Reduction of total E2F/DP activity induces senescence-like cell cycle arrest in cancer cells lacking functional pRB and p53

Abstract

E2F/DP complexes were originally identified as potent transcriptional activators required for cell proliferation. However, recent studies revised this notion by showing that inactivation of total E2F/DP activity by dominant-negative forms of E2F or DP does not prevent cellular proliferation, but rather abolishes tumor suppression pathways, such as cellular senescence. These observations suggest that blockage of total E2F/DP activity may increase the risk of cancer. Here, we provide evidence that depletion of DP by RNA interference, but not overexpression of dominant-negative form of E2F, efficiently reduces endogenous E2F/DP activity in human primary cells. Reduction of total E2F/DP activity results in a dramatic decrease in expression of many E2F target genes and causes a senescence-like cell cycle arrest. Importantly, similar results were observed in human cancer cells lacking functional p53 and pRB family proteins. These findings reveal that E2F/DP activity is indeed essential for cell proliferation and its reduction immediately provokes a senescence-like cell cycle arrest.

Full text

THE JOURNAL OF CELL BIOLOGY
JCB: REPORT
©
The Rockefeller University Press $8.00
The Journal of Cell Biology, Vol. 168, No. 4, February 14, 2005 553–560
http://www.jcb.org/cgi/doi/10.1083/jcb.200411093
JCB 553
Reduction of total E2F/DP activity induces
senescence-like cell cycle arrest in cancer cells
lacking functional pRB and p53
Kayoko Maehara,
1
Kimi Yamakoshi,
2
Naoko Ohtani,
2
Yoshiaki Kubo,
3
Akiko Takahashi,
2
Seiji Arase,
3
Nic Jones,
1
and Eiji Hara
1,2
1
Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, England, UK
2
Division of Protein Information, Institute for Genome Research, and
3
Department of Dermatology, School of Medicine, University of Tokushima, Tokushima 770-8503, Japan
2F/DP complexes were originally identified as po-
tent transcriptional activators required for cell prolif-
eration. However, recent studies revised this notion
by showing that inactivation of total E2F/DP activity by
dominant-negative forms of E2F or DP does not prevent
cellular proliferation, but rather abolishes tumor suppres-
sion pathways, such as cellular senescence. These obser-
vations suggest that blockage of total E2F/DP activity
may increase the risk of cancer. Here, we provide evi-
dence that depletion of DP by RNA interference, but not
E
overexpression of dominant-negative form of E2F, effi-
ciently reduces endogenous E2F/DP activity in human
primary cells. Reduction of total E2F/DP activity results in
a dramatic decrease in expression of many E2F target
genes and causes a senescence-like cell cycle arrest. Im-
portantly, similar results were observed in human cancer
cells lacking functional p53 and pRB family proteins.
These findings reveal that E2F/DP activity is indeed es-
sential for cell proliferation and its reduction immediately
provokes a senescence-like cell cycle arrest.
Introduction
As the major downstream mediator of the retinoblastoma protein
(pRB) tumor suppressor pathway, the E2F/DP transcription
factor complexes play a crucial role in cell cycle regulation
(Dyson, 1998). Recent studies suggest that E2F/DP complexes
can be broadly classified into two subgroups: a group of “acti-
vating” E2Fs (E2F1, E2F2, and E2F3) that are potent transcrip-
tional activators, and a second group of “repressive” E2Fs
(E2F4, E2F5, and E2F6) that appear to function primarily as
transcriptional repressors (Mann and Jones, 1996; Takahashi et
al., 2000; Trimarchi and Lees, 2002). This interpretation is
supported by chromatin immunoprecipitation (ChIP) analysis,
which reveals that activating E2Fs replace repressive E2Fs at
E2F-regulated promoters as cells progress from G0/G1 toward
S phase, a change that correlates with the induction of E2F-
dependent gene expression (Takahashi et al., 2000).
The combined ablation of E2F1, E2F2, and E2F3 genes
in primary mouse embryonic fibroblasts results in the reduction
of E2F target gene expression and block of cell proliferation
(Wu et al., 2001). However, inactivation of both E2F4 and
E2F5 does not inhibit cell proliferation, but instead renders
cells resistant to a p16
INK4a
-induced growth arrest (Gaubatz et
al., 2000; Ohtani et al., 2003). These results suggest that acti-
vating E2Fs and repressive E2Fs play opposing roles and that
the balance between activating E2Fs and repressive E2Fs is
likely to regulate cell cycle progression. Consistent with this
idea, inactivation of total E2F/DP activity by overexpression
of dominant-negative (dn-) forms of E2F or DP did not in-
hibit cellular proliferation, but rather abolished a variety of
growth arrest pathways, such as TGF-

–induced growth arrest,
p16
INK4a
-induced cell cycle arrest, contact inhibition, and cellular
senescence (Bargou et al., 1996; Zhang et al., 1999; Rowland
et al., 2002). These results suggest that activating E2Fs are only
required to counterbalance the effects of repressive E2Fs and
that total E2F/DP activity is not essential for cellular prolifera-
tion, but rather to promote tumor suppression mechanisms.
Thus, inactivation of total E2F/DP activity may actually increase
the risk of cancer. Because current therapeutic approaches that
target E2F/DP do not inactivate specific E2F family members,
but instead block all E2F/DP activity (Bandara et al., 1997), it
is vital to clarify the role of E2F/DP activity in normal and
cancerous human cells.
Correspondence to Eiji Hara: hara@genome.tokushima-u.ac.jp
Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; dn,
dominant negative; EMSA, electrophoretic mobility shift assay; HDF, human
diploid fibroblast; pRB, retinoblastoma protein; RNAi, RNA interference; SA-

-gal,
senescence-associated

-galactosidase; SAHF, senescence-associated hetero-
chromatic foci; shRNA, small hairpin RNA.
The online version of this article includes supplemental material.

JCB • VOLUME 168 • NUMBER 4 • 2005554
Figure 1. Effects of dn-E2F on cell growth. (A) The E2F1 and DP1 expression vectors (0.6 g) were cotransfected into 60-mm plates of SVts8 cells along
with a reporter plasmid and lacZ plasmid. Where indicated, cells were also cotransfected with an increasing amount of expression plasmid encoding
dn-E2F (lane 2: 0 g; lane 3: 0.1 g; lane 4: 0.3 g; lane 5; 0.6 g). Error bars indicate SD. (B) Early passage (45PDLs) TIG-3 cells infected with retro-
virus encoding dn-E2F or empty vector. Cells were analyzed for expression of E2F1 and dn-E2F protein by Western blot after selection with hygromycin.
The antibody against -actin was used as a loading control. (C) EMSAs were performed using a radiolabeled probe containing the E2F consensus sequence
of the adenovirus E2 gene promoter and extracts from cells infected with retrovirus encoding empty vector (lanes 1–5) or dn-E2F (lanes 6–10) in the
absence or presence of antibodies as indicated. The DNA-binding activity, which is not shifted by any available E2F antibodies, was marked by an asterisk.
(D and E) Cell extracts were prepared from cells at 60PDLs and subjected to RT-PCR (D) or Western blotting (E) with primers or antibodies shown at left.
E2F target genes reported previously were highlighted with an asterisk. CDK4 was used here as a loading control. (F) Early passage (45PDLs) TIG-3 cells
infected with retrovirus encoding dn-E2F or empty vector were selected for expression of the hygromycin selectable marker for 5 d and used in proliferation
curves performed in triplicate. Error bars indicate SD.

DEPLETION OF DP PROVOKES CELLULAR SENESCENCE • MAEHARA ET AL.
555
In this paper, we address the role of E2F/DP activity in
human primary and cancer cells by comparing two approaches
to inactivating total E2F/DP activity: the use of a dn mutant
and RNA interference (RNAi) (Brummelkamp et al., 2002).
Results and discussion
To delineate the role of E2F/DP on proliferation of human pri-
mary cells, we first tested the effect of overexpressing dn-E2F
(E2F-DB), which lacks both the transcriptional activation do-
main and the pRB-family protein binding domain (Zhang et al.,
1999). As reported previously (Zhang et al., 1999; Rowland et
al., 2002), increasing amounts of dn-E2F blocked the transacti-
vating activity of cotransfected E2Fs in human fibroblasts (Fig.
1 A). The dn-E2F was then retrovirally transduced into early
passage primary human diploid fibroblasts (HDFs), TIG-3
cells. Ectopic dn-E2F expression was more than 100 times
greater than that of the endogenous E2F1 (Fig. 1 B, lane 2). As
expected, the DNA-binding activity of endogenous E2F4 was
abolished and replaced by dn-E2F (Fig. 1 C, lanes 1 and 6). In
agreement with previous reports (Zhang et al., 1999; Rowland
et al., 2002), overexpression of dn-E2F did not reduce the ex-
pression of endogenous E2F target genes in TIG-3 cells (Fig. 1,
D and E). In addition, TIG-3 cells expressing dn-E2F grew
Figure 2. Depletion of DP1 causes reduction of E2F target gene expression in HDFs. (A) 20 g of total RNA prepared from TIG-3 (50PDLs), HeLa, U2OS,
HT29, and 293T cells transfected with or without DP2 expression plasmid were subjected to Northern blot analysis using the same activity of indicated
radiolabeled probes. (B and C) Early passage TIG-3 cells (45PDLs) were infected with retrovirus encoding DP1-shRNA (lane 2) or control-shRNA (lane 1).
Cell extracts were prepared from cells at 7 d after selection with puromycin, and were subjected to RT-PCR (B) or Western blotting (C) with primers or
antibodies shown at left. E2F target genes reported previously were highlighted with an asterisk. CDK4 was used here as a loading control. (D) EMSA
was performed using extracts from cells infected with retrovirus encoding control-shRNA (lanes 1 to 5) or DP1-shRNA (lanes 6 to 10) in the absence or
presence of antibodies shown at top. The DNA-binding activity, which is resistant to any available E2F antibodies, was marked by an asterisk.

JCB • VOLUME 168 • NUMBER 4 • 2005556
faster than control TIG-3 cells, especially at late passage (Fig.
1 F; unpublished data). These and earlier results (Rowland et
al., 2002) suggest that E2F/DP activity may not be essential for
cell proliferation in mammalian cells. However, these findings
could also be explained by an incomplete inhibition of endoge-
nous E2F/DP activity or by an unforeseen side effect of over-
expression of the dn-E2F. Therefore, we sought a different ap-
proach to inactivate total E2F/DP activity in HDFs.
Although the E2F family consists of six members, the DP
family contains only two (DP1 and DP2) (Trimarchi and Lees,
2002). Because the level of DP2 mRNA is very low in TIG-3
cells (Fig. 2 A) and heterodimerization with a DP protein is es-
sential for E2F activity, we generated a retrovirus vector en-
coding a small hairpin RNA (shRNA) directed against DP1 to
deplete E2F/DP complexes in primary human cells. Early pas-
sage TIG-3 cells were infected with retrovirus encoding either
a control sequence or shRNA specific for DP1. The levels of
DP1 mRNA and protein were significantly reduced within 7 d
of infection with the virus expressing the DP1-shRNA, but not
the control virus (Fig. 2, B and C). In addition, E2F-DNA–
binding activity was almost completely abolished in extracts of
TIG-3 cells expressing DP1-shRNA (Fig. 2 D, lanes 1 and 6).
In contrast to our results with dn-E2F, the expression level of
many E2F target genes required for S phase, such as cyclin A2,
thymidine kinase, and cdc6, was dramatically reduced in DP1
knock-down cells (Fig. 2, B and C). Such reduction has also
been reported in mouse embryonic fibroblasts lacking the acti-
vating E2F genes owing to genetic ablation (Wu et al., 2001).
The levels of cdc2 mRNA and protein, an essential component
of M phase progression, were also reduced in DP1 knock-down
cells. Similar results were also observed within 2 d of viral in-
fection, suggesting that the transcriptional changes directly re-
Figure 3. Depletion of DP1 causes severe growth arrest
in HDFs. (A and B) Early passage (45PDLs) TIG-3 cells
infected with retrovirus encoding DP1-shRNA or control-
shRNA were selected for expression of the puromycin-
selectable marker for 7d and used in a proliferation assay
performed in triplicate (A) or in cell cycle profile analysis
by FACS (B). Error bars indicate SD. (C and D) Early pas-
sage TIG-3 cells (45PDLs) were infected with retrovirus
encoding flag-tagged wild-type DP1 protein containing a
mutated shRNA cleavage site (lanes 3 and 4) or empty
vector (lanes 1 and 2). After selection with hygromycin,
cells were then super-infected with retrovirus encoding
DP1-shRNA (lanes 2 and 4) or control-shRNA (lanes 1
and 3). Expression of DP1, cyclin A, and CDC2 (C) and
BrdU incorporation (D) was examined 2 d after super-
infection. CDK4 was used here as a loading control. E2F
target genes reported previously were highlighted with
an asterisk. (E) TIG-3 cells infected with retrovirus encoding
DP1-shRNA or control-shRNA were examined for SA--gal
activity and SAHF.

DEPLETION OF DP PROVOKES CELLULAR SENESCENCE • MAEHARA ET AL.
557
sult from the decrease of E2F/DP activity (Fig. S1 A, available
at http://www.jcb.org/cgi/content/full/jcb.200411093/DC1). The
levels of PCNA and MCM3 were unchanged in DP1 knock-
down cells (Fig. 2, B and C). Thus, the expression of MCM3
and PCNA might be regulated by a balance between activating
E2Fs and repressive E2Fs, as seen in
Drosophila
cells (Frolov
et al., 2003). We failed to see any increase of interferon-induc-
ible genes (STAT1 and IFITM1) or p53 levels (Fig. 2, B and
C), arguing against the possibility that the DP1 shRNA induced
an interferon response or had nonspecific untargeted effects.
Next, we measured cell proliferation in DP1 knock-down
cells. To our surprise, DP1 depletion immediately caused cell
cycle arrest (Fig. 3 A) accompanied by a significant decrease in
the S phase population and an increase in the G1 phase and G2/
M phase populations (Fig. 3 B). To confirm that these effects
were actually due to the depletion of DP1 protein, a DP1 cDNA
containing a mutated shRNA cleavage site was retrovirally
transduced into TIG-3 cells. This mutated cDNA, which is re-
sistant to DP1-RNAi, maintained the level of flag-tagged wild-
type DP1 protein, despite expression of DP1-shRNA. Strik-
ingly, it also sustained expression of E2F target genes, such as
cyclin A2 and cdc2 (Fig. 3 C, lanes 2 and 4), and entry into S
phase (Fig. 3 D, lanes 2 and 4). These results confirm that the
reduction of E2F target gene expression and cell proliferation
by DP1-RNAi were specifically dependent on the depletion of
DP1 protein. Furthermore, several features of cellular senes-
cence, such as a substantial increase in senescence-associated

-galactosidase (SA-

-gal) activity (Dimri et al., 1995) and se-
nescence-associated heterochromatic foci (SAHF) (Narita et
al., 2003) were observed in DP1 knock-down cells (Fig. 3 E),
but not in cells expressing flag-tagged DP1 that is resistant to
RNAi (unpublished data). These results suggest that the main-
tenance E2F/DP activity is required for protecting cells from
onset of premature or stress-induced senescence.
DP1 knock-down and overexpression of dn-E2F were not
equivalent with respect to either E2F target gene expression or
cell proliferation. To understand the basis for the apparent dis-
crepancy, we performed ChIP assays using an antibody against
E2F3, the most abundant activating E2F in fibroblasts, and ex-
amined the precipitated DNA for the presence of cyclin A2
promoter sequences by PCR. Importantly, we found that a sig-
nificant amount of endogenous E2F3 remained bound to the
cyclin A2 promoter in TIG-3 cells expressing dn-E2F (Fig. 4
A, lanes 3 and 4). In contrast, E2F3 binding was significantly
reduced in DP1 knock-down TIG-3 cells (Fig. 4 B, lanes 3 and
4). Similar results were observed when we used an anti-E2F4
Figure 4. Incomplete blockage of E2F/DP activity in HDFs expressing dn-E2F. (A and B) ChIP assays were performed using TIG-3 cells expressing dn-E2F
(A), or DP1-shRNA (B) and the antibodies shown. The cyclin A2 promoter was recovered by PCR using primers flanking the E2F-binding sites in the human
cyclin A2 promoter. The human -actin promoter, which does not contain E2F-binding sites, was used as a negative control for PCR. (C) TIG-3 cells
(50PDLs) expressing dn-E2F (lanes 3 and 4) or empty vector (lanes 1 and 2) were super-infected with retrovirus encoding DP1-shRNA (lanes 2 and 4) or
control-shRNA (lanes 1 and 3). Total RNAs were prepared from cells at 7 d after selection with puromycin, and were subjected to RT-PCR with primers
shown on the left of the figure. E2F target genes reported previously were highlighted with an asterisk. (D) Cells described in C were used in a proliferation
assay performed in triplicate. Error bars indicate SD.

JCB • VOLUME 168 • NUMBER 4 • 2005558
antibody for the ChIP assay (Fig. 4, A and B; lanes 5 and 6).
These findings suggest that endogenous E2F/DP activity is ef-
fectively blocked by knockdown of DP1, but not by dn-E2F.
Indeed, depletion of DP1 from TIG-3 cells expressing dn-E2F
caused a significant reduction of E2F target gene expression
(Fig. 4 C, lanes 3 and 4) and consequent growth arrest (Fig. 4
D). These results confirm that a substantial level of endogenous
E2F/DP activity is indeed present in TIG-3 cells expressing
dn-E2F. Thus, the disparate results obtained by the two ap-
proaches can be explained by the insufficient blockage of en-
dogenous E2F/DP activity by dn-E2F in HDFs.
To verify our conclusions, we compared the effects of dn-
E2F with that of DP1-RNAi in human osteosarcoma U2OS
cells, which were used in the previous dn-E2F study (Zhang et
al., 1999). Consistent with our results in TIG-3 cells, DP1
knock-down caused a significant inhibition of cell growth (Fig.
5 A), whereas overexpression of dn-E2F had little effect (un-
published data). Additionally, the interpretation of results ob-
tained by dn-E2F has recently been questioned by a study
showing that dn-E2F (E2F-DB) contains a previously uncov-
ered pRB-binding domain that could potentially inactivate en-
dogenous pRB-family proteins (Dick and Dyson, 2003). In-
deed, we observed significant interaction between dn-E2F and
pRB in U2OS cells (Fig. S1 B). Moreover, although overex-
pression of dn-DP1 in human breast epithelial cells produced
effects similar to those produced by dn-E2F, namely an in-
creased incidence of tumors (Bargou et al., 1996), different re-
sults were observed depending on the particular dn-DP1 con-
struct that was used (Wu et al., 1996). Therefore, it is important
to stress that interpretation of experiments using dn-E2F or DP
requires careful evaluation.
A recently reported knock-out study of Kohn et al.
(2004) concludes that DP1 is dispensable for growth in vari-
ous mouse embryonic tissues. Because DP2 levels are very
low in human cells tested when compared with DP1 levels
(Fig. 2 A; Fig. S2 A, available at http://www.jcb.org/cgi/
Figure 5. Depletion of DP1 causes cellular
senescence in human cancer cell lines. (A–C)
U2OS (A), HT-29 (B), and HeLa cells (C) were
infected with retrovirus encoding DP1-shRNA
or control-shRNA and selected with puromycin
for 7 d. Cell numbers were then counted tak-
ing a time course in tetraplicate. The levels of
DP1 and CDK4 (loading control) were exam-
ined by Western blotting at d 3. (D) The HeLa
cells infected with retrovirus encoding either
control-shRNA or DP1-shRNA were used for
SA--gal assay 8 d after selection with puro-
mycin. The percentages of average SA--gal–
positive cells were indicated. (E) ChIP assays
were performed in HeLa cells with or without
DP1 knocking down as described in Fig. 4, A
and B. -Actin promoter, which does not con-
tain E2F-binding sites, was used as a negative
control for PCR. (F) Formation of subcutaneous
tumors in nude mice by HeLa cells expressing
shRNA against control sequence or DP1. For
each injection, 106 cells of the indicated pop-
ulations were injected subcutaneously in a
volume of 100 l. Mice were killed when the
tumors reached a diameter of 1 cm or after
10 wk of monitoring.

DEPLETION OF DP PROVOKES CELLULAR SENESCENCE • MAEHARA ET AL.
559
content/full/jcb.200411093/DC1), it is possible that the differ-
ent responses of mouse and human cells to DP1 loss may be
due to the DP2 status in the target cells. However, it is equally
possible that other explanations exist, such as cell type speci-
ficity, acute versus stable target loss, etc.
Next, we asked if DP1 knock-down inhibits the prolifera-
tion of other human cancer cell lines. DP1 knock-down signifi-
cantly inhibited cell proliferation in HT-29 cells, which lack
functional p53 (Fig. 5 B), and HeLa cells, in which pRB-family
proteins and p53 are inactivated by viral oncoproteins (Fig. 5 C).
Interestingly, reduction of DP1 protein levels induced several
features of cellular senescence in these cancer cells, including a
large, flat morphology and expression of SA-

-gal activity
(Fig. 5 D). HeLa cells expressing either DP1-shRNA or control
shRNA were injected subcutaneously into immunocompro-
mised mice. Although the control cells efficiently formed tu-
mors in 8 wk (19/20 cases), none of the DP1 knock-down cells
developed detectable tumors during the same time period (Fig.
5 F; Fig. S2 B). These results strongly suggest that E2F/DP
activity is required for tumor development.
Several lines of evidence suggest that pRB and p53 are
critical for induction of cellular senescence (Psyrri et al., 2004).
However, we have shown here that a senescence-like cell cycle
arrest can be induced by the reduction of total E2F/DP activity
without recovering the function of pRB and p53 in HeLa cells
(Fig. 5, C and D). Because the p16
INK4a
/RB tumor suppressor
pathway is frequently deregulated in a wide range of human
cancers, it is important to identify critical downstream targets
of this pathway for cancer therapy (Drayton and Peters, 2002;
Lowe and Sherr, 2003). Although additional studies are needed
to clarify the precise roles of E2F/DP complexes, our results
show that the proliferation of cancer cells could be controlled
through targeting the E2F/DP activity.
Materials and methods
Cell culture, transfection, and retrovirus production
TIG-3, SVts8, and HEK 293T cells were grown in DME supplemented with
10% FBS and penicillin/streptomycin. Retrovirus-shRNAs were generated
as described previously (Brummelkamp et al., 2002). To generate DP1-
resistant mutant against DP1 shRNA, three-point mutations, which do not
change encoding amino acids, were introduced into the shRNA cleavage
site of the DP1 cDNA. For growth rate analysis, cells were plated on the
gridded dish at concentration of 300 cells/1 cm
2
. Cell numbers were
counted in triplicate. BrdU incorporation was measured as described pre-
viously (Ohtani et al., 2003).
Antibodies and protein analyses
Immunoblotting was performed as described previously (Ohtani et al.,
2003) with primary antibodies against CDK4 (sc-601; Santa Cruz Bio-
technology, Inc.), cyclin A (sc-751; Santa Cruz Biotechnology, Inc.), E2F1
(sc-251; Santa Cruz Biotechnology, Inc.), MCM3 (sc-9849; Santa Cruz
Biotechnology, Inc.), PCNA (sc-56; Santa Cruz Biotechnology, Inc.), and
p107 (sc-318; Santa Cruz Biotechnology, Inc.), pRB (#554136; BD Bio-
sciences), FLAG (F3165; Sigma-Aldrich),

-actin (A5316; Sigma-Aldrich),
DP1 (#W32.3, Cancer Research UK; ab-11834, Abcam), CDC2 (#17;
Cancer Research UK), p16 (NA29; Oncogene Research Products), p53
(OP43; Calbiochem), p21 (sc-397; Santa Cruz Biotechnology, Inc.), and
p27 (sc-528; Santa Cruz Biotechnology, Inc.)
Electrophoretic mobility shift assays
Electrophoretic mobility shift assays (EMSAs) were performed as de-
scribed previously (Wu et al., 1995). The specificity of the protein–DNA
interactions was conformed by competition with wild-type oligonucleotides
or addition of the antibodies specific for E2F1 (sc-251), E2F2 (sc-633x),
E2F3 (sc-879x), E2F4 (sc-866), or GFP (sc-9996).
ChIP assay
ChIP assays were performed based on a modification of previously pub-
lished methods (Kanemaki et al., 2003; Ohtani et al., 2003). In brief, 5

10
6
cells were cross-linked by addition of formaldehyde to 1% final con-
centration, and then chromatin was sonicated and immunoprecipitation
was performed with Dynabeads protein A/G (Dynal), which were incu-
bated with antibody against E2F3 (sc-878x), E2F4 (sc-1082x), or Id3 (sc-
490) beforehand. Precipitates were washed and processed for DNA puri-
fication. DNA released from precipitated complexes was amplified using
sequence-specific primers by PCR (primer sequences: see online supple-
mental information).
Semiquantitative RT-PCR analyses
Total RNA was isolated using TRIzol (Invitrogen), and 5

g was reverse
transcribed with Super-scriptase (Invitrogen). Hot-start PCR was performed,
and the linear range of amplification was determined from PCRs run with
serially diluted cDNA. The results were verified by varying the number of
PCR cycles for each cDNA and set of primers (see online supplemental in-
formation). PCR products were separated on agarose gels and visualized
by ethidium bromide staining.
SA-

-gal and SAHF analysis
TIG-3 cells infected with retrovirus encoding DP1-shRNA or control-shRNA
were examined for SA-

-gal activity and SAHF as described previously
(Dimri et al., 1995; Narita et al., 2003). Slides for SA-

-gal were imaged
using a microscope (Axiovert 35M; Carl Zeiss MicroImaging, Inc.) with an
Achrostigmat objective (10

, 0.25 NA) and a digital camera (Axiocam
MR; Carl Zeiss MicroImaging, Inc.) and software (Zeiss Axiovision). Slides
for SAHF were imaged using a microscope (BX51; Olympus) with a Plan
Apochromat objective (60

, 1.4 NA) and a digital camera (Colorview
12; Soft-Imaging System) and a software (analysis; Soft-Imaging System).
Subsequent processing of TIFF files were undertaken in Adobe Photoshop
(version 7.0.1). Images were cropped and assembled into composites for
figures after minor adjustments for contrast and color balance were ap-
plied to all parts of each image.
Online supplemental material
Fig. S1 A shows the levels of E2F target gene expression after 2 d of DP1
knock-down. Fig. S1 B shows interaction between dn-E2F and pRB. Fig.
S2 A shows the levels of DP1 or DP2 mRNA expression in various human
cancer cells. Fig. S2 B shows an example of tumor formation assay. Sup-
plemental information shows primer sequences used for RNAi and PCR
analysis. Online supplemental material available at http://www.jcb.org/
cgi/content/full/jcb.200411093/DC1.
We thank Drs. R. Agami and R. Bernards (Netherlands Cancer Institute, Amster-
dam, Netherlands), S. Chellappan (University of South Florida, Tampa, FL), S.
Gaubatz (Philipps University, Marburg, Germany), D.M. Livingston Dana-Far-
ber Cancer Institute, Boston, MA), and E. Harlow (Harvard Medical School,
Boston, MA) for providing useful materials and K. Labib, J. Campisi, D. Mann,
G. Peters, and N. Dyson for valuable discussion. We are also grateful to Dr.
M. Kanemaki for help in ChIP assay and to members in the Paterson Institute for
Cancer Research for their various technical assists. We are indebted to Dr. K.
Helin and his colleagues for exchanging results before publication.
This work was supported by grants from Cancer Research UK, Associa-
tion for International Cancer Research, Yamanouchi Foundation for Research
on Metabolic Disorders, Takeda Science Foundation, and Ministry of Educa-
tion, Science, Sports, Culture and Technology of Japan to E. Hara.
Submitted: 16 November 2004
Accepted: 16 December 2004
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