High mobility group protein HMGA1 inhibits retinoblastoma protein-mediated cellular G0 arrest.

Abstract

Retinoblastoma protein (RB) acts as a tumor suppressor in many tissue types, by promoting cell arrest via E2F-mediated transcriptional repression. In addition to the aberrant forms of the RB gene found in different types of cancers, many viral oncoproteins including the simian virus 40 large T antigen target RB. However, cellular factors that inhibit RB function remain to be elucidated. Here, we report that RB interacts with the high mobility group protein A1 (HMGA1), a-non-histone architectural chromatin factor that is frequently overexpressed in cancer cells. HMGA1 binds the small pocket domain of RB, and competes with HDAC1. Subsequently, overexpression of HMGA1 abolishes the inhibitory effect of RB on E2F-activated transcription from the cyclin E promoter. Under serum starvation, T98G cells had been previously shown to be arrested in the G0 phase in an RB-mediated manner. The G0 phase was characterized by growth arrest and low levels of transcription, together with the hypophosphorylation of RB and the downregulation of HMGA1. In contrast, such serum-depleted G0 arrest was abrogated in T98G cells overexpressing HMGA1. The overexpressed HMGA1 was found to form complexes with cellular RB, suggesting that downregulation of HMGA1 is required for G0 arrest. There were no phenotypic changes in HMGA1-expressing T98G cells in the presence of serum, but the persistent expression of HMGA1 under serum starvation caused various nuclear abnormalities, which were similarly induced in T antigen-expressing T98G cells. Our present findings indicate that overexpression of HMGA1 disturbs RB-mediated cell arrest, suggesting a negative control of RB by HMGA1.

Full text

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doi: 10.1111/j.1349-7006.2007.00608.x
© 2007 Japanese Cancer Association
Cancer Sci| 2007
Blackwell Publishing Asia
High mobility group protein HMGA1 inhibits
retinoblastoma protein-mediated cellular G0 arrest
Yasuaki Ueda,1,2 Sugiko Watanabe,1 Shuchin Tei,1 Noriko Saitoh,1 Jun-ichi Kuratsu2 and Mitsuyoshi Nakao1,3
1Department of Regeneration Medicine, Institute of Molecular Embryology and Genetics, 2Department of Neurosurgery, Graduate School of Medical Sciences,
Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
(Received June 10, 2007/Revised July 24, 2007/Accepted August 4, 2007)
Retinoblastoma protein (RB) acts as a tumor suppressor in many
tissue types, by promoting cell arrest via E2F-mediated transcriptional
repression. In addition to the aberrant forms of the RB gene found
in different types of cancers, many viral oncoproteins including the
simian virus 40 large T antigen target RB. However, cellular factors
that inhibit RB function remain to be elucidated. Here, we report
that RB interacts with the high mobility group protein A1 (HMGA1),
a-non-histone architectural chromatin factor that is frequently
overexpressed in cancer cells. HMGA1 binds the small pocket domain
of RB, and competes with HDAC1. Subsequently, overexpression
of HMGA1 abolishes the inhibitory effect of RB on E2F-activated
transcription from the cyclin E promoter. Under serum starvation,
T98G cells had been previously shown to be arrested in the G0 phase
in an RB-mediated manner. The G0 phase was characterized by
growth arrest and low levels of transcription, together with the
hypophosphorylation of RB and the downregulation of HMGA1. In
contrast, such serum-depleted G0 arrest was abrogated in T98G cells
overexpressing HMGA1. The overexpressed HMGA1 was found to
form complexes with cellular RB, suggesting that downregulation
of HMGA1 is required for G0 arrest. There were no phenotypic
changes in HMGA1-expressing T98G cells in the presence of
serum, but the persistent expression of HMGA1 under serum
starvation caused various nuclear abnormalities, which were
similarly induced in T antigen-expressing T98G cells. Our present
findings indicate that overexpression of HMGA1 disturbs RB-
mediated cell arrest, suggesting a negative control of RB by HMGA1.
(Cancer Sci 2007)
T
implicated in tumor suppression and cell differentiation.(1–4)
The principal role of RB is in the control of the cell cycle
by repressing the E2F family of transcription factors, which
regulate the expression of a number of genes involved in DNA
synthesis and cell cycle progression.(5,6) RB suppresses the E2F-
mediated transcription of genes in the G1 to the S phase by at
least two mechanisms. First, RB binds to the transcriptional
activation domain of E2F, and blocks its ability to stimulate
gene expression.(6) Second, the formation of the RB-E2F
complex results in active repression by recruiting appropriate
co-repressors that remodel chromatin to be transcriptionally
inactive in the promoter region of E2F-targeted genes.
These co-repressors include histone deacetylases (HDACs),(7–10)
histone methyltransferases,(11,12) and DNA methyltransferases.(13,14)
RB also influences the accessibility of chromatin through the
recruitment of ATP-dependent chromatin remodeling factors
such as Brahma (BRM) and BRM-related gene (BRG).(15–18)
Thus, RB plays an important role in epigenetic gene regulation.
The ability of RB to repress E2F-mediated transcription
highly depends on the phosphorylation of RB by the cyclin-
dependent kinases (cdk),(2,19) indicating that RB itself is governed
by cell cycle machineries. In G0 and early G1 phases, RB is
primarily unphosphorylated or hypophosphorylated, and becomes
he retinoblastoma protein (RB) is known to be a key
regulator of cell proliferation and arrest, and is therefore
phosphorylated in the late G1 to S phase. Phosphorylation
gradually increases throughout the S and G2/M phases. The
initial phosphorylation occurs in the carboxyl terminal portion
of RB by cyclin D/cdk4 and cdk6, and displaces HDACs
from the pocket region of RB,(19) leading to the blockade of the
repressive role of RB. Subsequently, multiple phosphorylations
in the pocket region of RB by cyclin E/cdk2 disrupt binding to
E2F,(19) resulting in the dissociation of RB from E2F. On the
other hand, the G0 phase is a quiescent state where cells are
resting or terminally differentiated as being ‘out of the cycle’.(20)
It was reported that the acute inactivation of RB alone was
sufficient for G0-arrested cells to re-enter the cell cycle, suggesting
that RB is required for maintaining G0 arrest. Similarly, cyclin
C/cdk3 is involved in regulating the G0 to G1 transition (G0 exit)
through specific phosphorylation of RB.(20) However, the precise
control of RB in the G0 phase remains to be elucidated.
The RB gene has been shown to be inactivated not only in
retinoblastomas but also in a variety of cancer types.(4) Importantly,
several factors involved in the regulation of RB function, including
cyclin D, cdk4 or the p16 cdk inhibitor, are frequently altered
in many cancers,(4) indicating that the deregulation of the RB
pathway commonly contributes to tumor development. Recently,
the mitotic checkpoint protein Mad2 was reported as an E2F-
regulated gene. Inactivation of the RB pathway causes inappropriate
overexpression of Mad2 by E2F, and promotes aneuploidy in
tumorigenesis,(21,22) suggesting that RB is involved in the regulation
of mitotic events. Three members of the RB family (pocket
proteins) including RB, p107, and p130, have unique and over-
lapping functions.(23) Compared with p107 and p130, the loss of
RB function is closely linked to cancer. Viral oncoproteins such
as simian virus 40 (SV40) large T antigen, adenovirus E1A, and
human papilloma virus E7, target the pocket region of the RB
family proteins and thereby disrupt their function.(24,25) Like viral
oncoproteins, mammalian E1A-like inhibitor of differentiation 1
(EID-1) was found to bind RB for inactivation.(26) EID-1 is
highly and ubiquitously expressed in embryogenesis, and has
not been associated with cancer. Since both phosphorylated and
hypophosphorylated RB usually exist throughout the cell cycle,
the function of RB seems not to be determined by this modification
alone. Therefore, endogenous factors that inhibit RB may be
present in growing cells.
During our investigations, we found that RB interacted with
the high mobility group protein A1 (HMGA1), which is a non-
histone architectural chromatin protein, and participates in various
cell regulation mechanisms.(27) HMGA1 is characterized by the
presence of three DNA-binding motifs, called the adenine–
thymine (AT) hook that preferentially binds the stretches of
AT-rich DNA sequences.(27,28) In addition, this protein is reported
to interact with several transcriptional factors and chromatin
factors.(29–31) HMGA1 is highly expressed during embryonic
3To whom correspondence should be addressed. E-mail: mnakao@gpo.kumamoto-u.ac.jp

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© 2007 Japanese Cancer Association
development, and then its expression is downregulated in dif-
ferentiated adult cells.(32) It is important to note that HMGA1
is frequently overexpressed in many types of cancers.(33) The Myc
family of transcription factors particularly appears to stimulate
the expression of HMGA1.(34) Furthermore, HMGA1 is rapidly
induced by exposure to growth factors such as EGF.(35) In fact, the
expression levels of HMGA1 correlate with growth and meta-
static activities of malignant cells.(27) Transgenic mice over-
expressing HMGA1 in all tissues develop lymphomas, and similarly
to RB heterozygous mice, they have pituitary adenomas and
thyroid tumors.(36) These lines of evidence suggest that HMGA1
is a potential oncoprotein, and functionally cross-talks with the
RB pathway. Moreover, HMGA2, which is closely related to
HMGA1, was recently reported to induce pituitary tumorigenesis
by enhancing E2F1 activity.(37) However, the functional relation-
ship of overexpressed HMGA1 with RB remains unclear.
In the present study, we report that HMGA1 bound to the
pocket domain of RB, resulting in the competition with HDAC1
but not E2F1 for RB binding. Subsequently, the overexpression
of HMGA1 abolished the inhibitory effect of RB on E2F-
activated transcription from the cyclin E promoter. Under serum
starvation, RB-mediated G0 phase in T98G cells was characterized
by growth arrest and low levels of general transcription, together
with hypophosphorylation of RB and downregulation of
HMGA1. In contrast, serum-starved G0 arrest was abrogated by
exogenous expression of HMGA1 in cells. Exogenous HMGA1
was found to form complexes with cellular RB, suggesting that
downregulation of HMGA1 is required for RB-mediated G0
arrest. Together, our results indicate that HMGA1 is a cellular
inhibitor of RB, and overexpression of HMGA1 disturbs RB-
mediated cell arrest. These data shed light on a trans-acting
control of RB function by HMGA proteins.
Materials and Methods
Yeast two-hybrid screening. Yeast strain AH109 carrying the
pAS2-1-pocket region of mouse RB (amino acids 355–779)
was transformed with the mouse E11.5 and E17 whole embryo
cDNA libraries constructed in pACT2 (Clontech). Plasmids
harboring cDNA were recovered from both histidine- and adenine-
positive colonies, and were used for DNA sequencing analysis.
Plasmids. The human cDNA sequence for HMGA1 was cloned
into pcDNA3 (pcDNA3-FLAG-HMGA1), and into pCAG-EGFP-
IRES-Puro (pCAG-EGFP-HMGA1). pSG5L-HA-RB was
as previously described.(38)
Cell culture. T98G glioblastoma, Saos-2, and MCF-7 cells
were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s
minimum essential medium and Ham’s F-12 nutrient medium
(Sigma) supplied with 10% (v/v) heat-inactivated fetal bovine
serum (FBS). For synchronization studies, cells were incubated
in medium containing no FBS for 72 h, then were stimulated
with medium containing 10% FBS. Both total RNA and genomic
DNA were isolated using standard methods to determine RNA/
DNA ratios.
Transfection and cell treatment. T98G and Saos-2 cells were
transfected with plasmid DNAs using a liposome-mediated gene
transfer method. For the luciferase assay, Saos-2 cells (1 × 105
cells) were transfected with RB and HMGA1 expression vectors
(1 μg each) using FuGene6 (Roche Applied Science) in 6-well
plates and, after 48 h, the cells were used for the luciferase assay.
Protein expression. The pGEX-4T vector expressing glu-
tathione S-transferase (GST)-fused human RB (amino acids
301–928) was provided by Dr Y. Taya. cDNAs for human
HMGA1 were cloned into pET28a (Novagen). The expressions
of these proteins and His-fused MBD1(TRD) were verified as
described previously.(39)
Antibodies. The antibodies used were anti-RB from BD
Pharmingen; anti-HMGA1, anti-E2F1, anti-HDAC1, and anti-GFP
from Santa Cruz; anti-FLAG (M5) and anti-GFAP from Sigma;
anti-His tag from Qiagen; anti-GST from DAKO; and antiβ-
tubulin from Amersham.
Immunoprecipitation. For immunoprecipitation of RB complexes,
T98G cells were lyzed with a buffer (50 mM Tris (pH 8.0),
300 mM NaCl, 0.4% NP40, 10 mM MgCl2) supplemented with
a cocktail of protease and phosphatase inhibitors. Extracts were
cleared by centrifugation, and then diluted with a buffer
containing 50 mM Tris (pH 8.0), 0.4% NP40, and 2.5 mM
CaCl2. Extracts (250 μL) were precleared by incubation with
20 μL of protein A/G-agarose beads (Amersham Pharmacia and
Oncogene) for 30 min at 4°C. After incubation with the anti-RB
antibodies for 1 h at 4°C, 10 μL of protein A/G beads were added
to the extracts for 1 h at 4°C. Following extensive washes, bound
proteins were analyzed by Western blot analysis.
Chemical cross-linking immunoprecipitation was carried out
to examine cellular complexes of HMGA1 and RB. T98G cells
were treated with dimethyl 3,3′-dithiobispropionimidate-2HCl
(DTBP) (5 mM) (Pierce) in phosphate-buffered saline (PBS),
rinsed with an ice-cold buffer (100 mM Tris-HCl (pH 8.0), 150 mM
NaCl), lyzed with a buffer containing 10 mM Tris (pH 7.5),
500 mM NaCl, 1% NP-40, 5 mM EDTA, 5% glycerol, 0.1%
SDS, 1% sodium deoxycholate, and supplemented with a cocktail
of protease and phosphatase inhibitors. After being sonicated,
the supernatants (250 μL) were incubated for 1 h at 4°C with
specific antibodies or control immunoglobulin G (IgG), followed
by an another hour-incubation after the addition of 20 μL of
protein A/G agarose beads. The bound proteins were analyzed
by Western blot analysis.
In vitro binding and GST pull-down assay. Bacterially expressed
GST and GST-fused RB (1 μg) were immobilized on glutathione-
agarose beads, and incubated with His-tagged HMGA1 or
MBD1(TRD) (1 μg) in a buffer containing 0.05% Triton X-100,
50 mM HEPES (pH 7.4), 100 mM or 200 mM NaCl, 5%
glycerol, 10 μM ZnCl, 1 mM dithiothreitol, and protease inhibitors
for 1 h at 4°C. The input indicated 10% of the His-tagged
proteins. His-HMGA1 was used for a competition assay using
the RB immunoprecipitates.
Immunofluorescent analysis. After two washes with PBS,
T98G cells were fixed with 4% paraformaldehyde in PBS, for
15 min at room temperature; and then treated with 0.2% Triton
X-100 for 5 min at 4°C. After washing the cells with PBS
containing 0.5% bovine serum albumin (BSA), they were
incubated with specific antibodies in PBS containing 0.2%
BSA, for 1 h at room temperature. Samples were analyzed with
an Olympus IX71 microscope using the Lumina Vision software
(version 2.2).
Luciferase assay. Forty-eight hours after the transfection with
luciferase reporter pCycE-luc, together with pSG5L-HA-RB
and pcDNA3-FLAG-HMGA1, Saos-2 cells were lyzed in a
buffer provided by the manufacturer (Promega). pRL-TK and
insertless pcDNA3 were used as controls. Values reported are
means and standard deviations of the results from three
independent experiments.
Small interfering RNA-mediated knockdown. Small interfering
RNA (siRNA) duplexes were designed for targeting mRNAs
encoding human HMGA1 (Japan Bio Services), listed in
Table S1. The selected siRNA sequences were submitted to
human genome and EST databases to ensure the target
specificities. The siRNAs were transfected into the cells by
using lipofectamine RNAiMAX (Invitrogen).
Quantitative real-time reverse transcription-polymerase chain
reaction. Five micrograms of the total RNAs was reverse-
transcribed using Superscript III (Invitrogen) and random
hexamers (Operon). For quantification, real-time polymerase
chain reaction (PCR) analysis was carried out using Power
SYBR Green PCR Master Mix on an ABI Prism 7500 Sequence
Detector (Applied Biosystems). PCR amplification was repeated

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at least three times. The relative fold induction was quantified
by the comparative threshold cycle method, and β-actin was
used as an endogenous normalization control. Primer sets for
HMGA1 and the E2F target genes are listed in Table S2.
Results
RB interacts with HMGA1. To identify the factors that interact
with RB, we carried out a yeast two-hybrid screening using the
region including the small pocket A and B of mouse RB (amino
acids 355–779) as a bait (Fig. 1a). By screening approximately
3 × 106 independent transformants from 11.5 and 17-day-old
mouse embryo cDNA libraries, we isolated a total of 52 cDNA
clones encoding HMGA1 as well as previously known RB-
binding proteins including E2F1,(40) HDAC1, and HDAC2.(7–10)
The present screening did not isolate HMGA2. To confirm the
interaction between RB and HMGA1, we prepared His-tagged
human HMGA1, and subjected it to an in vitro pull-down
analysis (Fig. 1b). Briefly, GST and the GST-fused pocket
region of human RB (amino acids 301–928) were immobilized
on glutathione-agarose beads, and incubated with His-HMGA1
in a binding buffer containing 100 mM or 200 mM NaCl. GST-
fused RB bound to His-HMGA1, but not to methylated
DNA-binding protein MBD1 (TRD) fused to His-tag as control.
To check complex formation of RB and HMGA1 in vivo, we
carried out an immunoprecipitation analysis (Fig. 1c). Human
T98G cells were used in this study, and are known to express
wild-type RB and mutant-type p53, and to arrest in the G0
phase in an RB-mediated manner under serum starvation.(20,41)
Since currently available antibodies against HMGA1 were not
suitable for the immunoprecipitation experiments, FLAG-tagged
HMGA1 was transiently expressed in T98G cells and immuno-
precipitated by anti-RB and anti-FLAG antibodies. Western blot
analysis revealed that HMGA1 was present in the immuno-
precipitates with endogenous RB, but not in control immuno-
precipitates. RB was detected in the immunoprecipitates with
HMGA1. Two major forms for phosphorylated and hypophos-
phorylated RB were detected as upper and lower bands in input
lanes, respectively. Immunoprecipitation by anti-RB antibodies
efficiently concentrated both forms of RB, while hypophos-
phorylated RB (functionally active) predominantly bound to
HMGA1. Similar results were detected in MCF7 cells that also
expressed wild-type RB (data not shown). These results suggest
that HMGA1 interacts with RB in vivo.
HMGA1 overcomes the repressive effect of RB on E2F-mediated
transcription. RB inhibits E2F-activated transcription of cell
growth-regulated genes by recruiting co-repressor proteins.(7–10)
For example, as shown in Fig. 2(b), E2F binds its binding
motif in the promoter region of the cyclin E (CycE) gene,
and is targeted by the RB-HDAC complex for transcriptional
repression. Interestingly, biochemical and structural studies
revealed that HDAC1 binds to the pocket B of RB via the
‘L×C×E’ motif of this protein, while the opposite side of the
pockets A and B of RB are recognized by E2F.(42) Thus, RB-
binding proteins seem to interact with RB in different manners.
To test the effect of HMGA1 on RB function, we purified
endogenous RB-containing complexes in T98G cells, by
immunoprecipitation using anti-RB antibodies (Fig. 2a). The
precipitated complexes included E2F1, HDAC1, and RB, which
agreed with previous reports.(7–10) The RB-containing complexes
immobilized on glutathione-agarose beads were then used for a
competitive binding assay by adding recombinant His-HMGA1.
HDAC1 bound to RB was displaced by HMGA1 in a dose
(HMGA1)-dependent manner. In contrast, HMGA1 did not
affect the binding of E2F1 to RB. The results suggest that high
levels of HMGA1 disrupt the RB-HDAC1 repressive complex,
as was the case with HMGA2.(37) In addition, several chromatin-
associated factors including HDAC2, SUV39, BRM, and BRG,
have been reported to bind RB mechanistically alike HDAC1(42)
(See Discussion).
We tested whether HMGA1 influenced RB-mediated tran-
scriptional repression, using luciferase reporter experiments
(Fig. 2c). HA-tagged RB and FLAG-HMGA1 were expressed
in human Saos-2 cells that were null for RB genes. Western
blot analyzes revealed that HA-RB and FLAG-HMGA1 were
appropriately expressed in this assay (data not shown). The
Fig. 1.
group protein A1 (HMGA1). (a) Structures of RB and HMGA1. RB
contains a small pocket region containing A and B. It binds the
transcriptional factor E2F and histone deacetylase (HDAC). The pocket
region of mouse RB (amino acids 355–779) was used as bait in a yeast
two-hybrid screen, resulting in the identification of HMGA1 isoform
a (amino acids 1–107). HMGA1 (isoforms a and b) contains three
copies of the AT hook motif (AT). (b) Direct binding of HMGA1 to RB.
Recombinant GST and GST-fused human RB (amino acids 301–928) were
incubated with (His)6-fused HMGA1 or the carboxyl terminal region of
the methylated DNA binding protein MBD1, in a buffer containing
100 mM or 200 mM NaCl. Western blot analysis was carried out with
anti-His antibodies. The input shows 10% of each protein. (c) Complex
formation between RB and HMGA1 in vivo. FLAG-tagged HMGA1 was
transiently expressed in T98G cells that express wild-type RB, and
immunoprecipitated by anti-RB and anti-FLAG antibodies. Western
blot analysis of immunoprecipitates was carried out with anti-RB and
anti-FLAG antibodies. The input shows 10% of each lysate. The
phosphorylated and unphosphorylated/hypophosphorylated RB are
shown as pRB and RB, respectively.
Interaction of retinoblastoma protein (RB) with high mobility

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© 2007 Japanese Cancer Association
effects of the combined use of HA-RB and FLAG-HMGA1
were examined using a Photinus pyralis luciferase reporter
containing an E2F-binding motif of the human cyclin E gene
promoter (Fig. 2b). In the absence of HMGA1, RB decreased
cyclin E promoter activities by approximately five-fold (Fig. 2c,
left bars). HMGA1 alone tended to increase the luciferase activities
in a dose-dependent manner (Fig. 2c, white bars), suggesting a
direct effect of HMGA1 on promoter activities. RB-mediated
repression of cyclin E promoter activities was antagonized by
co-expression of HMGA1 (Fig. 2c, right bars).
To check the effect of HMGA1 on cell proliferation and endo-
genous E2F target genes, we knocked-down HMGA1 using syn-
thesized siRNAs. Western blot analysis confirmed that HMGA1
was depleted by the specific knockdown in T98G cells (Fig. 2d).
Using more than two independent assays, the depletion of HMGA1
significantly reduced the growth rate of the cells in comparison
with the control (Fig. 2e). To investigate the effect of HMGA1
knockdown on the E2F activities, we checked the expression of
E2F target genes (cyclin E1, cyclin A1, cyclin D1 and MAD2) using
a quantitative reverse transcription (RT)-PCR analysis (Fig. 2f).
By triplicate experiments, the mRNA levels in the control cells
were normalized to 1.0. The HMGA1 knockdown markedly
reduced the expression of these factors. These data suggest that
the overexpression of HMGA1 inhibits the repressive effect of
RB on E2F-mediated transcription, probably by disrupting the
RB-HDAC complexes, and that HMGA1 promotes cell growth
and induction of the E2F target genes.
Hypophosphorylation of RB and downregulation of HMGA1 in
cellular G0 phase. It is of great importance that RB represses
E2F-responsive genes to induce cell cycle arrest for tumor
suppression and cell differentiation.(3,4) The G0 phase is a
quiescent arrest state for mammalian cells where the cells are
not growing.(20,43) To investigate the cellular G0 entry and exit,
T98G cells were precultured in the presence of 10% FBS for
48 h, and then serum-depleted for 72 h. They were subsequently
cultured by adding serum (Fig. 3a). Cells mostly arrested at
12–24 h after serum starvation, and started to regrow 12–24 h
after serum addition. The quiescent state in the G0 phase is
well-characterized by low levels of general transcription
activities.(44) Therefore, we isolated total RNAs and genomic
DNAs at each cell cycle stage, and measured RNA/DNA ratios
(Fig. 3b). Twelve hours after serum-free culture, the RNA/DNA
ratio markedly decreased to less than two-fold (G0 entry), and
continued to decrease by 72 h (G0 phase). The addition of serum
in the culture medium at 72 h restored the RNA/DNA ratio at
96 h (G0 exit).
To examine the involvement of RB and HMGA1 in the
quiescent state, Western blot analysis was carried out using anti-
RB and anti-HMGA1 antibodies (Fig. 3c). There were both
phosphorylated and hypophosphorylated forms of RB under
growth-stimulated conditions (0 h). RB was dephosphorylated at
24–72 h after serum depletion, and then phosphorylated after
serum addition at 72 h. In addition, HMGA1 level remarkably
decreased under serum-free conditions. Thus, hypophosphorylated
RB coexisted with downregulated HMGA1 in the G0 phase of
serum-starved T98G cells. Immunostaining analysis using
anti-HMGA1 and antiglial fibrillary acidic protein (GFAP)
antibodies further emphasized the downregulation of HMGA1,
together with somewhat fibroblast-like morphologies of T98G cells,
72 h after serum depletion (Fig. 3d). These results suggest that
Fig. 2.
HMGA1 on E2F-RB-HDAC1 complexes. Endogenous RB-containing complexes in T98G cells were immunoprecipitated by anti-RB antibodies, and
immobilized on glutathione-agarose beads in a competitive binding assay using recombinant His-HMGA1. The asterisk shows the band of the
immunoglobulin. (b) Luciferase reporter assays. Hemagglutinin (HA)-tagged RB and FLAG-HMGA1 were expressed in human Saos-2 cells that do
not express RB genes. The effects of these combinations were examined using a Photinus pyralis luciferase reporter containing an E2F-binding
motif of the human cyclin E (CycE) gene promoter. (c) The effect of HMGA1 on RB-mediated repression of the cyclin E promoter. The luciferase
activities from insertless HA-mock were normalized to 1. The asterisk indicates statistical difference using a student t-test. (d) Specific siRNA-
mediated knockdown of HMGA1. β-tubulin is shown as a control. (e) The effect of HMGA1 knockdown on cell proliferation. The cell numbers were
determined on 0, 2, 4, and 6 days after the knockdown. Results were obtained from more than two independent experiments. Error bars
indicate standard deviation. (f) The effect of HMGA1 knockdown on the expression of E2F target genes. A quantitative reverse transcription-
polymerase chain reaction (RT-PCR) analysis showed that cyclin E1, cyclin A1, cyclin D1 and MAD2 were downregulated by depletion of HMGA1.
Using triplicate experiments, the mRNA levels in control cells are normalized to 1.
High mobility group protein A1 (HMGA1) inhibits retinoblastoma protein (RB)-mediated repression of E2F-target genes. (a) The effect of

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both hypophosphorylation of RB and downregulation of HMGA1
are characteristics of serum-starved G0 phase in T98G cells.
Persistent expression of HMGA1 abrogates serum-starved G0 arrest.
To check whether the downregulation of HMGA1 is required for
the quiescent state, we established green fluorescent protein
(GFP)-expressing T98G cells (T98G GFP), and GFP-fused
HMGA1-expressing T98G cells (T98G GFP-HMGA1) (Fig. 4a).
In addition to the immunostaining data, Western blot analysis
using anti-GFP antibodies showed that either GFP or GFP-HMGA1
was equally expressed in these cells (Fig. 4b). Using the
procedure indicated in Fig. 3(a), T98G GFP and T98G GFP-
HMGA1 cells were analyzed under serum starvation. Western
blot analysis was carried out to analyze the expressions of RB,
HMGA1, and GFP-HMGA1 in T98G GFP-HMGA1 cells (Fig. 4c).
GFP-HMGA1 was stably expressed using the viral cytomegalovirus
(CMV) promoter throughout the culture conditions. In contrast,
endogenous HMGA1 levels decreased under serum starvation,
as similarly shown in Fig. 3(c), suggesting that HMGA1 was
originally regulated by mitogenic stimulation. On the other
hand, dephosphorylation of RB slowed down in serum-starved
T98G GFP-HMGA1 cells, since phosphorylated RB still
remained 72 h after serum depletion. In addition, we confirmed
that HMGA1 was upregulated by epidermal growth factor or
17β-estradiol in MCF7 cells, and that it was downregulated by
retinoic acid-induced differentiation in mouse embryonic cells
(Fig. S1). These data suggest that HMGA1 expression highly
depends on proliferative and undifferentiated states. Furthermore,
as previously reported(45) MCF7 cells overexpressing GFP-HMGA1
formed colonies in soft agar (Fig. S2), suggesting that HMGA1
is potentially oncogenic.
We next examined the growth rates of T98G GFP-HMGA1
and T98G GFP cells in the presence and absence of serum
(Fig. 4d). Regardless of the expression of GFP-HMGA1, both
cells comparably proliferated under serum-containing conditions.
On the other hand, there were significant differences in growth
rate between these cells under serum-free conditions. T98G
GFP-HMGA1 cells continued to gradually grow even under
serum starvation, while T98G GFP cells as well as the original
T98G cells (data not shown) mostly arrested 24 h after incubation
in serum-free conditions. To examine whether T98G GFP-HMGA1
cells entered the G0 phase under serum depletion, we analyzed
the RNA/DNA ratio to assess general transcriptional activities
(Fig. 4e). In T98G GFP cells, the RNA/DNA ratio markedly
decreased 12–72 h after the start of serum starvation. In contrast,
the significant decrease in RNA/DNA ratio was lost in T98G
GFP-HMGA1 cells. These data suggest that the persistent
expression of HMGA1 abrogates RB-mediated G0 arrest.
Inhibition of RB-mediated G0 arrest by HMGA1 leads to chromosome
instability. To demonstrate whether persistently expressed
HMGA1 targets RB, we carried out an immunoprecipitation
analysis in T98G GFP-HMGA1 cells (Fig. 5a). Using T98G
GFP-HMGA1 cells, we examined the existence of GFP-
HMGA1 complexed with RB. As shown in Fig. 3(a), T98G
GFP-HMGA1 cells and T98G GFP cells were grown in serum-
containing medium, which was then replaced by serum-free
medium. Immunoprecipitation using anti-GFP and anti-RB
antibodies showed that RB was present in the precipitates with
GFP-HMGA1, and that GFP-HMGA1 coprecipitated with RB.
Hypophosphorylated RB, rather than its phosphorylated form,
tended to hbind GFP-HMGA1. The precipitates with species-
matched control IgG gave no band for GFP-HMGA1 or RB.
During our close observations of T98G-derived cells under
serum starvation, we unexpectedly found that various nuclear
structural abnormalities appeared in T98G GFP-HMGA1 cells,
but not in T98G or T98G GFP cells (Fig. 5b). As shown in
Fig. 4(a), GFP-HMGA1 constantly labeled the whole chromo-
somes in the cell cycle. Most of T98G GFP-HMGA1 cells had
a U-shape or had segmented nuclei 48 h after serum-free culture.
In addition, mitotic GFP-HMGA1 cells were detected with
condensed chromosomes (Fig. 5b, arrowheads). The nuclei of
the T98G GFP-HMGA1 cells further became multiple segmented,
and were enlarged with micronuclei at 72 and 96 h (Fig. 5c).
Fig. 3.
downregulation of high mobility group protein A1 (HMGA1) in serum-
starved G0 phase. (a) Culture of T98G cells. T98G cells were precultured
in the presence of 10% fetal bovine serum (FBS) for 48 h, serum-
depleted for 72 h, and subsequently grown by adding 10% serum.
White and black arrows show serum-free and serum addition,
respectively. The timings of G0 entry and exit are indicated. (b) Low
levels of general transcription activities in the quiescent G0 phase.
Total RNAs and genomic DNAs were isolated at each stage from the
serum-starved cells to measure RNA/DNA ratios. This ratio in growing
conditions (at 0 h) was normalized to 1. The asterisk indicates statistical
differences using a student t-test. (c) Expression statuses of RB and
HMGA1 in G0 arrest. Western blot analysis was carried out using anti-RB
and anti-HMGA1 antibodies. The phosphorylated and unphosphorylated/
hypophosphorylated RB are shown as pRB and RB, respectively. (d) Down-
regulation of HMGA1 in the G0 phase. Immunostaining analysis using
anti-HMGA1 and antiglial fibrillary acidic protein (GFAP) antibodies
was carried out in T98G cells.
Hypophosphorylated retinoblastoma protein (RB) and