Gene 247 (2000) 1–15
Cell-cycle inhibitors: three families united by a common cause
Anxo Vidal *, Andrew Koff
Laboratory of Cell Cycle Regulation, Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
Received 28 October 1999; received in revised form 3 February 2000; accepted 15 February 2000
Received by A.J. van Wijnen
In the cellular program leading to DNA synthesis, signals that drive cells into S-phase converge at the level of CDK activity.
The products of at least three different gene families, Ink4, Cip/Kip and the pRb pocket-protein family, suppress S-phase entry.
Ink4 proteins act by antagonizing the formation and activation of cyclin D-CDK4 complexes, of which the ultimate downstream
target as related to S-phase entry appears to be pRb. Cip/Kip inhibitors impinge upon that pathway by inhibiting CDK2 kinases
that participate in the inactivation of pRb and, like cyclin E, may also have roles independent of pRb. How the activities of these
three classes of proteins are coordinated remains obscure. In recent years, development of mouse models has accelerated the
elucidation of this complex network, showing roles that are sometimes cooperative and sometimes overlapping. We will discuss
the interrelationships between Cip/Kip inhibitors and the components of the pRb pathway, and how their activities ultimately
regulate cell proliferation. © 2000 Published by Elsevier Science B.V. All rights reserved.
Keywords: CDK; Cell cycle; CKI; Cyclin; Mouse models
1. Cell cycle: making decisions
pathways in G1, and during the remainder of the cell
cycle, is of capital importance. Do anti-mitogenic signals
interfere with mitogenic pathways at the level of the
signaling cascades, or do they act to block the mitogen-
induced cell-cycle machinery from carrying out its func-
tion (Fig. 1)? Likewise, do mitogens prevent cells from
responding to anti-mitogenic signals? Although still
incomplete, our knowledge of the cell-cycle machinery
offers an opportunity to determine how this decision is
made at the molecular level.
In a tissue, a cell simultaneously receives multiple
signals, some mitogenic and some anti-mitogenic. These
inputs exist as both soluble extracellular factors, such
as growth factors or hormones, and as physical forces
of interaction with other cells or with the substratum.
At a certain point in G1, the restriction point (Pardee,
1989), these signals culminate in a molecular mechanism
that allows only a binary decision—to either commit to
the mitotic cell cycle and enter S-phase, or to not commit
to the cell cycle and remain in a quiescent non-prolifera-
Commitment to the cell cycle is irreversible. How the
cell balances the mitogenic and anti-mitogenic signaling
1.1. Waves of kinases against a family of transcriptional
Several cyclin-dependent kinases play a role in the
G1-to-S transition. Each holoenzyme complex contains
a regulatory subunit, the cyclin, and a catalytic subunit,
the cyclin-dependent kinase or CDK. During pro-
gression through G1, the amount of D-type cyclins
increases, and, in a mitogen-regulated manner, these
proteins associate with, and activate, CDK4 or CDK6
(Matsushime et al., 1994).
The three D-type cyclins (D1, D2 and D3) are
expressed in a cell-type-specific manner (Sherr, 1993).
Abbreviations: CDK, cyclin dependent kinase; Cip, CDK inter-
acting protein; CKI, cyclin dependent kinase inhibitor; Ink4, inhibitor
of CDK4; Kip, Kinase inhibitory protein; LOH, loss of heterozygosity;
MEF, mouse embryo fibroblast; PP-1, protein phosphatase 1; pRb,
product of the retinoblastoma susceptibility gene; TGF, transforming
* Corresponding author. Tel.: +1-212-639-2355;
E-mail address: firstname.lastname@example.org (A. Vidal)
0378-1119/00/$ - see front matter © 2000 Published by Elsevier Science B.V. All rights reserved.
A. Vidal, A. Koff / Gene 247 (2000) 1–15
been proposed that phosphorylation of pRb by the
cyclin-associated kinases leads to different effects on
phorylation by the cyclin D-associated kinase releases a
histone deacetylase activity from the complex alleviating
transcriptional repression, and afterwards, the phos-
phorylation by the cyclin E-associated kinase can disrupt
the pocket domain of pRb dissociating the pRb–E2F
complex (Harbour et al., 1999). The interaction of the
pRb–E2F complexes with cell-cycle control genes has
been shown to be important for pRb-mediated cell-cycle
arrest (Zhang et al., 1999).
The E2F target genes induced as a consequence of
the disruption of pRb–E2F complexes include some of
the enzymes involved in DNA metabolism, some pro-
tooncogenes, and some cell-cycle regulatory proteins
(Dyson, 1998; Nevins, 1998). Interestingly, one E2F-
responsive gene is cyclin E (Ohtani et al., 1995; Botz
et al., 1996), and at least in some instances, expression
of cyclin E will substitute for cyclin D1 expression (Geng
et al., 1999). In one model, pRb and cyclin E-associated
kinase activity form a feedback loop that reinforces the
commitment of cells to S-phase. Once the inactivation
of pRb begins and E2F activity manifests itself, the
potential to increase cyclin E mRNA is realized. This
increase in cyclin E mRNA results in an increase in
cyclin E protein, an increase in cyclin E-CDK2 activity,
and phosphorylation of more pRb thus reinforcing the
cycle. However, it is important to note that cyclin E
mRNA is present even in the absence of E2F, so E2F
induces accumulation over a basal level.
The hyperphosphorylation of pRb may be maintained
during the cell cycle by cyclin A-CDK2. Resetting of
the pRb–E2F complex occurs in M-phase by dephos-
phorylation, probably by PP-1 (Durfee et al., 1993;
Ludlow et al., 1993), which would correlate well with
the time at which cyclin A is degraded.
Two other proteins, p130 (pRb2) and p107, are
members of the pRb ‘pocket protein’ gene family. The
pocket-protein family shares structural and biochemical
properties and interacts with a number of common
cellular proteins (Ewen, 1998; Mulligan and Jacks,
1998). Like pRb, p107 and p130 bind and modulate the
activity of E2F transcription factors, and they can
interact with histone deacetylases (Ferreira et al., 1998)
suggesting that they can also regulate transcription by
altering chromatin structure and availability of E2F.
There are preferences towards which pocket protein
interacts with which E2F family member. pRb preferen-
tially binds E2F1, E2F2, 3 or 4, and p107 and p130
appear to associate mainly with E2F4 and E2F5
(Yamasaki, 1998). Some of the complexes are regulated
in a cell-cycle phase-specific manner; for example, p130–
E2F complexes are generally found in growth arrested
cells, and p107–E2F complexes in S-phase cells (Moberg
Fig. 1. It is unclear how the cells integrate the positive and negative
inputs from the extracellular environment. The mitogenic and mito-
genic signals could cross-talk during the signaling cascade across the
cytoplasm,where one wouldoverride
Alternatively, the signals are fully transduced to and impinge on the
cell-cycle machinery, where the final decision between proliferation or
arrest is made. The quantitative and qualitative criteria that govern
that decision are largely unknown.
Often, their patterns of expression overlap in proliferat-
ing tissues; however, whether they have redundant func-
tions regulating G1 traverse is not always clear (Fantl
et al., 1995; Sicinski et al., 1995, 1996). Likewise, the
catalytic subunits, CDK4 and CDK6, are co-expressed
in many cell types. In this case, however, it is clear that
CDK6 will not fully compensate for the function of
CDK4 in most cells (Rane et al., 1999; Tsutsui et al.,
1999). This first wave of cyclin D-dependent kinase
activity is followed in late G1 by an increase in cyclin
E-CDK2 activity (Dulic et al., 1992; Koff et al., 1992).
Unlike the D-type cyclin-dependent kinase, both cyclin
E and CDK2 are expressed in all cell types, and their
assembly into an active kinase is not mitogen-dependent.
Current evidence suggests that the S-phase promoting
functions of cyclin D and cyclin E associated kinases
relate to their ability to phosphorylate pRb and release
the E2F transcription factors from an inactive or repres-
sive pRb–E2F complex (reviewed in Yamasaki, 1998;
see also, Weintraub et al., 1995, Brehm et al., 1998, Luo
et al., 1998, Magnaghi-Jaulin et al., 1998). In addition
to its role regulating proliferation, pRb has other func-
tions that might be required for differentiation (Sellers
et al., 1998; Tsai et al., 1998). The CDK enzymes
coordinately control the sequential phosphorylation of
pRb (Lundberg and Weinberg, 1998). Recently, it has
A. Vidal, A. Koff / Gene 247 (2000) 1–15
et al., 1996). However, the rules governing the formation
of different complexes are not yet clear.
Again like pRb, p107 and p130 are phosphorylated
in a cell-cycle-regulated manner (Grana et al., 1998). It
is likely that p107 and p130 are also substrates of CDKs,
and this controls their association with E2F. p130
phosphorylation occurs coincidentally with re-entry
of the cells into the cell cycle from quiescence.
Phosphorylation precedes a decrease in the abundance
of p130 protein, and because this reduction can be
blocked by proteasome inhibitors, it has been suggested
that phosphorylation of p130 might trigger its degrada-
tion by the ubiquitin-proteosome pathway (Stubdal
et al., 1997; Smith et al., 1998).
Not only are the rules governing the selectivity of
complex formation unknown, and the mechanisms regu-
lating the complexes yet to be precisely determined, their
roles in the cell cycle are still unclear. However, the cell-
cycle phase-dependent interactions suggest that p130
might have a role in the G0/G1 transition or mainte-
nance of the quiescent state, and p107 in late G1 and
S-phase, perhaps after the commitment. Analyses of
mice deficient in p107 and p130 have answered some of
these questions, but issues of redundancy and overlap-
ping functions (see below) require more precise applica-
tion of transgenic technologies in the future.
McConnell et al., 1999). Because of this specificity, the
inhibitory action of the Ink4 proteins is largely depen-
dent on the presence of pRb in the cell. In the absence
of pRb, cyclin E expression is increased, and inhibition
of cyclin D-CDK4 complexes does not inhibit S-phase
entry (Koh et al., 1995; Lukas et al., 1995; Medema
et al., 1995).
Ink4 proteins are expressed in a cell-type-specific
manner. However, the way in which their abundance is
controlled is largely unknown, with the exception of
p15Ink4b, which is induced by TGF-beta (Hannon and
Beach, 1994). Among this family, much attention has
been paid to the founder, p16Ink4a, because of its
correlation of mutation at this locus in melanoma
(Kamb et al., 1994). In fact, among all the CKIs, both
Ink4 and Cip/Kip, only p16Ink4a can be classified as a
tumor suppressor by the genetic criteria of LOH (Ruas
and Peters, 1998). Much less is known about p18Ink4c
and p19Ink4d. They are closely related and preferentially
bind CDK6,and their
differentially regulated during development (Roussel,
1.2.2. Cip/Kip family
Three proteins currently constitute the Cip/Kip
family (for Cdk interacting protein/ Kinase inhibitory
protein): p21Cip1, p27Kip1 and p57Kip2. These pro-
teins share a homologous inhibitory domain, which is
both necessary and sufficient for binding and inhibition
of CDK4- and CDK2-containing complexes. These pro-
teins act as stoichiometric inhibitors, and, although in
vitro, they inhibit all G1 complexes, they preferentially
act on CDK2 complexes in vivo. Indeed, cyclin D-CDK
complexes are not only unaffected by Cip/Kip proteins
in vivo (Soos et al., 1996), and in vitro (Blain et al.,
1997), they even require these CKIs to some extent for
proper formation (Cheng et al., 1999). This does not
exclude that under certain conditions, Cip/Kip proteins
will inhibit cyclin D-dependent activities (Kato et al.,
1994), and the understanding of how these proteins act
inthis typeof cell-context-dependent manner is
In one model, specifically geared to p27Kip1, place-
ment as an inhibitor of cyclin E-CDK2 sequestered in
cyclin D-CDK4 complexes appears quite reasonable
(Polyak et al., 1994a; Soos et al., 1996) and may explain
how anti-mitogenic signals affect CDK activity resulting
in cell-cycle arrest. For example, in TGFbeta-treated
cells, accumulation of p15Ink4b will lead to a reduction
in the amount of cyclin D-CDK4 complexes, which will
‘free’ p27Kip1, allowing it to interact with, and inhibit,
CDK2 (Reynisdottir et al., 1995). Further support for
this model is gathered from studies in cells obtained
from CDK4-p27 double null mice (Tsutsui et al., 1999).
CDK4 deficient cells have a delayed re-entry into
S-phase when released from quiescence. This can be
1.2. Cell-cycle inhibitors: opposing forces
The activity of the CDK4/6 and CDK2 kinases is
necessary for progression through G1 and entry into
S-phase. Control of these kinases is effected at multiple
levels: first, from the accumulation of the cyclin, second
at the level of assembly into a cyclin-CDK complex,
and third by specific phosphorylation and dephosphoryl-
ation events (Morgan, 1997). Additional regulation of
G1 CDK activity is effected by their association with
inhibitory proteins, the CKIs, that can either physically
(Pavletich, 1999). Some of these CKIs have been pos-
tulated to have additional functions (see below and
Sherr and Roberts, 1999 for a review). The known CKIs
are grouped into two gene families, Ink4 and Cip/Kip,
according to structural similarities.
1.2.1. Ink4 family
Four proteins currently make up the Ink4 (for inhibi-
tor of CDK4) family, p16Ink4a (Serrano et al., 1993),
p15Ink4b (Hannon and Beach, 1994) p18Ink4c (Guan
et al., 1994; Hirai et al., 1995) and p19Ink4d (Chan
et al., 1995; Hirai et al., 1995). These proteins share a
common structural feature — the presence of ankyrin
repeats. All of these proteins can inhibit cyclin
D-associated kinase activity. Generally, Ink4 proteins
compete with D-type cyclins for binding to the CDK
subunit (see, for example, Parry et al., 1995, 1999;
A. Vidal, A. Koff / Gene 247 (2000) 1–15
alleviated by p27 deficiency. This genetic complementa-
tion is consistent with the model that CDK4 complexes
sequester p27. An alternative interpretation is in wild-
type cells, the active CDK4 kinase initiates pRb phos-
phorylation enhancing cyclin E expression, which
eventually overcomes the p27 inhibitory barrier. Both
functions may be true, and the use of a catalytically
inactive CDK4 will differentiate between these possibilit-
ies. Final evidence for the model is that the cell-cycle
inhibitory properties of Ink4 proteins are somewhat
dependent on the ability of Cip/Kip proteins to inhibit
CDK2 kinase activity (Sherr and Roberts, 1999).
However, the fact that MEFs from mice lacking both
p21 and p27, which show impaired formation of cyclin
D complexes, are still partially responsive to p16Ink4a
inhibition shows that additional mechanisms exist
(Cheng et al., 1999), and we have a long way to go
towards understanding this relationship.
p21Cip1 was the first CKI identified, and its discovery
was achieved almost simultaneously by two different
approaches: genetically, as a CDK2 associated protein
in a two-hybrid system (Harper et al., 1993), and as a
growth inhibitor from senescent cells (Noda et al., 1994)
Additionally, p21 was biochemically isolated as a cyclin-
CDK2 binding protein (Gu et al., 1993; Xiong et al.,
1993). Reflecting such diversity, p21Cip1 is also named
Sdi1 (for senescent cell-derived inhibitor) and Waf1
(wild-type p53-activated fragment). Predominantly, the
regulation of p21 is at the message level, mostly tran-
scriptional (Gartel and Tyner, 1999), but mRNA sta-
bility may also be involved (Macleod et al., 1995; Wang
et al., 2000).
p21Cip1 is involved in p53-dependent DNA damage-
induced G1 arrest. The amount of p21Cip1 protein
increases following exposure to DNA damaging agents
in wild-type, but not in mutant p53-containing cells
(el-Deiry et al., 1993, 1994; Dulic et al., 1994).
Additionally, MEFs obtained from p21 null mice fail to
completely arrest in response
(Brugarolas et al., 1995; Deng et al., 1995). However,
although p21 is an effector of p53 at this checkpoint,
clearly, it is not the complete reason for a p53-dependent
In addition to the ability to associate with cyclin-
CDK complexes, p21Cip1 also associates with PCNA,
a subunit of DNA-polymerase delta. It is believed that
this is an additional mechanism by which p21Cip1 can
inhibit DNA synthesis (Zhang et al., 1993; Luo et al.,
1995). Both its involvement in the DNA-damage check-
point and its possible role in PCNA inactivation suggest
that p21Cip1 has a major role regulating replication in
committed cells. This is consistent with the observation
that p21Cip1 expression increases as cells enter the cell
cycle in several cellular systems.
p27Kip1 was first identified as a CDK2-inhibitory
activity detected in contact-inhibited or TGF-beta
treated cells (Koff et al., 1993; Polyak et al., 1994a,b).
At the same time, it was cloned in a tri-hybrid screen
as a cyclin D/CDK4 interacting protein (Toyoshima
and Hunter, 1994). Regulation of this protein is quite
complex, and reports on transcriptional (Kolluri et al.,
1999), translational (Hengst and Reed, 1996; Millard
et al., 1997), proteolytic (Pagano et al., 1995; Nguyen
et al., 1999), and localization (Soucek et al., 1998)
mechanisms abound in the literature with some represen-
tative examples given. Which, if any, mechanism is more
important for a specific function, or simply reflects cell-
type and signal-specific effects remains to be determined.
A number of studies have shown that many anti-
mitogenic signals induce the accumulation of p27Kip1,
including cell–cell contact, TGF-beta, cAMP, rapa-
mycin or lovastatin treatment (Hengst and Reed, 1998).
Other experiments demonstrated that a reduction of
p27kip1 in immortalized fibroblasts by anti-sense com-
prised serum-deprived induced withdrawal from the cell
cycle (Coats et al., 1996; Rivard et al., 1996). A number
of studies in p27 deficient mice (Tong et al., 1998; Drissi
et al., 1999; Lowenheim et al., 1999; Chen and Segil,
1999) and, using cells obtained from p27-deficient mice
(Casaccia-Bonnefil et al., 1997; Durand et al., 1998),
also demonstrated that the differentiation response is
impaired in the absence of p27. However, there was
little effect on serum-deprived induced withdrawal from
the cell cycle, a finding that still remains to be explained.
Together, these observations suggest that p27 plays a
central role in the decision to either commit to the cell
cycle or withdraw. However, it is important to realize
that although the amount of p27 can affect this decision,
it is not the only way in which a cell has to make the
decision. Indeed, the loss of p27 only delays the eventual
differentiation or withdrawal of cells. This is not wholly
unexpected as it is the kinase that drives the cell-cycle
engine, and p27 is one possible input on that engine.
Thus, it is convenient to think of p27 as one of the
immediate responses to withdrawal signals; in its
absence, withdrawal may be delayed because other pro-
cesses act (see, for example, Ewen et al., 1993; Florenes
et al., 1996; Iavarone and Massague, 1997; Carneiro
et al., 1998) or need time to ‘cover’ the absence of p27
with a redundant function, which is not clear.
p57Kip2 was cloned simultaneously by two groups
simply looking for homologues of p21 and p27 (Lee
et al., 1995; Matsuoka et al., 1995). Unlike the other
inhibitors, the p57 locus is imprinted in both mouse and
humans, and only the maternal allele is expressed
(Hatada and Mukai, 1995; Matsuoka et al., 1996).
p57Kip2 has been linked to Wilm’s tumors and
Beckwith–Wiedemann syndrome (Hatada et al., 1996;
Thompson et al., 1996). Targeted disruption of p57 in
mice showed a role for this CKI in the control of the
commitment/withdrawal decision as well as differentia-
A. Vidal, A. Koff / Gene 247 (2000) 1–15
tion and apoptosis in particular tissues (Yan et al., 1997;
Zhang et al., 1997).
1.3. pRb pathway and beyond
As statedbefore, both cyclinD- andcyclin
E-dependent activities cooperate in pRb inactivation
during G1, but they are clearly non-redundant. While
cyclin D activity is required for S-phase entry in pRb-
positive cells, it is dispensable in pRb-negative cells
(Koh et al., 1995; Lukas et al., 1995; Medema et al.,
1995). However, cyclin E is required in both pRb-
positive and negative cells (Ohtsubo et al., 1995). Cyclin
D and cyclin E are both rate-limiting for entry into
S-phase, but when coexpressed, they shorten the G1
phase even further than when expressed alone (Ohtsubo
and Roberts, 1993; Quelle et al., 1993; Resnitzky et al.,
1994). This suggested that they regulated overlapping,
yet also distinct, events. However, because cyclin E can
compensate for the loss of cyclin D1 (Geng et al., 1999),
and because cyclin E is a target for E2F activity, it is
clear that these proteins are in a common pathway, and
perhaps, the requirements for S-phase entry simply
bifurcate at cyclin E. Consistent with this, the overex-
pression of E2F is often not sufficient to promote
S-phase entry, and CDK2 activity is still required
(Hofmann and Livingston, 1996; Alevizopoulos et al.,
1997; Lukas et al., 1997). The most consistent conclusion
would be that cyclin E has more substrates that it must
phosphorylate, each of which carries out a different
rate-limiting function for S-phase entry. It is not clear
what these other substrates are, albeit candidates do
appear (Stillman, 1996; Zhao et al., 1998; Jiang et al.,
Fig. 2. Balanced model: the mitogenic stimuli converge to activate
cyclin D complexes, which, via pRb inactivation, allow E2F to
activate the expression of gene targets required for S-phase entry.
Antiproliferative signals, by means of still unclear mechanisms, affect
p27 which antagonizes cyclin E-CDK2 activity. Cyclin D complexes
negatively regulate p27 by sequestration, and p27 has a dual effect on
those, because it facilitates their activity to some extent, although it
can also inactivate them. The balance between the signals that activate
and those that inactivate cyclin E-dependent activity determines the
decision between progression to S-phase and growth arrest.
induce accumulation of Kips independently of Inks.
More slowly, they might affect the accumulation of
cyclin D-CDK4 (Ewen et al., 1993; Florenes et al., 1996;
Carneiro et al., 1998).
Now that the anti-mitogenic and mitogenic signals
that cells are exposed to have created a balance between
Kip and cyclin D-CDK4 such that cyclin E-CDK2 has
activated, the commitment decision must insure that
this is irreversible. Thus, we must ask: what is the
evidence that cyclin E-CDK2 activity is involved in
irreversibility? Although the story is still evolving, there
are observations that support the activation of cyclin
E-CDK2 as the ‘beginning of the end.’ If mitogenic
signals are influencing pRb pathway and anti-mitogenic
signals into levels of p27Kip1, cyclin E-CDK2 activation
can trigger a double-positive feedback, producing
increasing amounts of newly synthesized cyclin E and
triggering the destruction of p27Kip1 (Sheaff et al.,
1997; Vlach et al., 1997). This dual loop would make
the decision to enter S-phase fast and irreversible, and
the cell-cycle machinery refractory to further extracellu-
lar information, either positive or negative.
1.3.1. Non-return decision: a ‘balanced’ model
With the tentative viewpoint that cyclin E is a juncture
for commitment to S-phase (as it presumably phospho-
rylates multiple substrates required for independent
events), we should revisit the ideas of how the cell cycle
is made responsive to mitogens and anti-mitogens
(Fig. 2). Remember that the cyclin E mRNA is induced
by E2F, which is suppressed by pRb, which in turn is
inactivated by cyclin D-CDK4. The mitogen-dependent
activation of cyclin D-dependent kinases is remarkable.
Expression of cyclin D1, its assembly with CDK4, and
its turnover are all controlled by mitogen-dependent
events, largely dependent on Ras activation (reviewed
in Sherr and Roberts, 1999). Thus, mitogens can ‘feed’
into cyclin E-CDK2 via cyclin D-CDK4 inactivation of
pRb. Indeed, in pRb-negative cells, expression from a
dominant negative allele of Ras will not cause cell-cycle
arrest (Peeper et al., 1997). How do anti-mitogens get
there? To act rapidly, they can affect cyclin E-CDK2
activity either directly by inducing Ink4 proteins and
bouncing the Kips to the cyclin E-CDK2, or they could
A. Vidal, A. Koff / Gene 247 (2000) 1–15
Although our models could be interpreted to suggest
that cyclin E-CDK2 activity is the master regulator of
S-phase entry, it is noteworthy that elements still are
missing. In physiologic systems, i.e. T-cells obtained
from p27 null mice, the activity of CDK2 in quiescent
non-induced cells is quite high, comparable to that in
proliferating cells, but they remain in G0, with unphos-
phorylated pRb and presence of p130 (Coats et al.,
1999; T. Soos and A. Koff, unpublished results). Even
activation of cyclin D-CDK4, by cross-linking the T-cell
receptor, is not sufficient to promote S-phase entry in
these cells, even though p130 goes away and pRb is
now phosphorylated. Thus, without the mitogenic stimu-
lus of IL-2, CDK activity is not sufficient to promote
S-phase entry (T. Soos and A. Koff, unpublished
results). What is missing remains to be determined.
However, the ‘noise’ of the genetic alterations that led
to inmortalization or transformation can affect the
observations and cannot be ignored. Thus, studies car-
ried out in a more ‘normal’ biologic context are essential.
Targeted mutagenesis in mouse provides a powerful tool
for discovering the roles of cell-cycle regulators in devel-
opment and tumorigenesis. Induced mutant strains for
many of the genes involved in cell-cycle control are
available, and animals lacking one or more of CKIs as
well as of pocket proteins have been engineered
(Table 1). The promise of this approach is evident by
the observations that not all of the phenotypic changes
observed in mouse strains lacking cell-cycle regulators
have been due to defects in cell-cycle control. Suggested
roles of both the CKIs, like p21, and the pocket proteins
beyond cell proliferation, such as in cell death or in cell
differentiation, have been reinforced.
Moreover, animals with an induced mutation consti-
tute a source from which cells can been obtained and
cultured for in-vitro studies on proliferation or differen-
tiation. In fact, data from these in-vitro systems still
come several years after the targeted strains have been
made, and the detailed study of specific organs and
2. What mice tell us
Most of the biochemical work characterizing cell-
cycle machinery components comes from studies in cell
culture using immmortalized or transformed cell lines.
Mouse strains lacking one or more of the cell cycle inhibitors and their major phenotypic consequences. The pRb +/− strain is also included
because of its relevance. Additionally, p130−/− and p107−/− animals have been generated in different genetic backgrounds (LeCouter et al.,
Mouse strainPhenotypic consequences References
Embryonic lethality between d. 13.5 and 15.5. Defects in neurogenesis and
Animals are viable but develop thyroid and pituitary tumors. They die
around 340 days old.
Viable and fertile.
Viable and fertile.
Neonatal lethality. Impaired chondrocyte differentiation
Earlier embryonic lethality (d. 11.5) than in the Rb null strain.
Growth retardation. Increased mortality. Retinal dysplasia.
No evident phenotype. MEFs show defective G1 checkpoint
(Clarke et al., 1992; Jacks et al., 1992;
Lee et al., 1992)
(Hu et al., 1994; Harrison et al., 1995) Rb +/−
p130 −/− p107 −/−
Rb −/− p107 −/−
Rb +/− p107 −/−
(Cobrinik et al., 1996)
(Lee et al., 1996)
(Cobrinik et al., 1996)
(Lee et al., 1996)
(Lee et al., 1996)
(Brugarolas et al., 1995;
Deng et al., 1995)
(Fero et al., 1996; Kiyokawa et al., 1996;
Nakayama et al., 1996)
(Yan et al., 1997; Zhang et al., 1997)
Gigantism and organomegalia. Infertile females. Pituitary hyperplasia.
Some embryonic and neonatal lethality. Altered cell proliferation and
apoptosis in several tissues.
Altered lung development. Enhanced skeletal abnormalities. Skeletal muscle
Increased embryonic lethality. More severe lens alterations. Placental defects.
Normal development. General tumor predisposition.
Viable. No tumor predisposition
Gigantism and organomegalia. Pituitary hyperplasia.
Increased organomegalia. Earlier onset on pituitary adenomas.
Postnatal lethality at day 18. Ectopic neuronal divisions and apoptosis
leading to neurological defects.
Decreased survival. Earlier appearance of pituitary adenocarcinomas.
Decreased survival. Earlier onset and more aggressive pituitary tumors.
Viable and fertile.
p21 −/− p57 −/−
(Zhang et al., 1999)
p27 −/− p57 −/−
p15 −/− p18 −/−
p18 −/− p27 −/−
p19 −/− p27 −/−
(Zhang et al., 1998)
(Serrano et al., 1996)
(Franklin et al., 1998)
(Zindy et al., 2000)
(Franklin et al., 1998)
(Zindy et al., 1999)
p21 −/− Rb +/−
p27 −/− Rb +/−
p27 −/− p130 −/−
(Brugarolas et al., 1998)
(Park et al., 1999)
(Coats et al., 1999);
Vidal and Koff, unpublished.
A. Vidal, A. Koff / Gene 247 (2000) 1–15
Fig. 3. Members of three gene families, Ink4, Cip/Kip and pocket proteins, negatively regulate S-phase entry. Their specific and collaborative roles
in cell-cycle progression and tumorigenesis are being elucidated now using mouse models, which provide a powerful tool for genetic analysis and
a source of cell-culture systems for further biochemical analysis.
tissues will allow a further understanding of how cell-
cycle inhibitors work in particular situations (Fig. 3).
ther details can be found elsewhere (Lin et al., 1996;
Mulligan and Jacks, 1998).
2.1. pRb family null mice2.2. Targeting CKIs
Mice lacking functional CKI proteins display a vari-
ety of phenotypes. As single mutants, only p57Kip2 null
mice presented severe developmental abnormalities: they
show a high neonatal lethality, with altered cell prolifera-
tion in the lens and cartilage, as well as developmental
defects in several tissues (Yan et al., 1997; Zhang
et al., 1997).
As discussed before, p21Cip1 null mice did not show
any developmental defect (Brugarolas et al., 1995; Deng
et al., 1995), and analysis of MEFs indicated a role as
a checkpoint protein, rather than a regulator of
In contrast, studies on mice lacking p27Kip1 have
uncovered a prominent role in the decision to withdrawal
from the cell cycle. Importantly, different alleles of the
induced mutant are available: one a complete null (Fero
et al., 1996; Nakayama et al., 1996) and the other a
truncation that eliminated cyclin-CDK binding and inhi-
bition (Kiyokawa et al., 1996). These lines display the
same phenotypic spectrum: a gene-dose-dependent
increase in body size as a result of multiorgan hypercellu-
larity (Fero et al., 1996; Kiyokawa et al., 1996;
Analysis of the mouse strains lacking each member
of the pocket protein family has revealed important
differences in the relative contributions that each gene
makes to development. Embryos homozygous for
mutant Rb alleles die during embryogenesis, showing
defects arising as a consequence of abnormal prolifera-
tion and cell death in liver, lens and central nervous
system, as well as impaired erythropoiesis (Clarke et al.,
1992; Jacks et al., 1992; Lee et al., 1992).
Mice lacking p107 or p130 pocket proteins develop
normally, without any apparent phenotype (Cobrinik
et al., 1996; Lee et al., 1996). However, double mutant
mice for p107 and p130 show neonatal lethality, perhaps
as a consequence of excessive chondrocyte proliferation
leading to defective limb and rib formation and, thereby,
respiratory failure in newborn animals (Cobrinik et al.,
1996). This indicated an overlapping role of these two
proteins in at least some tissues. Overlapping of pRb
and p107 is also indicated because double null embryos
show earlier lethality and accelerated apoptosis in liver
and CNS (Lee et al., 1996). Knockout mice for the
pocket proteins have been extensively studied, and fur-
A. Vidal, A. Koff / Gene 247 (2000) 1–15
Nakayama et al., 1996), female infertility (Fero et al.,
1996; Kiyokawa et al., 1996; Nakayama et al., 1996),
and deafness (Chen and Segil, 1999; Lowenheim et al.,
1999). This appears to be a reflection of impaired
capacity to withdraw from the cell cycle in response to
anti-mitogenic signals, most completely demonstrated in
oligodendrocytes (Casaccia-Bonnefil et al., 1997), luteal
cells (Tong et al., 1998), osteoblasts (Drissi et al., 1999)
and hair cells of the organ of Corti (Chen and Segil,
1999; Lowenheim et al., 1999). According with this
impaired, although not abolished, capacity, cell cultures
derived from p27-deficient mice show a partial loss of
responsiveness to anti-mitogenic agents such as rapa-
mycin (Luo et al., 1996). However, unlike the cell lines
treated with antisense approaches (Coats et al., 1996;
Rivard et al., 1996), they do not display a restriction
point defect in response to serum.
A strikingly similar phenotype of gigantism and wide-
spread organomegaly was found in mice carrying a
targeted disruption of p18Ink4c (Franklin et al., 1998).
One model to account for this is that the lack of p18
allowed cyclin D complexes to sequester more p27, thus
recapitulating the p27 null mice phenotype. However,
p18 and p27 double null mice demonstrated cooperativ-
ity in these proteins, i.e. mice were larger, and pituitary
adenoma development was faster (Franklin et al., 1998).
Thus, these proteins may work in a common pathway,
or regulate two distinct pathways that cooperate in
controlling proliferation. It is attractive to speculate that
the two pathways are those regulated by cyclin D and
family members (Zalvide and DeCaprio, 1995; Stubdal
et al., 1997).
Evidence indicates that p27Kip1 can also be impor-
tant. First, although p27 null mice are not predisposed
to a general increase in tumor frequency, they do develop
pituitary adenomas (Fero et al., 1996; Kiyokawa et al.,
1996; Nakayama et al., 1996) and benign prostatic
hyperplasia (Cordon-Cardo et al., 1998) with high penet-
rance. Furthermore, the animals are hypersensitive to
radiation and chemical-carcinogen-induced tumorigene-
sis (Fero et al., 1998), in this case, displaying the
properties of a haploinsufficient gene. Second, although
inactivating mutations of the p27 gene are very infre-
quent, the postranscriptional mechanisms that govern
p27 expression could be targeted; in fact, low p27
expression has been reported in human breast and colon
carcinomas (Catzavelos et al., 1997; Loda et al., 1997;
Porter et al., 1997) and was attributed to increased
degradation. After those original studies, many others
have shown similar results in a broad variety of cancers
using immunohistochemical analysis. Interestingly, a
common feature is that low levels of p27 protein corre-
late with a poor prognosis of the tumor (Clurman and
Porter, 1998), for which p27 becomes an important
clinical marker of tumor progression.
It is interesting to note that both pRb and p16 lay in
a common route of cell proliferation, the pRb pathway.
Moreover, alterations in these or other members of the
pathway are common features detected in malignancies
(Sherr, 1996; Ruas and Peters, 1998). Those aberrations
include both those leading to a gain of function in the
positive regulators, like amplification of cyclin D or
CDK4 genes, and those that impinge in a loss of
inhibitory capacity, such as deletions or loss-of-function
mutations in Ink4 or Rb loci. As mitogen-activated
signals converge in the pRb pathway, the result of any
of these alterations is an overactivated proliferation-
stimulatory pathway, now independent of mitogenic
requirements. At the same time, as the oncogenic prolif-
eration promoting event, any overexpression of cyclin
D or CDK4, or the inactivation of p16, will result in an
increase in titratation of the Cip/Kip inhibitors, relieving
CDK2 from that constraint and provoking activation
of cyclin E/CDK2 activity. This consequence of the
ability of cyclin D/CDK4 complexes to sequestrate
Cip/Kip proteins might explain how alterations in the
pRb pathway, the ‘mitogen-dependent arm’ of the bal-
ance, are much more frequent in tumors, as they are
indirectly transmitted to the parallel ‘anti-mitogenic-
arm’ composed of p27. In contrast, alterations that only
affect a decrease in the p27 protein, by mutation or
alternative mechanisms, would not necessarily impinge
on the pRb pathway (note the T-cell observations
described above). Only in the context of another tumori-
genic hit would the loss of p27 cause any further
evolution of the malignancy. A useful analogy might be
2.3. Growth inhibitors and tumorigenesis
It becomes clear that proper activation and inactiva-
tion of those activities that drive cells in the cell cycle
are crucial for maintenance of a ordered normal prolifer-
ation and homeostasis in tissues, and alteration of this
order can lead to proliferative disorders. Thus, any
molecule with growth inhibitory properties becomes a
candidate for a tumor suppressor. Among them, p16,
p27 and pRb, are clearly in this class, with the others
being associated, but less prevalent.
The role of pRb as a tumor suppressor in human
tumors is not in any doubt since its identification in the
familial retinoblastoma. In agreement, Rb +/− mice
develop pituitary and thyroid carcinomas after LOH
occur (Hu et al., 1994; Harrison et al., 1995). The other
members of the pocket protein family have been associ-
ated with tumor development because they can restrain
cell proliferation. For example, Rb/p107-deficient chim-
eric mice develop retinoblastoma, indicating an impor-
tant growth control function in vivo for p107 (Robanus-
Maandag et al., 1998). Furthermore, the transformation
of mouse fibroblasts mediated by the viral oncoprotein
SV40 large T-antigen requires inactivation of the three
A. Vidal, A. Koff / Gene 247 (2000) 1–15
that the loss of the brake (p27) would be only important
once the car was already moving. If the car were parked
on a level surface, the absence of the brake would be
Targeted disruption of p16Ink4a in the mouse leads
to a high predisposition to tumor development (Serrano
et al., 1996), a result consistent with the tumor suppres-
sor role of p16Ink4a predicted from the clinical data.
However, this must await further challenge, because the
recent evidence indicates that the Ink4a locus encodes a
second reading frame, called ARF (p19 in mouse, p14
in humans). ARF is a cell-cycle regulatory protein, and
although it does not behave as a CKI, it does regulate
p53 function via Mdm2 (Sherr, 1998) and in a manner
related to myc. The complete phenotypic consequences
of p16Ink4a loss (mice are p16−/−ARF−/−) are
recapitulated in a mouse strain lacking ARF alone
(Kamijo et al., 1997). Despite the fact that ARF is
contributing to the phenotype of Ink4a null mice, point
mutations that do not compromise ARF function are
observed in human tumors (Ruas and Peters, 1998).
Consideration of ARF is beyond the scope of this
review, but the answer about the question of the contri-
bution of p16 deficiency to tumor development in mice
still awaits the publication of the phenotypes of the
p16−/− mice. One must wonder why it is taking
malignancies and thereby provides an unique animal
model for human tumors. Further characterization of
the growth and transformation properties of cells lacking
both pRb and p27 in cell culture should help to establish
how these proteins cooperate in tumor formation.
Meanwhile, a mouse strain lacking other Cip/Kip
inhibitor, p21, was engineered in a pRb defective back-
ground. Interestingly, MEFs derived from double null
embryos showed altered G1 regulation with respect to
those lacking only one gene. Moreover, they showed
anchorage-independent growth, one of the hallmarks
Furthermore, like in the Rb+/− p27−/− mouse strain,
the absence of the CKI contributed to a faster mortality
in the Rb+/− animals.
Both the Rb+/−p27−/− and Rb+/−p21−/−
studies support a model where inhibitors of the Cip/Kip
family cooperate with the tumor suppressor pRb in
tumor development. But do p27 and p21 deficiency act
in the same way to accelerate tumor development? The
answer to this awaits a more careful analysis.
2.5. Overlapping functions of inhibitory proteins
The absence of severe developmental defects in most
of the mouse strains lacking cell cycle inhibitors suggests
the existence of redundant roles between these proteins
or, alternatively, compensatory mechanisms that avoid
more severe phenotypes.
At this point, we can formally define three situations
where genetic redundancy may be observed, but the
functional implication of each of them is quite different
(Fig. 4). Two of them, which we will call pure redun-
dancy and compensatory redundancy, correlate with a
situation wherein the proteins share a biochemical func-
tion. The third, which we will call phenotypic redun-
dancy, is when proteins do not share a biochemical role.
It is important to consider that the redundancy can be
at the level of the molecules or at the processes that
they control (i.e. proliferation can be regulated at com-
mitment or at checkpoint levels).
In purely redundant mechanisms, if two proteins,
such as the cell-cycle inhibitors, perform overlapping
biochemical roles in a specific cell type and both are in
excess, the absence of either will be tolerable, and we
will only observe a phenotype if both are inactivated.
For example, in cells where both p27 and p21 are high
and are in excess to their CDK target, the loss of one
may have no phenotypic consequence.
Alternatively, in compenstory redundancy, the two
inhibitors are again expressed in the same cell and ‘can’
perform the same role, but normally would have
different roles in vivo. In this case, when one inhibitor
is removed, there is enough of the other to fulfill that
function, albeit that is not what it normally might do.
Thus, it has acquired a compensatory function. The case
2.4. Linking two families: an animal model for human
As mentioned above, Rb+/− mice develop tumors
in the intermediate lobe of the pituitary. This occurs
after loss of the remaining normal allele. Because mela-
notrophs are innervated by a dopaminergic neuron,
which negatively regulate their proliferation, abnormally
proliferating cells undergo apoptosis (Nikitin and Lee,
1996). Therefore, the tumor can only develop once those
cells become refractory to the inhibitory signal, and this
is consistent with a latency period observed in the
development of these tumors. However, interestingly,
p27 null mice show melanotroph hyperplasia, in
agreement with an impaired ability to respond to the
antiproliferative signal generated by the dopaminergic
neuron.Our group has
Rb+/−p27−/− mice develop melanotroph adenocar-
cinoma with LOH in the Rb locus, but these tumors
are more aggressive, and their onset occurs earlier (Park
et al., 1999), consistent with the model that a loss of
responsiveness to antimitogenic signals provides an addi-
tional growth advantage to the incipient tumor, thereby
shortening the period of latency before its apparent
This evidence indicates that the intermediate lobe
tumor development observed in pRb+/−p27−/− mice
recapitulates clinical observations on a broad range of
A. Vidal, A. Koff / Gene 247 (2000) 1–15
lacking p107 or p130 in an enriched Balb/cJ strain
display different and more severe phenotypes than those
(LeCouter et al., 1998a,b) argues against a full compen-
satory mechanism even between the two closest related
pocket proteins. Consistent with this, both p130 and
p107 are necessary in the bcl-2-dependent cell-cycle
arrest in MEFs (G. Vairo et al., unpublished results). It
is noteworthy that, in contrast with the model of com-
pensation for the pRb family, there is no biochemical
evidence of any functional replacement between CKIs
in cellular models from knockout mice.
Recently, a remarkable mechanism of compensation
involving genes from different
observed. Coats and coworkers reported that p130 can
act as a CKI inhibiting cyclin E-CDK2 in MEFs lacking
both p21 and p27 (Coats et al., 1999). This p130
function had been previously shown in vitro and is
shared by the related protein p107 (Zhu et al., 1995;
Woo et al., 1997; Castano et al., 1998). Again the cell-
type-specific nature of this redundant mechanism is
observed: p130 cannot compensate for the loss of p27
in T-cells, perhaps because its levels are lower than in
MEFs and are not sufficient to carry out both its E2F
regulatory and CKI functions. As is evident, redundancy
occurs in a very cell-type-specific fashion.
The third and most complex scenario is a situation
of phenotypic compensation. The phenotype is deter-
mined by the sum of several biochemical events, and a
block in any one of them may be enough to affect the
phenotype. In this scenario, the two proteins necessary
to yield a phenotype will have very different roles.
Moreover, it is important to define the ‘phenotypic
event’. The broader this is, the more proteins we will
find. Thus, if we define the phenotype as the appearance
of a molecule in a metabolic pathway, very few molecules
would be involved, but if the definition is the survival
of an animal, we can imagine that a lot of proteins
would be involved. Thus, phenotypic redundancy
between two knockout mouse strains should not be used
to suggest that both proteins have the same function or
impact on the same process. This is one of the most
confounding problems in cell-cycle research — what is
causal and what is effect.
Data from mice combining two targeted Cip/Kip
inhibitors have indeed shown phenotypic redundancy
on specific tissues. Thus, p21Cip1 and p57Kip2 appear
to cooperate in the control of skeletal muscle formation.
Double null mice for these genes exhibit a profound
defect in skeletal muscle as they fail to form myotubes
and display uncontrolled proliferation and cell death of
myoblasts, but neither of the single mutant animals
showed any of these defects (Zhang et al., 1999). Thus,
both CKI participate in the control of skeletal muscle,
but there is no direct evidence that they do so along the
families has been
Fig. 4. Models of redundant genetic relationships between cell-cycle
inhibitors. (A) Pure redundancy: molecules A and B, perform overlap-
ping roles inhibiting cell proliferation. Absence of inhibitor A has no
phenotypic consequences because molecule B is still able to inhibit the
pathway. (B) Compensatory redundancy: although inhibitors A and
B perform different roles, in the absence of one, A, the other, B, can
assume a new role compensating the loss. (C) Phenotypic redundancy:
inhibitors A and B control different, but sequential, steps in a pathway
leading to cell division. In the absence of inhibitor A, proliferation is
still impaired by the block caused by inhibitor B on the step under
of the pRb family members might be an example
(Mulligan and Jacks, 1998). In a physiological context,
when all pocket proteins are expressed, each regulates a
different subset of E2F-responsive genes (Hurford et al.,
1997). However, in the absence of one of the family
members, at least in some tissues, its role will be assumed
by another family member. This seems to be specially
true in the case of p130/E2F4-repressed genes: in T-cells,
loss of p130 can be fully compensated by p107 and pRb
(Mulligan et al., 1998) and in MEFs, by p107 (Cobrinik
et al., 1996). However, compensatory redundancy
between pocket proteins is not complete since pRb null
embryos die during development. One might assume
that this is because the lethality associated with Rb
deficiency might be independent of its cell-cycle control
of E2F, but the observation that E2F deficiency comple-
ments the pRB deficiency somewhat during embryogen-
esis would suggest that this is not true. Likewise, pRb
is not able to replace p130/p107 functions in chondrocyte
proliferation. Moreover, the recent report that mice
A. Vidal, A. Koff / Gene 247 (2000) 1–15
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and biochemical analyses must be performed to test the
results and the prediction of the other and, in this way,
will broaden our knowledge of cell-cycle inhibitors.
We apologize to those whose work has been cited
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publication and members of our laboratory by con-
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