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Département d’Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke (Québec), J1H 5N4, Canada
Address all correspondence and requests for reprints to: Dr. Nathalie Rivard, Département d’Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC, J1H5N4, Canada. E-mail: nrivard{at}courrier.usherb.ca
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The antiproliferative effect of somatostatin on the pancreas has been observed in vivo and in vitro, and this peptide may thus participate in the regulation of cell growth, both in normal and tumoral cells (4). Somatostatin and its stable analogues may affect tumor growth in vivo by inhibiting the action of hormones and growth factors (2, 8, 9, 10), although much evidence exists for direct mediated responses, as shown in human pancreatic cancer cells (11, 12, 13) and those of rodents (14). Recently, five somatostatin receptor subtypes, SST1 to SST5, have been cloned and functionally characterized (15, 16, 17, 18, 19). They all bind somatostatin-14 and somatostatin-28 with similar affinity but show major differences in their affinities for various somatostatin analogs (15, 16, 20). The somatostatin analog SMS 201–995 (SMS), or octreotide, has very low affinity for SST1 but has high binding affinity for the somatostatin receptor subtype SST2 and can induce stimulation of tyrosine phosphatase activity associated with inhibition of cell proliferation in those cells expressing SST2 (15, 16). Among the five somatostatin receptors, it was recently shown that SST2 selectively mediates the inhibitory effect of somatostatin on serum- or insulin-induced cell proliferation, by a mechanism involving stimulation of a phosphotyrosine phosphatase (PTP) recently identified as SHP-1 (21, 22).
The mechanisms of cell growth arrest by somatostatin are still poorly understood. Somatostatin analogues induce a G0/G1 cell cycle arrest and thus prevent DNA synthesis in GH3 rat pituitary tumor cells (23), whereas they induce a transient G2/M blockade and apoptosis in MCF7 human mammary tumor cells (24, 25). Progression through the G1 phase and the G1/S transition is orchestrated by complexes containing G1-specific cyclins and G1-specific cyclin-dependent kinases (Cdks): cyclin D family members are found associated with either cdk4 or cdk6 in early G1 phase, and cyclin E with cdk2 in late G1 (26). Biochemical and genetic data indicate that cyclin E-cdk2 activity is essential for entry into S phase (27). Activity of these complexes can be inhibited by the presence of a cyclin kinase inhibitor (cki), which belongs to an expanding new family of mammalian cell cycle modulators, including (so far) p21Cip, p27Kip1, and p57Kip2 in one subfamily and p16INK4A, p15INK4B, and p18INK4C in the other (26, 28). The INK4 proteins specifically inhibit cdk4 and cdk6, whereas the second family of inhibitors has less specificity being involved in the inhibition of cyclin D-cdk4, cyclin E-cdk2, cyclin A-cdk2, and the mitotic complex cyclin B-cdk1 (28). However, when overexpressed in cells, p21Cip and p27Kip1 cause only a G1 arrest, suggesting that, despite their ability to inhibit the mitotic cyclin B-cdk1 complex in vitro, they do not act on mitotic cdks in vivo (26, 28).
These observations raise the important question of whether somatostatin exerts its antiproliferative effect through the inhibition of cell cycle progression in pancreatic acinar cells. Here, we addressed the question of which, if any, cell cycle regulatory proteins are targeted by somatostatin. This study was therefore undertaken to evaluate activation of the MAP kinases p42/p44 and phosphatidylinositol 3- kinase and to analyze expression of some cell cycle regulatory proteins in relation to pancreatic acinar cell proliferation in vivo, as well as in a well-established pancreatic tumoral cell model, the AR4–2J cells. Our data suggest that, in rat pancreatic acinar cells, somatostatin-mediated growth inhibition is not mediated by a considerable modulation of MAP kinases activation but by inhibition of phosphatidylinositol 3-kinase activity and cyclin D1/cdk4 and cyclin E-cdk2 complexes activities. These reduced cyclin-cdk complexes activities may stem from the enhanced expression of p21Cip and p27Kip1 and thus may cause the cell cycle arrest.
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Materials and Methods |
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32P]ATP was from Amersham Pharmacia Biotech, Montréal, Canada. Caerulein was a gift from Dr.
Jean Morisset (University of Sherbrooke), and SMS was provided by
Sandoz Pharmaceuticals Corp. (Montréal, Canada) as a
stock solution of 0.5 mg/ml in acetate buffer, pH 4.2. Recombinant
human EGF were purchased from Life Technologies, Inc./BRL
(Burlington, Canada). Male Sprague Dawley rats (250–300 g) were from
Charles River Laboratories, Inc. (St. Constant, Canada).
All other materials were obtained from Sigma Aldrich Co. (Oakville, Canada) unless otherwise stated.
Animals
Surgical procedure. Male Sprague Dawley rats were fed Purina
rat chow ad libitum and kept in a room with controlled
temperature and light (20 C; 12 h light, 12 h darkness). They
were anesthetized with methoxyflurane and prepared with jugular
SILASTIC cannula (0.02 inch id x 0.037 inch od). The cannula was
routed under the skin to exit on the back of the animal. After surgery,
rats were kept in modified Bollmann-type restraint cages and fed Purina
rat chow and water ad libitum during a recovery period of 2
days, before the beginning of the experiments. All studies were
conducted in agreement with the principles and procedures outlined in
the Canadian Guidelines for Care and Use of Experimental Animals.
Experimental procedures. Experiments were performed during 2 days. Dilution of the stock solutions of caerulein and SMS were made in saline containing 0.05% BSA. Caerulein was infused iv at 0.25 µg/kg·h, whereas SMS was given at 0.5, 1, and 5 µg/kg·h at rates of 1 ml/h. The dose of caerulein was chosen on the basis of the observation that CCK-8, infused iv at 0.3 µg/kg·h for 24 h, resulted in plasma CCK levels of 6 pM, a value comparable with physiological postprandial concentrations in the rat (29). The dose of SMS was selected on the basis of a previous observation that its administration inhibited basal and stimulated pancreatic secretion (30). Control animals were infused with saline in 0.05% BSA. After 1, 18, 36, and 48 h of treatment, the animals were killed by CO2 inhalation. The pancreas was carefully dissected out, trimmed free of fat, mesentery, and lymph nodes, and weighed. A piece (100 mg) was homogenized in 0.6 N perchloric acid for DNA extraction and protein assay. A second piece (50 mg) was homogenized in RIPA (radioimmunoprecipitation analysis) buffer (50 mg tissue/ml). Half of the homogenate was mixed in Laemmli’s buffer, boiled for 5 min, and frozen until time for the Western blots; the other half, supplemented with protease inhibitors (0.1 mM phenylmethylsulfonylfluoride, 1 µg/ml pepstatin, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and the phosphatase inhibitor 0.2 mM orthovanadate), was saved and frozen for the Cdk2 assay. Proteins were determined by the modified Lowry procedure described by Peterson (31).
Cell culture
The rat acinar tumoral cell line AR4–2J (American Type Culture Collection, Manassas, VA) was cultured, as
previously described (32), in DMEM (from Life Technologies, Inc./BRL) supplemented with 10% FBS, 2
mM glutamine, and antibiotics (100 U/ml penicillin and 100
µg/ml streptomycin) at 37 C in a humid atmosphere (5%
CO2-95% air).
Growth assay
All experiments were performed starting with confluent cells
that were subsequently plated for growth assay in 35-mm diameter dishes
at 20,000 cells/ml. After an overnight attachment phase, the medium was
changed to serum-free medium and then supplemented daily with 10
nM EGF alone or in combination with 10 nM SMS.
Cell growth was measured after 2 and 4 days, by cell counting, as
previously described (32).
Immunoblotting
Fifty micrograms of protein from the Laemmli’s lysates were
loaded and separated through 10% SDS-PAGE for all proteins except for
retinoblastoma protein, which was run through a 7.5% SDS gel. After
migration, gels were electrotransferred onto nitrocellulose
(Amersham Pharmacia Biotech) for 2 h at 50 V. Western
blots were blocked, for 1 h at 37 C, with 10% low fat milk in PBS
+ 0.1% Tween 20, before the incubation for 2–3 h at room temperature
with the specific antibodies, then washed in PBS + 0.1% Tween 20
(4 x 10 min). This was sequentially followed by 1 h of
incubation at room temperature with either antirabbit (for polyclonal
Abs) or antimouse (for monoclonal Abs) IgG horseradish peroxidase
conjugated. Peroxidase activity was revealed using an enhanced
chemiluminescence kit from Amersham Pharmacia Biotech.
Quantitative analyses were carried out with a scanning densitometer
(Imagin Densitometer model GS-670; Bio-Rad Laboratories, Inc., Mississauga, Ontario, Canada).
Immunoprecipitation experiments and Cdk2 activity
The RIPA’s lysate (500 µg) was cleared by centrifugation
(10,000 x g, 10 min) before a 2-h incubation at 4 C
with protein A-Sepharose, preincubated for 1 h with the cdk2
antibody (4 µl; 1 µg/tube). The immunocomplex was first
washed four times with ice-cold buffer and three times with ice-cold
kinase buffer (20 mM
para-nitrophenylphosphate, 10 mM
MgCl2, 1 mM dithiothreitol,
in 20 mM HEPES, pH 7.4) before performing the
kinase assay as previously described (33). The reaction
was started by incubating the washed immunocomplex at 30 C for 30 min
in the presence of 5 µg histone H1, 2 µCi/assay
[32P] ATP, and 50 µM
cold ATP. After 30 min, the reaction was stopped by addition of
Laemmli’s 2x buffer. Radiolabeled histone H1 was separated from the
immunocomplex by 10% SDS-PAGE and was autoradiographed.
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Results |
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, pancreatic weights were significantly
increased above control values, by 133% (P < 0.05),
after 48 h of caerulein infusion. Increases of 45% in total DNA
contents were observed after 48 h, thus confirming the trophic
effect of caerulein (3). Pancreatic weight, in response to
the 48-h infusion of SMS, was constantly heavier than in controls, by
14% (P < 0.1). This same treatment, however, had no
significant effect on total DNA content. Interestingly, SMS completely
blocked the stimulatory effect of caerulein on pancreatic weight and
total DNA content.
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).
Inhibitory effects of similar magnitude were also observed in these
cells stimulated by somatostatin-14 (data not shown).
SMS moderately attenuates the long-term activation of p42/p44 MAP
kinases by caerulein in pancreas
To get insights on how SMS reduced pancreas growth and AR4–2J
cell proliferation, we examined some intracellular pathways known to be
associated with cell proliferation, and thus potential targets for a
somatostatin action. Because activation of the Ras/MAP kinase cascade
is required to pass the G1 restriction checkpoint to progress into the
cell cycle (35), this critical reaction was then first
investigated. As shown in Fig. 2A, using
a polyclonal antibody specific for the detection of the active
phosphorylated p42/p44 isoforms, control pancreas exhibited minimal
kinase activities throughout the entire time course study. In contrast,
caerulein infusion significantly increased p42/p44 MAP kinase
activities by 14-fold (P < 0.05, n = 3) as early
as 1 h after beginning the infusion. This activation was sustained
for at least 18 h (19-fold stimulation). Furthermore, SMS had no
significant effect on basal and caerulein-induced p42/p44 MAP kinase
activities after 1 h of infusion. However, after 18 h, SMS
consistently attenuated, by 2-fold (P < 0.05, n =
3), caerulein-induced p42/p44 MAP kinase activation, when compared with
caerulein control (Fig. 2A).
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). Paradoxically,
preincubation of AR4–2J cells during 30 min with SMS significantly
stimulated, by 2- to 3-fold (P < 0.05, n = 3),
basal and EGF-induced p42/p44 MAP kinase activities observed after 10
min and 3 h.
SMS abolishes caerulein-induced Akt phosphorylation in exocrine
pancreas
Activation of phosphatidylinositol 3-kinase signaling pathway
seems to be crucial for the G0-to-G1 phase progression
(36). Hence, we analyzed the effect of SMS on
phosphorylation of the kinase Akt, which is activated by the
3'-phosphorylated lipids products of phosphoinositol 3-kinase
(37). As shown in Fig. 3, using a polyclonal antibody specific for the detection of the active
phosphorylated Akt, a significant Akt activity was detectable in the
control pancreas throughout the entire time course study. In contrast,
caerulein infusion significantly increased Akt phosphorylation by
3-fold (P < 0.05, n = 3) as early as 1 h
after beginning the infusion. This activation was sustained for at
least 18 h. Interestingly, SMS had no significant effect on basal
Akt phosphorylation (P > 0.05, n = 3) but blocked
caerulein-induced Akt phosphorylation after 1 and 18 h (Fig. 3A).
Neither caerulein nor SMS treatments affected Akt expression, as
indicated with the Akt recognizing the phosphorylated and
unphosphorylated forms of the enzyme.
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, cyclin D1 is barely detectable in the
control animals, but expression of the protein became easily detectable
36 h after the beginning of caerulein infusion. Interestingly,
concomitant infusion of SMS dose-dependently attenuated
caerulein-induced cyclin D1 expression, with a maximal effect of SMS
observed at 5 µg/kg·h, a 32% inhibition (P <
0.05, n = 3). Expression of cyclin E was also significantly
induced, by 2- to 3-fold (P < 0.05, n = 3), after
caerulein infusion and was dose-dependently inhibited by the SMS
treatment.
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The specificity of SMS action was then assessed by analyzing the levels of cdk4 and cdk2, two proteins whose activities are required for G0/G1>S phase progression. The same SMS treatment did not induce a significant inhibition (P > 0.05, n = 3) of the protein expression of cdk4 or cdk2, a result indicative of a rather specific SMS action, because cdk4 protein expression was already described as being affected by TGFß, an inhibitor of DNA synthesis in epithelial cells (42).
Because cyclin D1 and cyclin E were recently described as being located
on distinct (although interdependent) pathways (43, 44, 45),
we then asked, whether the enzymatic activity of cyclin E-cdk2 was
sensitive to the SMS treatment. The results shown in Fig. 4A
unambiguously indicated that anti-cdk2 immunocomplexes exhibited much
more elevated activity in caerulein-stimulated pancreas than in
control pancreas. The enzymatic activities of anti-cdk2 immunocomplexes
were dose-dependently inhibited by SMS, 5–67% inhibition from 0.5–5
µg/kg·h (n = 3).
It is well known that activity of cyclin D/cdk4 and cyclin E/cdk2
complexes can be inhibited by the presence of a cdk inhibitor (cki).
The major role of ckis is to mediate cell cycle arrest in response to
antimitogenic signals and to ensure that specific cell cycle events do
not initiate before others are completed (28). Among these
inhibitors are the two structurally related
p21Cip and p27Kip1, which
inhibit cyclin/cdk complexes. As shown in Fig. 4A, p21Cip expression was not significantly affected
by caerulein infusion but was reproducibly increased by 2- to 4-fold
after 36 h of SMS infusion. In contrast, expression of
p27Kip1, which is quite high in control animals,
is significantly decreased by 65–75% (P < 0.05,
n = 3) after 36 h of caerulein infusion. Interestingly,
concomitant infusion of SMS dose-dependently rescued
p27Kip1 expression, up to the level of the
controls at the 5-µg/kg·h dose.
Biochemical and genetic data indicate that cyclin E-cdk2 activity is
essential for entry into S phase (27). Then, we next
analyzed the effect of SMS on cyclin E and cdk2 in AR4–2J cells. It is
noteworthy that the serum-deprived AR4–2J cells had a detectable basal
cdk2 activity (Fig. 4B) and that they grow in serum-free medium (Fig. 1). Treatment of serum-deprived cells with EGF significantly increased
cyclin E expression, by 2-fold (P < 0.05, n = 3),
and stimulated cdk2 activity by 3- to 4-fold (P <
0.05, n = 3). By contrast, the same treatment had a minimal effect
on p21Cip and p27Kip1
protein expression (P > 0.05, n = 3). Addition of
1–100 nM SMS strongly repressed the accumulation
of cyclin E and the stimulation of cdk2 elicited by EGF. Furthermore,
treatment of AR4–2J cells with 1–100 nM SMS
weakly increased p27Kip1 expression but
significantly enhanced the expression of p21Cip,
by 3-fold (P < 0.05, n = 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, three interesting mechanisms have been proposed to explain the growth-negative action of somatostatin. In the first one, the p42/p44 MAP kinase cascade was targeted for this inhibition (49). Indeed, somatostatin was shown to inhibit Raf-1, the upstream kinase of the MAP kinase module (50). Although this action was satisfying, in regard to the essential role played by the MAP kinase module in cell proliferation (35), no long-term experiments were performed to determine whether this inhibition was sustained enough to account for the growth inhibitory action of somatostatin. The second mechanism involved activation of a tyrosine phosphatase activity. Numerous studies reported that somatostatin can stimulate a tyrosine phosphatase activity and especially that of SHP-1, whose activation may be one of the early steps leading to its antiproliferative action (21, 22, 51). Furthermore, recent studies suggested a role of SHP-1 in terminating growth factor mitogenic signals by dephosphorylating critical molecules. Indeed, SHP-1 can dephosphorylate a variety of protein tyrosine kinase receptors when coexpressed in 293 cells (52) and down-regulate interleukin-3-induced tyrosine phosphorylation and mitogenesis in hematopoietic cells (53). The third, and perhaps the most appealing mechanism, remains the demonstration that somatostatin-mediated growth arrest could be related to an elevation of p27Kip1, a cki known to block cell cycle progression by targeting G1 cdks. This action was characterized in CHO expressing SST2 (54) and also in FRTL-5 thyroid cells (55).
Our data demonstrate that SMS specifically inhibited caerulein- and EGF-dependent cell cycle progression of pancreatic acinar cells, independently of considerable effect on p42/p44 MAP kinase activities. Hence, SMS seems to block pancreatic acinar cell proliferation in G1 phase without significantly affecting the hormone and growth factor-induced MAP kinase cascade. Although our data do not support the involvement of the MAP kinase cascade as a critical target of somatostatin, the possibility remains that somatostatin may inhibit other early signaling pathways. For instance, it is well known in many cell types that activation of the SST receptors is negatively coupled to adenylate cyclase with decreases in intracellular cAMP formation (18). In this study, this possibility is doubtful, because cAMP is a growth-suppressant in pancreatic cancer cells (56). Furthermore, Viguerie et al. (14). have already demonstrated that the antiproliferative effect of SMS on AR4–2J cells does not involve the GTP-binding protein-mediated negative coupling of somatostatin receptors to adenylate cyclase. Somatostatin analogs were also shown to inhibit the inositol phospholipid/calcium pathway in rat pancreas (57) and in clonal hamster ß-cells (58). Herein, we demonstrated, for the first time, that SMS completely inhibited the stimulatory effect of caerulein on Akt phosphorylation, suggesting that SMS interferes with growth factor-induced phosphatidylinositol 3-kinase signaling pathway. In this regard, Medina et al. (59) recently reported that, in FRTL-5 thyroid cells, somatostatin can exert its antiproliferative effect by inhibiting cyclin E expression, which appears to be dependent on the phosphatidylinositol 3-kinase pathway. Furthermore, the PACAP-induced increase in AR4–2J cell proliferation was inhibited by low concentrations of wortmannin (l nM), a known inhibitor of phosphatidylinositol 3-kinase (32). Hence, as previously described in other cell types (36), we put forward a hypothetical model of phosphatidylinositol 3-kinase inhibition-induced pancreatic acinar cell cycle arrest. It is noteworthy that signaling via SST2 apparently did not induce apoptosis in pancreas and in AR4–2J cells, because we could never observe any cleavage of poly-(ADPribosyl) polymerase, a well-known substrate for caspase-3 (60), after SMS treatment (data not shown).
Recently, Pagès et al. (54) demonstrated, by using a catalytically inactive mutant of SHP-1, that SHP-1 is necessary for SST2-induced cell cycle arrest. In the present study, we did not examine whether SMS activated SHP-1 activity, because we mainly focused on the p42/p44 MAP kinases and phosphatidylinositol 3-kinase pathways, two early signaling cascades that seem to be crucial for the G0-to-G1 phase progression. However, previous studies have already reported that somatostatin analogs stimulate tyrosine phosphatase activity in rat pancreas (3), in AR4–2J cells (15, 34), and in human pancreatic cancer MIA PaCa-2 cells (13) and especially that of SHP-1 in MCF-7 breast cancer cells (61) and in COS, CHO, and NIH 3T3 cells stably expressing the SST2 receptor subtype (16). Thus, these observations raise the possibility that SST2-activated SHP-1 activity interferes with growth factor-induced phosphatidylinositol 3-kinase signaling pathway, although further studies are needed to demonstrate this hypothesis, currently under investigation.
An obligatory step for the mitogen-stimulated passage of cells from the G1 to the S phase seems to be the induction of cyclin D1 and p27Kip1 down-regulation (33, 62, 63). Caerulein-stimulated induction of cyclin D1 is only slightly attenuated by the SMS treatment in exocrine pancreas. This reduction in cyclin D1 expression is probably attributable to the reduced p42/p44 MAP kinase activities observed after SMS treatment (62). In contrast, cyclin D1 seemed to be constitutively expressed in AR4–2J cells (data not shown), agreeing with the fact that the cyclin D1 gene is frequently overexpressed in pancreatic cancer cells (64). However, SMS has a strong inhibitory action on cyclin E induction by caerulein in exocrine pancreas, and this repression was similarly observed in EGF-stimulated AR4–2J cells. As expected from such a cyclin E repression, the resulting cyclin E-associated cdk2 activity, as well as pRb phosphorylation, was blunted by SMS treatment in both models. Similar effects of somatostatin were recently demonstrated in FRTL-5 thyroid cells, in which somatostatin inhibited TSH-induced cyclin E expression (59).
Another interesting point is the control of p27Kip1 levels by the SMS. As originally reported in FRTL-5 (55) and in CHO expressing SST2 (54), SMS has a remarkable effect in preventing caerulein-induced p27Kip1 down-regulation. This action certainly represents an attractive mechanism for somatostatin-mediated cell growth inhibition. Indeed, p27Kip1 was initially discovered as a cki induced by extracellular antimitogenic signals. Transforming growth factor-ß in mink epithelial cells (65), rapamycin in T lymphocytes (66), and cAMP in macrophages (40) and pancreatic cancer cells (56) prevented mitogen-induced p27Kip1 down-regulation and, therefore, cdk activation and G1 progression. We and others recently demonstrated that overexpression of p27Kip1 antisense cDNA allowed cells to grow for several generations in medium supplemented with insulin and transferrin (63) or in medium containing low concentrations of serum (67). Conversely, it was recently demonstrated that targeted disruption of the murine p27Kip1 gene enhanced growth of mice and led to striking enlargement of their thymus, pituitary, adrenal, and gonadal organs (68). These observations suggest that p27Kip1 can play a major role in controlling cell cycle exit and thus may be involved in somatostatin-mediated inhibition of normal pancreas and cancer cells growth. Recently, some studies indicated that inhibition of phosphatidylinositol 3-kinase signaling pathway actually inhibited Akt activity, up-regulated p27Kip1, and recruited cells in G1 (69). However, at this point, further studies are required to confirm such a link between the phosphatidylinositol 3-kinase signaling pathway and p27Kip1 in rat pancreatic acinar cells.
Intriguingly, EGF failed to efficiently down-regulate
p27Kip1 in AR4–2J cells but significantly
increased cyclin E-cdk2 activity. This failure in the ability of
mitogen to down-regulate p27Kip1 is rare but not
unique, because it was observed before in T cells from old mice
(70) and in TSH-stimulated primary thyroid epithelial
cells (71). Another interesting observation from the
present study remains that SMS increased p21Cip
levels in the normal pancreas and in AR4–2J cells. Previous studies
performed in CHO-K1 cells demonstrated that signaling via SST5 also
induced a marginal increase in p21 expression (72). Hence,
somatostatin-induced G1 arrest of pancreatic acinar cells may be caused
by the decreased cyclin E expression and by the enhanced levels of both
p21Cip and p27Kip1, which
associate and inhibit cyclin D/cdk4,6 and cyclin E-cdk2 complexes.
Interestingly, such up-regulation of p21Cip and
p27Kip1 by antiproliferative agents was described
before in other cell lines with interferon (73),
transforming growth factor-ß (74), and vitamin D
(75).
In conclusion, this report demonstrated that somatostatin-mediated growth inhibition of normal and cancer pancreatic acinar cells is triggered via: 1) an inhibition of phosphatidylinositol 3-kinase signaling pathway; 2) a suppression of mitogen-induced cyclin E expression and p27Kip1 down-regulation; and 3) a marginal induction of p21Cip expression. Interestingly, these actions are complementary to each other, because they cooperate toward inhibition of G1 cdks.
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Acknowledgments |
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Footnotes |
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2 A scholar from the Fonds de la Recherche en Santé du
Québec.
Received July 13, 2000.
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