Snail blocks the cell cycle
and confers resistance to cell death
Sonia Vega,1Aixa V. Morales,1,3Oscar H. Ocaña,1,3Francisco Valdés,2Isabel Fabregat,2
and M. Angela Nieto1,4
1Instituto Cajal, Consejo Superior de Investigaciones Cieutificas, 28002 Madrid, Spain;2Facultad de Farmacia, Universidad
Complutense, 28040 Madrid, Spain
The Snail zinc-finger transcription factors trigger epithelial-mesenchymal transitions (EMTs), endowing
epithelial cells with migratory and invasive properties during both embryonic development and tumor
progression. During EMT, Snail provokes the loss of epithelial markers, as well as changes in cell shape and
the expression of mesenchymal markers. Here, we show that in addition to inducing dramatic phenotypic
alterations, Snail attenuates the cell cycle and confers resistance to cell death induced by the withdrawal of
survival factors and by pro-apoptotic signals. Hence, Snail favors changes in cell shape versus cell division,
indicating that with respect to oncogenesis, although a deregulation/increase in proliferation is crucial for
tumor formation and growth, this may not be so for tumor malignization. Finally, the resistance to cell death
conferred by Snail provides a selective advantage to embryonic cells to migrate and colonize distant territories,
and to malignant cells to separate from the primary tumor, invade, and form metastasis.
[Keywords: Snail; cell cycle; cell death; malignization; chick embryo; mouse embryo]
Received December 5, 2003; revised version accepted April 5, 2004.
The Snail family members Snail and Slug trigger epithe-
lial-mesenchymal transitions (EMTs) during embryonic
development and tumor progression (Nieto 2002). Dur-
ing development, they are involved in the ingression of
the early mesodermal cells at gastrulation and in the
delamination of the neural crest from the neural tube
(Nieto 2002). Functional analyses in the chick (Nieto et
al. 1994) and a striking interchange in the expression
patterns at the sites of EMT in chicken and mouse em-
bryos (Sefton et al. 1998) suggested that Slug induces the
transition in the chick, whereas Snail triggers EMT in
the mouse. Indeed, in mammalian cells Snail induces
EMT and represses E-cadherin transcription (Batlle et al.
2000; Cano et al. 2000), and Snail mutant mice die at
gastrulation due to a defective EMT and maintained E-
cadherin expression (Carver et al. 2001). The analysis of
the gene family in all major vertebrate groups indicated
that Snail is higher in the gene hierarchy controlling
neural crest development in fish, amphibians, and mam-
mals (Locascio et al. 2002; Aybar et al. 2003).
Snail is also involved in the EMT that takes place con-
comitant with the acquisition of invasive properties in
tumors (Nieto 2002; Thiery 2002). It is expressed in the
invasive cells of tumors induced in the skin of mice
(Cano et al. 2000) and in biopsies from patients with
ductal breast carcinomas (Cheng et al. 2001; Blanco et al.
2002), gastric cancer (Rosivatz et al. 2002), and hepato-
cellular carcinomas (Sugimachi et al. 2003). Snail ap-
pears as an early marker of the malignant phenotype and
behaves as a prognostic factor (Blanco et al. 2002).
The process of EMT implies a dramatic phenotypic
change that includes the loss of epithelial markers, the
gain of mesenchymal markers, and changes in cell shape.
Because Snail is able to induce a complete EMT (Batlle et
al. 2000; Cano et al. 2000), it must have many targets.
Indeed, together with E-cadherin, other direct targets for
Snail repression that have been identified include the
epithelial Mucin-1 (Guaita et al. 2002) and the compo-
nents of the tight junctions claudins and occludin (Ike-
nouchi et al. 2003). Snail is upstream of molecules in-
volved in the degradation of the basement membrane
and extracellular matrix such as metalloproteinase 2
(MMP-2; Yokohama et al. 2003), the mesenchymal
markers vimentin and fibronectin (Cano et al. 2000;
Guaita et al. 2002), and other transcription factors such
as ZEB-1 and LEF-1 (Guaita et al. 2002). Although a di-
rect link between Snail expression and cytoskeletal pro-
teins has not been reported, RhoB, a small GTPase in-
volved in cytoskeletal actin rearrangements, lies down-
stream of Slug during chick neural crest delamination
(Del Barrio and Nieto 2002).
By analyzing epithelial cells transfected with Snail and
mouse and chick embryos, we show here that Snail also
regulates cell-cycle progression and survival. Snail regu-
lates components of the early to late G1 transition and
the G1/S checkpoint, including the repression of Cyclin
D2 transcription and the increase in p21/Cip1. Concomi-
3These authors contributed equally to this work.
E-MAIL email@example.com; FAX 34-91-585-4754.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/
GENES & DEVELOPMENT 18:1131–1143 © 2004 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/04; www.genesdev.org1131
tantly, Snail confers resistance to the lethal effects of
serum depletion or TNF-? administration by activating
the MAPK and PI3K survival pathways. These survival
properties confer a selective advantage to the invasive
and migratory cells during both embryonic development
and tumor dissemination.
Snail impairs cell-cycle progression
When stably expressed in different epithelial cell lines,
both mouse and human Snail dramatically decreased cell
growth, prompting us to analyze cell proliferation and
cycle progression of such cells. When transfected with
Snail (MDCK-Snail), MDCK cells underwent a complete
EMT (Cano et al. 2000) and incorporated lower levels of
BrdU after 24 h in culture (25% of that observed in
mock-transfected cells, Fig. 1A). Analysis by FACS (Fig.
1B) indicated that the vast majority of Snail-expressing
cells (93%) was in the G0/G1 phase of the cell cycle in
basal conditions after 72 h in culture. However, these
cells can respond to mitogens and proliferate, as ob-
served 16 h after they were replated with 10% serum
(Fig. 1B). The percentage of cells in G0/G1 was again
much higher in MDCK-Snail transfectants (61% vs.
36%) after 24 h in culture and at all other times analyzed
(Fig. 1B). To check whether Snail-expressing cells had
difficulties in progressing through the G1/S checkpoint,
we analyzed the expression of the Cip/Kip proteins, es-
sential to inhibit the activity of the cdk2–Cyclin E com-
plex, and responsible for the hyperphosphorylation of the
retinoblastoma protein (Rb) and the subsequent progres-
sion from late G1 to the S phase (Ortega et al. 2002).
Although the levels of p27 (Kip1) remained low in both
MDCK-Mock and MDCK-Snail cells, Snail greatly in-
creased the expression of p21 (Cip1) as shown in basal
conditions and after 8 h (Fig. 2A) and 12 h in culture (Fig.
2B). Nevertheless, a decrease was observed after 16 h,
coincident with Rb hyperphosphorylation (Fig. 2A) and
entry into S phase (Fig. 1B). When we analyzed the levels
of p21 at later times, we found that they increased again
in Snail-expressing cells after 24 h and were always
maintained at higher levels than in control transfectants
(Fig. 2B). Thus, p21 expression is tightly regulated and
maintained at a high level except for during short periods
of time when cells are exposed to a high concentration of
mitogens and respond by progressing through the cycle.
Snail represses Cyclin D2 transcription
The G1/S checkpoint requires the activity of Cyclin E
and the activity of the Cyclins D helps to progress from
early to late G1 passing the restriction point (R) where
cells are committed to another round of division. Thus,
we decided to analyze the expression of Cyclins D and E
in the two cell lines. Although we could not detect sig-
nificant differences in the amount of Cyclin E (data not
shown) or Cyclin D3 (Fig. 3A) in MDCK Snail-expressing
versus nonexpressing cells, we observed a decrease in the
levels of Cyclins D1 and D2 (Fig. 3A). In addition, we
also found that the expression of their partner, the cyc-
lin-dependent kinase cdk4, was decreased in MDCK-
Snail cells (Fig. 3A). These results indicate that Snail-
expressing cells, with limited amounts of Cyclins D,
have an impairment to progress through the restriction
After we had observed that Snail-expressing cells con-
tain low levels of Cyclins D, we wanted to assess
whether they can be direct targets of Snail transcrip-
tional repression. Although we found that the levels of
Cyclin D1 mRNA were lower in cells expressing Snail
compared to mock-transfected cells, those of Cyclin D2
were strongly down-regulated (Fig. 3B). Because the Cy-
clin D2 promoter contains two E-box consensuses for
Snail binding (Mauhin et al. 1993) that are conserved
among mouse, rat, and human (Bouchard et al. 1999), we
analyzed the effect of Snail on Cyclin D2 transcription.
We expressed mouse Snail cDNA together with reporter
constructs of the human Cyclin D2 promoter in the ke-
ratinocyte cell line MCA3D, previously used to study
the proximal E-cadherin promoter (Cano et al. 2000).
Snail repressed the wild-type promoter activity of Cyclin
D2 to ∼55% of its activity (Fig. 3C) but did not affect that
tion. (A) BrdU incorporation after a 1-h pulse in
MDCK cells stably transfected with Snail (MDCK-
Snail) or the empty vector (MDCK-Mock). Bright-
field images after 24 h in culture. (B) FACS analysis
of the cell cycle in MDCK-Mock and MDCK-Snail
cells after different times in culture.
Snail expression impairs cell prolifera-
Vega et al.
1132GENES & DEVELOPMENT
of promoter constructs carrying either a mutated or de-
leted distal E-box (located at −1600). Reporter activity
was significantly decreased (to ∼40%) when the proximal
E-box (located at −1400) was mutated or deleted. As this
construct is insensitive to Snail when the distal box is
intact, both E-boxes are needed to mediate repression.
The proximal box and sequences located immediately 3?
to it must be essential for promoter activity, because the
−1303 D2-Luc deletion provoked a 10-fold decrease in
activity (Fig. 3C). Similar results were obtained when
human Snail cDNA constructs were used (data not
shown). Thus, Snail can repress the activity of the Cyclin
D2 promoter in epithelial cells. The presence of the
proximal E-box (−1400) is needed for the distal box
(−1600) to repress, and the region between −1400 and
−1300 that includes the proximal box acts as an en-
Snail and the cell cycle in the mouse embryo
Having seen that Snail repressed Cyclin D2 expression in
cultured cells, we set out to determine whether the same
could occur during embryonic development. Thus, we
compared the expression of Cyclins D1 and D2 with that
of Snail in 8.5-d postcoitum (dpc) mouse embryos (Fig. 4).
At this developmental stage, Snail is expressed in re-
gions undergoing EMT such as the premigratory neural
crest and the primitive streak (Nieto et al. 1992; Smith et
al. 1992; Sefton et al. 1998), and in mesodermal deriva-
tives including the decondensing somites and the allan-
tois (Fig. 4A,D,G,J). Cyclin D2 transcripts were readily
detected at high levels in the neural plate (Fig. 4F) and
the neural tube (Fig. 4I), whereas they were absent from
Snail-expressing regions (Fig. 4F,I,L), evidencing a clear
inverse correlation in their expression patterns. Cyclin
D1 was also expressed in the neural tube (Fig. 4E,H) and
absent from regions expressing high levels of Snail tran-
scripts such as the allantois (Fig. 4K). However, Cyclin
D1 transcripts were observed in regions where Snail was
only moderately expressed, including the decondensing
somites (Fig. 4H). Thus, our data are compatible with
Snail being a strong repressor of Cyclin D2 transcription,
and are in agreement with the results obtained from
Northern analysis of both Cyclin D1 and D2 and with
those of the Cyclin D2 promoter activity in epithelial
The impairment in cell-cycle progression in Snail-ex-
pressing cells was due not only to the maintained down-
regulation of Cyclin D2 transcription but also to the
changes in the expression of additional components of
the G1/S checkpoint. Indeed, the expression of Snail was
correlated with an increase of the proportion of cells in
G0/G1 (Fig. 1). To assess whether a similar change oc-
curred in vivo, we compared the pattern of Snail expres-
sion with that of BrdU incorporation in mouse embryos
to visualize cells in the S phase of the cycle (Fig. 5A,B). In
agreement with the data from cell cultures, in the re-
gions of the embryo where Snail was expressed, a much
lower proportion of nuclei accumulated BrdU. In general,
it was possible to discern a complementary pattern of
Snail expression and BrdU incorporation within the em-
progression from G1 to S phase. Western blot analysis of
MDCK-Mock and MDCK-Snail cells. (A) Levels of the G1
checkpoint molecules, the cdk inhibitors p21 and p27, and the
degree of Rb phosphorylation. (B) Levels of p21 after different
times in culture. Rb, retinoblastoma protein; pRb, hypophos-
phorylated state; ppRb, hyperphosphorylated state. Representa-
tive experiments are shown (n = 4).
Snail alters the expression of proteins involved in
of D cyclins and their partner cdk4. Immunoblotting of total
erk2 was used as a control of gel loading. (B) Analysis of Cyclins
D1 and D2 transcription by Northern blot of RNA extracted
from MDCK-Mock and MDCK-Snail cells after different times
in culture. The GAPDH probe was used as a control of loading.
(C) Activity of the Cyclin D2 promoter. Luciferase reporter con-
structs carrying the wild-type human Cyclin D2 promoter
(−1624) or independent deletions/mutations in the two E-boxes
were transfected into MCA3D cells together with a mouse Snail
expression vector or the empty vector (pcDNA3) as a control.
Luciferase activity was assayed 40 h after transfection. Activity
is expressed relative to that of the wild-type construct. Results
are the mean values ± S.E. of duplicates from four independent
Snail represses Cyclin D2 transcription. (A) Analysis
Snail in proliferation and survival
GENES & DEVELOPMENT1133
bryo (Fig. 5A,B). On the whole, as BrdU incorporation
increased, Snail expression decreased (Fig. 5, cf. A and B
[along the proximodistal axis of the allantois], C and D
[in the section taken through the forebrain]). We also
analyzed the phosphorylation of histone H3 to quantify
the cells undergoing mitosis (Prigent and Dimitrov 2003)
in the developing nervous system of 8.5-dpc mouse em-
bryos. We observed high levels of phospho-H3 in the ven-
tricular surface of the neural epithelium, where nuclei
undergo mitosis (Fig. 5G,H). When quantified, at both
the head and trunk levels, an approximately fivefold de-
crease in labeling was observed in the Snail-expressing
regions compared to similar-sized regions located in the
intermediate or ventral areas. These results indicate that
Snail-expressing cells are difficult to find undergoing
DNA synthesis or mitosis both in culture and in em-
Snail confers resistance
to serum depletion-induced cell death
In several studies of hepatocytes (Valdés et al. 2002) and
cancer cells progressing towards malignancy (Thiery
2002; Siegel and Massagué 2003), the transition to the
mesenchymal phenotype is associated with a reduced
susceptibility to apoptosis. Because Snail induces a com-
plete EMT in MDCK cells (Batlle et al. 2000; Cano et al.
2000), accompanies tumor malignancy (Blanco et al.
2002; Sugimachi et al. 2003), and is induced in hepato-
cytes undergoing EMT (Spagnoli et al. 2000; Valdés et al.
2002), we tested whether Snail conferred resistance to
cell death. Two main pathways can provoke apoptosis,
the stress pathway triggered by developmental cues or
intracellular damage (?-irradiation, cytokine depriva-
tion, etc.), and the activation of death receptors such as
those of the TNF family mediated by extracellular sig-
nals, among them TNF- ?, Fas ligand, and Trail.
To analyze the response to cellular stress, cells were
maintained in the absence of serum. MDCK-Snail cells
survived for at least 7 d, whereas a large amount of cell
death was seen in mock-transfected cells after 2–3 d of
serum deprivation (Fig. 6A). After 48 h in culture, label-
ing with propidium iodide showed that whereas mock
transfectants had 10.3 ± 0.1% positive nuclei, only
3.8 ± 0.2% of the nuclei of Snail-expressing cells were
labeled. Caspase-3 is involved in the cell death response
and was three times more active in mock-transfected
cells than in the corresponding Snail-expressing cells
To check whether Snail may confer resistance to the
death induced by developmental cues, we compared the
pattern of cell death with that of Snail expression in
mouse embryos (Fig. 6C). Although a clear pattern of cell
death has been identified in rhombomeres (r) 2, 3, and 5
in the developing hindbrain of the chick (Graham et al.
1996; Ellies et al. 2000), the situation is not so clear in
the mouse (Trainor et al. 2002). By comparing with the
known expression of Krox-20 in the presumptive rhom-
bomeres (pr) 3 and 5 at this developmental stage (Fig.
6C), we observed numerous dying cells in pr2, 3, 5, and 6
D2 and Snail expression in mouse embryos. Whole-
mount in situ hybridization of 8.5-dpc mouse embryos
(A–C) and transverse paraffin sections of the same em-
bryos taken at the level of the posterior hindbrain (D–F),
the trunk (G–I), and the allantois (J–L). Snail expression
can be observed at the edges of the neural plate (D, pnc)
corresponding to premigratory crest cells undergoing
EMT. Snail expression is maintained in crest cells after
delamination (G, nc), and it is also apparent in the de-
condensing somites (G, s) and in the allantois (J, al). An
inverse correlation between Snail and Cyclin D2 tran-
scripts is readily observed in all the tissues analyzed (cf.
D,G,J and F,I,L). (K) Although this correlation is not so
striking for Snail and Cyclin D1, note that Cyclin D1
expression is not detected in regions with high levels of
Snail transcripts such as the allantois. (al) Allantois;
(hb) hindbrain; (nc) neural crest; (np) neural plate; (nt)
neural tube; (pnc) premigratory neural crest; (s) somite.
An inverse correlation exists between Cyclin
Vega et al.
1134GENES & DEVELOPMENT
(NBS; Fig. 6C). Conversely, pr4 and the anterior spinal
cord are regions where cell death was not apparent, co-
inciding with areas of Snail expression (Fig. 6C, high
magnification). The absence of Snail expression from
other regions of intense cell death was also apparent in
the midbrain and forebrain, and in other tissues outside
of the central nervous system such as the developing
heart (Fig. 6C). In contrast, mesenchymal tissues of the
head, including the migratory neural crest cells, showed
high levels of Snail transcripts and no cell death. In sum-
mary, an inverse correlation between cell death and
Snail expression existed in the embryos analyzed (Fig.
Given that Snail-expressing cells were resistant to se-
rum depletion, we studied the activity of different sur-
vival pathways in these cells. Both the MAPK and PI3K
pathways are highly active in Snail-expressing cells (Fig.
7A,B). In agreement with the higher levels of PI3K activ-
ity, we also found a much greater phosphorylation of its
downstream effector Akt (Fig. 7B). Both the MEK/Erk
and PI3-K/Akt pathways can mediate the up-regulation
of Bcl-xLexpression (Ramljak et al. 2003), a death-inhibi-
tory member of the Bcl-2 family that blocks the stress-
induced release of cytochrome c from the mitochondria.
Thus, we looked at the expression of Bcl-xLin MDCK-
Snail cells and found that it was increased at all times
analyzed (Fig. 7C). These data indicate that the activa-
tion of the MEK/Erk and PI3-K/Akt pathways may ex-
plain the survival properties associated with Snail ex-
pression following serum depletion.
Snail protects from TNF-?-induced cell death
Because Snail expression conferred resistance to stress-
induced cell death, we checked whether Snail-expressing
cells might also be resistant to that induced by pro-
apoptotic signals that activate the death receptor path-
way such as TNF-?. Indeed, whereas mock-transfected
MDCK cells died within 24 h of treatment, MDCK-Snail
cells survived (Fig. 8A). Caspase-8 is specifically re-
cruited to the death receptors upon ligand binding and is
then auto-activated initiating the apoptotic pathway.
Accordingly, we found that treatment with TNF-? in-
duced the activation of caspase-8 in mock-transfected
cells. An approximately fivefold decrease in the activity
of caspase-8 was observed in Snail-expressing cells after
24 h of treatment compared with that of mock transfec-
tants. This result substantiates that the death observed
in MDCK cells was mediated by the activation of this
pathway (Fig. 8B). As expected, in both cell types the
activity of the initiator, caspase-8, correlated with that of
the effector, caspase-3 (Fig. 8C), confirming that the ex-
pression of Snail protects the MDCK cells from TNF-?-
Slug behaves in the chick as Snail in the mouse
We showed previously that the Snail family member
Slug induces EMT in the chick embryo (Nieto et al.
1994) and that in general, the expression patterns of
these two family members are interchanged at the sites
of EMT in birds and mammals (Sefton et al. 1998). In-
deed, the cells undergoing EMT in the neural tube and
the primitive streak express Snail in the mouse and Slug
in the chick. Nevertheless, Snail can induce EMT when
ectopically expressed in the chick embryo hindbrain (Del
Barrio and Nieto 2002), indicating that the two proteins
can be functionally equivalent during embryonic devel-
opment. However, even though they can be functionally
equivalent, only one gene is expressed in each corre-
sponding embryo and thus, Snail could not play a role in
the control of cell division or survival in the neural tube
of the chick, because it is not expressed there. Thus, this
functional equivalence and the striking interchange in
the expression patterns led us to analyze whether the
family member expressed in the chick, Slug, can regulate
cell-cycle progression and survival in the neural tube.
and Snail expression in mouse embryos. The embryos in A and
B show a side-by-side comparison between Snail expression and
BrdU incorporation as a measure of cell proliferation in the
whole embryo in culture. An overall complementary pattern is
observed, that can be better examined in the sections taken at
the level of the forebrain (C,D) and the base of the allantois (E,F).
G and H show sections at the trunk level to compare Snail
expression with histone H3 phosphorylation, taken as a mea-
sure of cells undergoing mitosis. The squares mark the Snail-
expressing region of the neural epithelium. (a) Amnion; (al) al-
lantois; (fb) forebrain; (h) heart; (hb) hindbrain.
An inverse correlation exists between proliferation
Snail in proliferation and survival
GENES & DEVELOPMENT1135
To analyze the cell cycle we measured both BrdU in-
corporation and phospho-H3 expression in chick em-
bryos as we had performed for the mouse. We observed
high levels of cell proliferation in the basal half of the
neural tube epithelium, where the nuclei undergo DNA
synthesis. In contrast, and as previously described
(Burstyn-Cohen and Kalcheim 2002), the levels of incor-
poration were much lower in the dorsal region of the
neural tube at the level of the epithelial somites (Fig.
9A). Interestingly, the dorsal neural tube is occupied by
the premigratory neural crest, which expresses high lev-
els of Slug transcripts (Fig. 9B). Quantification of BrdU-
positive cells indicated that the proportion of cells in the
S phase of the cycle was approximately three times less
in the Slug-expressing area compared to that found in
nonexpressing regions of a similar size (data not shown).
When we quantified the cells containing phosho-H3 all
along the dorsoventral axis of the neural tube, we found
that in the Slug-expressing area, positive cells were only
∼15% of those found in adjacent regions (data not
shown). Thus, with respect to the cell cycle, Slug-ex-
pressing cells in the developing neural tube of the chick
embryo behave as Snail-expressing cells in the mouse
embryo and similarly, they are difficult to find undergo-
ing DNA synthesis or mitosis.
As already mentioned, cell death has been analyzed in
detail in the chick embryonic hindbrain (Ellies et al.
2000), with cell death in neural crest cells from r3 and r5
being crucial for the patterning of the branchial region
(Graham et al. 1996; Ellies et al. 2002; Trainor et al.
2002). We carried out a careful analysis of cell death by
both TUNEL and NBS staining and compared the data
with the expression of Slug. We found that r4, which is
naturally protected from cell death, expressed much
higher levels of Slug transcripts than the adjacent rhom-
bomeres (Fig. 9C–E). This result is reminiscent of the
data regarding cell death and Snail expression from
mouse embryos (see Fig. 6C).
sis induced by serum deprivation. (A) Cell vi-
ability was assessed by propidium iodide
staining 48 h after serum depletion. (B)
Caspase-3 activity at different times after se-
rum removal represented as mean values ±
S.E. from three independent experiments car-
ried out with duplicate dishes. Note the low
levels of activity in Snail-expressing cells 48h
after serum deprivation compared to the
mock-transfected cells. (C) Cell death visual-
ized using Nile Blue Sulphate staining is com-
pared side-by-side with Snail expression in
the head of an 8.5-dpc mouse embryo. The
pattern of cell death assessed by Nile Blue
Sulphate (NBS) staining (stars) is complemen-
tary to that of Snail (brackets). A similar em-
bryo hybridized with Krox-20 to indicate the
relative position of pre-rhombomeres (pr) 3
and 5 in the hindbrain to help compare the
pattern of cell death and Snail expression.
The inverse correlation can be better assessed
in the high-power photographs. (fb) Forebrain;
(h) heart; (mb) midbrain; (sc) anterior spinal
Snail confers resistance to apopto-
Vega et al.
1136GENES & DEVELOPMENT
Taking advantage of the amenability of the chick em-
bryo to experimental manipulation, we further studied
the role of Slug in promoting cell survival by overex-
pressing it in the neural tube in ovo. High levels of GFP
(and Slug, data not shown) expression could be achieved
by co-electroporation of Slug- and GFP-encoding vectors
into the right-hand side of the developing neural tube
(Fig. 9F). Several streams of electroporated neural crest
cells were observed migrating from the hindbrain (Fig.
9F). NBS staining of the same embryo shows that the
area of Slug overexpression presents a dramatic reduc-
tion of the naturally occurring cell death (Fig. 9G,H).
Rescue of cell death could be observed in 53% of the
electroporated embryos (n = 30), indicating that Slug can
act as a survival factor in the chick embryo hindbrain. Is
it worth noting here that the amount of naturally occur-
ring cell death in the different rhombomeres correlates
with the balance in the expression of the death inducer
BMP (Graham et al. 1994) and the survival factor Slug.
Indeed, r4 expresses high levels of Slug and very low
levels of BMP4. This balance also explains why in our
overexpression experiments the death of r2 cells (with
low levels of both BMP4 and Slug) can be more effectively
rescued by Slug overexpression than that of r5 (which
expresses very high levels of BMP4; data not shown).
Although some of the migratory Slug-expressing cells
pass close to the otic vesicles, the dying cells indicated
by black stars in Figure 9H are nonelectroporated ecto-
dermal cells (as assessed in sections, data not shown).
These results highlight the specificity of this rescue in
the electroporated cells, and confirm that Slug confers
resistance to cell death in the developing embryo.
Snail genes impair cell-cycle progression
in cultured cells and in developing embryos
The epithelial mesenchymal transition (EMT) confers
migratory and invasive properties to epithelial cells,
critical for the generation of cells that originate at a dis-
tance from their final destination (Nieto 2002) during
embryonic development. When the transition to a mes-
enchymal phenotype occurs in the adult, it is usually
associated with pathological processes such as tumor
progression (Thiery 2002). Concomitant with the acqui-
sition of motility, cells undergo dramatic changes in cell
adhesion properties and cell shape. Snail induces a com-
plete EMT in epithelial cells concurrent with the afore-
mentioned changes (Batlle et al. 2000; Cano et al. 2000)
and is associated with the process of EMT in both physi-
ological and pathological conditions (Nieto 2002; Thiery
2002; Yáñez-Mo et al. 2003). The conversion to mesen-
chyme implies a profound reorganization of the cyto-
skeleton that may be incompatible with a highly prolif-
erative estate. Here we demonstrate that cell prolifera-
tion is impaired in Snail-expressing cells, supporting this
different survival pathways were analyzed in cells cultured in
the absence of serum and collected at different times. The levels
of active ERKs (phospho-erk1 and phospho-erk2; A), active Akt
(B), and Bcl-xL(C) were analyzed by Western blot and found to
be increased in Snail-expressing cells. Total erk2 was used as a
control for gel loading. (B) Higher levels of PI3K activity were
also detected in Snail-expressing cells as analyzed by thin-layer
chromatography, compatible with the higher levels of phos-
phorylation found for Akt.
Snail activates survival pathways. Molecules from
TNF-?. Mock and MDCK-Snail-expressing cells were treated
with TNF-? (5 ng/mL) after being pretreated with cyclohexi-
mide (0.5 µg/mL for 30 min) to prevent the induction of the
survival protein NF?B. (A) Photographs of the cultures taken
after 16 or 24 h of treatment. (B,C) The activity of the death
receptors-specific caspase-8 and effector caspase-3, respectively,
are shown from one representative experiment. Note the low
activity of both caspases in Snail-expressing cells, explaining
the healthy appearance observed in A.
Snail confers resistance to the cell death induced by
Snail in proliferation and survival
GENES & DEVELOPMENT1137
hypothesis. Further evidence that profound morphologi-
cal changes are incompatible with high proliferation can
be seen in the low BrdU incorporation in the premigra-
tory neural crest (Burstyn-Cohen and Kalcheim 2002; the
present study), in the mitotic arrest in the cells of the
ventral furrow during Drosophila gastrulation (Foe
1989), the low proliferation in the invasive front of car-
cinomas (Jung et al. 2001), and the link between high p21
expression and poor prognosis in breast carcinomas
(Yang et al. 2003). Significantly, Snail is expressed and
triggers EMT in all of these territories and circumstances
(for review, see Nieto 2002).
We have found that Snail impairs the transition from
early to late G1 by maintaining low levels of Cyclins D
and can block the G1/S transition by maintaining high
levels of p21. However, Snail-expressing cells can re-
spond to mitogenic signals by transiently decreasing p21
expression, which favors the transition to the S phase.
Interestingly, neural crest cells synchronously enter into
the S phase upon delamination from the neural tube
(Burstyn-Cohen and Kalcheim 2002), which occurs after
the process of EMT has been completed. Thus, when
Snail (and Slug in the chick) induces EMT in the premi-
gratory neural crest cells, it probably blocks prolifera-
tion, synchronizing them in G1 and allowing morpho-
logical changes to occur. Subsequently, cells will enter S
phase upon delamination. Interestingly, the bladder epi-
thelial carcinoma cell line NBT-2 is receptive only to
FGF-induced EMT at G1 (Bonneton et al. 1999), and lym-
phocyte migration and hepatoma cell invasion occur
only in G1 (Ratner 1992; Iwasaki et al. 1995). Thus, al-
though a deregulation/increase in cell division is crucial
for tumor formation and growth, this is not so for tumor
malignization. The behavior of invasive cells allows tu-
mor proliferation to be dissociated from malignancy.
Snail genes protect cultured cells and embryos
from cell death induced by both the retrieval
of survival factors and apoptotic signals
Selective cell death is of crucial importance for sculpting
the embryo and maintaining tissue homeostasis. How-
ever, deregulation of programmed cell death can be criti-
cal in pathological processes such as cancer. Several
studies have correlated the conversion to a mesenchy-
mal phenotype with cell survival. Indeed, EMT protects
fetal hepatocytes from the death induced by TGF-?
(Valdés et al. 2002). Interestingly, TGF-? has multiple
effects on cellular behavior, from inducing growth arrest
and death to triggering EMT, survival, and tumor pro-
gression (Siegel and Massagué 2003). Indeed, when ex-
pressed in the skin of mice, TGF-? inhibits the forma-
tion of benign tumors but increases the frequency of in-
vasive spindle carcinomas (Cui et al. 1996). Members of
the TGF-? superfamily including TGF-?s and BMPs in-
duce Snail/Slug expression in several systems such as
hepatocytes (Spagnoli et al. 2000; Gotzmann et al. 2002;
Valdés et al. 2002), epithelial and mesothelial cells (Pei-
nado et al. 2003; Yáñez-Mo et al. 2003), and in the de-
veloping embryo (Dickinson et al. 1995; Liem et al. 1995;
Romano and Runyan 2000; Piedra and Ros 2002). When
Snail genes are induced by TGF-?, EMT is triggered and
cells become resistant to TGF-?-induced cell death
(Valdés et al. 2002), consistent with Snail activating the
Mek/Erk and PI3K/Akt survival pathways. TGF-? is also
capable of inducing the progression towards invasive car-
and protects the developing neural tube from physiological cell
death in the chick. (A) In ovo BrdU incorporation was analyzed
in transverse sections. One such section from the trunk of a
stage 11 chicken embryo is shown. (B) A similar section hybrid-
ized with a Slug probe. Note the absence of BrdU in the premi-
gratory neural crest, showing high levels of Slug expression.
(C,D) The pattern of cell death in the hindbrain region of a stage
12 chick embryo as assessed by NBS and TUNEL staining, re-
spectively. Compare the pattern of cell death (blue and brown
stars in C,D) with that of Slug transcripts (E). As previously
described, r4 shows very few apoptotic cells, coinciding with
high levels of Slug transcripts (brackets). (F–H) An embryo
electroporated with plasmids containing chick Slug and GFP
cDNAs at stage 8 and analyzed 15 h later (stage 12). (F) GFP (and
thus, Slug) expression is observed in the right-hand side of the
neural tube and in cells migrating from it. (G) NBS staining of
the same embryo shows a striking decrease in cell death in the
side where Slug is overexpressed. (H) A higher-magnification
picture that allows a better assessment of the region protected
from cell death. The dotted lines demarcate the borders of the
neural tube and the otic vesicle. The black stars indicate a re-
gion of ectodermal cell death that appears symmetrical on both
sides of the embryo (see text). (hb) Hindbrain; (mb) midbrain;
(nc) neural crest; (nt) neural tube; (ov) otic vesicle, (s) somite.
Slug expression correlates with little proliferation
Vega et al.
1138GENES & DEVELOPMENT
cinoma (Cui et al. 1996; Siegel and Massagué 2003), and
this is usually explained by the tumor cells overcoming
the TGF?-induced growth arrest (Siegel and Massagué
2003). However, in light of our data, it seems more likely
that these invasive tumor cells overcome cell death and
not growth arrest. Indeed, TGF-? induces EMT in epi-
thelial cells and hepatocytes concomitant with the inhi-
bition of apoptosis but without altering the growth re-
tardation effect (Lehmann et al. 2000; Valdés et al. 2002;
Peinado et al. 2003).
With respect to embryonic development, we show
here that the expression of Snail genes in the chick and
the mouse are inversely correlated with cell death in
different developing tissues. Significantly, Slug overex-
pression protects the neural crest from the naturally oc-
curring cell death in the chick hindbrain. Interestingly,
members of the BMP family induce Snail or Slug expres-
sion in the hindbrain (Nieto 2002), and BMPs are also
responsible for the cell death induced in particular rhom-
bomeres (Graham et al. 1996). This situation appears to
be similar to the response of hepatocytes to TGF-?,
where this factor induces cell death in half of the cell
population and induces EMT and resistance to TGF-?-
induced cell death in the other half (Valdés et al. 2002).
TGF-?-induced EMT and survival in both hepatocytes
and epithelial cells occurs concomitantly with Snail in-
duction (Valdés et al. 2002; Peinado et al. 2003). Alto-
gether, these data are in agreement with the notion that
high levels of Snail/Slug expression in both mouse and
chick hindbrains are sufficient to overcome the BMP-
induced cell death.
Other members of the Snail superfamily are thought to
mediate cell survival. In Caenorhabditis elegans, Ces-2
represses the Scratch homolog Ces-1 (Metzstein and
Horwitz 1999), promoting the physiological death of a
particular class of neurons. In humans, a translocation
converts the Ces-2 homolog (HLF) in an activator that in
turn induces Slug expression, leading to aberrant cell
survival and leukemogenesis (Inukai et al. 1999). Fur-
thermore, hematopoietic progenitors in Slug null mu-
tant mice show an increased sensitivity to the death in-
duced by ?-irradiation (Inoue et al. 2002; Pérez-Losada et
al. 2003). However, there is no indication that Scratch
protects cells in C. elegans physiology (Metzstein and
Horwitz 1999; Thellmann et al. 2003), or that Slug pro-
motes survival in physiological circumstances in mam-
mals. The evidence in these cases points to an anti-apo-
ptotic function following DNA damage (Inoue et al.
2002; Pérez-Losada et al. 2003). It thus seems likely that
Snail in mammals and Slug in avians may be more effi-
cient in conferring resistance to cell death, with the
other family member maintaining certain activity of this
ancestrally inherited property. The ancestral condition
associated with the Snail superfamily is also supported
by the fact that Scratch is the member acting as such in
C. elegans, as the nematode does not seem to express any
functional Snail protein (Manzanares et al. 2001).
In conclusion, Snail favors changes in cell shape rather
than proliferation in cells that become migratory, in ac-
cordance with the often neglected low rates of prolifera-
tion observed at the invasive front of tumors. Concomi-
tantly, Snail offers protection from both stress-induced
cell death and that provoked by pro-apoptotic signals. In
this way, Snail confers a selective advantage to invasive
cells to migrate through hostile territories. This resis-
tance to cell death is essential in the embryo for migra-
tory cells to reach their final destinations and in the
adult for malignant cells to disseminate and form me-
Materials and methods
Cell lines and antibodies
Canine MDCK (Madin-Darby canine kidney) and mouse epider-
mal keratinocyte MCA3D cells were grown in Dulbecco’s
modified Eagle medium (DMEM) and Ham’s F12, respectively,
supplemented with 10% fetal calf serum (FCS). The following
antibodies were used: Polyclonal antisera against Cyclins D1,
D2, and D3, cdk4, p21, p27, Erk2, and Bcl-x (Santa Cruz Bio-
technology), anti-phospho Akt (New England Biolabs), and
Phosphohistone-3 (Upstate Biotechnology); monoclonal p21/
Cip1 and retinoblastoma protein (Pharmingen BD Biosciences)
and anti-MAPK (p44/p42; Cell Signaling Technology) antibod-
ies. As secondary antibodies, both peroxidase-conjugated (anti-
mouse and anti-rabbit) were used (Bio-Rad Laboratories) to-
gether with a biotinylated anti-rabbit serum (Vector).
Mouse embryos were obtained from natural matings of Balb-C
mice from the animal facility at the Cajal Institute. Fertilized
hen eggs were obtained from Granja Santa Isabel, Córdoba,
Spain. The age of mouse embryos was determined as days post-
coitum (dpc), the day on which the vaginal plug was detected
being designated 0.5 dpc. Eggs were incubated and opened, and
the embryos staged according to Hamburger and Hamilton
Analysis of cell proliferation through BrdU incorporation
and phosphohistone-3 immunohistochemistry
To measure the percentage that incorporated 5-bromo-2?-deoxy-
uridine (BrdU), cells were grown on coverslips in DMEM plus
10% FCS. BrdU (10 µM in PBS) was added at different times, and
cells were fixed and stained 1 h after addition according to the
Detection kit II (Roche). Cells that had incorporated BrdU were
visualized and quantified using a Leica DMR microscope. For in
ovo labeling, BrdU (10 mM) containing fast green (0.25 µL/mL)
in PBS was injected into chick embryos 1 h before sacrificing.
Mouse embryos were dissected in DMEM plus 10% FCS at 8.5
dpc, leaving the extraembryonic membranes intact. BrdU (10
mM) was immediately injected into the amniotic cavity, and
the embryos were cultured for 1 h. They were fixed overnight in
4% paraformaldehyde at 4°C, dehydrated through a series of
ethanol, and rehydrated. In the case of chick embryos, after
fixation, dehydration, and rehydration, immunohistochemistry
was carried out on 15-µm paraffin sections (Fibrowax, BDH).
After paraffin removal with HistoClear, the chick embryo sec-
tions were rehydrated, and both the chick sections and mouse
embryos were processed to develop BrdU incorporation follow-
ing the manufacturer’s instructions (Roche).
Cells undergoing mitosis were identified by the presence of
phospho-histone-3 (Prigent and Dimitrov 2003) in 50-µm vibra-
tome sections obtained from mouse and chicken embryos. The
Snail in proliferation and survival
GENES & DEVELOPMENT1139
sections were incubated overnight with PBT (PBS, 0.5% Triton
X-100) at 4°C and subsequently treated with 0.1% H2O2for 4 h
at room temperature. After washing, they were blocked with
10% FCS and 1 mg/mL BSA in PBT for 3 h at 4°C. Incubation
with primary and secondary (biotinylated) antibodies was per-
formed overnight at 4°C. After washing, the sections were de-
veloped with the ABC kit (Pierce).
Analysis of DNA content by flow cytometry
Cells were detached from dishes with trypsin, fixed in 70%
ethanol (−20°C) for 1 min, and treated with RNAse (1 mg/mL)
for 15 min at 37°C. After propidium iodide staining (0.05 mg/
mL in PBS 15 min at room temperature in the dark), the cellular
DNA content was evaluated in a FACS flow cytometer (Becton-
Dickinson). For computer analysis, only signals from single
cells were considered (10,000 cells/assay).
Western blots and immunoprecipitation assays
Cells were scraped off the plates after washing with cold PBS
and lysed at 4°C in the following buffer: 20 mM Tris-HCl at pH
7.4, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 1 mM NaF,
100 mM ?-glycerophosphate, 1 mM EGTA, 5 mM NaPPi, 5
µg/mL leupeptin, 1 mM sodium o-vanadate, and 1 mM PMSF.
Proteins were separated by SDS-PAGE on 12% or 7.5% (for
retinoblastoma protein), and transferred to PVDF membranes
(Millipore) that were then blocked in TTBS (TBS plus 0.05%
Tween-20) containing 5% nonfat dried milk. The membranes
were incubated for 2 h with the corresponding primary antibody
at room temperature in blocking solution. After washing, they
were incubated with peroxidase-conjugated anti-mouse or anti-
rabbit immunoglobulin for 1 h at room temperature (1:3000).
Antibody binding was visualized by ECL (Amersham Biosci-
For immunoprecipitation assays to detect levels of p21 and
p27, the cells were lysed and the proteins purified as above.
Equal amounts of total protein (400 µg) were incubated with 1
µg of the appropriate antibody (p21/Cip1 or p27/Kip1) and pro-
tein A-agarose beads (Sigma) for 3 h at 4°C, and the beads were
washed twice with lysis buffer, centrifuged, resolved by SDS-
PAGE, and subjected to immunoblot analysis as described above.
RT–PCR analysis and primer sequence
Poly(A)+mRNA was isolated from MDCK cells using the Mi-
crofast Track isolation kit (Invitrogen) and treated with DNAse
I before cDNA synthesis. Reverse transcription was carried out
as described (Sefton et al. 1998), and PCR to amplify coding
fragments for canine Cyclins D1 and D2 was performed over 35
cycles at an annealing temperature of 55°C using primers as
follows. For Cyclin D1, primers derived from the mouse se-
quence were used: forward, 5?-CTGCGAAGTGGAGACCAT
CCG-3?; and reverse, 5?-GTCCGGGTCACACTTGATGAC-3?
(mouse-specific primers). For Cyclin D2, degenerate primers
were used: forward, 5?-GAA/GGAA/GC/AGITAT/CT/CTIC
CICAA/GTG-3?; and reverse, 5?-GAA/GTACATIGCA/GAAT/
CTTA/GAAA/GTC-3?. The amplified fragments were sub-
cloned in pGEMT-easy and sequenced. After sequencing, dog-
specific primers were designed and after a similar amplification
protocol, the fragment was subcloned, sequenced, and used for
Northern blot analysis.
Total RNA was isolated as described by Chomczynski and Sac-
chi (1987). For each assay, 20 µg of denatured RNA was used per
lane. The coding fragments for canine Cyclins D1 and D2 ob-
tained by RT–PCR as described above were labeled using the
Rediprime II kit (Amersham Biosciences). A human GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) probe was used as
a control for the quantity of RNA.
Transient transfections and promoter analysis
To analyze the Cyclin D2 promoter, mouse epidermal keratino-
cyte MCA3D cells were cotransfected with 40 ng of Renilla
vector and 400 ng of pGL3 vector (Promega) containing the Cy-
clin D2 promoter fused to the Luciferase reporter gene together
with 25 ng of human SNAI1 or mouse Snail complete coding
sequences in pZeo (Invitrogen) and pcDNA3 (Invitrogen) vec-
tors, respectively. Control transfections were performed with
the empty plasmids (Mock). Transfections were carried out us-
ing Lipofectamine (Life Technologies). Luciferase and Renilla
activities were assayed using the dual-luciferase reporter system
kit (Promega), and the activity normalized to that of the pro-
moter cotransfected with the control vectors. Human D2-Luc
(1624 base pairs) and the deletions −1303 D2-Luc and −444 D2-
Luc were kindly provided by Brad H. Nelson (Martino et al.
2001). The D2-Luc (−1624) was used to delete and mutate the
two E-boxes present in the human Cyclin D2 promoter with a
Quickchange Site Directed Mutagenesis Kit (Stratagene). The
core sequence 5?-GCACGTGC-3? contained in the two E-boxes
of the human Cyclin D2 promoter was independently mutated
to 5?-TTACGTGC-3?. These two point mutations (GC to TT)
abolish the E-box and were shown to derepress the proximal
mouse E-cadherin promoter (Cano et al. 2000).
In situ hybridization
Whole-mount in situ hybridization was carried out in chick and
mouse embryos at several stages of development as described
(Nieto et al. 1996). Digoxigenin-labeled probes were synthesized
from the full-length cDNA of mouse Snail and chick Slug.
Probes for the mouse Cyclins D1 and D2 were synthesized from
cDNAs obtained from the mouse epithelial cell line NMuMG
and amplified by RT–PCR with the primers and conditions de-
scribed above. Following hybridization, the embryos were incu-
bated with alkaline phosphatase-conjugated anti-digoxigenin
antibody. The alkaline phosphatase activity was detected by
incubation with NBT/BCIP substrates (Roche). After hybridiza-
tion, embryos were fixed in 4% paraformaldehyde in PBS,
washed, and photographed in whole mount under a Leica M10
dissecting scope. Subsequently, mouse embryos were embedded
in paraffin (Fibrowax), sectioned at 15 µm, and photographed
using a Leica DMR microscope.
Cell death analysis in cell lines and embryos
Cells were grown on coverslips in 6-cm cell culture dishes and
stained with propidium iodide (0.05 mg/mL) in PBS for 15 min
at room temperature in the dark. Only cells with altered mem-
brane permeability were stained and could be visualized using a
Leica DMR microscope.
For the analysis of apoptosis in embryos, DNA fragmentation
was detected using the TUNEL in situ cell death detection kit
(Roche) or by staining with Nile Blue Sulphate (NBS; Sigma)
that marks dying cells. For TUNEL detection, whole embryos
were fixed in 4% paraformaldehyde in PBT at 4°C, dehydrated
and treated for 2 h at room temperature in 100% methanol plus
1% H2O2, and washed several times in methanol. After rehy-
dration the embryos were digested with proteinase K (10 µg/mL
for 3 min) at room temperature, washed, and fixed for 30 min in
Vega et al.
1140GENES & DEVELOPMENT
4% paraformaldehyde. After several washes, they were treated
with the reaction mix for 2 h at 37°C, washed, blocked for 2 h
with blocking solution (KTBT 0.1% Triton X-100, 15% FCS,
0.7% blocking powder from Roche), and incubated with the
POD-converter (1 h at 37°C). Embryos were developed in the
dark with DAB (3,3?-Diaminobenzidine, Sigma) containing
0.03% H2O2. After washing, embryos were photographed and
embedded in 0.5% gelatin (Sigma) prior to being sectioned at 40
µm on a vibratome. NBS staining was carried out on whole
embryos immediately after dissection. The embryos were incu-
bated in NBS (20 µg/mL in PBS) containing 0.1% Tween 20 for
30 min at room temperature, briefly washed in PBS, and imme-
diately photographed in 4% PFA.
Analysis of caspase-3 and caspase-8 activities
Cells attached to the dish and those in the supernatant were
collected and lysed at 4°C in 5 mM Tris-HCl at pH 8.0, 20 mM
EDTA, and 0.5% Triton X-100. Ac-DEVD-AMC and Ac-IETD-
AFC were used as substrates to measure the enzymatic activi-
ties of caspase-3 and caspase-8, respectively (Herrera et al. 2001).
Analyses were performed in a Luminescence Spectrophotom-
eter (Perkin-Elmer LS-50). A unit of caspase activity was defined
as the amount of active enzyme necessary to produce an in-
crease of 1 arbitrary luminescence unit in 2 h. The protein con-
centration of the cell lysates was determined with the Bio-Rad
assay kit, and the results are presented as units of caspase ac-
tivity per µg of protein.
After solubilization of cells in lysis buffer (10 mM Tris-HCl, 5
mM EDTA, 50 mM NaCl, 30 mM NaPPi, 50 mM NaF, 100 µM
sodium o-vanadate, 1% Triton X-100 at pH 7.6) containing leu-
peptin (10 µg/mL), aprotinin (10 µg/mL) and 1 mM PMSF, ly-
sates were clarified by centrifugation, and the proteins were
immunoprecipitated with a monoclonal anti-Tyr-Phosphate an-
tibody (Py72). Immunoprecipitates were used to analyze PI 3-ki-
nase activity by in vitro phosphorylation of phosphatidylinosi-
tol as described (Valdés et al. 2004).
Chick embryo electroporation
In ovo electroporation was essentially carried out as described
(Del Barrio and Nieto 2002) with the following modifications:
pCX-EGFP construct (1 mg/mL; Ikawa et al. 1995) was co-elec-
troporated with pCX-Slug (1.5 mg/mL) containing the full-
length chick Slug cDNA or with the empty pCX vector as a
control. The DNAs were injected into stage 8 chick hindbrains
in ovo and electroporated using two 50-msec 10 V pulses. The
embryos were allowed to develop for a further 14–16 h. In all the
experiments the control side was to the left. Embryos were pho-
tographed in ovo to record GFP expression and processed for
We thank members of the Nieto lab for helpful discussions
throughout the completion of this work, B. Nelson for Cyclin
D2 constructs, J. Miyazaki and M. Okabe for providing the
pCX-EGFP plasmid, A. Vázquez for help with the flow cytom-
etry analysis, and M. Sefton for critical reading of the manu-
script and editorial assistance. This work was supported by
grants from the Spanish Ministry of Science and Technology
(DGICYT-BMC2002-0383 to M.A.N.), the Ministry of Health
(FIS-01/985 to M.A.N. and FIS-01/0797 to I.F.), and the Comu-
nidad Autónoma de Madrid (CAM 08.1/0044/2000 and 08.1/
0049.1/2003 to M.A.N. and CAM 08.1/0078/2000 and 08.1/
0003.1/2003 to I.F.). A.V.M. was supported by Advancell S.L.
and the I3P Program (European Social Fund/Spanish Ministry of
Science and Technology), and O.H.O. was the recipient of a
predoctoral fellowship form the Spanish Ministry of Education
The publication costs of this article were defrayed in part by
payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 USC section
1734 solely to indicate this fact.
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