RB’s original CIN?
Julien Sage1,2,4and Aaron F. Straight3,5
1Department of Pediatrics, Stanford University, Stanford, California 94305, USA;2Department of Genetics, Stanford University,
Stanford, California 94305, USA;3Department of Biochemistry, Stanford University, Stanford, California 94305, USA
The retinoblastoma tumor suppressor RB is the down-
stream mediator of a cellular pathway that is thought to
prevent cancer by controlling the ability of cells to enter
or exit the cell cycle in G0/G1. Recently, however, ac-
cumulating evidence has suggested that RB, its family
members p107 and p130, and their partners, the E2F
family of transcription factors, may have important cel-
lular functions beyond the G1/S transition of the cell
cycle, including during DNA replication and at the tran-
sition into mitosis. In this issue of Genes & Develop-
ment, three studies demonstrate a critical role for RB
in proper chromosome condensation, centromeric func-
tion, and chromosome stability in mammalian cells, and
link these cellular functions of RB to tumor suppression
in mice. Here we discuss how transcriptional and post-
transcriptional mechanisms under the control of the RB
pathway ensure accurate progression through mitosis,
thereby preventing cancer development.
The RB tumor suppressor was first identified in familial
cases of retinoblastoma, a pediatric cancer of the eye. RB
was then found to be mutated in a large number of human
tumors, including osteosarcoma; small-cell lung carci-
noma; and breast, bladder, or prostate cancers. When RB
itself is not mutated in human cancer cells, these cells
nearly always carry alterations in upstream regulators of
RB function, such as the p16Ink4acell cycle inhibitor or
the CyclinD/Cdk4,6 kinase complexes. A vast number of
experiments have assigned a potent role for the RB path-
way at the G1/S transition of the cell cycle, and provide
a model to explain why loss of RB function may lead to
cancer: In G0/G1, RB is mostly hypophosphorylated, and
is able to bind and control the expression of critical cell
cycle genes by interacting with E2F transcription factors
and chromatin remodeling complexes; as cells enter the
cell cycle, RB becomes inactivated by phosphorylation,
allowing E2F to transactivate the expression of genes
essential for DNA replication and cell cycle progression.
RB inactivation in cancer cells by direct mutation or con-
stitutive hyperphosphorylation leads to loss of an impor-
tant checkpoint at the G1/S transition of their cell cycle,
and mutant cells proliferate even under cytostatic condi-
tions. This model explains why the RB pathway is such
a strong tumor suppressor pathway, but it does not ex-
clude that RB has other tumor suppressor activities; for
instance, RB may also directly promote differentiation,
which could play a role in inhibiting cancer development
(for review, see Burkhart and Sage 2008).
Loss of RB function and chromosomal instability (CIN)
Human tumor cells often show signs of genomic insta-
bility (Negrini et al. 2010). Accumulating evidence has
indicated that loss of RB function may play a role in this
process; for example, by inducing defects during the rep-
lication of DNA, and potentially by causing abnormal
segregation of chromosomes during mitosis (Kennedy
et al. 2000; Foijer et al. 2005; Eguchi et al. 2007). Em-
bryonic stem cells (ESCs), which are largely devoid of a
G1 checkpoint, provide a good system to investigate a
potential role for the RB pathway in G2/M (Conklin and
Sage 2009). Deletion of both RB alleles in mouse ESCs
results in increased chromosomal alterations (Zheng
et al. 2002). In mouse embryonic fibroblasts (MEFs), RB
inactivation leads to polyploidy (Srinivasan et al. 2007). In
mouse hepatocytes, loss of RB function promotes aneu-
ploidy (Mayhew et al. 2005). Similarly, knockdown of RB
in human primary cells promotes aneuploidy via micro-
nuclei formation (Amato et al. 2009).
Interestingly, several studies, including the analysis of
gene expression profiles, have identified E2F target genes
involved not only in the G1/S transition and DNA rep-
lication, but also in DNA repair, in G2, and during mi-
tosis in mammalian cells (Ishida et al. 2001; Ren et al.
2002; Polager and Ginsberg 2003). The regulation of G2/M
genes by E2F may be further induced in cells under
genotoxic stress (Eguchi et al. 2007; Plesca et al. 2007).
Different E2F family members may play different roles in
this process, and they interact functionally with other
specific transcription factors in G1/S and G2/M (Zhu
et al. 2004). Among all these potential targets, the reg-
ulation of the expression of the mitotic checkpoint com-
ponent MAD2 by RB/E2F transcriptional regulators has
been shown to play an important role in the control of
chromosomal stability (Fig. 1; Hernando et al. 2004).
MAD2 normally prevents cell cycle progression by
[Keywords: Chromosome instability; centromere; chromosome conden-
sation; cell cycle; Rb; sister chromatid cohesion]
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Article published online ahead of print. Article and publication date are
online at http://www.genesdev.org/cgi/doi/10.1101/gad.1948010.
GENES & DEVELOPMENT 24:1329–1333 ? 2010 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/10; www.genesdev.org 1329
blocking activation of the key E3 ubiquitin ligase, the
anaphase-promoting complex (APC), which ubiquitylates
Cyclin proteins and the chromosome cohesion regulator
Securin. In the absence of APC activity, Cyclins and
Securin remain stable, and cells arrest in metaphase until
the mitotic checkpoint is satisfied. Overexpression of
MAD2 is sufficient to induce CIN and cancer in mice
(Sotillo et al. 2007). Related to the transcriptional control
of Mad2 levels by RB/E2F complexes, the control of the
expression of other negative regulators of APC activity,
Emi1 and BubR1, by E2F (Hsu et al. 2002; Lehman et al.
2007) indicates that both increased E2F-mediated tran-
scription and inappropriate protein stability may cooper-
ate in the genomic instability downstream from loss of
In addition to its role in regulating the transcription of
specific genes important for G2 and M, RB may prevent
genomic instability by controlling the expression of
chromatin- and DNA-modifying enzymes themselves
(McCabe et al. 2005; Siddiqui et al. 2007), and by directly
influencing chromatin structure, including in centro-
meric and telomeric regions; for instance, RB directly
binds the histone H4 Lys 20 (H4K20) methyltransferases
SUV4-20H1 and SUV4-20H2, and is thought to recruit
those enzymes to pericentric heterochromatin to main-
tain pericentric histone H4 methylation (Gonzalo and
Blasco 2005; Isaac et al. 2006; Siddiqui et al. 2007). Loss of
SUV4-20H1,2 activity has been associated with increased
frequencies of telomere recombination (Benetti et al.
2007), and loss of H4K20 methylation in general has been
connected to chromosome condensation defects, G2/M
checkpoint arrest, and DNA damage (Heit et al. 2009;
Oda et al. 2009), but a direct link between RB loss, loss of
these methylation marks, and CIN is still missing.
While these observations have provided strong sup-
port for a role of the RB pathway in the maintenance of
chromosomal stability, the exact mechanisms underlying
this role are still poorly understood, and a direct link be-
tween loss of RB function, increased instability, and can-
cer has not been clearly demonstrated. Three studies
published in this issue of Genes & Development (Coschi
et al. 2010; Manning et al. 2010; van Harn et al. 2010) now
provide novel mechanistic advances regarding the role of
RB in the maintenance of chromosomal stability and can-
Accumulation of DNA damage and CIN in RB family
mutant cells under stress conditions
In mammalian cells, RB belongs to a family of structur-
ally and functionally related proteins with p107 and p130.
Combined deletion of the three RB family genes in MEFs
blocks the ability of these triple-knockout (TKO) cells to
arrest in G1 (Dannenberg et al. 2000; Sage et al. 2000).
Hein te Riele and colleagues (van Harn et al. 2010) had
already shown that TKO MEFs expressing Bcl2 (to pre-
vent apoptotic cell death) arrest in G2 after serum dep-
rivation; this arrest is transient, and the cells can com-
plete their cell cycle upon serum stimulation (Foijer et al.
2005). van Harn et al. (2010) took this observation one
step further, and found that inhibition of the sensor of
DNA damage, ATM, accelerated mitotic entry of serum-
stimulated TKO-Bcl2 MEFs that were arrested in G2,
suggesting that a DNA damage response was slowing cell
cycle re-entry in G2. Indeed, G2-arrested TKO-Bcl2 MEFs
displayed an increase in the presence of DNA double-
strand breaks (DSBs) compared with cycling controls.
Furthermore, some DSBs persisted after mitosis, indi-
cating that TKO cells did not fully repair the damage to
their DNA before re-entering the cell cycle. van Harn
et al. (2010) then analyzed the chromosomes of TKO-Bcl2
MEFs that had been arrested in G2 in low serum and
then had resumed proliferation in full serum. They found
that these mutant cells displayed chromatid breaks,
railroad chromosomes, and loss of tight centromeric co-
hesion. Clones derived from these cells were analyzed by
by several mechanisms. Inactivation of RB
and its family members, p107 and p130, re-
sults in deregulated activation of E2F tran-
scription factors. Targets of E2F include
MAD2, a critical regulator of the spindle as-
sembly checkpoint in mitosis, as well as
enzymes that modify the structure of the
DNA, such as the DNMT1 methyltransfer-
ase. In addition, loss of RB function may
alter the activity of some of its binding
partners involved in the control of chro-
matin structure, such as SUV4-20H. Simi-
larly, loss of RB affects the function of
complexes, such as condensin II complexes,
involved in mitotic chromosome assembly.
Finally, loss of RB family function may lead
to the accumulation of DSBs due to defects
during DNA replication, including under
stress conditions. Altogether, this defines
Loss of RB function triggers CIN
a complex regulatory network of transcriptional and post-transcriptional elements by which the RB pathway controls chromosomal
stability at the G2/M transition of the cell cycle.
Sage and Straight
1330GENES & DEVELOPMENT
tiplex FISH (M-FISH), showing that copy number alter-
ations (CNAs) were increased compared with control cells
(van Harn et al. 2010).
Together, these studies provide convincing evidence
that, in mitogen-deprived conditions, TKO-Bcl2 MEFs
exhibit an increase in DSBs that are not fully repaired
before these cells divide, which eventually leads to CIN
when cells are allowed to re-enter the cell cycle. In the
model proposed by van Harn et al. (2010), cells with
alterations in pathways controlling the G1/S transition of
the cell cycle and cell survival may accumulate genomic
instability, providing a mechanism for cancer progression
(Fig. 1). Studies in Schizosaccharomyces pombe have
shown that H4K20 methylation is important for recruit-
ing the DNA damage checkpoint response protein Crb2
to sites of DSBs, but that loss of H4K20 does not affect
pericentric heterochromatin as it does in vertebrates
(Sanders et al. 2004). It will be interesting to determine
whether the DNA damage phenotypes in TKO-Bcl2 cells
are analogous to the phenotypes seen in S. pombe,
whether stalled or collapsed DNA replication forks or
the DSBs that result from replication defects are directly
linked to the chromosome cohesion defects observed in
the mutant cells, or whether cohesion defects arise from
alternative functions of RB.
Centromeric dysfunction in RB mutant human cells
Data from van Harn et al. (2010) with RB/p107 double-
mutant MEFs suggest that the G2/M defects observed in
TKO cells may exist in cells in which the function of only
one or two family members is abrogated, but RB-deficient
MEFs arrest in G1 upon serum removal, preventing the
analysis of its role in CIN in the assay developed by these
investigators. In contrast, recent experiments in Dro-
sophila and human cells with only mutations in RB in-
dicate that RB may normally promote chromosomal con-
densation and preserve chromosomal stability through
an interaction with condensin II complexes (Longworth
et al. 2008). Nick Dyson and colleagues (Manning et al.
(2010) pursued this analysis, and investigated the conse-
quences of depleting RB function in human RPE-1 cells
for centromere function and chromosomal stability. Con-
firming the findings in TKO MEFs, this analysis in hu-
man cells shows that RB knockdown causes a significant
increase in chromosome missegregation, as measured by
FISH, at a rate similar to what is seen in human tumor
cell lines with a CIN phenotype. In a series of elegant
experiments, Manning et al. (2010) dissected the mitotic
phenotypes of RB knockdown cells; they found that the
mutant cells have a higher mitotic index with more cells
in prometaphase despite a normal spindle checkpoint
and no signs of centrosome overduplication. In contrast,
they observed increased intercentromeric distance, de-
fects in chromosomal alignment in a tight metaphase
plate, bioriented sister kinetochores that often deviate
from the pole-to-pole axis, and premature loss of sister
chromatid cohesion, especially when cells were delayed
in mitosis, similar to the study in TKO MEFs. Together,
these observations led Manning et al. (2010) to further
explore centromeric defects in RB mutant cells.
Similar to what was observed in TKO MEFs, loss of RB
in RPE-1 cells did not affect the levels of the cohesin pro-
tein RAD21 (Peters et al. 2008). However, RAD21 punc-
tate pattern on the DNAwas decreased in RB knockdown
cells after nuclear envelope breakdown. The same obser-
vation was made in polytene chromosomes of RB mutant
flies. Lack of evidence for increased RAD21 cleavage sug-
gested that these defects are probably loading or mainte-
nance defects of cohesin at the centromere, rather than
excessive removal. Furthermore, Manning et al. (2010)
found that the amount of chromatin-associated CAP-D3
condensin was reduced in RB knockdown cells, espe-
cially at the centromere. Cells depleted of RB or CAP-D3
by siRNA showed a similar increase in intercentromeric
distance (Fig. 1; Manning et al. 2010). A particularly in-
teresting aspect of this study is the fact that the pheno-
types of loss of RB function are subtle, still allowing the
mutant cells to divide while accumulating instability,
providing a mechanism for the development of cancer
upon loss of RB function.
Cancer development due to CIN in RB mutant cells
Preliminary evidence provided by van Harn et al. (2010)
suggests that the arrest in G2 and the re-entry with
unrepaired DNA in TKO MEFs may be a step toward
cancerous transformation in Ras transformation assays.
However, this study (van Harn et al. 2010) and the study
from the Dyson group (Manning et al. 2010) did not di-
rectly address whether the genomic instability resulting
from loss of RB function is linked to cancer development.
RB binds to several chromatin modifiers through the
LXCXE-binding domain, and recent evidence by Fred
Dick andcolleagues (Coschiet al. 2010) hasdemonstrated
that an LXCXE-binding mutant that retains E2F-binding
capability still contributes to CIN through its failure to
regulate appropriate pericentric heterochromatin forma-
tion (Isaac et al. 2006). Coschi et al. (2010) further ex-
amined this mutant allele of RB (DL) and evaluated its
role in tumorigenesis. They first examined mitotic events
in wild-type and RBDL/DLmutant MEFs and ESCs and
found an increase in centromere interactions, delayed
ging chromosomes in the mutant cells. Because these
defects were reminiscent of defects in chromosome con-
densation and/or cohesion, Coschi et al. (2010) measured
the protein levels of the components of the condensin and
cohesin complexes. Similar to what Manning et al. (2010)
had observed in RB mutant human cells, Coschi et al.
(2010) found decreased levels of chromatin-associated
condensin II in the RBDLmutant cells. Depletion of the
condensin II subunit CAP-D3 resulted in defects similar
to those observed in DL mutant cells, and similar to what
2003; Hirota et al. 2004). Inaddition,the DL form of RB was
not able to interact with CAP-D3, suggesting a functional
link between RB and condensin II complexes (Fig. 1). Then
Manning et al. (2010) tested the functional role of the DL
RB suppresses cancer in G2/M
GENES & DEVELOPMENT1331
allele in cancer in the context of p53 deficiency. Double-
mutant mice died faster than p53 mutant mice, with
more tumors that were more aggressive and more meta-
static. Interestingly, double-mutant thymic lymphomas
display increased genomic instability compared with p53
mutant tumors. Furthermore, p53+/?;RBDL/DLmutant
mice died faster than p53+/?mice, suggesting that the
DL allele leads to faster loss of the wild-type p53 allele,
and providing a mechanism for the accumulation of
genomic instability and tumor progression in RB mutant
Conclusions and future directions
A number of studies on the mechanisms of action of
cohesin and condensin complexes, and more generally on
the molecular mechanisms ensuring progression through
G2 and M, have been performed in tumor cell lines in
which the function of the RB family of proteins pathway
is nearly always compromised. The data presented in
Coschi et al. (2010), Manning et al. (2010), and van Harn
et al. (2010) showing a role for RB in the control of
condensin II complexes and CIN raise the question of
whether these experiments have given a full and accurate
description of the cellular functions of these complexes.
These studies also open new avenues of research in the
RB field. First, the direct binding partner(s) of RB in the
condensin II complexes remain to be identified. Second,
RB’s role for condensin II recruitment seems to be trans-
ferred to protein phosphatase II A (PP2A) once cells are in
metaphase (Takemoto et al. 2009). Understanding the re-
lationship between these two mechanisms for condensin
recruitment provides an interesting avenue for further
study. Third, loss of RB function and condensin recruit-
ment is likely to contribute to CIN by perturbing centro-
mere structure and inducing merotelic kinetochore at-
Salmon et al. 2005; Cimini 2008; Samoshkin et al. 2009).
However, the relationship between RB and sister chro-
matid cohesion is still unclear, given that the known
phenotypes of condensin mutations, with the exception
of an increased distance between sister centromeres in
mitosis, do not exactly mimic known sister chromatid
cohesion mutants. In vertebrate systems, condensin II
does not appear to play a major role in centromere co-
hesion, while condensin I is important for the resolution
of sister chromosome arms (Ono et al. 2003; Hirota et al.
2004). In Drosophila, condensin complexes are important
for sister chromosome resolution, and defects in condensin
function lead to chromosome missegregation (Coelho
et al. 2003; Dej et al. 2004; Savvidou et al. 2005). In this
context, loss of condensin function would not be pre-
dicted to cause premature chromosome separation, but to
inhibit the proper separation of sister chromatids, and
this is the primary phenotype observed in metazoan
condensin mutant cells (for review, see Hudson et al.
2009). Thus, one interesting possibility that can be tested
in future studies is that the cohesion defects seen by
Coschi et al. (2010), Manning et al. (2010), and van Harn
et al. (2010) in RB mutant cells represent an alternative
pathway that is at least partly independent of condensin II
recruitment to chromosomes, and instead is more directly
related to cohesin function. Coschi et al. (2010), Manning
et al. (2010), and van Harn et al. (2010) demonstrated that
RB disruption did not dramatically alter cohesin binding
to chromatin; future experiments to determine if the
chromatin-bound cohesin in RB mutant cells is produc-
tively mediating chromosome cohesion will likely pro-
vide important insights into RB-dependent cohesion phe-
notypes and RB’s role in the prevention of CIN.
We thank Jamie Conklin for helpful discussions on the manu-
script. We also apologize to our colleagues whose work was not
cited due space limitations. The work in J.S.’s laboratory is sup-
ported by NIH/NCI grant R01 CA114102-01A1, ACS grant RSG-
10-071-01-TBG, and CIRM grant RB1-01385. The work in A.F.S’s
laboratory is supported by grant GM074728 from the NIH.
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