Oncogene (2003) 22, 5208–5219
& 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00
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Cell cycle regulation and neural differentiation
Umberto Galderisi1,2, Francesco Paolo Jori3 and Antonio Giordano*,2
1
Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, School of Medicine, Second University of
Naples, Naples, Italy; 2Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple
University, Philadelphia, PA, USA; 3Department of Neurological Sciences, School of Medicine, Second University of Naples, Naples,
Italy
The general mechanisms that control the cell cycle in
mammalian cells have been studied in depth and several
proteins that are involved in the tight regulation of cell
cycle progression have been identified. However, the
analysis of which molecules participate in cell cycle exit
of specific cell lineages is not exhaustive yet. Moreover,
the strict relation between cell cycle exit and induction of
differentiation has not been fully understood and seems to
depend on the cell type. Several in vivo and in vitro studies
have been performed in the last few years to address these
issues in cells of the nervous system. In this review, we
focus our attention on cyclin–cyclin-dependent kinase
complexes, cyclin kinase inhibitors, genes of the retinoblastoma family, p53 and N-Myc, and we aim to
summarize the latest evidence indicating their involvement
in the control of the cell cycle and induction of
differentiation in different cell types of the peripheral
and central nervous systems. Studies on nervous system
tumors and a possible contributory role in tumorigenesis
of polyomavirus T antigen are reported to point out the
critical contribution of some cell cycle regulators to
normal neural and glial development.
Oncogene (2003) 22, 5208–5219. doi:10.1038/sj.onc.1206558
Keywords: neurogenesis; cyclin-dependent kinases; cyclin
kinase inhibitors; Rb family; p53; N-Myc
Introduction
In order for an organism to develop, two kinds of
processes are fundamental: cell division and cell
differentiation. Both of these formative pathways must
be carefully regulated and coordinated for normal
growth to occur. The mechanisms that control cell
growth and differentiation are now beginning to be
understood. This is mainly because of the identification
of molecules that orchestrate the cell cycle (Hunter,
1993; Sherr, 1994).
In the recent years, great efforts have been dedicated
to address the contribution of cell cycle exit to the
*Correspondence: A Giordano, Sbarro Institute for Cancer Research
and Molecular Medicine, College of Science and Technology, Temple
University, BioLife Science Bldg, Suite 333, 1900 N 12th Street,
Philadelphia, PA 19122,USA; E-mail: [email protected]
acquisition of neural cell identity. The differentiation of
multipotent neural progenitor cells into neurons or glial
cells occurs over a protracted period and appears to be
accompanied by a progressive restriction in the range of
fates available to individual cells (Figure 1) (Edlund and
Jessell, 1999). The fate of certain classes of neural cells
appears to be restricted several divisions before cell cycle
exit; however, for other cell types this restriction occurs
much closer to the last mitosis, and for yet others only
after they have reached the postmitotic stage (Edlund
and Jessell, 1999).
Immature cells in the developing spinal cord represent
an example of restriction in developmental potential in
the final progenitor cell cycle. The extrinsic signal Sonic
hedgehog (Shh) directs the fate of several different
neuronal types (Ericson et al., 1996). Motor neuron
progenitors require an early phase of Shh signaling;
then, in their final cell cycle, they progress to a state of
independence from Shh and at this point appear to
commit to a motor neuron phenotype (Ericson et al.,
1996; Tanabe et al., 1998). Another example is provided
by multipotent cells of the telencephalic ventricular area,
which give rise to cells that exit the cell cycle and then
undergo differentiation and migration (Rakic, 1974;
Bayer et al., 1991). However, the progenitor cells located
within the anterior part of the subventricular area of the
postnatal forebrain migrate to the olfactory bulb and
concurrently undergo cell division, while expressing a
neuronal phenotype (Luskin, 1993; Menezes and Luskin, 1994; Coskun and Luskin, 2001). Also, certain
neural crest-derived progenitor cells are indeed committed to a specific fate several cell divisions before their
exit from the cell cycle (Edlund and Jessell, 1999). In
some cases, the specification of neural cell identity
occurs after the last cell mitosis. A subset of sympathetic
neurons, innervating sweat gland cells, undergo a
developmental switch in their neural properties: they
lose their initial noradrenergic transmitter phenotype
and acquire cholinergic properties (Landis, 1990).
Despite the above-described examples, cell cycle exit
represents the fundamental step to trigger differentiation
of cells and induction of a novel program of gene
expression leading to the elaboration of a specialized
phenotype (Hofbauer and Denhardt, 1991; Duronio and
O’Farrell, 1994; Lassar et al., 1994; Boulikas, 1995;
Edlund and Jessell, 1999). Moreover, there is evidence
demonstrating that several components of the cell cycle
Cell cycle regulation and neural differentiation
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STEM CELLS
NEURAL PROGENITORS
GLIAL PROGENITORS
O-2A CELLS
(cyclin) and catalytic CDK subunits. CDKs are initially
activated through a series of steps, beginning with the
association with a cyclin subunit followed by phosphorylation/dephosphorylation of specific amino acids.
The G1/S transition is the key step for cell cycle
progression and is controlled by D-type cyclins/CDK4,
D-type cyclins/CDK6, which act in mid-G1, and by
cyclin E/CDK2, which operates in late G1 (Figure 2)
(Kasten and Giordano, 1998; Stiegler et al., 1998;
Harbour et al., 1999; Sherr and Roberts, 1999;
Watanabe et al., 1999; Pucci et al., 2000).
One key substrate of CDKs/cyclins is the nuclear
tumor suppressor pRb (and its related proteins pRb2/
p130 and p107), which is phosphorylated on serine and
threonine residues during G1 phase. pRb phosphorylation results in the liberation of E2F factors, whose
ASTROCYTES
NEURONS
OLIGODENDROCYTES
Figure 1 A simplified model of neural stem cell (NSC) differentiation. NSCs can be committed toward either neural- or glialrestricted progenitors. Neuronal precursors differentiate into
several types of mature neurons. Glial precursors can give rise
directly to astrocytes or to O-2A progenitor cells, which in turn
differentiate either in astrocytes or oligodendrocytes
machinery play a major role also in neural cell
specification and differentiation (Massague and Polyak,
1995; Bartek et al., 1997; Piette, 1997; ElShamy et al.,
1998; Horsfield et al., 1998).
Studies of myogenic differentiation provide a model
for understanding how neural cell cycle exit coincides
with the program of cell fate specification (Piette, 1997).
In this system, the activation of the MyoD family of
bHLH proteins regulates muscle fate and differentiation. However, the execution of an overt myogenic
differentiation program depends on cell cycle exit and
reflects the inhibition of MyoD activity under conditions
of cell proliferation and high cyclin-dependent kinase
(CDK) activity (Lassar et al., 1994; Skapek et al., 1995;
Walsh and Perlman, 1997; Zabludoff et al., 1998). Thus,
it appears that terminal mitosis and terminal differentiation are intimately coupled.
In this review, we will mainly focus on some
mechanisms that regulate cell cycle exit and neural/glial
differentiation.
Cell cycle regulation
The decision as to whether somatic cells become
quiescent or resume active proliferation is dictated by
extracellular and intracellular factors that act on the cell
cycle machinery. This is composed of several factors that
control cell cycle progression. The players in this
scenario are holoenzymes composed of regulatory
Figure 2 Cell cycle regulation and differentiation. G1/S cyclin/
CDK complexes and some of the proteins involved in their
regulation are depicted in the picture. Ink4 CKI act on D-type
cyclins/CDKs, while Cip/Kip proteins inhibit both cyclin D/CDKs
and cyclin E/CDKs. Myc proteins positively regulate cell cycle
progression by allowing the transcription of several components of
the ‘core cell cycle machinery’. On the contrary, p53 inhibits cell
proliferation. This task is accomplished mainly through the
induction of p21. Cyclin/CDK complexes phosphorylate and
inhibit Rb proteins. Rb could promote cell differentiation by
inactivating the E2F-dependent transcription of genes involved in
cell proliferation and, at the same time, relieve Id2 inhibition of
bHLH transcription factors that promote the expression of lineagespecific genes involved in differentiation
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activity is required for entry into S phase (Figure 2)
(Kasten and Giordano, 1998; Stiegler et al., 1998;
Harbour et al., 1999; Sherr and Roberts, 1999;
Watanabe et al., 1999; Pucci et al., 2000).
Two classes of molecules can bind to CDKs and
inhibit their kinase activity, by arresting cells in G1. One
class includes p16INK4a, p15 INK4b, p18 INK4c and p19 INK4d,
all of which contain characteristic fourfold ankyrin
repeats. The second group of CDK inhibitors includes p21Cip1, p27Kip1 and p57kip2. The Ink4 proteins
specifically interact with CDK4 and CDK6 and
impair their interaction with D-type cyclins, whereas
Cip/Kip molecules act on cyclin/CDK by forming a
ternary complex (Figure 2) (Kasten and Giordano,
1998; Stiegler et al., 1998; Harbour et al., 1999; Sherr
and Roberts, 1999; Watanabe et al., 1999; Pucci et al.,
2000).
Cyclins, CDKs and differentiation
Terminally differentiated neural cells are arrested in the
G0 phase and some key positive regulators of the cell
cycle are downexpressed. However, one of the most
important cyclins in the late G1 phase, cyclin D1, has
been found to increase in some models of neural
differentiation (Hayes et al., 1991; Okano et al., 1993;
Yan and Ziff, 1995; van Grunsven et al., 1996). In vivo
analysis of cyclin D1 showed that its level increases
during rat brain maturation and reduced cyclin D1
expression correlates with neural development delay
induced by nutritional deprivation (Tamaru et al., 1993;
Tamaru et al., 1994; Shambaugh et al., 1996). On the
other hand, disruption of the cyclin D1 gene in mice
revealed neurological defects, such as abnormal limb
reflex, similar to those observed by targeted disruption
of genes important in neuronal development or function
(Sicinski et al., 1995).
The role of cyclin D1 has been studied in PC12
pheochromocytoma cells, which are one of the most
common models utilized to study neuronal differentiation. Surprisingly, Yan and Ziff (1995) demonstrated
that cyclin D1 was highly increased by inducers of
differentiation, such as NGF and FGF, and not by
inducers of proliferation (EGF and insulin). Although
they observed an increase of cyclin D1/CDK2 and cyclin
D1/CDK4 complexes in cells induced to differentiate,
the associated kinase activity decreased (Yan and Ziff,
1995). These results are in agreement with those
reported by Xiong et al. (1997). They investigated the
role of cyclin D1 in bFGF-induced differentiation of a
rat immortalized hippocampal cell line. After treatment
with bFGF, there was a 10-fold increase of cyclin D1
protein that was localized primarily in the nucleus
(Xiong et al., 1997). However, overexpression of cyclin
D1 per se did not promote neuronal differentiation.
Furthermore, antisense inhibition of cyclin D1 expression did not produce differences in the kinetics or extent
of cellular differentiation (Xiong et al., 1997). All of the
above researches do not explain why cyclin D1 increases
Oncogene
during neuronal development. It has been suggested that
cyclin D1 is required for growth arrest prior to
commitment to differentiation (Yan and Ziff, 1995;
Xiong et al., 1997).
CDK5 is a CDK that is highly homologous to the
other CDK proteins. However, its kinase activity is only
detectable in postmitotic neurons of the central nervous
system (Nikolic et al., 1996). Unlike cyclin D1, which
forms a complex with CDK5 both in vivo and in vitro
without any known activity, a neuron-specific cyclin,
p35, associates with and activates CDK5 (Xiong et al.,
1992; Ishiguro et al., 1994; Lew et al., 1994; Tsai et al.,
1994; Lee et al., 1996a; Nikolic et al., 1996). Cyclin p35
is detected exclusively in postmitotic neurons of the
central nervous system and its expression pattern
parallels cortical neuron development (Nikolic et al.,
1996). In agreement with these observations, p35
knockout experiments in mice demonstrated that
animals lacking p35 exhibit abnormal cortical lamination, probably because of abnormal neuronal migration
(Chae et al., 1997). In a rat hippocampal neuronal
cell line, the overexpression of p35-induced cell differentiation and neurite outgrowth. These phenomena
were blocked by coexpression of dominant-negative
(dn) CDK5 protein, suggesting that p35 promotes
neurite outgrowth through activation of CDK5 (Xiong
et al., 1997). Nevertheless, further experiments have
shown that, in these cells, differentiation induced by
FGF was not repressed by the inhibition of p35
expression obtained with antisense molecules (Xiong
et al., 1997).
Besides p35, another cyclin, called p39, has been
shown to activate CDK5. Cyclin p39 has 57% aminoacid identity with p35 and is specifically detected in rat
cerebrum and cerebellum (Tang et al., 1995). It has been
demonstrated that p39 ectopic expression in a rat
neuronal cell line leads to neurite outgrowth and that
differentiation induced by FGF is arrested by the
inhibition of p39 expression obtained with antisense
molecules. These studies suggest that p35 is sufficient
but not required, and that p39 is both necessary and
sufficient for neural differentiation in this experimental
model (Xiong et al., 1997).
Another intriguing study on the role of CDKs in
neural development was carried out by Ferguson et al.
(2000). They infected cultures of mouse cortical neural
progenitors with adenoviruses expressing dn CDK
mutants to determine which cyclin/CDK complex plays
a major role in neural cell cycle progression, and
consequently must be inhibited to induce terminal
mitosis and differentiation. Infection of neural cells
with either dnCDK2 or dnCDK4/6 resulted in the arrest
of cell cycle progression. In a second round of
experiments, they infected Rb-deficient neural progenitors with either dnCDK2 or dnCDK4/6. The results
demonstrated that both cyclin/CDK complexes are
essential for cell cycle progression, and that cell growth
regulation by CDK4/6 activity was entirely dependent
on the Rb pathway. In contrast, it appeared that CDK2
functions only partially through an Rb-dependent
pathway (Ferguson et al., 2000).
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Cyclin kinase inhibitors
p27Kip1
Differentiation is associated with a reduction in the
overall amount of CDKs activity in G1. Consistent with
this event, the accumulation of cyclin kinase inhibitors
(CKIs) has been observed in many differentiated cell
types in mice (Matsuoka et al., 1994; Parker et al., 1995).
In particular, several in vivo and in vitro studies have
demonstrated that p27Kip1 has a key role during neuronal
differentiation.
In rat embryonic forebrains, the expression of p27
protein (detected by immunocytochemical staining) was
weak in the ventricular zone of the cerebral cortex, a
layer containing mainly proliferating neural progenitors.
In agreement, in the cortical plate and preplate, which
are composed mostly of postmitotic neurons, the
expression of p27 was very high. Strong p27 expression
was observed also in the neurons located in the basal
telencephalon and the diencephalon (Lee et al., 1996a).
These data suggest that high p27 expression is characteristic of postmitotic neurons. High levels of p27 in
postmitotic neurons correlated with p27 binding to and
inactivation of CDK2. However, also cyclin p35/CDK5
complexes, which play a role in the control of
cytoskeletal function in neurons, were present in these
cells. The presence of an active cyclin p35/CDK5
complex raises the question of how CDKs could escape
p27 inhibition. It has been suggested that p35/CDK5
could be refractory to p27 inhibition. This hypothesis is
in accordance with the observation that no p27-bound
CDK5 was detected by Western blotting or immunoprecipitation experiments performed on cell lysates
obtained from rat postmitotic neurons (Lee et al.,
1996a).
In a multipotent embryonal carcinoma cell line
induced to differentiate toward a neuronal phenotype
by retinoic acid treatment, p27Kip1 expression strictly
correlated with the differentiation grade of neuronal
cells. In this cell system, the growth arrest was preceded
by accumulation of p27 and not of other CKIs
(Baldassarre et al., 2000). Moreover, evidence causally
linking retinoic acid-dependent growth arrest and
differentiation was provided by inhibition of retinoic
acid-induced p27 upregulation by transfection of cell
cultures with a plasmid expressing antisense p27 molecules. This treatment resulted in failure to block cell
growth and to induce differentiation (Baldassarre et al.,
2000).
In the N2a-beta neuroblastoma cell line, the induction
of neuronal differentiation by tri-iodothyronine (T3)
treatment relies also upon p27 activity. T3-mediated
growth arrest and differentiation were associated with
augmented expression of p27 protein, which in turn led
to a significant increase in p27/CDK2 complexes and
marked inhibition of CDK2 kinase activity (Perez-Juste
and Aranda, 1999).
The most detailed information on the regulation and
role of p27 protein in vertebrate neural cells emerged
from studies on oligodendrocyte commitment and
differentiation. A useful model for studying glial cell
development is the O-2A bipotential progenitor cell,
which can differentiate into either type 2 astrocytes or
oligodendrocytes (Figure 1) (Raff et al., 1983). In O-2A
cell cultures, differentiation may be induced either by
withdrawing mitogens or by an intrinsic program, called
‘internal clock’. This program is activated after a
discrete number of divisions in the presence of mitogens,
such as PDGF, and hydrophobic extracellular signals,
such as the thyroid hormone (Durand et al., 1997). The
‘clock mechanism’ postulates the existence of a molecular component (the counter) that is sensitive to the
number of cell divisions. The counter must be linked to
an ‘effector’, which triggers cell cycle arrest and
differentiation (Durand et al., 1997). The molecular
nature of the counter has not been fully elucidated and it
is not clear whether the ‘internal clock’ primarily
controls the onset of differentiation, with the following
arrest of the cell cycle or vice versa. However, it is
evident that at some point the ‘clock’ must interact with
regulators of cell cycle progression (Durand et al., 1997).
It was demonstrated that p27 protein progressively
increases in O-2A cells in culture and reaches a plateau
when cells stop dividing and differentiate. The time
course of this increase is consistent with the possibility
that p27 accumulation has a role both in the ‘counter’
and in the ‘effector’ activity (Durand et al., 1997).
The role of p27 in cell cycle exit and differentiation of
oligodendrocyte precursors was further investigated
following the in vitro differentiation of O-2A cells
obtained from p27-deficient mice. Under conditions
that promote differentiation of wild-type cells, O-2A
p27-deficient cells showed impaired growth arrest after
mitogen starvation. However, a fraction of these cells
was still able to undergo cell cycle exit and to perform a
proper differentiation program (Casaccia-Bonnefil et al.,
1997). Impaired growth arrest correlated with continued
progression of 0-2A cells into S phase. This observation
suggests that p27 is a component of the mechanism,
which regulates terminal mitosis of O-2A cells, but at the
same time this protein has no role in the differentiation
process (Casaccia-Bonnefil et al., 1997).
The cell cycle exit, which is believed to precede the
differentiation toward type 2 astrocytes of O-2A
precursor cells, appears also to be regulated by p27
protein. In fact, in CG-4 cells (a clonal glial cell line with
characteristics of O-2A cells) the differentiation toward
mature astrocytes is accompanied by a progressive
increase in p27 expression, which in turn blocks the
activity of G1CDKs (Tikoo et al., 1997).
The p27 protein is also involved in the control of
cerebellar granule cell precursors (GCPs) proliferation.
p27 expression was analysed in the developing cerebellum of mouse embryos (Miyazawa et al., 2000). p27 was
expressed at higher levels in the GCPs of the deeper
areas of external germinal layers than in cells of the
middle zone. This finding suggests that p27 gradually
accumulates in GCPs as they proliferate, and not after
cell cycle exit. Furthermore, in cultured GCPs it was
demonstrated that there was an inverse correlation
between p27 expression and bromo-deoxyuridine
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incorporation (a hallmark of cell cycle progression). All
these data suggest that p27 could be part of the
intracellular mechanism regulating the cell cycle exit
that precedes cell differentiation, as observed in
oligodendrocyte precursors (Miyazawa et al., 2000).
These data suggested that these two CKI proteins play
nonredundant roles in oligodendrocyte maturation: p27
appears to be required for cell cycle arrest and p21 could
have a role in differentiation, independent by its ability
to control cell cycle progression (Zezula et al., 2001).
p21Cip1
p19INK4d
Evidence showing that p21Cip1 participates in the
regulation of neural differentiation has been provided
by in vitro studies on PC12 pheochromocytoma cells. It
was demonstrated that the in vitro differentiation of
PC12 cells induced by NGF is accompanied by a huge
increase in p21 protein levels (Erhardt and Pittman,
1998). In agreement with this observation, it was
demonstrated that the ectopic expression of p21 in this
cell line resulted in growth arrest associated with an
increase in cyclin D1 and cyclin E levels along with a
decrease in cyclin A, cyclin B, Cdc2 and CDK4 levels.
The cell cycle exit induced by p21 ectopic expression
triggered the differentiation of PC12 cells toward a
mature neuronal phenotype. Overall, the cell cycle
changes and the induction of differentiation observed
following p21 overexpression mimicked the changes in
PC12 cell biology induced by NGF treatment (Erhardt
and Pittman, 1998). This observation further lends
credit to the hypothesis that p21 plays a major role in
neural precursor cell cycle exit and differentiation.
Basic helix–loop–helix (bHLH) transcription factors
participate at different stages of neural stem cell (NSC)
differentiation. It has been demonstrated that astroglial
differentiation of NSCs requires the expression of the
HES proteins, which are members of the bHLH family.
The inhibition of their expression by the antisense
technique results in the triggering of neuronal differentiation. The block of HES activity affects two
different stages of neuronal differentiation. The first is
the selection of neuronal versus glial fate and the second
is the selection of a specific neuronal phenotype (Kabos
et al., 2002). The inhibition of HES expression and the
subsequent neuronal differentiation are associated with
a huge increase in p21 protein levels and p21 was the
only CKI, which showed a significant change in its
expression during this process. These results suggest that
bHLH regulation of NSC differentiation is interconnected with a p21Cip1-dependent pathway (Kabos et al.,
2002).
A study on the in vitro maturation of O-2A cells to the
oligodendrocyte phenotype demonstrated that p21 and
p27 are both required for proper differentiation of these
precursor cells. However, these two CKIs control
different aspects of the differentiation process. O-2A
cells isolated from either p21- or p27-deficient mice were
cultured in differentiation medium for several days. It
was observed that p21- and p27-deficient cells failed to
differentiate with wild-type kinetics and required longer
times (Zezula et al., 2001). It was also demonstrated, by
bromo-deoxyuridine incorporation, that while p21deficient cells exited the cell cycle within the first day
in culture as the wild-type cells, p27-deficient cells
continued to proliferate even after 5 days in culture.
Among the Ink4 family members, only p18INK4c and
p19INK4d are expressed in fetal mouse development, and
typical expression patterns have been observed in the
central nervous system from embryonic E11.5 developmental day onward (Zindy et al., 1997a, b). In
particular, during the development of the neocortex,
neuronal precursors exit the cell cycle, migrate to their
final position in the cortex and differentiate (Caviness
et al., 1995). In these cells, p18 expression is turned off
as they exit from the cell cycle and is replaced by p19,
whose expression continues in postmitotic neurons and
is maintained into adulthood (Zindy et al., 1997a). The
persistence of p19 expression may therefore keep
neurons in a postmitotic state, thereby playing a role
in the final phase of cell differentiation.
The regulation of p19 expression could decide
whether neuronal differentiation starts before or after
cell cycle exit (Coskun and Luskin, 2001). As reported
above, neuronal precursors located in the posterior
regions of the telencephalic ventricular areas differentiate after cell cycle exit. In contrast, the precursors
located within the anterior part of the subventricular
area start differentiation while they are still cycling
(Rakic, 1974; Bayer et al., 1991; Luskin, 1993; Menezes
and Luskin, 1994; Coskun and Luskin, 2001).
The analysis of p19 expression pattern in developing
mouse brains has demonstrated that in the telencephalic
ventricular zone the increase in p19 expression, observed
at the time of cell cycle exit, persists also in postmitotic
neurons. On the contrary, neuronal progenitor cells
located in the anterior part of the subventricular zone
have exhibited a different developmental pattern of p19
expression. It has been suggested that these cells
undergo multiple rounds of cell division by repetitively
downregulating their p19 expression (Coskun and Luskin, 2001). These data indicate that in the developing
cerebral cortex, neuronal precursor cells leave the cell
cycle, differentiate and remain forever postmitotic. In
contrast, neurons generated in the anterior part of the
subventricular area may successively downregulate their
p19 expression and re-enter the cell cycle before
becoming permanently postmitotic neurons (Coskun
and Luskin, 2001).
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Proteins associated with the adenovirus 5 E1A
oncoprotein and neural differentiation
DNA tumor viruses have been shown to be effective
tools in the identification of protein factors that regulate
fundamental cellular processes such as transcription,
RNA processing and DNA replication. The transforming gene products of these viruses, such as the E1A
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U Galderisi et al
5213
oncoprotein of adenovirus have also helped to identify
cellular factors involved in neoplastic transformation as
well as in physiological activities. The importance of the
retinoblastoma (RB) family (pRb/p105, p107 and pRb2/
p130) in the inhibition of proliferation and their direct
or indirect role in the cell cycle are evidenced by the
necessity of a number of oncogenic DNA viruses to
encode functional oncoproteins including E1A, T
antigen and E7, which can effectively bind and sequester
RB family members, in order to elicit a transformed
phenotype in infected cells. The role of RB family in cell
cycle regulation has been strictly linked to promoting
cell differentiation.
Rb-, p53- and Myc-pathways are among the main
molecular mechanisms that contribute to regulating the
functions of cell cycle machinery, and are also intimately
interconnected with differentiation pathways. Some
examples on these topics are reported in the following
paragraphs.
RB family
The RB family genes, RB, RB2/p130 and p107, play a
major role in controlling the cell cycle. This function is
mainly accomplished by the differential binding of RB
family proteins to the members of the E2F family of
nuclear factors, which in turn regulate the transcription
of several genes involved in DNA synthesis and cell
cycle progression (Figure 2). RB family proteins are the
substrate of different cyclin–CDK complexes and hence
undergo cyclic phosphorylation and dephosphorylation:
in the hypophosphorylated active status, they bind E2F
family members and inhibit their function, while they
are inactive when hyperphosphorylated (Paggi et al.,
1996; Kasten and Giordano, 1998; Stiegler et al., 1998).
Although some data indicate that pRb, pRb2/p130 and
p107 are able to compensate each other, their specific
binding properties suggest that each RB family protein
plays a distinct role in cell cycle regulation, depending
on cell maturation state and type (Lee et al., 1992;
Claudio et al., 1994; Hurford et al., 1997; LeCouter
et al., 1998a, b).
The RB gene family members are also involved in the
onset of differentiation in a wide variety of cell types
(Garriga et al., 1998; Stiegler et al., 1998; Lipinski and
Jacks, 1999). Several data indicate that pRb and pRb2/
p130 play a crucial role in neurogenesis. In mouse
embryos that lack both copies of the RB gene, dividing
neural precursor cells are found outside of the normal
neurogenic regions in both the central and peripheral
nervous systems. Many of the ectopically dividing cells
die by apoptosis; moreover, the expression of several
neural differentiation markers is greatly reduced (Clarke
et al., 1992; Jacks et al., 1992; Lee et al., 1992, 1994).
Mice lacking either pRb2/p130 or p107 in a mixed 129/
Sv : C57Bl/6J genetic background exhibited no overt
phenotype; furthermore, they were viable and fertile
(Lee et al., 1994; Cobrinik et al., 1996; Herrera et al.,
1996; Hurford et al., 1997). Embryos lacking both
pRb2/p130 and p107 died in utero 2 days earlier than
Rb-deficient embryos and exhibited apoptosis in the
liver and central nervous system, suggesting some
redundancy in function (Cobrinik et al., 1996; Lee
et al., 1996b). However, in a Balb/CJ genetic background the knockdown of pRb2/p130 produced embryonic lethality associated with increased cellular
proliferation and apoptosis in the neural tube (LeCouter
et al., 1998b). In detail, histological analysis revealed
varying degrees of disorganization in neural structures
and a poorly formed notochord. The differentiation of
motoneurons in the ventral horn was impaired and there
was a decreased number of sensory neurons within a
poorly demarcated dorsal root ganglia. Moreover, the
neural epithelium in the neural tube failed to produce a
basement membrane (LeCouter et al., 1998b).
Several in vitro studies confirmed that the RB family
plays a major role in neural cell differentiation. P19
embryonal carcinoma multipotent cells can be induced
to differentiate in vitro into neurons and astrocytes when
treated with retinoic acid. During this neuroectodermal
differentiation, the level of RB family proteins rises
dramatically from barely detectable levels in the
undifferentiated cells (Slack et al., 1995). The functional
inhibition of RB family members by ectopic expression
of a mutated adenovirus E1A protein in P19 cultured
cells caused a huge increase in apoptosis of differentiating neurons and astrocytes (Slack et al., 1995).
The role of pRb in neurogenesis was further
investigated by introducing a neuronal marker gene
into mice lacking a functional RB gene. The marker
transgene consisted of the neuron-specific promoter
alpha-tubulin driving a lacZ reporter gene that was
induced as progenitor cells were committed to a
neuronal fate (Slack et al., 1998; Gloster et al., 1994,
1999). These studies demonstrated that in pRb-deficient
mice, several aberrations in the developing nervous
system occur. Moreover, the timing of marker gene
expression suggested that pRb becomes essential immediately following commitment to a neuronal fate and
that in its absence a huge cell death phenomenon occurs
(Slack et al., 1998).
Other studies appeared to confute that pRb plays a
major role in neurogenesis. In fact, primary neuron cell
cultures, obtained from mice lacking the RB gene,
survived and differentiated as the wild-type counterpart
(Lee et al., 1994; Slack et al., 1998; Callaghan et al.,
1999). However, it was demonstrated that cortical
progenitor cells from RB-null mice exhibited delayed
terminal mitosis and a compensatory increased p107
expression. In spite of this gene compensation, a
deregulation of E2F1 and E2F3 activity was observed
in Rb-deficient cells (Callaghan et al., 1999).
Our research group (also in collaboration with Paggi
and colleagues) has performed several studies on the role
of RB family members in differentiation. We determined
the expression and phosphorylation status of RB family
gene products during DMSO-induced differentiation of
N1E-115 murine neuroblastoma cells (Raschella et al.,
1998). In this system, pRb2/p130 was strongly upregulated during mid-late differentiation stages, while, on the
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Cell cycle regulation and neural differentiation
U Galderisi et al
5214
contrary, pRb and p107 were found to be markedly
decreased at the late stages. Differentiating N1E-115
cells also showed a progressive decrease in B-myb levels,
a proliferation-related protein whose constitutive expression inhibits neuronal differentiation. Transfection
of each of the RB family genes in these cells was able, at
different degrees, to induce neuronal differentiation,
inhibit [3 H] thymidine incorporation and downregulate
the activity of the B-myb promoter (Raschella et al.,
1998). Since neuroblastoma cell lines are heterogeneous
and could behave in different way following ectopic
gene regulation or drug treatment, we analysed the role
of the RB family in neural differentiation by studying
also the human neuroblastoma cell line LAN-5
(Raschella et al., 1997). We demonstrated that pRb2/
p130 expression levels increase during differentiation of
LAN-5 neuroblastoma cells. On the other hand, both
pRb and p107 decreased and underwent progressive
dephosphorylation at late differentiation times
(Raschella et al., 1997). The expression of B-myb and
c-myb, two targets of the RB family proteins, was
downregulated in association with the increase of pRb2/
p130, which was detected as the major component of the
complexes with E2F on the E2F-binding site of the Bmyb promoter in differentiated cells. Interestingly,
E2F4, a preferential partner of p107 and pRb2/p130,
was upregulated and underwent changes in cellular
localization during differentiation (Raschella et al.,
1997).
Other studies have been aimed at understanding the
role of RB2/p130 in commitment and differentiation of
neuroblastoma neural crest stem cells. Neuroblastoma
cells are a convenient model for studies on neural crest
progenitor cells (Jori et al., 2001). These tumor cells
show various phenotypes that represent different neural
populations: the neuroblastic (N) phenotype, the
Schwann/glial/melanocytic-like (S) phenotype and
the intermediate (I) phenotype, which has been
demonstrated to represent a multipotent embryonic
precursor cell of the peripheral nervous system (Ross
et al., 1995).
We demonstrated that RB2/p130 ectopic expression
induces morphological and molecular modifications,
promoting differentiation of I phenotype SK-N-BE(2)C neuroblastoma cells toward a neuroblastic (N) rather
than a Schwann/glial/melanocytic (S) phenotype. These
modifications are stable as they persist even after
treatment with an S phenotype inducer. RB2/p130
ectopic expression also induces a more differentiated
phenotype in N-type SH-SY-5Y cells. Further, this
function appears to be independent from cell cycle
withdrawal. The data reported suggest that pRb2/p130
is able to induce neuronal lineage specification and
differentiation in neural crest stem and committed
neuroblastoma cells, respectively. Thus, pRb2/p130
seems to be required throughout the full neural
maturation process (Jori et al., 2001).
The role of pRb and pRb2/p130 in glial genesis is less
characterized. These findings are hindered also by the
fact that both RB- and RB2/p130-deficient mouse
embryos die early in the development, before the
Oncogene
complete genesis of glial cells (Lee et al., 1994; LeCouter
et al., 1998b; Callaghan et al., 1999).
We contributed to elucidating the role of pRb2/p130
in astrocyte development. For this purpose, we ectopically expressed the pRb2/p130 protein in astrocytoma
and normal astrocyte cultures by adenoviral-mediated
gene transfer. In astrocytoma cells, RB2/p130 ectopic
expression resulted in a significant reduction of cell
growth and in an increased G0/G1 cell population. We
did not observe any induction of programmed cell death
as determined by TUNEL reaction. Interestingly, RB2/
p130 ectopic expression induced astrocyte differentiation. Astrocyte cell cycle arrest and differentiation
seemed to proceed through a pathway that was
independent from p53 (Galderisi et al., 2001).
While a major role for pRb and pRb2/p130 in neural
differentiation has been demonstrated, how their function is accomplished remains to be determined. At least
two different mechanisms could trigger RB familymediated differentiation. The first relies upon transcriptional repression of E2F-responsive promoters; the
other one could be ascribed to the activation of bHLH
proteins.
The E2F transcription factor family is composed of
six members, from E2F1 to E2F6. These factors
heterodimerize with either DP-1 or DP-2 proteins and
bind specific DNA consensus sequences located in
promoters of several genes involved in cell cycle
progression (Sidle et al., 1996; Dyson, 1998). Most of
the E2F factors (except E2F6) bind to the RB family
proteins. However, these interactions are restricted; in
fact, pRb binds preferentially to E2F1, E2F2 and E2F3,
while p107 binds to E2F4 and pRb2/p130 to E2F4 and
E2F5. Binding of the RB family proteins to E2F
activation domain blocks transactivation by E2Fs (Sidle
et al., 1996; Dyson, 1998; Kasten and Giordano, 1998;
Stiegler et al., 1998).
In several systems, terminal differentiation is accompanied by exit from the cell cycle, dephosphorylation of
RB family members along with loss of free E2F
complexes (not associated with RB proteins). As
differentiation proceeds, there is an increase in pRb–
E2F and pRB2/p130–E2F complexes (Sidle et al., 1996;
Dyson, 1998; Stiegler et al., 1998). A strong increase in
pRB2/p130 levels and a reduction of p107 was observed
during retinoic acid-induced differentiation of the
multipotent carcinoma cell line P19 into mature
neurons. These changes were reflected by a quantitative
shift of E2F-site binding complexes containing p107 to
complexes containing pRb2/p130 (Corbeil et al., 1995;
Sidle et al., 1996). It has been suggested that while E2F1,
E2F2 and E2F3 act mainly as inducers of cell cycle
progression, E2F4 and E2F5 are important during the
differentiation process (Sidle et al., 1996; Dyson, 1998;
Stiegler et al., 1998). This hypothesis agrees partially
with the E2F expression pattern during mouse neural
development. The expression of E2F1, E2F2 and E2F5
has been detected in proliferating neuronal precursors of
developing mouse brain; as the neurons differentiated,
the expression of these factors greatly decreased
(Dagnino et al., 1997). Other research performed on
Cell cycle regulation and neural differentiation
U Galderisi et al
5215
PC12 pheochromocytoma cells has demonstrated that
E2F4 plays a role in neuronal differentiation. Following
NGF-induced neuronal differentiation, there was a huge
increase of E2F4 expression level along with an
increased amount of pRb2/p130–E2F4 complexes (Persengiev et al., 1999).
The bHLH family of transcription factors regulates
gene expression, orchestrating cell cycle control, cell
lineage commitment and cell differentiation. The HLH
domain primarily mediates homo- or heterodimerizations, which are essential for DNA binding and
transcriptional regulation (Norton, 2000). Many positively acting bHLH proteins are expressed in the
developing central nervous system and play a crucial
role in neural differentiation (Lee, 1997). Among these,
the bHLH proteins NeuroDs are required for the proper
differentiation of neurons present in the cerebellum and
in the hippocampus of mouse embryos; the Math
proteins are essential for the genesis of cerebellar
granule neurons in mice (Ben-Arie et al., 1997; Miyata
et al., 1999; Schwab et al., 2000).
Toma et al. (2000) demonstrated that bHLH proteins
collaborate with pRb to regulate cortical neurogenesis.
They inhibited all bHLH transcriptional activity by
overexpressing Id2, a member of the Id family, in
primary cultures of cortical progenitors and neurons.
The members of the Id family (Id1, Id2, Id3 and Id4) do
not possess a basic DNA-binding domain and function
as dn regulators of bHLH proteins (Benezra et al., 1990;
Christy et al., 1991; Sun et al., 1991). Furthermore, Id2
function has been coupled to cell cycle machinery
through a direct interaction with pRb, pRb2/130 and
p107 (Figure 2) (Iavarone et al., 1994; Lasorella et al.,
1996). The ectopic expression of Id2 by adenovirus
infection of cortical progenitor cultures arrested neuronal differentiation and triggered apoptosis. These
phenomena were inhibited by coexpression of a constitutively activated pRb mutant, which restored the
proper differentiation process (Toma et al., 2000). How
does pRb rescue neuronal differentiation in cells over
expressing Id2? Toma et al. proposed that pRb, by
binding to Id2, inhibits its ability to bind to and inhibit
positively acting bHLH proteins.
RB family and viral oncoproteins in nervous system
tumors
Q1
The importance of RB family in neural and glial cell
development and differentiation is further underscored
by the observation that alterations in the Rb pathways
are found in several types of tumors, occurring both in
the peripheral and the central nervous system.
Mutations in the genes of Rb family members have
been described in human astrocytomas, glioblastomas
and gliosarcomas (Ueki et al., 1996; Louis, 1997; Nozaki
et al., 1999; Rathore et al., 1999; Puduvalli et al., 2000;
Reis et al., 2000; Wechsler-Reya and Scott, 2001). The
loss of Rb activity determines cell growth deregulation
along with improper cell differentiation. In the last few
years, it has been observed that inhibition of Rb
pathways by viral oncoproteins could contribute to
neuroblastoma and glioblastoma development. In studies on 18 human neuroblastomas and five normal
human adrenal glands, BK polyomavirus DNA was
detected in all tumor samples but in none of the normal
adrenal glands (Flaegstad et al., 1999). The expression
of BK virus T antigen could contribute to tumor
development, inhibiting the activity of tumor-suppressor
genes, such as RB family members and p53. Another
study demonstrated the presence of human JC polyomavirus T antigen in glioblastoma samples (Gordon
et al., 1998; Del Valle et al., 2002), further suggesting
that neurotropic viruses could play a role in neural cell
tumorigenesis and that the RB family is crucial for
control of normal neuronal and glial development. In
particular, we demonstrated that induction of pRb2/
p130 in a glioblastoma cell line expressing the polyomavirus T antigen was able to overcome JCV T-Agmediated cellular aberrant proliferation (Howard et al.,
1998). The same results were obtained with in vivo
injection of tumor cells into nude mice: the induction of
pRb2/p130 expression brought about a 3.2-fold reduction in final tumor mass (Howard et al., 1998). pRb2/
p130 counteracts the neoplastic transformation induced
by the JCV T antigen through the inhibition of cyclin Aand cyclin E-associated kinase activity and, by doing so,
induces p27Kip (Howard et al., 2000).
p53 pathway
The tumor-suppressor p53 gene is involved in cell cycle
regulation and differentiation in several biological
systems. p53 is a transcription factor that can transactivate genes of importance both for apoptosis (such as
BAX) and for G1 arrest (such as p21) (Giaccia and
Kastan, 1998). The molecular mechanisms for p53mediated differentiation are not clear but appear to rely
on the induction of p21 or on cell cycle arrest mediated
by the hypophosphorylation of pRb (Ehinger et al.,
1997; Giaccia and Kastan, 1998; Chylicki et al., 2000a, b).
Whatever the molecular pathway for p53-induced
differentiation, several studies have reported an involvement of p53 in neural cell differentiation.
Eizenberg et al. (1996) analysed the expression and
subcellular localization of p53 in cultures of hippocampal neurons. Soon after in vitro plating, the p53 protein
was detected mainly in cell nuclei and was barely seen in
the cytoplasms. The maturation process of these cells
was associated with a gradual reduction of p53
expression in the nucleus. Finally, the fully differentiated neurons showed p53 expression localized only
into the cytoplasm (Eizenberg et al., 1996). However,
the neurons utilized in these experiments were already
committed to differentiation. Thus, the authors performed the same study on the PC12 cell line, which is
composed of uncommitted neuronal progenitors. Immunostaining experiments showed that p53 was barely
detected in the nuclei of proliferating PC12 cells. Then,
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Cell cycle regulation and neural differentiation
U Galderisi et al
5216
when cells were induced to differentiate by NGF
treatment, high levels of p53 were found in the nuclei.
Finally, when cells were fully differentiated p53 was
detected only in the cytoplasms. All these studies
suggest that at early stage of neuronal differentiation,
p53 accumulates in the nucleus, where it may activate genes involved in cell differentiation. Once cells
become committed toward maturation, p53 is probably no longer needed in the nucleus (Eizenberg et al.,
1996).
The involvement of p53 in neural differentiation has
been further demonstrated by the treatment of PC12 cell
cultures with retroviruses expressing a dn form of p53
(dn-p53). These experiments showed a substantial
reduction in the percentage of differentiating neurons
in cultures expressing the dn form of p53 compared to
controls (Eizenberg et al., 1996).
p53 contributes also to oligodendrocyte differentiation. Cultures of O-2A cells, obtained from the optic
nerve of P7 rats and infected with retroviruses encoding
a dn form of p53, were induced to differentiate either by
mitogen withdrawal or by treatment with hydrophobic
extracellular signals (thyroid hormone). The expression
of dn-p53 completely inhibited both cell cycle withdrawal and differentiation induced by thyroid hormone.
This finding suggests that p53 is part of the effector
component utilized by the ‘internal clock mechanism’ of
O-2A cells to induce cell differentiation. In contrast, dnp53 had no effect on oligodendrocyte differentiation
following mitogen starvation. As a consequence, differentiation obtained by mitogen deprivation is believed to
proceed through a p53-independent pathway (Tokumoto et al., 2001).
N-Myc in neurogenesis
The Myc family (c-, N-, L- and S-Myc) of bHLH-Zip
transcription factors have been implicated in the control
of cell proliferation and differentiation (Queva et al.,
1998; Luscher and Larsson, 1999). However, while cMyc is fairly ubiquitous, the other members of the
family are expressed only in specific tissues and organs.
In particular, N-Myc has a restricted spatio-temporal
expression. It is expressed mainly in the central and
peripheral nervous systems, kidney, lung and spleen.
Moreover, its expression is high during early stages of
development and decreases in adult organisms (Queva
et al., 1998).
N-Myc-deficient mice have shown severe abnormalities in organs where the gene is mainly expressed
(Stanton et al., 1992; Sawai et al., 1993). For example,
they showed great reduction in the number of mature
neurons of the dorsal root ganglia and sympathetic
ganglia, indicating the importance of N-Myc expression
in the production of neurons, especially those derived
from the neural crest (Stanton et al., 1992; Sawai et al.,
1993).
Analysis of N-Myc expression in mouse embryos has
indicated that this protein is expressed rather uniformly
Oncogene
in the migrating crest cells. However, during the
progression of embryo development, its expression
becomes restricted to cells undergoing neuronal differentiation, suggesting that N-Myc expression has a
determining effect on the neuronal/non-neuronal choice
of neural crest cells (Wakamatsu et al., 1997).
Avian embryos represent a useful model to study
neural crest development; for this reason, the expression
of N-Myc protein in the trunk neural crest cells has been
examined in chicken and quail embryos (Wakamatsu
et al., 1997). N-Myc protein was detected in the nuclei of
all neural crest cells before and after their migration
from the dorsal margins of the neural tube. It was
noteworthy that, as neuronal maturation proceeds, NMyc localization switches from nucleus to cytoplasm
(Wakamatsu et al., 1997). Although the significance of
cytoplasmic N-Myc protein in gene regulation has not
been fully understood, it may be hypothesized, as it was
postulated for p53 protein, that at early stages of
neuronal differentiation, N-Myc accumulates in the
nucleus, where it could be involved in the regulation of
cell proliferation and differentiation. Once cells become
committed toward the neuronal phenotype, N-Myc is
probably no longer needed in the nucleus.
To analyse the role played by N-Myc in the
development of neural crest cells, we inhibited its
expression in three different neuroblastoma cell phenotypes by the antisense technique (Galderisi et al., 1999).
The downregulation of N-Myc protein in neuroblastoma stem cells with an (I) phenotype resulted in the
inhibition of cell proliferation, while no induction
toward either Schwann–glial or neuronal phenotype
was observed. On the contrary, the inhibition of N-myc
activity in cells committed toward (N)- or (S)-phenotypes resulted in the induction of differentiation along
with a decrease in the proliferation rate (Galderisi et al.,
1999). These data suggest that N-Myc expression could
regulate the proliferation of neural multipotent crest
cells. Moreover, once cells are committed to a specific
phenotype, N-Myc expression should decrease to allow
a complete differentiation to be reached.
Other studies support this hypothesis. In cultures of
neuronal precursor cells derived from null N-Myc
mouse embryos, a severe reduction of cell proliferation
with a huge diminution of S phase and mitotic cells was
observed. Moreover, the differentiation process was
impressively increased (Knoepfler et al., 2002).
Conclusions
It is clear that interactions among extrinsic and intrinsic
signals determine cell commitment and differentiation.
Thus, for a given cell, the decision about its fate arises
from a unique and specific combination of these signals.
Nevertheless, it is mandatory to rely upon components
of cell cycle machinery to execute the order. For these
reasons, these molecules do not simply regulate cell
division; rather they are part of the ‘committee’ that
decides cell fate.
Cell cycle regulation and neural differentiation
U Galderisi et al
5217
Another point should be stressed. By studying
proteins involved in cell cycle machinery, one could
argue that their job is almost the same in all biological
systems: they have to regulate (positively or negatively)
cell cycle progression. Indeed, an in-depth look at the
‘cell cycle world’ allows us to understand that, for
example, even if the CKIs work everywhere to block cell
cycle progression, each of them could preferentially act
in a specific system rather than in another. As reported
in this review, among the CKIs, p27 appears to be a
keystone protein in neurogenesis. Moreover, the tight
regulation of cell proliferation and cell differentiation
needed for the correct development of an organism is
afforded by the complex interplay between the molecules involved in cell cycle control and the bHLH family
of transcription factors responsible for the expression of
lineage-specific genes.
Acknowledgements
NIH grants and the SBARRO Health Research Organization
(AG).
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