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Oncogene (2003) 22, 52085219

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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 acquisition of neural cell identity. The differentiation of
mammalian cells have been studied in depth and several multipotent neural progenitor cells into neurons or glial
proteins that are involved in the tight regulation of cell cells occurs over a protracted period and appears to be
cycle progression have been identied. However, the accompanied by a progressive restriction in the range of
analysis of which molecules participate in cell cycle exit fates available to individual cells (Figure 1) (Edlund and
of specic cell lineages is not exhaustive yet. Moreover, Jessell, 1999). The fate of certain classes of neural cells
the strict relation between cell cycle exit and induction of appears to be restricted several divisions before cell cycle
differentiation has not been fully understood and seems to exit; however, for other cell types this restriction occurs
depend on the cell type. Several in vivo and in vitro studies much closer to the last mitosis, and for yet others only
have been performed in the last few years to address these after they have reached the postmitotic stage (Edlund
issues in cells of the nervous system. In this review, we and Jessell, 1999).
focus our attention on cyclincyclin-dependent kinase Immature cells in the developing spinal cord represent
complexes, cyclin kinase inhibitors, genes of the retino- an example of restriction in developmental potential in
blastoma family, p53 and N-Myc, and we aim to the nal progenitor cell cycle. The extrinsic signal Sonic
summarize the latest evidence indicating their involvement hedgehog (Shh) directs the fate of several different
in the control of the cell cycle and induction of neuronal types (Ericson et al., 1996). Motor neuron
differentiation in different cell types of the peripheral progenitors require an early phase of Shh signaling;
and central nervous systems. Studies on nervous system then, in their nal cell cycle, they progress to a state of
tumors and a possible contributory role in tumorigenesis independence from Shh and at this point appear to
of polyomavirus T antigen are reported to point out the commit to a motor neuron phenotype (Ericson et al.,
critical contribution of some cell cycle regulators to 1996; Tanabe et al., 1998). Another example is provided
normal neural and glial development. by multipotent cells of the telencephalic ventricular area,
Oncogene (2003) 22, 52085219. doi:10.1038/sj.onc.1206558 which give rise to cells that exit the cell cycle and then
undergo differentiation and migration (Rakic, 1974;
Keywords: neurogenesis; cyclin-dependent kinases; cyclin Bayer et al., 1991). However, the progenitor cells located
kinase inhibitors; Rb family; p53; N-Myc 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 Lu-
Introduction skin, 1994; Coskun and Luskin, 2001). Also, certain
neural crest-derived progenitor cells are indeed com-
In order for an organism to develop, two kinds of mitted to a specic fate several cell divisions before their
processes are fundamental: cell division and cell exit from the cell cycle (Edlund and Jessell, 1999). In
differentiation. Both of these formative pathways must some cases, the specication of neural cell identity
be carefully regulated and coordinated for normal occurs after the last cell mitosis. A subset of sympathetic
growth to occur. The mechanisms that control cell neurons, innervating sweat gland cells, undergo a
growth and differentiation are now beginning to be developmental switch in their neural properties: they
understood. This is mainly because of the identication lose their initial noradrenergic transmitter phenotype
of molecules that orchestrate the cell cycle (Hunter, and acquire cholinergic properties (Landis, 1990).
1993; Sherr, 1994). Despite the above-described examples, cell cycle exit
In the recent years, great efforts have been dedicated represents the fundamental step to trigger differentiation
to address the contribution of cell cycle exit to the of cells and induction of a novel program of gene
expression leading to the elaboration of a specialized
*Correspondence: A Giordano, Sbarro Institute for Cancer Research phenotype (Hofbauer and Denhardt, 1991; Duronio and
and Molecular Medicine, College of Science and Technology, Temple
University, BioLife Science Bldg, Suite 333, 1900 N 12th Street,
OFarrell, 1994; Lassar et al., 1994; Boulikas, 1995;
Philadelphia, PA 19122,USA; E-mail: Giordano@temple.edu Edlund and Jessell, 1999). Moreover, there is evidence
demonstrating that several components of the cell cycle
Cell cycle regulation and neural differentiation
U Galderisi et al
5209
STEM 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 phosphor-
ylation/dephosphorylation of specic amino acids.
NEURAL PROGENITORS GLIAL PROGENITORS 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
O-2A CELLS
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 phosphoryla-
tion results in the liberation of E2F factors, whose

ASTROCYTES

NEURONS

OLIGODENDROCYTES

Figure 1 A simplied model of neural stem cell (NSC) differentia-


tion. NSCs can be committed toward either neural- or glial-
restricted 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


specication and differentiation (Massague and Polyak,
1995; Bartek et al., 1997; Piette, 1997; ElShamy et al.,
1998; Horseld et al., 1998).
Studies of myogenic differentiation provide a model
for understanding how neural cell cycle exit coincides
with the program of cell fate specication (Piette, 1997).
In this system, the activation of the MyoD family of
bHLH proteins regulates muscle fate and differentia-
tion. However, the execution of an overt myogenic
differentiation program depends on cell cycle exit and
reects 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 differentia-
tion are intimately coupled.
In this review, we will mainly focus on some
mechanisms that regulate cell cycle exit and neural/glial
differentiation. 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
Cell cycle regulation progression by allowing the transcription of several components of
the core cell cycle machinery. On the contrary, p53 inhibits cell
The decision as to whether somatic cells become proliferation. This task is accomplished mainly through the
quiescent or resume active proliferation is dictated by induction of p21. Cyclin/CDK complexes phosphorylate and
extracellular and intracellular factors that act on the cell inhibit Rb proteins. Rb could promote cell differentiation by
inactivating the E2F-dependent transcription of genes involved in
cycle machinery. This is composed of several factors that cell proliferation and, at the same time, relieve Id2 inhibition of
control cell cycle progression. The players in this bHLH transcription factors that promote the expression of lineage-
scenario are holoenzymes composed of regulatory specic genes involved in differentiation

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Cell cycle regulation and neural differentiation
U Galderisi et al
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activity is required for entry into S phase (Figure 2) during neuronal development. It has been suggested that
(Kasten and Giordano, 1998; Stiegler et al., 1998; cyclin D1 is required for growth arrest prior to
Harbour et al., 1999; Sherr and Roberts, 1999; commitment to differentiation (Yan and Ziff, 1995;
Watanabe et al., 1999; Pucci et al., 2000). Xiong et al., 1997).
Two classes of molecules can bind to CDKs and CDK5 is a CDK that is highly homologous to the
inhibit their kinase activity, by arresting cells in G1. One other CDK proteins. However, its kinase activity is only
class includes p16INK4a, p15 INK4b, p18 INK4c and p19 INK4d, detectable in postmitotic neurons of the central nervous
all of which contain characteristic fourfold ankyrin system (Nikolic et al., 1996). Unlike cyclin D1, which
repeats. The second group of CDK inhibitors in- forms a complex with CDK5 both in vivo and in vitro
cludes p21Cip1, p27Kip1 and p57kip2. The Ink4 proteins without any known activity, a neuron-specic cyclin,
specically interact with CDK4 and CDK6 and p35, associates with and activates CDK5 (Xiong et al.,
impair their interaction with D-type cyclins, whereas 1992; Ishiguro et al., 1994; Lew et al., 1994; Tsai et al.,
Cip/Kip molecules act on cyclin/CDK by forming a 1994; Lee et al., 1996a; Nikolic et al., 1996). Cyclin p35
ternary complex (Figure 2) (Kasten and Giordano, is detected exclusively in postmitotic neurons of the
1998; Stiegler et al., 1998; Harbour et al., 1999; Sherr central nervous system and its expression pattern
and Roberts, 1999; Watanabe et al., 1999; Pucci et al., parallels cortical neuron development (Nikolic et al.,
2000). 1996). In agreement with these observations, p35
knockout experiments in mice demonstrated that
animals lacking p35 exhibit abnormal cortical lamina-
tion, probably because of abnormal neuronal migration
Cyclins, CDKs and differentiation (Chae et al., 1997). In a rat hippocampal neuronal
cell line, the overexpression of p35-induced cell differ-
Terminally differentiated neural cells are arrested in the entiation and neurite outgrowth. These phenomena
G0 phase and some key positive regulators of the cell were blocked by coexpression of dominant-negative
cycle are downexpressed. However, one of the most (dn) CDK5 protein, suggesting that p35 promotes
important cyclins in the late G1 phase, cyclin D1, has neurite outgrowth through activation of CDK5 (Xiong
been found to increase in some models of neural et al., 1997). Nevertheless, further experiments have
differentiation (Hayes et al., 1991; Okano et al., 1993; shown that, in these cells, differentiation induced by
Yan and Ziff, 1995; van Grunsven et al., 1996). In vivo FGF was not repressed by the inhibition of p35
analysis of cyclin D1 showed that its level increases expression obtained with antisense molecules (Xiong
during rat brain maturation and reduced cyclin D1 et al., 1997).
expression correlates with neural development delay Besides p35, another cyclin, called p39, has been
induced by nutritional deprivation (Tamaru et al., 1993; shown to activate CDK5. Cyclin p39 has 57% amino-
Tamaru et al., 1994; Shambaugh et al., 1996). On the acid identity with p35 and is specically detected in rat
other hand, disruption of the cyclin D1 gene in mice cerebrum and cerebellum (Tang et al., 1995). It has been
revealed neurological defects, such as abnormal limb demonstrated that p39 ectopic expression in a rat
reex, similar to those observed by targeted disruption neuronal cell line leads to neurite outgrowth and that
of genes important in neuronal development or function differentiation induced by FGF is arrested by the
(Sicinski et al., 1995). inhibition of p39 expression obtained with antisense
The role of cyclin D1 has been studied in PC12 molecules. These studies suggest that p35 is sufcient
pheochromocytoma cells, which are one of the most but not required, and that p39 is both necessary and
common models utilized to study neuronal differentia- sufcient for neural differentiation in this experimental
tion. Surprisingly, Yan and Ziff (1995) demonstrated model (Xiong et al., 1997).
that cyclin D1 was highly increased by inducers of Another intriguing study on the role of CDKs in
differentiation, such as NGF and FGF, and not by neural development was carried out by Ferguson et al.
inducers of proliferation (EGF and insulin). Although (2000). They infected cultures of mouse cortical neural
they observed an increase of cyclin D1/CDK2 and cyclin progenitors with adenoviruses expressing dn CDK
D1/CDK4 complexes in cells induced to differentiate, mutants to determine which cyclin/CDK complex plays
the associated kinase activity decreased (Yan and Ziff, a major role in neural cell cycle progression, and
1995). These results are in agreement with those consequently must be inhibited to induce terminal
reported by Xiong et al. (1997). They investigated the mitosis and differentiation. Infection of neural cells
role of cyclin D1 in bFGF-induced differentiation of a with either dnCDK2 or dnCDK4/6 resulted in the arrest
rat immortalized hippocampal cell line. After treatment of cell cycle progression. In a second round of
with bFGF, there was a 10-fold increase of cyclin D1 experiments, they infected Rb-decient neural progeni-
protein that was localized primarily in the nucleus tors with either dnCDK2 or dnCDK4/6. The results
(Xiong et al., 1997). However, overexpression of cyclin demonstrated that both cyclin/CDK complexes are
D1 per se did not promote neuronal differentiation. essential for cell cycle progression, and that cell growth
Furthermore, antisense inhibition of cyclin D1 expres- regulation by CDK4/6 activity was entirely dependent
sion did not produce differences in the kinetics or extent on the Rb pathway. In contrast, it appeared that CDK2
of cellular differentiation (Xiong et al., 1997). All of the functions only partially through an Rb-dependent
above researches do not explain why cyclin D1 increases pathway (Ferguson et al., 2000).
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Cyclin kinase inhibitors differentiation. A useful model for studying glial cell
development is the O-2A bipotential progenitor cell,
p27Kip1 which can differentiate into either type 2 astrocytes or
oligodendrocytes (Figure 1) (Raff et al., 1983). In O-2A
Differentiation is associated with a reduction in the
cell cultures, differentiation may be induced either by
overall amount of CDKs activity in G1. Consistent with
withdrawing mitogens or by an intrinsic program, called
this event, the accumulation of cyclin kinase inhibitors
internal clock. This program is activated after a
(CKIs) has been observed in many differentiated cell
discrete number of divisions in the presence of mitogens,
types in mice (Matsuoka et al., 1994; Parker et al., 1995).
such as PDGF, and hydrophobic extracellular signals,
In particular, several in vivo and in vitro studies have
such as the thyroid hormone (Durand et al., 1997). The
demonstrated that p27Kip1 has a key role during neuronal clock mechanism postulates the existence of a mole-
differentiation.
cular component (the counter) that is sensitive to the
In rat embryonic forebrains, the expression of p27
number of cell divisions. The counter must be linked to
protein (detected by immunocytochemical staining) was an effector, which triggers cell cycle arrest and
weak in the ventricular zone of the cerebral cortex, a
differentiation (Durand et al., 1997). The molecular
layer containing mainly proliferating neural progenitors.
nature of the counter has not been fully elucidated and it
In agreement, in the cortical plate and preplate, which
is not clear whether the internal clock primarily
are composed mostly of postmitotic neurons, the
controls the onset of differentiation, with the following
expression of p27 was very high. Strong p27 expression
arrest of the cell cycle or vice versa. However, it is
was observed also in the neurons located in the basal
evident that at some point the clock must interact with
telencephalon and the diencephalon (Lee et al., 1996a).
regulators of cell cycle progression (Durand et al., 1997).
These data suggest that high p27 expression is char-
It was demonstrated that p27 protein progressively
acteristic of postmitotic neurons. High levels of p27 in
increases in O-2A cells in culture and reaches a plateau
postmitotic neurons correlated with p27 binding to and when cells stop dividing and differentiate. The time
inactivation of CDK2. However, also cyclin p35/CDK5
course of this increase is consistent with the possibility
complexes, which play a role in the control of
that p27 accumulation has a role both in the counter
cytoskeletal function in neurons, were present in these
and in the effector activity (Durand et al., 1997).
cells. The presence of an active cyclin p35/CDK5
The role of p27 in cell cycle exit and differentiation of
complex raises the question of how CDKs could escape
oligodendrocyte precursors was further investigated
p27 inhibition. It has been suggested that p35/CDK5
following the in vitro differentiation of O-2A cells
could be refractory to p27 inhibition. This hypothesis is
obtained from p27-decient mice. Under conditions
in accordance with the observation that no p27-bound
that promote differentiation of wild-type cells, O-2A
CDK5 was detected by Western blotting or immuno-
p27-decient cells showed impaired growth arrest after
precipitation experiments performed on cell lysates mitogen starvation. However, a fraction of these cells
obtained from rat postmitotic neurons (Lee et al.,
was still able to undergo cell cycle exit and to perform a
1996a).
proper differentiation program (Casaccia-Bonnel et al.,
In a multipotent embryonal carcinoma cell line 1997). Impaired growth arrest correlated with continued
induced to differentiate toward a neuronal phenotype
progression of 0-2A cells into S phase. This observation
by retinoic acid treatment, p27Kip1 expression strictly
suggests that p27 is a component of the mechanism,
correlated with the differentiation grade of neuronal
which regulates terminal mitosis of O-2A cells, but at the
cells. In this cell system, the growth arrest was preceded
same time this protein has no role in the differentiation
by accumulation of p27 and not of other CKIs
process (Casaccia-Bonnel et al., 1997).
(Baldassarre et al., 2000). Moreover, evidence causally
The cell cycle exit, which is believed to precede the
linking retinoic acid-dependent growth arrest and
differentiation toward type 2 astrocytes of O-2A
differentiation was provided by inhibition of retinoic
precursor cells, appears also to be regulated by p27
acid-induced p27 upregulation by transfection of cell
protein. In fact, in CG-4 cells (a clonal glial cell line with
cultures with a plasmid expressing antisense p27 mole- characteristics of O-2A cells) the differentiation toward
cules. This treatment resulted in failure to block cell
mature astrocytes is accompanied by a progressive
growth and to induce differentiation (Baldassarre et al.,
increase in p27 expression, which in turn blocks the
2000). activity of G1CDKs (Tikoo et al., 1997).
In the N2a-beta neuroblastoma cell line, the induction
The p27 protein is also involved in the control of
of neuronal differentiation by tri-iodothyronine (T3)
cerebellar granule cell precursors (GCPs) proliferation.
treatment relies also upon p27 activity. T3-mediated
p27 expression was analysed in the developing cerebel-
growth arrest and differentiation were associated with
lum of mouse embryos (Miyazawa et al., 2000). p27 was
augmented expression of p27 protein, which in turn led
expressed at higher levels in the GCPs of the deeper
to a signicant increase in p27/CDK2 complexes and
areas of external germinal layers than in cells of the
marked inhibition of CDK2 kinase activity (Perez-Juste
middle zone. This nding suggests that p27 gradually
and Aranda, 1999).
accumulates in GCPs as they proliferate, and not after
The most detailed information on the regulation and
cell cycle exit. Furthermore, in cultured GCPs it was
role of p27 protein in vertebrate neural cells emerged demonstrated that there was an inverse correlation
from studies on oligodendrocyte commitment and
between p27 expression and bromo-deoxyuridine
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incorporation (a hallmark of cell cycle progression). All These data suggested that these two CKI proteins play
these data suggest that p27 could be part of the nonredundant roles in oligodendrocyte maturation: p27
intracellular mechanism regulating the cell cycle exit appears to be required for cell cycle arrest and p21 could
that precedes cell differentiation, as observed in have a role in differentiation, independent by its ability
oligodendrocyte precursors (Miyazawa et al., 2000). to control cell cycle progression (Zezula et al., 2001).

p21Cip1 p19INK4d

Evidence showing that p21Cip1 participates in the Among the Ink4 family members, only p18INK4c and
regulation of neural differentiation has been provided p19INK4d are expressed in fetal mouse development, and
by in vitro studies on PC12 pheochromocytoma cells. It typical expression patterns have been observed in the
was demonstrated that the in vitro differentiation of central nervous system from embryonic E11.5 develop-
PC12 cells induced by NGF is accompanied by a huge mental day onward (Zindy et al., 1997a, b). In
increase in p21 protein levels (Erhardt and Pittman, particular, during the development of the neocortex,
1998). In agreement with this observation, it was neuronal precursors exit the cell cycle, migrate to their
demonstrated that the ectopic expression of p21 in this nal position in the cortex and differentiate (Caviness
cell line resulted in growth arrest associated with an et al., 1995). In these cells, p18 expression is turned off
increase in cyclin D1 and cyclin E levels along with a as they exit from the cell cycle and is replaced by p19,
decrease in cyclin A, cyclin B, Cdc2 and CDK4 levels. whose expression continues in postmitotic neurons and
The cell cycle exit induced by p21 ectopic expression is maintained into adulthood (Zindy et al., 1997a). The
triggered the differentiation of PC12 cells toward a persistence of p19 expression may therefore keep
mature neuronal phenotype. Overall, the cell cycle neurons in a postmitotic state, thereby playing a role
changes and the induction of differentiation observed in the nal phase of cell differentiation.
following p21 overexpression mimicked the changes in The regulation of p19 expression could decide
PC12 cell biology induced by NGF treatment (Erhardt whether neuronal differentiation starts before or after
and Pittman, 1998). This observation further lends cell cycle exit (Coskun and Luskin, 2001). As reported
credit to the hypothesis that p21 plays a major role in above, neuronal precursors located in the posterior
neural precursor cell cycle exit and differentiation. regions of the telencephalic ventricular areas differenti-
Basic helixloophelix (bHLH) transcription factors ate after cell cycle exit. In contrast, the precursors
participate at different stages of neural stem cell (NSC) located within the anterior part of the subventricular
differentiation. It has been demonstrated that astroglial area start differentiation while they are still cycling
differentiation of NSCs requires the expression of the (Rakic, 1974; Bayer et al., 1991; Luskin, 1993; Menezes
HES proteins, which are members of the bHLH family. and Luskin, 1994; Coskun and Luskin, 2001).
The inhibition of their expression by the antisense The analysis of p19 expression pattern in developing
technique results in the triggering of neuronal differ- mouse brains has demonstrated that in the telencephalic
entiation. The block of HES activity affects two ventricular zone the increase in p19 expression, observed
different stages of neuronal differentiation. The rst is at the time of cell cycle exit, persists also in postmitotic
the selection of neuronal versus glial fate and the second neurons. On the contrary, neuronal progenitor cells
is the selection of a specic neuronal phenotype (Kabos located in the anterior part of the subventricular zone
et al., 2002). The inhibition of HES expression and the have exhibited a different developmental pattern of p19
subsequent neuronal differentiation are associated with expression. It has been suggested that these cells
a huge increase in p21 protein levels and p21 was the undergo multiple rounds of cell division by repetitively
only CKI, which showed a signicant change in its downregulating their p19 expression (Coskun and Lu-
expression during this process. These results suggest that skin, 2001). These data indicate that in the developing
bHLH regulation of NSC differentiation is intercon- cerebral cortex, neuronal precursor cells leave the cell
nected with a p21Cip1-dependent pathway (Kabos et al., cycle, differentiate and remain forever postmitotic. In
2002). contrast, neurons generated in the anterior part of the
A study on the in vitro maturation of O-2A cells to the subventricular area may successively downregulate their
oligodendrocyte phenotype demonstrated that p21 and p19 expression and re-enter the cell cycle before
p27 are both required for proper differentiation of these becoming permanently postmitotic neurons (Coskun
precursor cells. However, these two CKIs control and Luskin, 2001).
different aspects of the differentiation process. O-2A
cells isolated from either p21- or p27-decient mice were
cultured in differentiation medium for several days. It Proteins associated with the adenovirus 5 E1A
was observed that p21- and p27-decient cells failed to oncoprotein and neural differentiation
differentiate with wild-type kinetics and required longer
times (Zezula et al., 2001). It was also demonstrated, by DNA tumor viruses have been shown to be effective
bromo-deoxyuridine incorporation, that while p21- tools in the identication of protein factors that regulate
decient cells exited the cell cycle within the rst day fundamental cellular processes such as transcription,
in culture as the wild-type cells, p27-decient cells RNA processing and DNA replication. The transform-
continued to proliferate even after 5 days in culture. ing gene products of these viruses, such as the E1A
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oncoprotein of adenovirus have also helped to identify pRb2/p130 and p107 died in utero 2 days earlier than
cellular factors involved in neoplastic transformation as Rb-decient embryos and exhibited apoptosis in the
well as in physiological activities. The importance of the liver and central nervous system, suggesting some
retinoblastoma (RB) family (pRb/p105, p107 and pRb2/ redundancy in function (Cobrinik et al., 1996; Lee
p130) in the inhibition of proliferation and their direct et al., 1996b). However, in a Balb/CJ genetic back-
or indirect role in the cell cycle are evidenced by the ground the knockdown of pRb2/p130 produced em-
necessity of a number of oncogenic DNA viruses to bryonic lethality associated with increased cellular
encode functional oncoproteins including E1A, T proliferation and apoptosis in the neural tube (LeCouter
antigen and E7, which can effectively bind and sequester et al., 1998b). In detail, histological analysis revealed
RB family members, in order to elicit a transformed varying degrees of disorganization in neural structures
phenotype in infected cells. The role of RB family in cell and a poorly formed notochord. The differentiation of
cycle regulation has been strictly linked to promoting motoneurons in the ventral horn was impaired and there
cell differentiation. was a decreased number of sensory neurons within a
Rb-, p53- and Myc-pathways are among the main poorly demarcated dorsal root ganglia. Moreover, the
molecular mechanisms that contribute to regulating the neural epithelium in the neural tube failed to produce a
functions of cell cycle machinery, and are also intimately basement membrane (LeCouter et al., 1998b).
interconnected with differentiation pathways. Some Several in vitro studies conrmed that the RB family
examples on these topics are reported in the following plays a major role in neural cell differentiation. P19
paragraphs. 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
RB family dramatically from barely detectable levels in the
undifferentiated cells (Slack et al., 1995). The functional
The RB family genes, RB, RB2/p130 and p107, play a inhibition of RB family members by ectopic expression
major role in controlling the cell cycle. This function is of a mutated adenovirus E1A protein in P19 cultured
mainly accomplished by the differential binding of RB cells caused a huge increase in apoptosis of differentiat-
family proteins to the members of the E2F family of ing neurons and astrocytes (Slack et al., 1995).
nuclear factors, which in turn regulate the transcription The role of pRb in neurogenesis was further
of several genes involved in DNA synthesis and cell investigated by introducing a neuronal marker gene
cycle progression (Figure 2). RB family proteins are the into mice lacking a functional RB gene. The marker
substrate of different cyclinCDK complexes and hence transgene consisted of the neuron-specic promoter
undergo cyclic phosphorylation and dephosphorylation: alpha-tubulin driving a lacZ reporter gene that was
in the hypophosphorylated active status, they bind E2F induced as progenitor cells were committed to a
family members and inhibit their function, while they neuronal fate (Slack et al., 1998; Gloster et al., 1994,
are inactive when hyperphosphorylated (Paggi et al., 1999). These studies demonstrated that in pRb-decient
1996; Kasten and Giordano, 1998; Stiegler et al., 1998). mice, several aberrations in the developing nervous
Although some data indicate that pRb, pRb2/p130 and system occur. Moreover, the timing of marker gene
p107 are able to compensate each other, their specic expression suggested that pRb becomes essential im-
binding properties suggest that each RB family protein mediately following commitment to a neuronal fate and
plays a distinct role in cell cycle regulation, depending that in its absence a huge cell death phenomenon occurs
on cell maturation state and type (Lee et al., 1992; (Slack et al., 1998).
Claudio et al., 1994; Hurford et al., 1997; LeCouter Other studies appeared to confute that pRb plays a
et al., 1998a, b). major role in neurogenesis. In fact, primary neuron cell
The RB gene family members are also involved in the cultures, obtained from mice lacking the RB gene,
onset of differentiation in a wide variety of cell types survived and differentiated as the wild-type counterpart
(Garriga et al., 1998; Stiegler et al., 1998; Lipinski and (Lee et al., 1994; Slack et al., 1998; Callaghan et al.,
Jacks, 1999). Several data indicate that pRb and pRb2/ 1999). However, it was demonstrated that cortical
p130 play a crucial role in neurogenesis. In mouse progenitor cells from RB-null mice exhibited delayed
embryos that lack both copies of the RB gene, dividing terminal mitosis and a compensatory increased p107
neural precursor cells are found outside of the normal expression. In spite of this gene compensation, a
neurogenic regions in both the central and peripheral deregulation of E2F1 and E2F3 activity was observed
nervous systems. Many of the ectopically dividing cells in Rb-decient cells (Callaghan et al., 1999).
die by apoptosis; moreover, the expression of several Our research group (also in collaboration with Paggi
neural differentiation markers is greatly reduced (Clarke and colleagues) has performed several studies on the role
et al., 1992; Jacks et al., 1992; Lee et al., 1992, 1994). of RB family members in differentiation. We determined
Mice lacking either pRb2/p130 or p107 in a mixed 129/ the expression and phosphorylation status of RB family
Sv : C57Bl/6J genetic background exhibited no overt gene products during DMSO-induced differentiation of
phenotype; furthermore, they were viable and fertile N1E-115 murine neuroblastoma cells (Raschella et al.,
(Lee et al., 1994; Cobrinik et al., 1996; Herrera et al., 1998). In this system, pRb2/p130 was strongly upregu-
1996; Hurford et al., 1997). Embryos lacking both lated during mid-late differentiation stages, while, on the
Oncogene
Cell cycle regulation and neural differentiation
U Galderisi et al
5214
contrary, pRb and p107 were found to be markedly complete genesis of glial cells (Lee et al., 1994; LeCouter
decreased at the late stages. Differentiating N1E-115 et al., 1998b; Callaghan et al., 1999).
cells also showed a progressive decrease in B-myb levels, We contributed to elucidating the role of pRb2/p130
a proliferation-related protein whose constitutive ex- in astrocyte development. For this purpose, we ectopi-
pression inhibits neuronal differentiation. Transfection cally expressed the pRb2/p130 protein in astrocytoma
of each of the RB family genes in these cells was able, at and normal astrocyte cultures by adenoviral-mediated
different degrees, to induce neuronal differentiation, gene transfer. In astrocytoma cells, RB2/p130 ectopic
inhibit [3 H] thymidine incorporation and downregulate expression resulted in a signicant reduction of cell
the activity of the B-myb promoter (Raschella et al., growth and in an increased G0/G1 cell population. We
1998). Since neuroblastoma cell lines are heterogeneous did not observe any induction of programmed cell death
and could behave in different way following ectopic as determined by TUNEL reaction. Interestingly, RB2/
gene regulation or drug treatment, we analysed the role p130 ectopic expression induced astrocyte differentia-
of the RB family in neural differentiation by studying tion. Astrocyte cell cycle arrest and differentiation
also the human neuroblastoma cell line LAN-5 seemed to proceed through a pathway that was
(Raschella et al., 1997). We demonstrated that pRb2/ independent from p53 (Galderisi et al., 2001).
p130 expression levels increase during differentiation of While a major role for pRb and pRb2/p130 in neural
LAN-5 neuroblastoma cells. On the other hand, both differentiation has been demonstrated, how their func-
pRb and p107 decreased and underwent progressive tion is accomplished remains to be determined. At least
dephosphorylation at late differentiation times two different mechanisms could trigger RB family-
(Raschella et al., 1997). The expression of B-myb and mediated differentiation. The rst relies upon transcrip-
c-myb, two targets of the RB family proteins, was tional repression of E2F-responsive promoters; the
downregulated in association with the increase of pRb2/ other one could be ascribed to the activation of bHLH
p130, which was detected as the major component of the proteins.
complexes with E2F on the E2F-binding site of the B- The E2F transcription factor family is composed of
myb promoter in differentiated cells. Interestingly, six members, from E2F1 to E2F6. These factors
E2F4, a preferential partner of p107 and pRb2/p130, heterodimerize with either DP-1 or DP-2 proteins and
was upregulated and underwent changes in cellular bind specic DNA consensus sequences located in
localization during differentiation (Raschella et al., promoters of several genes involved in cell cycle
1997). progression (Sidle et al., 1996; Dyson, 1998). Most of
Other studies have been aimed at understanding the the E2F factors (except E2F6) bind to the RB family
role of RB2/p130 in commitment and differentiation of proteins. However, these interactions are restricted; in
neuroblastoma neural crest stem cells. Neuroblastoma fact, pRb binds preferentially to E2F1, E2F2 and E2F3,
cells are a convenient model for studies on neural crest while p107 binds to E2F4 and pRb2/p130 to E2F4 and
progenitor cells (Jori et al., 2001). These tumor cells E2F5. Binding of the RB family proteins to E2F
show various phenotypes that represent different neural activation domain blocks transactivation by E2Fs (Sidle
populations: the neuroblastic (N) phenotype, the et al., 1996; Dyson, 1998; Kasten and Giordano, 1998;
Schwann/glial/melanocytic-like (S) phenotype and Stiegler et al., 1998).
the intermediate (I) phenotype, which has been In several systems, terminal differentiation is accom-
demonstrated to represent a multipotent embryonic panied by exit from the cell cycle, dephosphorylation of
precursor cell of the peripheral nervous system (Ross RB family members along with loss of free E2F
et al., 1995). complexes (not associated with RB proteins). As
We demonstrated that RB2/p130 ectopic expression differentiation proceeds, there is an increase in pRb
induces morphological and molecular modications, E2F and pRB2/p130E2F complexes (Sidle et al., 1996;
promoting differentiation of I phenotype SK-N-BE(2)- Dyson, 1998; Stiegler et al., 1998). A strong increase in
C neuroblastoma cells toward a neuroblastic (N) rather pRB2/p130 levels and a reduction of p107 was observed
than a Schwann/glial/melanocytic (S) phenotype. These during retinoic acid-induced differentiation of the
modications are stable as they persist even after multipotent carcinoma cell line P19 into mature
treatment with an S phenotype inducer. RB2/p130 neurons. These changes were reected by a quantitative
ectopic expression also induces a more differentiated shift of E2F-site binding complexes containing p107 to
phenotype in N-type SH-SY-5Y cells. Further, this complexes containing pRb2/p130 (Corbeil et al., 1995;
function appears to be independent from cell cycle Sidle et al., 1996). It has been suggested that while E2F1,
withdrawal. The data reported suggest that pRb2/p130 E2F2 and E2F3 act mainly as inducers of cell cycle
is able to induce neuronal lineage specication and progression, E2F4 and E2F5 are important during the
differentiation in neural crest stem and committed differentiation process (Sidle et al., 1996; Dyson, 1998;
neuroblastoma cells, respectively. Thus, pRb2/p130 Stiegler et al., 1998). This hypothesis agrees partially
seems to be required throughout the full neural with the E2F expression pattern during mouse neural
maturation process (Jori et al., 2001). development. The expression of E2F1, E2F2 and E2F5
The role of pRb and pRb2/p130 in glial genesis is less has been detected in proliferating neuronal precursors of
characterized. These ndings are hindered also by the developing mouse brain; as the neurons differentiated,
fact that both RB- and RB2/p130-decient mouse the expression of these factors greatly decreased
embryos die early in the development, before the (Dagnino et al., 1997). Other research performed on
Oncogene
Cell cycle regulation and neural differentiation
U Galderisi et al
5215
PC12 pheochromocytoma cells has demonstrated that years, it has been observed that inhibition of Rb
E2F4 plays a role in neuronal differentiation. Following pathways by viral oncoproteins could contribute to
NGF-induced neuronal differentiation, there was a huge neuroblastoma and glioblastoma development. In stu-
increase of E2F4 expression level along with an dies on 18 human neuroblastomas and ve normal
increased amount of pRb2/p130E2F4 complexes (Per- human adrenal glands, BK polyomavirus DNA was
sengiev et al., 1999). detected in all tumor samples but in none of the normal
The bHLH family of transcription factors regulates adrenal glands (Flaegstad et al., 1999). The expression
gene expression, orchestrating cell cycle control, cell of BK virus T antigen could contribute to tumor
lineage commitment and cell differentiation. The HLH development, inhibiting the activity of tumor-suppressor
domain primarily mediates homo- or heterodimeriza- genes, such as RB family members and p53. Another
tions, which are essential for DNA binding and study demonstrated the presence of human JC poly-
transcriptional regulation (Norton, 2000). Many posi- omavirus T antigen in glioblastoma samples (Gordon
tively acting bHLH proteins are expressed in the et al., 1998; Del Valle et al., 2002), further suggesting
developing central nervous system and play a crucial that neurotropic viruses could play a role in neural cell
role in neural differentiation (Lee, 1997). Among these, tumorigenesis and that the RB family is crucial for
the bHLH proteins NeuroDs are required for the proper control of normal neuronal and glial development. In
differentiation of neurons present in the cerebellum and particular, we demonstrated that induction of pRb2/
in the hippocampus of mouse embryos; the Math p130 in a glioblastoma cell line expressing the poly-
proteins are essential for the genesis of cerebellar omavirus T antigen was able to overcome JCV T-Ag-
granule neurons in mice (Ben-Arie et al., 1997; Miyata mediated cellular aberrant proliferation (Howard et al.,
et al., 1999; Schwab et al., 2000). 1998). The same results were obtained with in vivo
Toma et al. (2000) demonstrated that bHLH proteins injection of tumor cells into nude mice: the induction of
collaborate with pRb to regulate cortical neurogenesis. pRb2/p130 expression brought about a 3.2-fold reduc-
They inhibited all bHLH transcriptional activity by tion in nal tumor mass (Howard et al., 1998). pRb2/
overexpressing Id2, a member of the Id family, in p130 counteracts the neoplastic transformation induced
primary cultures of cortical progenitors and neurons. by the JCV T antigen through the inhibition of cyclin A-
The members of the Id family (Id1, Id2, Id3 and Id4) do and cyclin E-associated kinase activity and, by doing so,
not possess a basic DNA-binding domain and function induces p27Kip (Howard et al., 2000).
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 p53 pathway
p107 (Figure 2) (Iavarone et al., 1994; Lasorella et al.,
1996). The ectopic expression of Id2 by adenovirus The tumor-suppressor p53 gene is involved in cell cycle
infection of cortical progenitor cultures arrested neuro- regulation and differentiation in several biological
nal differentiation and triggered apoptosis. These systems. p53 is a transcription factor that can transacti-
phenomena were inhibited by coexpression of a con- vate genes of importance both for apoptosis (such as
stitutively activated pRb mutant, which restored the BAX) and for G1 arrest (such as p21) (Giaccia and
proper differentiation process (Toma et al., 2000). How Kastan, 1998). The molecular mechanisms for p53-
does pRb rescue neuronal differentiation in cells over mediated differentiation are not clear but appear to rely
expressing Id2? Toma et al. proposed that pRb, by on the induction of p21 or on cell cycle arrest mediated
binding to Id2, inhibits its ability to bind to and inhibit by the hypophosphorylation of pRb (Ehinger et al.,
positively acting bHLH proteins. 1997; Giaccia and Kastan, 1998; Chylicki et al., 2000a, b).
Whatever the molecular pathway for p53-induced
differentiation, several studies have reported an involve-
ment of p53 in neural cell differentiation.
RB family and viral oncoproteins in nervous system Eizenberg et al. (1996) analysed the expression and
tumors subcellular localization of p53 in cultures of hippocam-
pal neurons. Soon after in vitro plating, the p53 protein
The importance of RB family in neural and glial cell was detected mainly in cell nuclei and was barely seen in
development and differentiation is further underscored the cytoplasms. The maturation process of these cells
by the observation that alterations in the Rb pathways was associated with a gradual reduction of p53
are found in several types of tumors, occurring both in expression in the nucleus. Finally, the fully differen-
the peripheral and the central nervous system. tiated neurons showed p53 expression localized only
Mutations in the genes of Rb family members have into the cytoplasm (Eizenberg et al., 1996). However,
been described in human astrocytomas, glioblastomas the neurons utilized in these experiments were already
Q1 and gliosarcomas (Ueki et al., 1996; Louis, 1997; Nozaki committed to differentiation. Thus, the authors per-
et al., 1999; Rathore et al., 1999; Puduvalli et al., 2000; formed the same study on the PC12 cell line, which is
Reis et al., 2000; Wechsler-Reya and Scott, 2001). The composed of uncommitted neuronal progenitors. Im-
loss of Rb activity determines cell growth deregulation munostaining experiments showed that p53 was barely
along with improper cell differentiation. In the last few detected in the nuclei of proliferating PC12 cells. Then,
Oncogene
Cell cycle regulation and neural differentiation
U Galderisi et al
5216
when cells were induced to differentiate by NGF in the migrating crest cells. However, during the
treatment, high levels of p53 were found in the nuclei. progression of embryo development, its expression
Finally, when cells were fully differentiated p53 was becomes restricted to cells undergoing neuronal differ-
detected only in the cytoplasms. All these studies entiation, suggesting that N-Myc expression has a
suggest that at early stage of neuronal differentiation, determining effect on the neuronal/non-neuronal choice
p53 accumulates in the nucleus, where it may acti- of neural crest cells (Wakamatsu et al., 1997).
vate genes involved in cell differentiation. Once cells Avian embryos represent a useful model to study
become committed toward maturation, p53 is pro- neural crest development; for this reason, the expression
bably no longer needed in the nucleus (Eizenberg et al., of N-Myc protein in the trunk neural crest cells has been
1996). examined in chicken and quail embryos (Wakamatsu
The involvement of p53 in neural differentiation has et al., 1997). N-Myc protein was detected in the nuclei of
been further demonstrated by the treatment of PC12 cell all neural crest cells before and after their migration
cultures with retroviruses expressing a dn form of p53 from the dorsal margins of the neural tube. It was
(dn-p53). These experiments showed a substantial noteworthy that, as neuronal maturation proceeds, N-
reduction in the percentage of differentiating neurons Myc localization switches from nucleus to cytoplasm
in cultures expressing the dn form of p53 compared to (Wakamatsu et al., 1997). Although the signicance of
controls (Eizenberg et al., 1996). cytoplasmic N-Myc protein in gene regulation has not
p53 contributes also to oligodendrocyte differentia- been fully understood, it may be hypothesized, as it was
tion. Cultures of O-2A cells, obtained from the optic postulated for p53 protein, that at early stages of
nerve of P7 rats and infected with retroviruses encoding neuronal differentiation, N-Myc accumulates in the
a dn form of p53, were induced to differentiate either by nucleus, where it could be involved in the regulation of
mitogen withdrawal or by treatment with hydrophobic cell proliferation and differentiation. Once cells become
extracellular signals (thyroid hormone). The expression committed toward the neuronal phenotype, N-Myc is
of dn-p53 completely inhibited both cell cycle with- probably no longer needed in the nucleus.
drawal and differentiation induced by thyroid hormone. To analyse the role played by N-Myc in the
This nding suggests that p53 is part of the effector development of neural crest cells, we inhibited its
component utilized by the internal clock mechanism of expression in three different neuroblastoma cell pheno-
O-2A cells to induce cell differentiation. In contrast, dn- types by the antisense technique (Galderisi et al., 1999).
p53 had no effect on oligodendrocyte differentiation The downregulation of N-Myc protein in neuroblasto-
following mitogen starvation. As a consequence, differ- ma stem cells with an (I) phenotype resulted in the
entiation obtained by mitogen deprivation is believed to inhibition of cell proliferation, while no induction
proceed through a p53-independent pathway (Tokumo- toward either Schwannglial or neuronal phenotype
to et al., 2001). was observed. On the contrary, the inhibition of N-myc
activity in cells committed toward (N)- or (S)-pheno-
types resulted in the induction of differentiation along
with a decrease in the proliferation rate (Galderisi et al.,
N-Myc in neurogenesis 1999). These data suggest that N-Myc expression could
regulate the proliferation of neural multipotent crest
The Myc family (c-, N-, L- and S-Myc) of bHLH-Zip cells. Moreover, once cells are committed to a specic
transcription factors have been implicated in the control phenotype, N-Myc expression should decrease to allow
of cell proliferation and differentiation (Queva et al., a complete differentiation to be reached.
1998; Luscher and Larsson, 1999). However, while c- Other studies support this hypothesis. In cultures of
Myc is fairly ubiquitous, the other members of the neuronal precursor cells derived from null N-Myc
family are expressed only in specic tissues and organs. mouse embryos, a severe reduction of cell proliferation
In particular, N-Myc has a restricted spatio-temporal with a huge diminution of S phase and mitotic cells was
expression. It is expressed mainly in the central and observed. Moreover, the differentiation process was
peripheral nervous systems, kidney, lung and spleen. impressively increased (Knoeper et al., 2002).
Moreover, its expression is high during early stages of
development and decreases in adult organisms (Queva
et al., 1998).
N-Myc-decient mice have shown severe abnormal- Conclusions
ities in organs where the gene is mainly expressed
(Stanton et al., 1992; Sawai et al., 1993). For example, It is clear that interactions among extrinsic and intrinsic
they showed great reduction in the number of mature signals determine cell commitment and differentiation.
neurons of the dorsal root ganglia and sympathetic Thus, for a given cell, the decision about its fate arises
ganglia, indicating the importance of N-Myc expression from a unique and specic combination of these signals.
in the production of neurons, especially those derived Nevertheless, it is mandatory to rely upon components
from the neural crest (Stanton et al., 1992; Sawai et al., of cell cycle machinery to execute the order. For these
1993). reasons, these molecules do not simply regulate cell
Analysis of N-Myc expression in mouse embryos has division; rather they are part of the committee that
indicated that this protein is expressed rather uniformly decides cell fate.
Oncogene
Cell cycle regulation and neural differentiation
U Galderisi et al
5217
Another point should be stressed. By studying regulation of cell proliferation and cell differentiation
proteins involved in cell cycle machinery, one could needed for the correct development of an organism is
argue that their job is almost the same in all biological afforded by the complex interplay between the mole-
systems: they have to regulate (positively or negatively) cules involved in cell cycle control and the bHLH family
cell cycle progression. Indeed, an in-depth look at the of transcription factors responsible for the expression of
cell cycle world allows us to understand that, for lineage-specic genes.
example, even if the CKIs work everywhere to block cell
cycle progression, each of them could preferentially act
Acknowledgements
in a specic system rather than in another. As reported
NIH grants and the SBARRO Health Research Organization
in this review, among the CKIs, p27 appears to be a (AG).
keystone protein in neurogenesis. Moreover, the tight

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