative prostaglandins
have been reported to arrest cell growth without cyclic AMP
elevation(47) , cyclic AMP may not be a mediator of PGA
growth inhibition. Furthermore, recent studies suggest that
cyclic AMP exerts its cell growth inhibitory effect by protein kinase
A-dependent phosphorylation of either the regulatory domain or the
kinase domain of Raf protein, resulting in either disruption of the
association between Ras and Raf (48, 49, 50) or inhibition of the Raf kinase
activity including v-Raf(51) , respectively. Since MAP kinase
activation requires Raf
activity(48, 50, 52) , and since PGA2 did not
inhibit the serum-induced MAP kinase activation, neither of cyclic
AMP-dependent Raf inactivation mechanisms appears to be involved in
PGA
growth arrest.
While it is unlikely that PGA functions through cyclic AMP, there are numerous signaling
systems functioning within the cell which affect proliferation. Some of
these, such as the stress kinase family, are likely to affect
proliferative signaling(53) . Others, such as small GTP-binding
proteins of the Rho family, are likely to be required for proliferation
in some cells(54) . Finally, the cytokine signaling pathway
might function either to enhance or interfere with proliferative
signaling. The evidence above makes it clear that PGA
affects a molecule closely related to the point of connection
between the oncogene signaling pathway and the action of cyclins and
related proteins. This might be accomplished by its ability to interact
directly with a molecule whose action is closely related to the linkage
between these two cell proliferation control systems, or it might be
due to its ability to modify a separate signaling system which
ultimately affects such a linkage molecule(s). Our data raise the
possibility that p21
might be one of such
linkage molecules. In any case, it is likely that PGA
will
provide a valuable tool in unraveling the important mechanism by which
the signal from proto-oncogenes alters the activity of cyclins and
their associating proteins, thereby directly controlling passage
through the cell cycle.