Mice thrive without Cdk4 and Cdk2.

Abstract

Mammalian cell division is thought to be driven by sequential activation of several Cyclin-dependent kinases (Cdk), mainly Cdk4, Cdk6, Cdk2 and Cdk1. Since mice lacking Cdk4, Cdk6 or Cdk2 are viable, it has been proposed that they play compensatory roles. We report here that mice lacking Cdk4 and Cdk2 complete embryonic development to die shortly thereafter presumably due to heart failure. However, conditional ablation of Cdk2 in adult mice lacking Cdk4 does not result in obvious abnormalities. Moreover, these double mutant mice recover normally after partial hepatectomy. In culture, Cdk4(-/-);Cdk2(-/-) embryonic fibroblasts become immortal, display robust pRb phosphorylation and have normal S phase kinetics. These observations indicate that Cdk4 and Cdk2 are dispensable for the mammalian cell cycle and for adult homeostasis.

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Mice thrive without Cdk4 and Cdk2
Ce ´dric Barrie `rea,c,1, David Santamarı ´aa,1, Antonio Cerqueiraa, Javier Gala ´na,
Alberto Martı ´na, Sagrario Ortegab, Marcos Malumbresa, Pierre Dubusc,
Mariano Barbacida,*
aMolecular Oncology, Centro Nacional de Investigaciones Oncolo ´gicas, E-28029 Madrid, Spain
bBiotechnology Program, Centro Nacional de Investigaciones Oncolo ´gicas, E-28029 Madrid, Spain
cEA2406 University of Bordeaux 2, F-33076 Bordeaux, France
A R T I C L EI N F O
Article history:
Received 22 December 2006
Received in revised form
15 February 2007
Accepted 15 February 2007
Available online 14 March 2007
Keywords:
Cyclin-dependent kinases
Mouse development
Cell proliferation
Conditional knock outs
Liver regeneration
A B S T R A C T
Mammalian cell division is thought to be driven by sequential activation of several Cyclin-
dependent kinases (Cdk), mainly Cdk4, Cdk6, Cdk2 and Cdk1. Since mice lacking Cdk4,
Cdk6 or Cdk2 are viable, it has been proposed that they play compensatory roles. We report
here that mice lacking Cdk4 and Cdk2 complete embryonic development to die shortly
thereafter presumably due to heart failure. However, conditional ablation of Cdk2 in adult
mice lacking Cdk4 does not result in obvious abnormalities. Moreover, these double mutant
mice recover normally after partial hepatectomy. In culture, Cdk4?/?;Cdk2?/?embryonic
fibroblasts become immortal, display robust pRb phosphorylation and have normal S phase
kinetics. These observations indicate that Cdk4 and Cdk2 are dispensable for the mam-
malian cell cycle and for adult homeostasis.
ª 2007 Federation of European Biochemical Societies.
Published by Elsevier B.V. All rights reserved.
1. Introduction
The widely accepted model for the mammalian cell cycle
involves sequential activation of related heterodimeric pro-
tein kinases, composed of a catalytic subunit, the Cyclin-
dependent kinase (Cdk), and a regulatory subunit known as
Cyclin (reviewed in Malumbres and Barbacid, 2005). Two of
these Cdks, Cdk4 and Cdk6, are activated by the D-type
Cyclins and have been implicated in the early phases of the
cycle, particularly during exit from quiescence. Cdk4/6-Cyclin
D heterodimeric kinases are supposed to promote re-entry
into the cycle by initiating phosphorylation of the retinoblas-
toma (Rb) protein family, pRb, p107 and p130 (reviewed in
Adams, 2001; Sherr and Roberts, 1999). Rb phosphorylation
results in the liberation of transcription factors, such as mem-
bers of the E2F family (reviewed in Dyson, 1998; Trimarchi and
Lees, 2002), which are bound to the hypophosphorylated Rb
proteins in non-proliferating cells. These transcription factors
are responsible for directing the expression of a variety of
genes essential for advancing cells though the S phase of the
cell cycle (Adams, 2001). Two of these genes encode Cyclins
E1 and E2 which specifically bind to Cdk2. Active Cdk2–Cyclin
E complexes further phosphorylate the Rb protein family,
resulting in their complete inactivation. This process is con-
sidered to be essential for the liberation of transcription fac-
tors that mediate the synthesis of other cell cycle regulators
such as the A-type Cyclins and Cdk1. Sequential activation
of Cdk2 and Cdk1 by the A-type Cyclins is believed to be
* Corresponding author. Centro Nacional de Investigaciones Oncolo ´gicas (CNIO), Melchor Ferna ´ndez Almagro 3, E-28029 Madrid, Spain.
Tel.: þ34 91 2246900; Fax: þ34 91 7328033.
E-mail address: mbarbacid@cnio.es (M. Barbacid).
1These authors contributed equally to this work.
1574-7891/$ – see front matter ª 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.molonc.2007.03.001
available at www.sciencedirect.com
www.elsevier.com/locate/molonc
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essential for the successful duplication of the cellular genome
during the S phase and for progression into mitosis (Dyson,
1998; Trimarchi and Lees, 2002).
This model, mainly deduced from biochemical evidence,
has not sustained genetic scrutiny. For instance, all mouse
cell types, with the exception of pancreatic beta cells and pitu-
itary lactotrophs proliferate normally in the absence of Cdk4
(Rane et al., 1999; Tsutsui et al., 1999). Likewise, ablation of
Cdk6onlyresultsinreductionofasubsetofhematopoieticcells
(Malumbres et al., 2004). Loss of both of these enzymes causes
a much more dramatic phenotype that limits the proliferation
of hematopoietic cell lineagesleading to late embryonic lethal-
ity (Malumbres et al., 2004). Yet, double mutant embryos show
normalproliferationratesinothertissues,indicatingthatCdk4
and Cdk6 only play compensatory roles in cells of hemato-
poietic lineages. In agreement with these observations, mouse
embryonicfibroblasts(MEFs)lackingCdk4andCdk6proliferate
well and become immortal upon continuous culture in vitro.
Moreimportantly,theyexitquiescenceuponmitogenicstimuli
andenterSphasewithnormalkinetics(Malumbresetal.,2004).
Similar results have been obtained in mice lacking the three
D-typeCyclins (Kozaretal.,2004).Likewise,Cdk2,akinasepre-
viouslythoughttobeessentialfordrivingcellsthroughtheG1/S
transition, is dispensable for normal embryonic development
and adult homeostasis (Berthet et al., 2003; Ortega et al.,
2003). Unexpectedly, Cdk2 is essential for the first meiotic divi-
sionofbothmaleandfemalegermcells,anactivitythatcannot
be compensated by any of the other Cdks (Ortega et al., 2003).
These observations have been attributed, at least in part to
compensatory activities between Cdk2 and Cdk4. In this man-
uscript, we report the generation and characterization of mice
carrying germ line as well as conditional mutations in the loci
encoding these kinases. Mice lacking Cdk4 and Cdk2 in the
germ line complete embryonic development and are born
alive. Although they die soon thereafter possibly due to their
limited numbers of cardiomyocytes, the rest of the tissues dis-
play normallevels of cell proliferation. More importantly, con-
ditional ablation of Cdk2 in adult Cdk4knock out mice does not
result in detectable abnormalities even in highly proliferating
tissues. Indeed, these double mutant mice efficiently regener-
ate their livers after partial hepatectomy (PH). These observa-
tions indicate that Cdk4 and Cdk2 are dispensable for
mammalian cell division and raise further questions about
their proposed role in driving the mammalian cell cycle.
2. Results
2.1.
absence of Cdk4 and Cdk2
Complete embryonic development in the
We have generated double mutant Cdk4þ/?;Cdk2þ/loxand
Cdk4þ/?;Cdk2þ/?mice by targeting the Cdk4 locus in ES cells
carrying a conditional Cdk2loxallele (Figure 1). Intercrosses
between Cdk4þ/?;Cdk2þ/?double heterozygous animals result
in the generation of midgestation (E13.5) Cdk4?/?;Cdk2?/?
Figure 1 – Generation of Cdk4L/L;Cdk2lox/loxand Cdk4L/L;Cdk2L/Lmice. (A) Targeting strategy. The targeting vector carries three frt sequences
flanking exons 2 and 4 as well as a PGK-HygR cassette used for positive selection. The vector also contains a PGK-TK cassette used for negative
selection. Recombinant ES cell clones containing a floxed Cdk2loxallele were used to generate mice carrying the Cdk43frtand Cdk2loxalleles in the
same chromosome. These mice were sequentially crossed with transgenic mice expressing the Flpe (pCAG-Flpe) and Cre (CMV-Cre)
recombinases to generate heterozygous Cdk4L/L;Cdk2L/Lmice. Cdk4 coding sequences are indicated by hatched boxes. Cdk4 non-coding exons
are indicated by open boxes. Cdk2 coding sequences are indicated by black boxes. Cdk2 non-coding exons are indicated by grey boxes. frt and loxP
sites are indicated by open and closed triangles, respectively. Only the restriction sites used in these diagnostic hybridizations are indicated. The
location of probes a and b used in Southern blot analysis is indicated by a thick line. (B) Southern blot analysis to identify the Cdk43frtrecombinant
allele. (C) Southern blot analysis to identify the Cdk4Lnull allele.
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embryos with Mendelian ratios (41/165, 25%). More impor-
tantly, a significant percentage of these embryos (about 40%)
complete embryonic development. Newborn Cdk4?/?;Cdk2?/?
mice weigh 25–40% less than their wild type littermates, a
reduction in weight slightly more pronounced than that
reported for mice lacking Cdk4 alone (20–25%) (Tsutsui et al.,
1999; our unpublished observations). Importantly, neonatal
Cdk4?/?;Cdk2?/?animals move normally and ingest milk.
Yet, they die within 24 h after birth.
Histological analysis indicate that organogenesis is not sig-
nificantly affected in newborn Cdk4?/?;Cdk2?/?mice. More-
over, they display normal levels of proliferating (Ki67) and
apoptotic (TUNEL, active Caspase 3) cells in all tissues exam-
ined (data not shown), except in heart (see below). Previous
studies with double Cdk mutant mice have revealed strong
compensatory activities in the hematopoietic system between
Cdk4 and Cdk6, but not between Cdk2 and Cdk6 (Malumbres
et al., 2004). To determine the extent of compensation
between Cdk2 and Cdk4 in hematopoietic cells we analyzed
peripheral blood and fetal liver from late embryos for com-
parative purposes. Cdk4?/?;Cdk6?/?embryos die during late
embryonic development due to anemia caused by limited
proliferation of erythroid progenitors (Malumbres et al.,
2004). In contrast, the relative levels of white and red blood
cells in E17.5 Cdk4?/?;Cdk2?/?embryos were basically normal
except for a small decrease in the number of red blood cells
(1.27?106?0.14 RBC/mm3versus 1.45?106?0.11 RBC/mm3
in wild type mice, n ¼3) that did not affect viability. Analysis
of hematopoietic precursors by flow cytometry revealed that
the relative percentages of hematopoietic stem cells (HSC),
granulocyte-macrophage progenitors (GMP), common mye-
loid progenitors (CMP) and megakaryocyte-erythroid progeni-
tors (MEP) in Cdk4?/?;Cdk2?/?embryos were very similar to
those of wild type littermates (Figure 2A). Similar results
were obtained with P1 neonatal mutants (data not shown).
Cdk4?/?;Cdk2?/?fetal livers displayed about 60% the number
of cells of livers isolated from wild type embryos, a reduction
proportional to the smaller size of these double mutant em-
bryos (Figure2B, left).Accordingly, the totalnumbersof hema-
topoietic progenitors per liver was also reduced to a similar
extent (Figure 2B, right). These observations contrast with
those observed in embryos defective for Cdk4 and Cdk6 which
displayed about one-tenth the numbers of GMP and CMP pre-
cursors and about half of MEP progenitors (Malumbres et al.,
2004). Thus, Cdk4 and Cdk2 do not play compensatory roles
in embryonic and neonatal hematopoietic cells, indicating
that these cells proliferate well with just one interphase
kinase, either Cdk4 (in Cdk6 and Cdk2 double knock out
mice) or Cdk6 (in the Cdk4 and Cdk2 double knock out mice
described here).
Figure 2 – Analysis of hematopoietic stem cells and lineage committed progenitors in late gestation embryos. (A) Cells were isolated from fetal
livers of E17.5 embryos and analysed by flow cytometry. Relative percentages of hematopoietic stem cells (HSC), granulocyte-macrophage
progenitors (GMP), common myeloid progenitors (CMP) and megakaryocyte-erythroid progenitors (MEP) are shown. (B) Left panel shows
absolute cell numbers per fetal liver of wild type (empty boxes) and Cdk4L/L;Cdk2L/L(solid boxes) embryos. Right panel shows total numbers of
GMP, CMP and MEP populations calculated per liver. Solid boxes represent samples obtained from Cdk4L/L;Cdk2L/Lembryos and empty boxes
represent wild type littermates. Error bars: SD.
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Newborn Cdk4?/?;Cdk2?/?mice show thinning of their ven-
tricular walls due to a decrease in the number of proliferating
cardiomyocytes (Figure 3). Quantitative analysis of Ki67 stain-
ing revealed about one-third the number of proliferating cells
in the ventricular walls of mutant hearts (Figure 3). No signif-
icant differences in apoptosis levels were observed between
mutant and wild type hearts (data not shown). In some cases,
Cdk4?/?;Cdk2?/?cardiomyocytes had an enlarged phenotype
suggesting a hypertrophic, adaptive response (Figure 3).
Moreover, these cardiomyocytes were partially disorganized
by prominent capillaries. These observations suggest that
Cdk4?/?;Cdk2?/?mice may not be able to sustain the major he-
modynamic changes that take place at birth and die of cardiac
failure. Indeed, newborn Cdk4?/?;Cdk2?/?mice have conges-
tive livers (data not shown), a defect that reinforces the car-
diac failure hypothesis.
2.2. Expression of cell cycle regulators
To understand the molecular mechanisms responsible for
the absence of major developmental and proliferative defects
in Cdk4?/?;Cdk2?/?double mutant mice, we examined the
expression levels of other cell cycle regulators. To prevent
possible indirect effects of damage suffered during delivery,
we used late embryos (E17.5 or E18.5) to carry out these stud-
ies. As illustrated in Figure 4A, concomitant loss of Cdk4 and
Cdk2 does not result in significant changes in the levels of
Cdk6 and Cdk1, the two additional kinases implicated in
driving the cell cycle. Likewise, all the Cyclins tested, includ-
ing Cyclin D1 and D2, Cyclin E1 and Cyclin A2, display normal
levels of expression. The steady state levels of the cell cycle
inhibitors p21Cip1
andp27Kip1
(Figure 4A).
alsoremainunchanged
We also examined how the absence of Cdk4 and Cdk2
affected the formation of Cyclin–Cdk complexes and the
inactivation of pRb, a requirement for progression into
the S phase. As shown in Figure 4B, we observed increased
levels of Cyclin D2 complexed with Cdk6 and increased
binding of Cyclin E1 to Cdk1, a non-canonical Cyclin–Cdk
complex recently reported by Aleem et al. (2005). Surpris-
ingly, these double mutant embryos displayed normal
levels of pRb phosphorylation. Moreover, phosphorylation
of pRb occurs at residues S807/811 and S780, three sites
known to be specifically phosphorylated by Cyclin D–Cdk4
complexes (Figure 4C). Likewise, pRb is also phosphorylated
at residue S608 in Cdk4?/?;Cdk2?/?late embryos, a residue
known to be the target of Cdk2 (Figure 4C). These observa-
tionssuggestthat CyclinD–Cdk6
complexes may contribute to drive cell division by phos-
phorylating pRb at canonical sites in the absence of Cdk4
and Cdk2.
andCyclinE–Cdk1
2.3.Immortalization of mouse embryonic fibroblasts
Proliferation of primary MEFs lacking either Cdk4 or Cdk2 is
less robust than proliferation of wild type cells (Berthet et al.,
2003; Ortega et al., 2003; Rane et al., 1999; Tsutsui et al.,
1999). Concomitant loss of both kinases results in further de-
crease in their proliferation rate (Figure 5A, left). Primary
Cdk4?/?;Cdk2?/?MEFs display delayed entry into S phase, al-
though the percentage of cells that enter S phase eventually
equals that of wild type MEFs (Figure 4B, top). More impor-
tantly,mostofourcultures,8outof10embryosunderclassical
culture conditions using 20% oxygen and four out of four em-
bryos in low percent (3%) oxygen, became immortal upon con-
tinuous passage following a standard 3T3 protocol (Figure 4A,
Figure 3 – Mice lacking Cdk4 and Cdk2 develop to term but die soon after birth due to cardiac failure. (Left) Representative images of Cdk4D/D;
Cdk2D/D(top) and Cdk4L/L;Cdk2L/L(bottom) P1 mice. Microscopic images from these mice showing (center left) H&E staining (3200) and
(center right) Ki67 immunostaining of serial sections of ventricular walls (3200), and (right) H&E staining of the myocardium showing partially
disorganized and enlarged cardiomyocytes with prominent capillaries (3400).
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Figure 5 – Proliferative properties of primary and immortal MEFs. (A) Left panel shows proliferation of primary MEFs at passage 2. Center panel
shows immortalization of primary MEFs by continuous passage following a classical 3T3 protocol. Cdk4D/D;Cdk2D/D(open circles) and Cdk4L/L;
Cdk2L/L(filled circles). Left panel shows proliferation of immortal Cdk4L/L;Cdk2lox/loxMEFs infected with empty virus (open circles) or with
virus expressing Cre recombinase (filled circles). Insert shows a Southern blot depicting complete cleavage of the Cdk2loxallele in MEFs infected
with virus expressing Cre recombinase (E, empty infected). (B) Percentage of quiescent Cdk4D/D;Cdk2D/D(open bars) and Cdk4L/L;Cdk2L/L
(filled bars) MEFs entering S phase upon addition of serum in the presence of 50 mM BrdU. Top panel shows results with primary MEFs. Bottom
panel shows results with immortal MEFs. Error bars: SD. (C) Expression of Cdk1, Cyclin A2 (Cyc A2), hypophosphorylated (Rb) and
phosphorylated (P-Rb) forms of pRb in immortal MEFs at the indicated times after the addition of serum. b-Actin serves as loading control.
Figure 4 – Biochemical characterization of cell cycle regulators in embryos lacking Cdk4 and Cdk2. (A) Expression levels of cell cycle regulators in
wild type (Cdk4D/D;Cdk2D/D) and mutant (Cdk4L/L;Cdk2L/L) E17.5 embryos. Protein extracts were analyzed by immunoblotting with antibodies
elicited against the indicated proteins. (B) Cyclin D2/Cdk6 and Cyclin E1–Cdk1 complexes were immunoprecipitated with antibodies against
Cyclin D2 or Cyclin E1 and analyzed by immunoblotting using antisera against Cdk6 or Cdk1, respectively. M: mock immunoprecipitates; WCE:
whole cell extract at 1:20 of starting material prior to immunoprecipitation. (C) Analysis of pRb phosphorylation levels using antibodies specific for
phosphorylated residues S807/811, S780 or S608.
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middle). These observations indicate that embryonic cells in
culturecanproliferateandbecomeimmortalinspiteoflacking
the two main Cdks implicated in the interphase (G1 phase,
G1/S transition and S phase) of the cell cycle.
To rule out that the ability of these cells to proliferate in
culture was due to the rapid accumulation of mutations
and/or epigenetic alterations during the immortalization pro-
tocol, we acutely ablated the Cdk2 locus in immortal Cdk4?/?;
Cdk2lox/loxMEFs. These cells do not contain mutations in
either the INK4a/Arf or the P53 loci (data not shown). As illus-
tratedinFigure5A(right),acuteremovalofthefloxedCdk2locus
by infecting Cdk4?/?;Cdk2lox/loxMEFs with a retrovirus express-
ingthebacteriophageCrerecombinasefailedtosignificantlyre-
duce their proliferation rate. Thus, Cdk4 and Cdk2 ameliorate
the adaptation process, also known as ‘‘culture shock’’, that
MEFs must endure when transferred from the developing em-
bryo to a Petri dish. However, these kinases are dispensable
for proper proliferation of these cells once they have become
adapted to grow under in vitro culture conditions.
Immortal Cdk4?/?;Cdk2?/?MEFs respond efficiently to mi-
togenic signals as illustrated by their normal kinetics in enter-
ing S phase (Figure 5B, bottom). These double mutant cells
also display robust andtimely
following serum stimulation (Figure 5C). E2F targets such as
Cyclin A2 are also expressed with kinetics indistinguishable
from wild type controls. Interestingly, induction of Cdk1,
which has been shown to be partially dependent on E2F
activity, also occurs normally (Figure 5C).
pRbphosphorylation
2.4. Ablation of Cdk4 and Cdk2 in adult mice
Most studies using genetic approaches in mice are limited to
embryonic development, in particular when the targeted mu-
tations result in a lethal phenotype. Thus, we examined
whether the early postnatal death of Cdk4?/?;Cdk2?/?double
mutant mice was due to an intrinsic cell cycle deficiency or
to a developmental defect in specific lineages such as cardio-
myocytes by ablating the Cdk2 locus in adult mice. To this end,
we generated compound Cdk4?/?;Cdk2lox/lox;RERTert/ertani-
mals (see Section 4) in which the Cdk2loxalleles can be
knocked out upon activation of the inducible CreERT2 recom-
binase (Feil et al., 1996) encoded by the ert allele. This allele
drives CreERT2 expression from the locus encoding the large
subunit of RNA polymerase II by an IRES-dependent bi-cis-
tronic strategy and it is expressed in most, if not all cell types
(Guerra et al., 2003).
Weaned (P21) Cdk4?/?;Cdk2lox/lox;RERTert/ertmice were ex-
posed to 4-hydroxy-tamoxifen (4OHT) for 4 months (1 mg,
twice a week) to allow efficient cleavage of the conditional
Cdk2loxalleles. As illustrated in Figure 6A, ?95% of the condi-
tional Cdk2loxhas recombined to generate the null allele in
many tissues, including colon, pancreas, skin, small intestine,
spleen, stomach and thymus. Other tissues such as heart,
kidney, lung, testis and white adipose tissue displayed
recombination levels ranging from 70 to 90%. Only brain con-
tained minimal levels of recombined Cdk2?allele, a conse-
quence of the limited penetrability of 4OHT through the
blood/brain barrier (Figure 6A). Ablation of Cdk2 expression
in some of the tissues displaying high percentage of Cdk2loxre-
combination, including colon, liver, lung and pancreas was
confirmed by Western blot analysis (Figure 6B). These 4OHT-
treated Cdk4?/?;Cdk2lox/lox;RERTert/ertmice will be designated
from now on as Cdk4?/?;Cdk2D/D;RERTert/ert. Cdk4þ/þ;Cdk2þ/þ;
RERTert/ertanimals submitted to the same 4OHT treatment
were used as controls.
Despitecompleteornear completeablation of Cdk2in most
tissues, Cdk4?/?;Cdk2D/D;RERTert/ertmice did not display obvi-
ous phenotypic deficiencies when compared to control
Cdk4þ/þ;Cdk2þ/þ;RERTert/ertmice submitted to the same 4OHT
treatment (Figure 7A), except for those defects associated
with loss of Cdk4 such as small size and diabetes (Rane
et al., 1999; Tsutsui et al., 1999). Moreover, comparative
Figure 6 – Generation of adult mice lacking Cdk4 and Cdk2. (A) Levels of excision of the conditional Cdk2loxalleles in Cdk4L/L;Cdk2lox/lox;
RERTert/ertexposed to 4OHT for 4 months. DNA was isolated from the indicated tissues of the resulting Cdk4L/L;Cdk2D/D;RERTert/ertmice and
analyzed by Southern blot. The migration of the conditional Cdk2loxand null Cdk2Lalleles is indicated by arrowheads. The percentage of
recombined Cdk2Lallele in each of the tissues is indicated at the bottom. (B) Immunoblot analysis of the levels of expression Cdk4 and Cdk2 in
some of the tissues shown in (A). As controls, we used extracts from the same tissues obtained from Cdk4D/D;Cdk2D/D;RERTert/ertmice treated
with 4OHT. b-Actin served as loading control.
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analysis of the proliferation rates in all tissues examined by
Ki67 immunostaining did not reveal significant differences.
These results were most illustrative in tissues such as the
esophagus (stratum germinativum) or intestine known for
their active epithelial cell renewal (Figure 7B).
2.5.
Cdk4 and Cdk2
Adult hematopoiesis in the absence of
Likewise, lympho-hematopoietic organs such as spleen and
thymus known for their continuous production of lymphoid
Figure 7 – Normal adult homeostasis and cell proliferation levels in tissues of Cdk4L/L;Cdk2D/D;RERTert/ertmice. (A) Five-month-old Cdk4D/D;
Cdk2D/D;RERTert/ert, Cdk4L/L;Cdk2D/D;RERTert/ertand Cdk4L/L;Cdk2lox/lox;RERTert/ertmice. The Cdk4D/D;Cdk2D/D;RERTert/ertand the
Cdk4L/L;Cdk2D/D;RERTert/ertmice were treated with 4OHT, whereas the Cdk4L/L;Cdk2lox/lox;RERTert/ertmouse was treated with oil. (B) Ki67
immunostaining of the indicated tissues obtained from 5-month-old 4OHT-treated Cdk4D/D;Cdk2D/D;RERTert/ertand Cdk4L/L;Cdk2D/D;
RERTert/ertmice. Esophagus: most of the stratum germinativum cells are proliferative (3400). Small intestine: proliferative cells are restricted to
the lower part of the intestinal crypts (3200). Spleen: strong proliferation in the red pulp indicative of an active hematopoiesis. Most of the
lymphoid cells in the white pulp are quiescent except those forming germinal centers (350). Thymus: most thymocytes in the cortex are positive for
Ki67 immunostaining (3100). The presence of the ert alleles in the description of the genotypes in the figure has been omitted for clarity.
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or hematopoietic cells, also display normalproliferation levels
(Figure 7B). In the spleen, the red pulp displayed the strongest
Ki67 staining regardless of the presence or absence of Cdk4
and Cdk2 revealing that active erythropoiesis was not af-
fected. Moreover, in the white pulp most of the lymphocytes
appeared quiescent with the exception of several germinal
centers. Other secondary lymphoid follicles present in the in-
testinal wall (Peyer’s patches) and in lymph nodes displayed
normalproliferationrates(datanotshown),suggestingafunc-
tional B-cell response in Cdk4?/?;Cdk2D/D;RERTert/ertanimals.
Moreover, in the thymus, one of the tissues with faster cell
turnover and most sensitive to cell division defects, almost
all cortical thymocytes were positive for Ki67 immunostain-
ing, indicating that the continuous and sustained production
of naive T cells was preserved in the absence of Cdk4 and
Cdk2.
In depth analysis of the adult hematopoietic system of
Cdk4?/?;Cdk2D/D;RERTert/ertmice revealed that Cdk4 and Cdk2
were dispensable for proliferation and differentiation of all
lineages. As illustrated in Figure S1A, bone marrow of Cdk4?/?;
Cdk2D/D;RERTert/ertmice had the same number of burst-
forming erythroid units, and colony-forming units for granu-
locyte, macrophage, and mixed granulocyte/macrophage
lineages as wild type controls. To rule out the possibility
that these colonies arose from stem cells that retained Cdk2lox
alleles, we performed semi-quantitative PCR analysis on ge-
nomic DNA extracted from individual colonies. In all cases,
we found complete excision of the floxed Cdk2 sequences
(data not shown). Next, we examined the contributions of
these hematopoietic precursors to the various cell lineages
by staining bone marrow cells with lineage-specific anti-
bodies. Flow cytometry analysis indicated that the percentage
of B-cells (B220 antibodies), NK-cells (DX5a antibodies), T-cells
(CD3 antibodies) and granulocyte/monocytes (GR1 and CD11b
antibodies) were basically the same as in the bone marrow of
Cdk4þ/þ;Cdk2þ/þ;RERTert/ertmice (Figure S1B).
The percentage of lymphoid B-cells (B220 antibodies),
T-cells (CD3 antibodies) and NK-cells (DX5a antibodies) were
also similar to those obtained in control spleen (Figure S1C).
Likewise loss of Cdk4 and Cdk2 did not alter cell proliferation
in the thymus. Moreover, ablation of these genes did not
appear to have a negative effect on the maturation program of
T lymphocytes since the percentages of double negative, dou-
ble positive and single positive populations were the same as
in wild type mice (Figure S1D). These results conclusively
demonstrate that adult hematopoiesis is not dependent on
the kinase activity of both Cdk4 and Cdk2.
2.6.
Cdk4 and Cdk2
Normal liver regeneration in the absence of
Liver regeneration following PH is considered one of the most
stringent assays to assess the proliferative capacity of adult
tissues. In this assay, adult hepatocytes regain proliferative
properties to restore hepatic function as a response to the
reduction in liver mass. Thus, we decided to test the prolifera-
tive properties of Cdk4?/?;Cdk2D/D;RERTert/ertmice by resecting
two-thirds of their livers and measuring hepatic regeneration
9 days later. Liver mass in the mixed 129/SvJ?C57BL/6J back-
ground in which the Cdk4 and Cdk2 strains are maintained is
around 2.5% of the total body weight. Regenerated livers of
4-OHT-treated Cdk4þ/þ;Cdk2þ/þ;RERTert/ertand Cdk4?/?;Cdk2D/D;
RERTert/ertmice (n¼3) displayed similar overall appearance
and size (2.70?0.14% in the control mice and 2.55?0.21% in
Cdk4?/?;Cdk2D/D;RERTert/ertanimals). Histological characteriza-
tion of sections from the regenerated livers showed normal
morphologyregardlessofwhethertissueregenerationoccurred
in the presence or absence of Cdk4 and Cdk2 (Figure 8A). More-
over, the levels of cell proliferation, determined by Ki67 immu-
nostaining, were similar in the regenerated livers of control
and Cdk4?/?;Cdk2D/D;RERTert/ertmice (Figure 8A).
To eliminate the possibility that liver regeneration in mu-
tant mice may originate from a small subpopulation of stem
cells that retained the conditional Cdk2loxallele, we isolated
DNA from regenerated livers of control and mutant mice and
submitted them to Southern blot analysis. As shown in
Figure 8B, DNA isolated from two independent mice only
displayed the DNA band corresponding to the null Cdk2?
allele, thus indicating that hepatic regeneration occurred from
Cdk4?/?;Cdk2?/?hepatocytes. Finally, western blot analysis
of liver protein extracts obtained from the regenerated livers
also showed absence of Cdk2 expression (Figure 8C). Inter-
estingly,regenerated livers
RERTert/ertmice expressed Cdk1 levels similar or possibly
higher than those of control mice. These observations indi-
cate that induction of Cdk1 expression in response to mito-
genic signaling in proliferating tissues is independent of
the presence of the two G1/S kinases, Cdk4 and Cdk2. These
findings, taken together, provide convincing evidence that
adult mammalian cells proliferate normally in the absence
of Cdk4 and Cdk2.
of mutantCdk4?/?;Cdk2D/D;
3. Discussion
During evolution, eukaryotes have increased the number of
Cdks according to their structural and functional complexity.
Whereas yeasts have a single cell cycle Cdk, Cdc2/Cdc28, the
mammalian genome has accumulated at least five loci encod-
ing cell cycle Cdks (reviewed in Malumbres and Barbacid
(2005)). They include Cdk1, most likely the functional ortho-
logue of yeast Cdc2/Cdc28, Cdk2, Cdk3, Cdk4 and Cdk6. Widely
accepted models propose that the increased Cdk complexity
of higher eukaryotes is due to their need to have a more pre-
cise control of the various phases of the cell cycle. According
to this model, Cdk3, Cdk4 and Cdk6 would be responsible for
getting cells out of quiescence and driving them through G1,
whereas Cdk2 and Cdk1 would be essential for the two most
fundamental aspects of cell division, DNA synthesis and mito-
sis, respectively.
Recent genetic evidence, however, has challenged this
model. We and others have targeted the genes encoding
Cdk2, Cdk4 and Cdk6, to show that they are dispensable for
cell proliferation, as well as for embryonic development and
adult homeostasis (Berthet et al., 2003; Malumbres et al.,
2004; Ortega et al., 2003; Rane et al., 1999; Tsutsui et al., 1999;
Ye et al., 2001). Exceptions include the essential role of Cdk2
for meiotic cell division (Ortega et al., 2003), a fact often ig-
nored when studying this kinase, and of Cdk4 for postnatal
proliferation of specialized endocrine cells types such as
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insulin producing pancreatic beta cells and pituitary lacto-
trophs responsible for production of prolactin (Martin et al.,
2003; Moons et al., 2002; Rane et al., 1999; Tsutsui et al., 1999).
It has been argued that the lack of requirement of individ-
ual Cdks for mammalian cell division is due to functional
compensation by other Cdks. Indeed, concomitant ablation
of the genes encoding Cdk4 and Cdk6 in the mouse germ
line results in embryonic lethality, a more dramatic pheno-
type than that induced by ablating each locus independently
(Malumbres et al., 2004). Cdk4 and Cdk6 are highly related
kinases activated by the same D-type Cyclins (Malumbres
and Barbacid, 2005). Cdk4?/?;Cdk6?/?embryos die during late
gestation due to limited proliferation of hematopoietic
precursors, particularly those responsible for populating the
erythroid lineage (Malumbres et al., 2004). Yet, Cdk4?/?;
Cdk6?/?embryos display normal rates of cell proliferation in
other tissues. Moreover, MEFs derived from these mutant em-
bryos grow well in culture and enter the cell cycle upon mito-
genic stimuli with normal kinetics (Malumbres et al., 2004).
Similar results have been obtained in mice lacking the three
D-type Cyclins (Kozar et al., 2004). Thus, Cdk4 and Cdk6 play
compensatory roles but only in highly specialized cell types.
In contrast, a recent report by Berthet et al. (2006) indicates
that Cdk4 and Cdk2, the main G1/S kinases, have significant
overlapping roles. In this study, Cdk4?/?;Cdk2?/?embryos die
during midgestation due to heart defects not too different
from those described here for newborn Cdk4?/?;Cdk2?/?
mice. However, according to this report, combined absence
of Cdk4 and Cdk2 results in limited phosphorylation, and
hence deficient inactivation of pRb. This defect leads to
limited expression of E2F targets essential for cell cycle pro-
gression such as Cyclin A and Cdk1. Indeed, in this study,
Cdk4?/?;Cdk2?/?MEFs proliferate poorly and do not become
immortal upon continuous passage in culture. Interestingly,
the proliferative defects observed in these double mutant
mice only occurred after midgestation, since E12.5 Cdk4?/?;
Cdk2?/?embryos appeared to be normal (Berthet et al., 2006).
Theauthorsspeculatedthatduringearlyembryogenesis,either
Cdk6 complexed to D-type Cyclins or Cdk1 can inactivate
pRb. A decrease in Cyclin D1 during midgestation would result
in impaired pRb phosphorylation with subsequent inhibition
of E2F-dependent Cdk1 expression, leading to an overall
decrease in cell proliferation that becomes incompatible with
life (Berthet et al., 2006).
Our resultsareat variancewith thesefindings.Whereas we
observed embryonic lethality of Cdk4?/?;Cdk2?/?embryos,
a significant percentage of them develop to term. Newborn
Cdk4?/?;Cdk2?/?mice die shortly thereafter presumably due
to cardiac insufficiency, a consequence of their limited num-
ber of cardiomyocytes. Yet, all other tissues, including those
of hematopoietic origin, display normal proliferative levels.
These observations indicate that Cdk2 does not play signifi-
cant compensatory roles with Cdk4 in hematopoietic cells or
any other cell type except for embryonic cardiomyocytes.
Cdk4?/?;Cdk2?/?embryos are slightly smaller than those lack-
ing Cdk4 alone (Rane et al., 1999; Tsutsui et al., 1999). However,
the mixed background used in these studies prevents us to
ascribe this small difference to loss of Cdk2.
Loss of viability during embryonic or early postnatal devel-
opment often masks the overall phenotypic consequences of
Figure 8 – Liver regeneration in Cdk4L/L;Cdk2D/D;RERTert/ertmice. (A) 4OHT-treated Cdk4D/D;Cdk2D/D;RERTert/ertand Cdk4L/L;Cdk2D/D;
RERTert/ertmice were submitted to partial hepatectomy (PH) as indicated in Section 4 and the regenerated livers analyzed 9 days later by H&E
and Ki67 immunostaining. A small hematopoietic cluster is indicated by arrowheads. (B) Southern blot analysis of the excision of conditional
Cdk2loxalleles in resected (Pre) and regenerated (Pos) liver samples from two independent Cdk4L/L; Cdk2D/D;RERTert/ertmice. DNAs from brain
(Br) and testis (Ts) tissue from one of these mice were also tested as controls. These tissues are known to undergo limited cleavage of the Cdk2lox
allele (see Figure 5). (C) Immunoblot analysis of the expression of Cdk4, Cdk2 and Cdk1 in resected (Pre) and regenerated (Pos) livers of 4OHT-
treated Cdk4D/D;Cdk2D/D;RERTert/ertand Cdk4L/L; Cdk2D/D;RERTert/ertmice. b-Actin served as loading control. The presence of the ert alleles
in the description of the genotypes has been omitted for clarity.
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gene targeting. Generation of mice carrying a conditional mu-
tation in the Cdk2 locus in a Cdk4 null background has allowed
us to overcome this limitation by ablating Cdk2 in adult tis-
sues. The absence of any overt phenotype in Cdk4?/?;Cdk2D/D;
RERTert/ertmice, other than those observed in single Cdk4 and
Cdk2 null animals, is not compatible with the central role pro-
posed for these kinases during interphase. It could be argued
that Cdk4?/?;Cdk2D/D;RERTert/ertmice do not display obvious
abnormalities due to the low proliferation rates of most adult
tissues. However, as illustrated here, a battery of highly prolif-
erating adult tissues such as skin or small intestine as well as
hematopoietic organs, display normal proliferation rates.
Even hematopoietic precursors proliferate normally and re-
tain the capacity to generate a normal mature hematopoietic
system in the absence of Cdk4 and Cdk2. Moreover, adult he-
patocytes deficient for these kinases can respond to severe
stress conditions such as liver regeneration upon PH.
At the present time we can only speculate about the rea-
sons for the different results obtained by us and Berthet
et al. (2006). Both mice have similar genetic backgrounds,
composed mainly of those of 129J and C57Bl6 origin, albeit
our animals contain a limited contribution from CD1 mice.
Whether this difference accounts for survival beyond mid-
gestation remains to be determined. It is also possible that
the mice described by Berthet et al. have accumulated addi-
tional mutations either by chance or during selection of
mice that underwent the rare recombination required to
co-segregate the neighboring Cdk4 and Cdk2 targeted loci. In
any case, our results illustrate that mice can thrive without
Cdk4 and Cdk2, specially during adult homeostasis.
Our in vitro studies with MEFs provide additional evidence
that Cdk4 and Cdk2 are dispensable for the mammalian cell
cycle. Cdk4?/?;Cdk2?/?MEFs become immortal with high
(>80%) frequency when maintained in culture. These immor-
tal MEFs have normal proliferation rates including S phase
kinetics. Indeed, the only substantial difference between
wild type and Cdk4?/?;Cdk2?/?MEFs resides in their increased
susceptibility to the stress imposed by their adaptation to in
vitro culture conditions. Under these stress conditions, MEFs
lacking Cdk4 and Cdk2 proliferate significantly more slowly
than MEFs expressing both enzymes. Yet, once Cdk4?/?;
Cdk2?/?MEFs become adapted to culture, they behave as
wild type cells. Additional proof of the selective requirement
for Cdk4 or Cdk2 during adaptation to culture conditions
was provided by the efficient proliferation of immortal
Cdk4?/?;Cdk2lox/lox
MEFs upon
Although the molecular mechanisms by which Cdk4 and
Cdk2 facilitate cell division under in vitro stress conditions
remain to be worked out, our results clearly illustrate that
Cdk4 and Cdk2 are dispensable for mammalian cell division.
In summary, our results provide genetic evidence that
Cdk4 and Cdk2 are dispensable for mammalian cell division,
both during embryonic development and in adult homeosta-
sis. These results are reminiscent of those previously reported
for mice lacking Cdk6 and Cdk2 (Malumbres et al., 2004). Only
loss of Cdk4 and Cdk6 expression is incompatible with life, at
least during embryogenesis. Yet, most cell types proliferate
well in the absence of these kinases, indicating that they
play a developmental role in hematopoietic lineages, but are
not essential for the basic process of mammalian cell division
acuteremovalof Cdk2.
(Malumbres et al., 2004). Next, it will be necessary to deter-
mine to what extent interphase Cdks are essential for
mammalian cell division by generating triple mutant mice
deficient for Cdk4, Cdk6 and Cdk2. Targeting the Cdk3 locus
will not be necessary since the strains used for these studies
already carry a germ line mutation for Cdk3 (Ye et al., 2001).
In addition, it will essential to determine whether Cdk1, the
proposed orthologue for the yeast Cdc2/Cdc28 kinase, is also
indispensable for mammalian cell division or can be compen-
sated by other Cdks, by targeting this locus. This information
will help us to better understand the role of Cdks in driving
mammalian cell proliferation.
4.Experimental procedures
4.1.Gene targeting and mouse strains
The Cdk4 targeting vector was prepared as described in
Figure 1. Briefly, a 6.4 kbp EcoRI–BamHI genomic fragment con-
taining the Cdk4 locus was isolated from a 129Sv/J genomic
library and subcloned in pBluescript (Rane et al., 1999). An frt
site was inserted 230 bp upstream of exon 2. In addition, an
frt-PGK-HygR-frt hygromycin resistance cassette was inserted
115 bp downstream of exon 4. A PGK-thymidine kinase (TK)
cassettewasincorporatedintothetargetingvectorfornegative
selectionaspreviouslydescribed(Raneetal.,1999).Thetarget-
ing vector was electroporated into ES cells carrying a Cdk2
conditional knock out allele (Ortega et al., 2003). Southern blot
analysisof120HygR/GanRclonesidentifiedfourrecombinants
(Figure 1B,C) that were aggregated to CD1 morulas. Male
chimeras derived from two clones (ESDS1.15 and ESDS1.66)
co-segregated the targeted Cdk4 (Cdk43frt) and Cdk2 (Cdk2lox)
alleles to their offspring. The resulting Cdk4þ/3frt;Cdk2þ/lox
mice were crossed to pCAG-Flpe transgenics (Rodriguez et al.,
2000) to excise the PGK-HygR cassette and the exons 2-4
(Figure 1A). The resulting Cdk4þ/?;Cdk2þ/loxmice were crossed
to CMV-Cretransgenics(Schwenket al., 1995) to generatedou-
ble heterozygous Cdk4þ/?;Cdk2þ/?animals. These heterozy-
gous mice were crossed with wild type C57Bl6 animals to
segregate the Flpe and Cre transgenes. Cdk4þ/?;Cdk2þ/loxmice
were also mated with RERTert/ertmice (Guerra et al., 2003) to
generate the Cdk4?/?;Cdk2lox/lox;RERTert/ertanimals used to
ablate Cdk2 postnatally. These mice were also crossed with
wild type C57Bl6 animals to segregate the Flpe transgene.
Cdk4?/?;Cdk2lox/lox;RERTert/ertmice were treated at weaning
(P25)with4-OHTfor2–4months(1 mg,twiceaweek)to ensure
efficient ablation of the conditional Cdk2loxallele. These
treated mice were designated as Cdk4?/?;Cdk2D/D;RERTert/ert.
Routine genotyping of Cdk4?, Cdk2?and Cdk2loxalleles was
carriedoutbyPCRamplificationusingspecificoligonucleotides
(primer sequences are available from the authors upon re-
quest). Mice were maintained according to the animal care
standards established by national and international institu-
tions including the European Union.
4.2.Histopathology and immunohistochemistry
Embryos or organs dissected from mice were fixed in 10%-
buffered formalin (Sigma) and embedded in paraffin. Three- or
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five-micrometer-thick sections were stained with hema-
toxylin and eosin (H&E). For proliferation studies, tissues or
embryos were stained with Ki67 specific antibodies (MIB-1;
Dako). Detection of apoptotic cells on tissue sections was
carried out using the TUNEL assay (Apoptag Peroxidase,
Intergen) or anti-active Caspase 3 antibodies (R&D Systems).
4.3. Cell culture assays
Mouse embryonic fibroblasts (MEFs) were isolated from
E13.5 embryos and cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 2 mM glutamine, 1%
penicillin/streptomycin and 10% fetal bovine serum (FBS).
MEFs were propagated in culture according to standard 3T3
protocols. For proliferation assays, 5? 104cells were plated
on 6-well plates in duplicate essentially as previously
described (Martı ´n et al., 2005). To analyse S phase entry,
MEFs (106cells/10 cm dish) were deprived of serum for
72 h in DMEMþ0.1% FBS and re-stimulated with 10% FBS
to enter the cell cycle. Cells were continuously labeled
with 50 mM bromodeoxyuridine (BrdU; Sigma), harvested at
the indicated times and stained with anti-BrdU antibodies
(Becton Dickinson) and propidium iodide. DNA content was
analyzed by flow cytometry (FACSScalibur from Becton-
Dickinson). MEFs were infected with retroviral vectors
expressing the Cre recombinase as previously described
(Martı ´n et al., 2005).
To study proliferation of hematopoietic precursors, livers
were collected from E17.5 embryos, single cell suspensions
prepared and 5 ?104cells plated in methylcellulose (Stem
Cell Technologies #03434) as suggested by the manufacturer.
For each sample, two duplicate dishes were analyzed 9 days
after plating.
4.4.FACS analysis
Livers were collected from E17.5 embryos and single cell sus-
pensions were prepared. 2 ?106cells/liver were immuno-
stained and hematopoietic progenitors were identified as
previously described (Malumbres et al., 2004). HSCs were
gated as Lin?IL7Ra?c-KitþSca1þ; CMPs as Lin?IL7Ra?c-
KitþSca1?FcgRlowCD34þ; GMPs as Lin?IL7Ra?c-KitþSca1?
FcgRhiCD34þ; and MEPs as Lin?IL7Ra?c-KitþSca1?FcgRlow
CD34?. Cell surface markers used to identify the specific
hematopoietic populations in adult bone marrow, thymus or
spleen include CD3, CD4, CD8, CD11B, CD16/32, CD19, CD45,
B220, DX5a, GR1 and Ter119 (all from Pharmingen). Their
relative numbers were quantified using FACSScalibur and
FACSAria (Becton Dickinson) cytometers.
4.5.Liver regeneration
Animals were anesthetized using inhalation of 2% isoflourane
after which resection of two-thirds of total liver mass (left lat-
eral, left median and right median lobectomy) was performed.
All animals were treated with 0.05 mg/kg of buprenorphine
following PH. The mass of the resected liver tissue was
measured right after surgery, and that of the regenerated
liver was determined after sacrificing the animals 9 days after
the PH.
4.6.Protein analysis
Protein lysates were isolated and used for protein analysis
by immunoblotting as previously described (Martı ´n et al.,
2005). Antibodies against the following proteins were used:
Cdk2 (M2; Santa Cruz), Cdk1 (17; Santa Cruz), Cdk4 (C22;
Santa Cruz), Cyclin A (H432; Santa Cruz), Cyclin E (M20;
Santa Cruz), Cyclin D1 (DCS6; Neo Markers), Cyclin D2 (Santa
Cruz), p21Cip1(C19; Santa Cruz), p27Kip1(Transduction Labo-
ratories), b-Actin (AC15, Sigma) and pRb (BD Pharmingen).
pRb phosphospecificantibodies
(#9307) and S807/811 (#9308) were from Cell Signalling. As
secondary antibodies,peroxidase-conjugated
were used, followed by chemiluminescence detection (ECL;
Amhersam).
to S608(#2181),S780
IgG(Dako)
Acknowledgements
We thank Rut Gonza ´lez, Marta San Roma ´n, Blanca Velasco,
and Raquel Villar for excellent technical assistance and
Arancha Garcı ´a for invaluable help with the cytometer. We
also value the excellent support provided by the Transgenic
and Comparative Pathology Units of the CNIO. This work was
supported by grants from the Plan Nacional de Investigacio ´n
Cientı ´fica to D.S. (GEN2003-20243-C08-02), M.M. (BMC2003-
06098) and M.B. (SAF2000-0009-CO2-01, SAF2002-10374-E,
SAF1999-1825-CE and SAF2004-20477-E), Fondo de Investiga-
cio ´n Sanitaria to D.S. (PI031527), V Framework Programme of
the European Union to M.B. (QLK3-1999-00875, QLG2-CT-
2002-00930, LSHC-CT-2004-503438), INSERM and Association
pour la Recherche contre le Cancer (Re ´gion Aquitaine) to P.D.
C.B. and A.C. were supported by fellowships from la Ligue
contre le Cancer (Comite ´ de la Dordogne) and FPI (Ministerio
de Educacio ´n y Ciencia), respectively. The CNIO was partially
supported by the RTICCC (Red de Centros de Ca ´ncer; FIS
C03/10).
Appendix A. Supplementary material
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.molonc.2007.03.001.
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