From the Division of Molecular Oncology, Institute
for Genetic Medicine, Hokkaido University, Sapporo 060-0815, the
§ Division of Nephrology and Endocrinology, Department of
Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo
113-8655, the ¶ Department of Orthopedic Surgery, and the
Department of Surgical Oncology, Hokkaido University Graduate
School of Medicine, Sapporo 060-8638, Japan
Received for publication, October 23, 2002, and in revised form, January 27, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The retinoblastoma protein (pRB) and its
homologues, p107 and p130, prevent cell cycle progression from
G0/G1 to S phase by forming complexes
with E2F transcription factors. Upon phosphorylation by G1
cyclin-cyclin-dependent kinase (Cdk) complexes such as
cyclin D1-Cdk4/6 and cyclin E-Cdk2, they lose the ability to bind E2F, and cells are thereby allowed to progress into S phase. Functional loss
of one or more of the pRB family members, as a result of genetic
mutation or deregulated phosphorylation, is considered to be an
essential prerequisite for cellular transformation. In this study, we
found that pRB family proteins have the ability to stimulate
cyclin D1 transcription by activation of the NF- The retinoblastoma protein
(pRB)1 and its homologues,
p107 and p130, are important regulators of the mammalian cell cycle
(1-11). pRB (and probably p107 as well) plays an essential role in the growth decision-making at the late G1 restriction point,
whereas p130 is thought to be involved in
G0-to-G1 phase transition (10, 11). Results of
recent studies have further suggested that pRB and p107 play a
regulatory role in the S phase in response to DNA damage (12, 13). The
cell cycle-controlling activities of the pRB family proteins are
dependent on the functions of the shared structure termed the
"pocket" that is composed of the A-box, the spacer region, the
B-box, and the C-terminal region (10, 14-17). The pocket is capable of
binding a number of cellular proteins (18). Among those, E2F family
transcriptional factors are thought to be physiologically relevant
targets of pRB family proteins (10, 19-21). Upon complex formation,
pRB family proteins inhibit transcriptional activation of
E2F-dependent genes, whose products are essentially
required for cell cycle progression. A pRB family protein-E2F complex
also acts as a repressor against promoters containing E2F-binding
sites, thereby actively repressing transcription of E2F-responsive
genes in the G0/G1 phase (20-22). Stimulation of resting (or G0) cells with mitogenic signals gives rise
to the induction of G1 cyclins (cyclin D1, D2, D3, and E),
which in turn activate cyclin-dependent kinases (Cdk) such
as Cdk2, 4, and 6 (1, 2, 23). The resulting G1 cyclin-Cdk
complexes phosphorylate pRB family proteins and abolish their ability
to form physical complexes with E2F, thereby leading to cell cycle progression and subsequent cell division.
The function of pRB is lost through mutations of the RB gene
in retinoblastomas, small cell lung carcinomas, osteosarcomas, and
bladder carcinomas (1, 2, 24). Although mutations of the RB
gene occur in less than 20% of whole cancer cell types, loss of
p16INK4A, a specific inhibitor of cyclin D1-Cdk4/6, cyclin
D1 overexpression, or production of Cdk4 mutants that cannot bind
p16INK4A is observed in most, if not all, cancer cells
(25-30). All of these changes lead to inappropriate phosphorylation
and, hence, functional inactivation of pRB family proteins. Thus, the
frequent alterations in upstream regulators that constitute the
"p16INK4A-pRB family" pathway appear to be an essential
step in cancer development.
Cyclin D1 is inducibly expressed upon mitogenic stimulation and forms a
physical complex with Cdk4 or Cdk6 (2, 31, 32). Since the cyclin
D1-Cdk4/6 complex is a major kinase for pRB family proteins, cyclin D1
is recognized as an upstream regulator of pRB family proteins.
Furthermore, cyclin D1 binds pRB through the pocket domain (33, 34),
indicating that cyclin D1-Cdk4/6 may be a downstream target of pRB as
well. A complex interaction between cyclin D1 and pRB has also been
suggested by the finding that pRB negatively regulates
p16INK4A (35). These findings indicate the presence of a
regulatory network among pRB family proteins, p16INK4A and
cyclin D1. Such a regulatory loop may play a crucial role in the
physiological regulation of pRB family proteins.
In addition to the above described interaction between pRB and cyclin
D1, Müller et al. reported that pRB has the ability to
induce cyclin D1 (36). We have extended their work by demonstrating that all of the pRB family proteins are capable of inducing cyclin D1.
We also found that pRB family proteins transcriptionally
activate the cyclin D1 gene by stimulating the NF- Cells--
The human osteosarcoma line SAOS-2 was provided by
Dr. Phil Hinds (Harvard Medical School). The human cervical carcinoma
line C33A and the human osteosarcoma line U2-OS were obtained from the
American Type Culture Collection (Manassas, VA). The cells were
cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS).
cDNAs and Plasmids--
Expression vectors for the human
pRB, p107, and p130 were constructed as described previously (37). A
cDNA encoding the phosphorylation-resistant pRB mutant,
pRB Transfection and Immunoblotting--
Expression plasmids (18 µg) were transfected together with the 2 µg of the puromycin
resistance gene (pBabe-puro) into 1.5 × 106 SAOS-2 cells or 4 × 106 C33A cells in
a 100-mm plate by the calcium phosphate precipitation method as
described previously (43). In I Transfection of Small Interfering RNAs (siRNAs)--
Synthetic
siRNAs were purchased from Greiner Bio-One. The pRB-specific siRNA
molecules used in this study have the following sequences:
5'-cuguggggaaucuguaucu TT and 3' TT-gacaccccuuagacauaga. The
U2-OS cells were grown in DMEM supplemented with 10% FCS. Synthetic
siRNA transfection was carried out in a 60-mm dish (1.5 × 105 cells/4 ml of DMEM with 10% FCS) by using
Oligofectamine Reagent (Invitrogen) according to the manufacturer's
instructions. At 72 h after the transfection, cells were harvested
and cell lysates were prepared. Aliquots of the cell extracts
containing equal amounts of proteins were analyzed by immunoblotting.
Luciferase Assay--
SAOS-2 cells or C33A cells were
transiently co-transfected with a luciferase reporter plasmid and an
expression vector by the calcium phosphate precipitation method.
Transfected cells were cultured in DMEM plus 10% FCS for 36 h,
and luciferase activities were measured using the luciferase reporter
assay system (Promega) as described previously (44).
pRB, p107, and p130 Stimulate Expression of Cyclin D1--
Ectopic
expression of pRB in RB-deficient cells has been reported to
induce cyclin D1 (36). We carried out an experiment to determine
whether this cyclin D1-inducing activity is a biological property
shared by other pRB family members. An expression vector for pRB, p107,
or p130 was transiently transfected together with the puromycin
resistance gene (pBabe-puro) into the
RB-defective human osteosarcoma line SAOS-2. Transfected
cells were selected by puromycin, and the cell lysates were prepared
72 h after the drug selection. Anti-cyclin D1 immunoblotting of
the cell lysates exhibited that, in addition to pRB, ectopic expression
of p107 or p130 is capable of inducing cyclin D1 (Fig.
1A). To exclude the
possibility that the cyclin D1 induction is simply due to G1 cell cycle arrest provoked by pRB family proteins, we
ectopically expressed a p27Kip1 Cdk inhibitor, which exerts
a potent action to arrest the cell cycle at the G1 phase,
and confirmed that p27Kip1 did not induce cyclin D1 in
SAOS-2 cells (Fig. 1B). An increase in cyclin D1 protein
levels induced by pRB family proteins was also observed in the human
cervical carcinoma cell line C33A (Fig. 1A).
Effect of pRB-specific siRNA on Cyclin D1 Expression--
We next
used U2-OS human osteosarcoma cells in an experiment to determine
whether the level of cyclin D1 is reduced by a decrease in endogenous
pRB level. In U2-OS cells, virtually all of the pRB species are present
in the hyperphosphorylated form (Fig. 2A) due to loss of the
p16INK4A Cdk inhibitor as previously reported (37, 45).
Treatment of U2-OS cells with pRB-specific siRNA (pRB-siRNA) resulted
in specific inhibition of pRB expression (Fig. 2B).
Quantitation of the pRB bands using the Cdk2 bands as controls revealed
that pRB expression was reduced by 50% in cells treated with
pRB-siRNA. This pRB inhibition was concomitantly associated with
reduced levels of cyclin D1 (~50% reduction) (Fig. 2B).
The result indicates that endogenous pRB plays an important role in the
maintenance of cellular cyclin D1 levels. Since pRB-siRNA treatment did
not alter the phosphorylation status of pRB, the result also suggests that hyperphosphorylated pRB, which is incapable of binding to E2F and
several other pocket-binding proteins, retains the ability to induce
cyclin D1.
Activation of the Cyclin D1 Promoter by pRB, p107, and
p130--
pRB stimulates cyclin D1 through transcriptional activation
of the cyclin D1 gene (36). To determine whether the same is also true in the cases of p107 and p130, we examined their effects on
the human cyclin D1 promoter. Transient co-expression of a reporter
plasmid that has a luciferase gene fused to the human the cyclin D1
promoter together with each of pRB family expression vectors in C33A
cells revealed that, like pRB, both p107 and p130 are capable of
transactivating the cyclin D1 promoter (Fig.
3A). Since these
proteins are characterized by a shared structure termed the
"pocket" that is involved in their interaction with target proteins, the results indicate that pRB family proteins induce cyclin
D1 through a shared pocket function, most probably through pocket-protein interaction.
Involvement of NF-
The above observation suggested that the NF-
Since activity of NF- Structural Requirement for pRB in the Induction of Cyclin
D1--
To determine whether the cyclin D1-inducing activity of pRB is
due to the pocket domain that is shared among pRB family proteins, we
examined the effect of adenovirus E1A 12S product, which specifically binds to the pockets of all of the pRB family proteins (50-52). As
shown in Fig. 5A,
ectopic co-expression of E1A completely inhibited induction of cyclin
D1 by pRB. On the other hand, a mutant E1A (E1A 928) defective for pRB
binding (53) did not exhibit any effect on the cyclin D1-inducing
activity of pRB. The results indicate that the pocket structure is
required for the cyclin D1-inducing activity of pRB (and probably that
of p107 and p130 as well) and suggest the involvement of a cellular
protein(s) that physically interacts with the pocket for cyclin D1
induction. This notion was further supported by the observation that
NF-IL-6 (CCAAT/enhancer-binding protein-
To elucidate the structural basis of pRB involved in cyclin D1
induction, we generated two pRB mutants: pRBN392, which consists of
N-terminal amino acid residues 1-392 of pRB, and pRB Tumor-derived pRB Pocket Mutants Retain the Ability to Induce
Cyclin D1--
The pRB pocket mutants described above are incapable of
binding E2F and are "functionally inactive" in inhibiting cell
growth. It is therefore thought that the cyclin D1-inducing activity of pRB does not require structural integrity of the pocket domain that is
specifically involved in cell growth inhibition. This notion prompted
us to investigate the activity of tumor-derived pRB pocket mutants,
pRB In this work, we demonstrated that the retinoblastoma family of
pocket proteins, consisting of pRB, p107, and p130, are capable of
inducing cyclin D1. Our finding extends the results of previous work by
Müller et al. (36) showing that pRB induces cyclin D1
and reveals the existence of a "pRB-NF- Induction of cyclin D1 by pRB family proteins is due to transcriptional
activation of the cyclin D1 gene. Whereas the cyclin D1
promoter is regulated by multiple transcription factors, pRB family-dependent induction is specifically mediated by the
NF- The cell cycle effect of ectopic pRB family proteins depends on cell
context. In cells such as SAOS-2 and C33A that possess high levels of
p16INK4A due to a lack of endogenous pRB (35), ectopic
expression of pRB, p107, or p130 induces cyclin D1 but arrests cells in
G1 (37). This G1 block is mediated by the pRB
family proteins, which are kept in their hypophosphorylated forms as a
result of cyclin D1-Cdk4/6 inhibition by elevated p16INK4A.
In such cells, ectopically expressed pRB family proteins may be in
molar excess to E2F and therefore not only neutralize E2F but also
interact with other pocket-binding proteins, including those involved
in NF- The potential role of the hyperphosphorylated pRB in cyclin D1
induction is indicated by the observation that knockdown of pRB by RNA
interference in U2-OS cells, in which pRB is totally hyperphosphorylated, significantly reduced cyclin D1 levels.
Furthermore, pRB pocket mutants such as pRB Given that both the growth-suppressive activity and the cyclin
D1-inducing activity of pRB require the pocket domain, through which
pRB binds multiple cellular targets, the two seemingly counterintuitive pRB activities may be mutually exclusive. Furthermore, the cyclin D1-inducing activity of pRB family proteins may be insensitive to
phosphorylation. Accordingly, we suggest that pRB family proteins are
converted from inhibitors to stimulators of the cell cycle and
vice versa, depending on their phosphorylation status. In hypophosphorylated forms, they block cell cycle progression at G0/G1 by interacting with E2F as well as other
cell cycle-regulating proteins via the pocket domain. Once cells have
been committed to cell cycle progression, they undergo
hyperphosphorylation, release E2Fs, and then interact with a distinct
cellular protein(s) via the pocket domain. By doing so, pRB family
proteins may activate NF- Finally, our results suggest a heretofore unexplored function of pRB in
tumor development. Certain tumor-derived pRB pocket mutants, such as
pRB706CF and pRBB transcription
factor. The cyclin D1-inducing activity of pRB is abolished by
adenovirus E1A oncoprotein but not by the deletion of the A-box, the
B-box, or the C-terminal region of the pocket, indicating that multiple
pocket sequences are independently involved in cyclin D1 activation.
Intriguingly, tumor-derived pRB pocket mutants retain the cyclin
D1-inducing activity. Our results reveal a novel role of pRB family
proteins as potential activators of NF-
B and inducers of
G1 cyclin. Certain pRB pocket mutants may give rise to a
cellular situation in which deregulated E2F and cyclin D1 cooperatively
promote abnormal cell proliferation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
transcription factor. The cyclin D1-inducing activity is dependent on
the pocket structure but is independent of pocket function for
inhibition of cell growth. Intriguingly, tumor-derived pRB pocket
mutants, which cannot bind E2F and therefore cannot inhibit progression
of the cell cycle, are still capable of inducing cyclin D1. Stimulation
of cyclin D1 by pRB family proteins may play an important role in the
physiological regulation of cell cycle progression as well as in the
development of cancer.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S/T-P, was described previously (38). cDNAs encoding
tumor-derived pocket mutants, pRB
22 and pRB706CF, were described
previously (39). The pRB
N-HA and pRBN392 mutants were generated by
subcloning pRB fragments generated by PCR using human RB
cDNA as a substrate. The PCR primers employed were as follows: for
pRB
N-HA, sense primer 5'-GGGCGGCCGCGCCGCCATGGCAAGTGAT-3' and
antisense primer 5'-CTGGGTCTGGAAGGCTGAGGTTGC-3'; for pRBN392, sense primer 5'-GGTTCACCTCGAACACCCAGGCGAGG-3' and antisense primer 5'-GGGGTACCTCATGCAGAATTTAAAATCATCATTAATTGTTGG-3'. A series of deletion mutants for the pRB pocket were generated by
oligonucleotide-mediated mutagenesis with the use of the Chameleon
site-directed mutagenesis system (Stratagene). The RB mutant
cDNAs were subcloned into pSP65-SR
vector (40). The human cyclin
D1 promoter-luciferase construct, pGL2-944 cycD1-luc, was obtained
from Rolf Müller (Philipps-Universitat Marburg, Germany).
Construction of the cyclin D1 promoter mutants (pGL2-707
NF-
B2, -229
STAT2, -95
SP1, -23
NF-
B3,
-229
STAT2/CREB mut, and -229
STAT2/NF-
B3 mut) has been
described previously (41). p65 NF-
B cDNA and p55-Ig
-luc
reporter plasmid were gifts from Dr. Takashi Fujita (Tokyo Metropolitan
Institute for Medical Science, Tokyo, Japan) (42). pME-I
B
and
pME-I
B
S32AS36A were gifts from Dr. Jun-Ichiro Inoue (University
of Tokyo, Tokyo, Japan). pSV-E1A-12S and pSV-E1A-928 were described
previously (37).
B experiment, 5 µg of
pSP65-SR
-pRB and 15 µg of pME-I
B
or pME-I
B
S32AS36A
were transfected together with the 2 µg of pBabe-puro into
1.5 × 106 SAOS-2 cells. In the E1A experiment, 10 µg of pSP65-SR
-pRB and 10 µg of pSV-E1A-12S or pSV-E1A-928
mutant were transfected together with the 2 µg of
pBabe-puro into 1.5 × 106 SAOS-2 cells.
After 72 h of culture in DMEM with 10% FCS in the presence of 0.5 and 2 µg/ml puromycin for SAOS-2 cells and C33A cells, respectively,
the transfected cells were harvested and lysed in E1A cell lysis buffer
as described (43). Cell lysates were subjected to SDS-PAGE, transferred
to polyvinylidene difluoride filters (Millipore Corp.), and
immunoblotted with appropriate antibodies. Proteins were visualized
using the enhanced chemiluminescence detection system (ECL; PerkinElmer
Life Sciences). The antibodies used were anti-cyclin D1 (H-295, sc-753;
Santa Cruz Biotechnology), anti-pRB (G3-245; Pharmingen), anti-HA
monoclonal antibody (12CA5), and anti-Cdk2 (M2, sc-163; Santa Cruz
Biotechnology). Intensities of chemiluminescence on the immunoblotted
filters were quantitated by using a luminescent image analyzer
(LAS-1000; Fuji).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
Induction of cyclin D1 by ectopic expression
of pRB, p107, and p130. A, whole cell extracts were
prepared from SAOS-2 cells or C33A cells transfected with each of the
pRB family expression vector and were immunoblotted with the described
antibodies. The plasmid-directed pRB family proteins were HA
epitope-tagged at the C terminus and thus were detectable by anti-HA.
B, whole cell extracts were prepared from SAOS-2 cells
transfected with HA-tagged p27Kip1 (p27-HA) or a control empty
vector. Cell lysates prepared were immunoblotted with the described
antibodies.
View larger version (24K):
[in a new window]
Fig. 2.
Effect of pRB knockdown by siRNA on the
levels of cyclin D1. A, phosphorylation status of pRB
in U2-OS cells. Whole cell extracts were prepared from U2-OS cells
transfected with control vector (control) or expression vector for the
phosphorylation-resistant pRB, pRB S/T-P, and were immunoblotted with
anti-pRB antibody. The positions of the hypophosphorylated pRB
(pRB) and the hyperphosphorylated pRB (ppRB) as
determined by the band corresponding to pRB
S/T-P are indicated.
B, whole cell extracts were prepared from U2-OS cells
treated with (+) or without (
) pRB-specific siRNA for 72 h and
were immunoblotted with the indicated antibodies.
View larger version (29K):
[in a new window]
Fig. 3.
pRB family proteins transcriptionally
activate cyclin D1 through a proximal NF- B
site. A, C33A cells were transiently cotransfected with
a reporter plasmid containing the cyclin D1 promoter-luciferase gene
(
944 cycD1-luc) and pRB-HA, p107-HA, p130-HA, or a control empty
vector. The promoter activation was shown as a ratio of the luciferase
activities in the presence and absence of individual pRB family
protein. The graphs represent the means and 2× S.D. values
from three individual experiments. B, schematic
representations of cis-elements in the human cyclin D1
promoter as well as the mutant cyclin D1 promoter-luciferase constructs
used in the experiment (upper panel). SAOS-2 or C33A cells
were transiently cotransfected with a reporter plasmid containing the
mutant cyclin D1 promoter-luciferase gene and pRB-HA or a
control empty vector. The promoter activation was shown as a ratio of
the luciferase activities between the presence and absence of pRB-HA.
The graphs represent the means and 2× S.D. values from
three individual experiments (lower panel). C,
C33A cells were transiently cotransfected with a reporter construct
with three tandemly repeated
B motifs upstream of a minimal
interferon-
promoter (p55-Ig
-luc) and pRB-HA, p107-HA, p130-HA,
or a control empty vector. The promoter activation was shown as a ratio
of the luciferase activities in the presence and absence of individual
pRB family members. The graphs represent the means and 2×
S.D. values from three individual experiments.
B in Cyclin D1 Induction by pRB--
The
cyclin D1 promoter is known to be regulated by multiple
cis-acting elements, each of which plays a distinct role in
promoter activation. They include SP1 sites, a cAMP-responsive element (CRE), and distal and proximal NF-
B sites. To delineate
cis elements that are required for transcriptional
activation of the cyclin D1 promoter by pRB, we transfected a series of
luciferase reporter constructs (41) that contained various lengths of
the cyclin D1 promoter together with the RB expression
vector in SAOS-2 cells. As shown in Fig. 3B, the 5'-boundary
of the functional sequence required for cyclin D1 induction was located
between
95 and
23 from the transcription initiation site of the
cyclin D1 gene. The identified sequence contained CRE and
the proximal NF-
B site, both of which are reported to be important
for cyclin D1 induction in other cell types. Accordingly, we next
examined a pGL2/
229 derivative in which either the CRE or NF-
B
site was specifically destroyed by introducing a point mutation. The
promoter activity was not affected when CRE was mutated but was totally
abolished by mutation of the proximal NF-
B site (Fig. 3B,
lower panel, left). Our results thus indicate
that the proximal NF-
B binding site on the cyclin D1 promoter is
required for the induction of cyclin D1 by pRB. The same conclusion was
also obtained with the use of C33A cells (Fig. 3B,
lower panel, right).
B transcription factor
is involved in the induction of cyclin D1 by pRB family proteins. To
investigate this, we co-transfected p55-Ig
-luc, which has a
luciferase reporter gene fused to three tandem repeats of the NF-
B
binding sites from the immunoglobulin
light chain enhancer, with
the expression vector for pRB, p107, or p130 into C33A cells. Upon
ectopic expression, all of the pRB family proteins were capable of
stimulating the NF-
B-dependent promoter, indicating that
the pocket proteins indeed stimulated transcriptional activity of
NF-
B (Fig. 3C). As reported (46-48), ectopic expression
of the p65 (RelA) subunit of NF-
B transactivated the cyclin D1
promoter and increased cyclin D1 protein levels in C33A
cells.2
B is inhibited by interaction with I
B, we
next addressed the effect of I
B
on the induction of cyclin D1 by
pRB. Co-expression of the NF-
B inhibitor, I
B
, together with
pRB inhibited pRB-dependent cyclin D1 induction (Fig.
4). An I
B up-mutant, I
B
S32AS36A, which has serine-to-alanine substitutions at the amino acid
residues 32 and 36 and thus is resistant to phosphorylation-dependent degradation (49), exhibited
greater activity to inhibit cyclin D1 induction by pRB than wild-type I
B. Based on these observations, we concluded that pRB, and probably the pRB-related p107 and p130 as well, stimulated NF-
B and that the
stimulated NF-
B in turn induced transcriptional activation of the
cyclin D1 gene.
View larger version (42K):
[in a new window]
Fig. 4.
Inhibition of pRB-dependent
cyclin D1 induction by I B
. SAOS-2 cells were transfected
with expression plasmids encoding pRB and I
B
or its up-mutant,
I
B
S32AS36A, in which serine 32 and serine 36 were replaced by
alanine residues, together with the puromycin resistance gene. After
selection with puromycin for 72 h, cells were harvested, and the
cell lysates prepared were immunoblotted with anti-cyclin D1 and
anti-Cdk2, respectively.
), which also binds to the
pRB pocket (54-56), again competitively inhibited the induction of cyclin D1 by pRB.3
View larger version (17K):
[in a new window]
Fig. 5.
Involvement of the pRB pocket domain for
cyclin D1 induction. A, SAOS-2 cells were cotransfected
with pRB and adenovirus E1A (either 12 S product or 928 mutant)
expression plasmids. Cell lysates were immunoblotted with anti-cyclin
D1 and anti-Cdk2, respectively. B, schematic representations of pRB N-HA
and pRBN392 (upper panel). Black and
gray rectangles represent the A-box and the B-box
of pRB, respectively. HA indicates the HA-epitope tag. Whole cell
lysates were prepared from SAOS-2 cells transfected with pRB-HA,
pRB
N-HA, pRBN392, or a control empty vector and were subjected to
immunoblottings with the described antibodies (middle and
lower panels). Note that pRBN392 does not have the HA
epitope and therefore is detected by anti-pRB but not by anti-HA.
C, C33A cells were transiently cotransfected with
944
cycD1-luc and pRB-HA, pRB
N-HA, pRBN392, or a control empty vector.
The cyclin D1 promoter activation was shown as a ratio of the
luciferase activities in the presence and absence of wild-type or each
pRB mutant. The graphs represent the means and 2× S.D.
values from three individual experiments.
N-HA, which is
composed of the pocket spanning amino acid residues 392-928 of pRB
(Fig. 5B, upper panel). The pRB
N-HA mutant
retained the ability to induce the formation of flat cells (37, 57), a well known pocket activity, when expressed in SAOS-2 cells, and, as
expected from the results of the E1A experiment, it was also capable of
stimulating cyclin D1. In contrast, pRBN392 did not have the cyclin
D1-inducing activity (Fig. 5, B (middle,
lower panel) and C). To further clarify the pRB
regions that are responsible for cyclin D1 induction, we generated a
series of pocket deletion mutants (Fig.
6A). These pRB mutants were
expressed in SAOS-2 cells, and their activity to induce cyclin D1 was
examined by the use of anti-cyclin D1 immunoblotting. Surprisingly,
each of the pRB pocket mutants, which independently lacks the A-box,
the spacer region, the B-box, or the C-terminal region, was still capable of inducing cyclin D1 (Fig. 6B). These pRB mutants
exhibited comparable levels of cyclin D1 induction, although the
expression levels of the
C mutant were significantly higher than
those of other mutants. Hence,
C may be less effective in inducing
cyclin D1 than other pocket mutants. To determine the role of the
C-terminal region of pRB in cyclin D1 induction, we also generated
pRB
AB (Fig. 7A), in which
the A-box and the B-box were simultaneously deleted. As shown in Fig.
7, B and C, the mutant retained the ability to
induce cyclin D1. These results indicate that the "A/B-boxes" and
the "C-terminal region" are independently capable of inducing cyclin D1.
View larger version (23K):
[in a new window]
Fig. 6.
Determination of the pRB pocket subregions
required for cyclin D1 induction. A, schematic
representations of pRB and its pocket mutants. Black and
gray rectangles represent the A-box and the B-box
of pRB, respectively. B, total cell lysates were prepared
from SAOS-2 cells transfected with each of the pRB deletion
mutants or a control empty vector and were immunoblotted with the
described antibodies.
View larger version (16K):
[in a new window]
Fig. 7.
The role of the pRB C-terminal
region in cyclin D1 induction. A, schematic
representations of pRB and pRB AB. Black and
gray rectangles represent the A-box and the B-box
of pRB, respectively. B, whole cell extracts were prepared
from SAOS-2 cells transfected with pRB, pRB
AB, or a control empty
vector and were immunoblotted with the described antibodies.
C, C33A cells were transiently co-transfected with
944
cycD1-luc and pRB, pRB
AB, or a control empty vector. The promoter
activation was shown as a ratio of the luciferase activities in the
presence and absence of wild-type pRB or pRB
AB. The
graphs represent the means and 2× S.D. values from three
individual experiments.
22 and pRB706CF, in inducing cyclin D1. pRB
22 is an exon
22-skipping pRB mutant, and pRB706CF has a cysteine-to-phenylalanine point mutation at residue 706. Upon expression in SAOS-2, both the
pRB
22 and pRB706CF mutants were capable of inducing cyclin D1 (Fig.
8A). Consistent with this
observation, both of the mutants activated NF-
B and stimulated the
cyclin D1 promoter (Fig. 8, B and C).
Accordingly, we concluded that the tumor-derived pRB mutants, while
incapable of inhibiting cell proliferation, still possess the ability
to activate cyclin D1 transcription.
View larger version (24K):
[in a new window]
Fig. 8.
Induction of cyclin D1 by tumor-derived pRB
pocket mutants. A, whole cell extracts were prepared
from SAOS-2 cells transfected with pRB, pRB 22, pRB706CF, or a
control empty vector and were immunoblotted with the described
antibodies. B, C33A cells were transiently co-transfected
with
944 cycD1-luc and pRB, pRB
22, pRB706CF, or a control empty
vector. The promoter activation was shown as a ratio of the luciferase
activities between the presence and absence of wild-type or the
tumor-derived pRB pocket mutant. The graphs represent the
means and 2× S.D. value from three individual experiments.
C, C33A cells were transiently co-transfected with
p55-Ig
-luc and pRB, pRB
22, pRB706CF, or a control empty vector.
The promoter activation was shown as a ratio of the luciferase
activities in the presence and absence of wild type or the
tumor-derived pRB pocket mutant. The graphs represent the
means and 2× S.D. values from three individual experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-cyclin D1" pathway. Since cyclin D1 forms complexes with Cdk4 or Cdk6 and inactivates the
growth-suppressive activity of pRB family proteins by phosphorylation, our results indicate the existence of a complicated functional interplay between pRB family proteins and cyclin D1-Cdk4/6. From the
results of overexpression and knockdown experiments, it appears that
pRB plays the greatest role in regulation of cyclin D1 among the pRB
family proteins. Our present work therefore provides a molecular basis
for the reduction in the level of cyclin D1 expression in
RB-deficient cells (35, 58-62) as well as in cells
expressing simian virus 40 large T antigen, adenovirus E1A, or human
papilloma virus E7 oncoprotein (36).
B transcription factor through the proximal NF-
B binding site
on the cyclin D1 promoter. Consistent with our observation, recent studies have shown the importance of NF-
B in the activation of cyclin D1 (46-48, 63-65). Proteins of the pRB family can
cooperate with transcription factors, such as SP-1 (66-68),
CCAAT/enhancer-binding protein family members (54-56), and MyoD (69),
to transcriptionally activate genes. Although NF-
B has also been
reported to interact with pRB (70), we were not able to detect complex
formation between the p65 subunit of NF-
B and
pRB4 (data not shown).
Accordingly, the molecular mechanisms through which pRB family members
stimulate NF-
B are still not known. However, the finding that the
adenovirus E1A 12 S RNA product, which specifically interacts with the
pocket domains of pRB family proteins (50-52), inhibited cyclin D1
induction by pRB indicates that the activity requires a cellular
molecule(s) that physically interacts with the pocket domain.
B activation. As a result, cells arrest in G1 but
induce cyclin D1. On the other hand, in cells such as U2-OS that do not
express p16INK4A (45), ectopically expressed pRB family
proteins are instantly hyperphosphorylated and inactivated by
deregulated cyclin D1-Cdk4/6 and thus cannot halt the cell cycle
(71).
22 and pRB706CF, both of
which are derived from cancer patients, retain the ability to induce cyclin D1. Like hyperphosphorylated pRB, these tumor-derived mutants are unable to bind to E2F and thus are unable to inhibit cell growth.
Hence, the cyclin D1-inducing activity of a pRB family member is
separable from the pocket function that has been well characterized as
growth-suppressive activity. Accordingly, hyperphosphorylated pRB
and certain pRB pocket mutants may share a
"phosphorylation-insensitive" pocket structure that is involved in
cyclin D1 induction. The results of a series of pRB pocket mutant
analyses suggested that the presence of either A/B-boxes or the
C-terminal region is sufficient for the induction of cyclin D1.
Müller et al. (36) reported that a pRB mutant
that lacks the B-box and the C-terminal region failed to induce cyclin
D1. Hence, the presence of the A-box alone may not be sufficient for
cyclin D1 stimulation. These observations collectively suggest that a
pocket-binding protein(s) involved in cyclin D1 induction, if it
exists, has at least two independent pocket-binding sites, one for the
A/B-boxes and the other for the C-terminal region. Binding to one of
these pRB sites may be sufficient for significant activation of
NF-
B.
B and thereby induce cyclin D1, enforcing
irreversible cell cycle progression to the S phase in cells that have
passed the G1 restriction point. In this regard, recent
studies have indicated that non-pRB substrates of cyclin D1-Cdk4/6 play
crucial roles in G1/S transition (72, 73). Elevated cyclin
D1-Cdk4/6 may also promote cell cycle progression by squelching Cdk
inhibitors such as p27Kip1 (74, 75).
22 mutants, are unable to bind to E2F and are thus
unable to induce cell growth inhibition, yet they are biologically
active in inducing cyclin D1. Accordingly, in cells harboring such a
pRB mutant, cyclin D1 levels may be higher than those in
RB-null cells. Whereas inactivation of pRB as a cell growth
inhibitor may be sufficient to provoke cellular transformation, certain
levels of cyclin D1 induced by pRB pocket mutants should accelerate
abnormal cell proliferation by phosphorylation of p107, p130, and other
non-pRB family substrates. Hence, such RB pocket mutants may
be more potent than RB-null mutants in promoting transformation. Furthermore, increases in the levels of pRB have been
reported in colorectal carcinomas (76). In such cells, constitutive
hyperphosphorylation of elevated pRB, occasionally due to loss of
p16INK4A, may also play a role in cellular transformation
by aberrantly increasing cyclin D1 levels.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Takashi Fujita for p65 NF-B
cDNA and Ig
-reporter plasmids, Drs. Rolf Müller and Brian
Elenbaas for cyclin D1 promoter, Dr. Jun-Ichiro Inoue for I
B
cDNAs, and Yuhki Ishikawa for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by grants-in-aid for science research from the Ministry of Education, Science, Sports, and Culture of Japan, by a research grant from the Human Frontier Science Program Organization, and by a grant of the Virtual Research Institute of Aging of Nippon Boehringer Ingelheim.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. Tel./Fax: 81-11-706-7544; E-mail: mhata@imm.hokudai.ac.jp.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M210849200
2 T. Takebayashi, H. Sudo, and M. Hatakeyama, unpublished observation.
3 Y. Ishikawa, H. Sudo, T. Takebayashi, and M. Hatakeyama, unpublished observation.
4 Y. Ishikawa, H. Sudo, T. Takebayashi, and M. Hatakeyama, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: pRB, retinoblastoma protein; Cdk, cyclin-dependent kinase; HA, hemagglutinin; siRNA, small interfering RNA; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; CRE, cAMP-response element.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Sherr, C. J.
(1996)
Science
274,
1672-1677 |
2. | Weinberg, R. A. (1995) Cell 81, 323-330[Medline] [Order article via Infotrieve] |
3. | Buchkovich, K., Duffy, L. A., and Harlow, E. (1989) Cell 58, 1097-1105[Medline] [Order article via Infotrieve] |
4. | Chen, P. L., Scully, P., Shew, J. Y., Wang, J. Y., and Lee, W. H. (1989) Cell 58, 1193-1198[Medline] [Order article via Infotrieve] |
5. | DeCaprio, J. A., Ludlow, J. W., Lynch, D., Furukawa, Y., Griffin, J., Piwnica- Worms, H., Huang, C. M., and Livingston, D. M. (1989) Cell 58, 1085-1095[Medline] [Order article via Infotrieve] |
6. | Mihara, K., Cao, X. R., Yen, A., Chandler, S., Driscoll, B., Murphree, A. L., T'Ang, A., and Fung, Y. K. (1989) Science 246, 1300-1303[Medline] [Order article via Infotrieve] |
7. | Beijersbergen, R. L., Carlee, L., Kerkhoven, R. M., and Bernards, R. (1995) Genes Dev. 9, 1340-1353[Abstract] |
8. | Mayol, X., Garriga, J., and Grana, X. (1995) Oncogene 11, 801-808[Medline] [Order article via Infotrieve] |
9. |
Xiao, Z. X.,
Ginsberg, D.,
Ewen, M.,
and Livingston, D. M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4633-4637 |
10. | Grana, X., Garriga, J., and Mayol, X. (1998) Oncogene 17, 3365-3383[CrossRef][Medline] [Order article via Infotrieve] |
11. | Mayol, X., Garriga, J., and Grana, X. (1996) Oncogene 13, 237-246[Medline] [Order article via Infotrieve] |
12. |
Kondo, T.,
Higashi, H.,
Nishizawa, H.,
Ishikawa, S.,
Ashizawa, S.,
Yamada, M.,
Makita, Z.,
Koike, T.,
and Hatakeyama, M.
(2001)
J. Biol. Chem.
276,
17559-17567 |
13. | Chew, Y. P., Ellis, M., Wilkie, S., and Mittnacht, S. (1998) Oncogene 17, 2177-2186[CrossRef][Medline] [Order article via Infotrieve] |
14. | Ewen, M. E., Xing, Y. G., Lawrence, J. B., and Livingston, D. M. (1991) Cell 66, 1155-1164[Medline] [Order article via Infotrieve] |
15. | Hannon, G. J., Demetrick, D., and Beach, D. (1993) Genes Dev. 7, 2378-2391[Abstract] |
16. | Li, Y., Graham, C., Lacy, S., Duncan, A. M., and Whyte, P. (1993) Genes Dev. 7, 2366-2377[Abstract] |
17. | Mayol, X., Grana, X., Baldi, A., Sang, N., Hu, Q., and Giordano, A. (1993) Oncogene 8, 2561-2566[Medline] [Order article via Infotrieve] |
18. | Morris, E. J., and Dyson, N. J. (2001) Adv. Cancer Res. 82, 1-54[Medline] [Order article via Infotrieve] |
19. | Chellappan, S. P., Hiebert, S., Mudryj, M., Horowitz, J. M., and Nevins, J. R. (1991) Cell 65, 1053-1061[Medline] [Order article via Infotrieve] |
20. |
Dyson, N.
(1998)
Genes Dev.
12,
2245-2262 |
21. | Nevins, J. R. (1992) Science 258, 424-429[Medline] [Order article via Infotrieve] |
22. | Weintraub, S. J., Chow, K. N., Luo, R. X., Zhang, S. H., He, S., and Dean, D. C. (1995) Nature 375, 812-815[CrossRef][Medline] [Order article via Infotrieve] |
23. | Sherr, C. J. (1995) Trends. Biol. Sci. 20, 187-190[CrossRef] |
24. | Horowitz, J. M., Park, S. H., Bogenmann, E., Cheng, J. C., Yandell, D. W., Kaye, F. J., Minna, J. D., Dryja, T. P., and Weinberg, R. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2775-2779[Abstract] |
25. | Serrano, M., Hannon, G. J., and Beach, D. (1993) Nature 366, 704-707[CrossRef][Medline] [Order article via Infotrieve] |
26. | Delmer, A., Tang, R., Senamaud-Beaufort, C., Paterlini, P., Brechot, C., and Zittoun, R. (1995) Leukemia 9, 1240-1245[Medline] [Order article via Infotrieve] |
27. | Ruas, M., Brookes, S., McDonald, N. Q., and Peters, G. (1999) Oncogene 18, 5423-5434[CrossRef][Medline] [Order article via Infotrieve] |
28. | Ruas, M., and Peters, G. (1998) Biochim. Biophys. Acta. 1378, F115-F177[Medline] [Order article via Infotrieve] |
29. | Russo, A. A., Tong, L., Lee, J. O., Jeffrey, P. D., and Pavletich, N. P. (1998) Nature 395, 237-243[CrossRef][Medline] [Order article via Infotrieve] |
30. | Serrano, M. (1997) Exp. Cell Res. 237, 7-13[CrossRef][Medline] [Order article via Infotrieve] |
31. | Matsushime, H., Ewen, M. E., Strom, D. K., Kato, J. Y., Hanks, S. K., Roussel, M. F., and Sherr, C. J. (1992) Cell 71, 323-334[Medline] [Order article via Infotrieve] |
32. | Meyerson, M., and Harlow, E. (1994) Mol. Cell. Biol. 14, 2077-2086[Abstract] |
33. | Dowdy, S. F., Hinds, P. W., Louie, K., Reed, S. I., Arnold, A., and Weinberg, R. A. (1993) Cell 73, 499-511[Medline] [Order article via Infotrieve] |
34. | Ewen, M. E., Sluss, H. K., Sherr, C. J., Matsushime, H., Kato, J., and Livingston, D. M. (1993) Cell 73, 487-497[Medline] [Order article via Infotrieve] |
35. | Parry, D., Bates, S., Mann, D. J., and Peters, G. (1995) EMBO J. 14, 503-511[Abstract] |
36. | Müller, H., Lukas, J., Schneider, A., Warthoe, P., Bartek, J., Eilers, M., and Strauss, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2945-2949[Abstract] |
37. |
Ashizawa, S.,
Nishizawa, H.,
Yamada, M.,
Higashi, H.,
Kondo, T.,
Ozawa, H.,
Kakita, A.,
and Hatakeyama, M.
(2001)
J. Biol. Chem.
276,
11362-11370 |
38. |
Hoshikawa, Y.,
Mori, A.,
Amimoto, K.,
Iwabe, K.,
and Hatakeyama, M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8574-8579 |
39. | Hatakeyama, M., Brill, J. A., Fink, G. R., and Weinberg, R. A. (1994) Genes Dev. 8, 1759-1771[Abstract] |
40. | Takebe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai, K., Yoshida, M., and Arai, N. (1988) Mol. Cell. Biol. 8, 466-472[Medline] [Order article via Infotrieve] |
41. |
Nagata, D.,
Suzuki, E.,
Nishimatsu, H.,
Satonaka, H.,
Goto, A.,
Omata, M.,
and Hirata, Y.
(2001)
J. Biol. Chem.
276,
662-669 |
42. | Fujita, T., Nolan, G. P., Liou, H. C., Scott, M. L., and Baltimore, D. (1993) Genes Dev. 7, 1354-1363[Abstract] |
43. | Hinds, P. W., Mittnacht, S., Dulic, V., Arnold, A., Reed, S. I., and Weinberg, R. A. (1992) Cell 70, 993-1006[Medline] [Order article via Infotrieve] |
44. |
Mizuguchi, R.,
and Hatakeyama, M.
(1998)
J. Biol. Chem.
273,
32297-32303 |
45. | Suzuki, T. I., Higashi, H., Yoshida, E., Nishimura, S., and Kitagawa, M. (1997) Biochem. Biophys. Res. Commun. 234, 386-392[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Hinz, M.,
Krappmann, D.,
Eichten, A.,
Heder, A.,
Scheidereit, C.,
and Strauss, M.
(1999)
Mol. Cell. Biol.
19,
2690-2698 |
47. |
Guttridge, D. C.,
Albanese, C.,
Reuther, J. Y.,
Pestell, R. G.,
and Baldwin, A. S., Jr.
(1999)
Mol. Cell. Biol.
19,
5785-5799 |
48. |
Joyce, D.,
Bouzahzah, B.,
Fu, M.,
Albanese, C.,
D'Amico, M.,
Steer, J.,
Klein, J. U.,
Lee, R. J.,
Segall, J. E.,
Westwick, J. K.,
Der, C. J.,
and Pestell, R. G.
(1999)
J. Biol. Chem.
274,
25245-25249 |
49. | Brown, K., Gerstberger, S., Carlson, L., Franzoso, G., and Siebenlist, U. (1995) Science 267, 1485-1488[Medline] [Order article via Infotrieve] |
50. | Whyte, P., Williamson, N. M., and Harlow, E. (1989) Cell 56, 67-75[Medline] [Order article via Infotrieve] |
51. | Hu, Q. J., Dyson, N., and Harlow, E. (1990) EMBO J. 9, 1147-1155[Abstract] |
52. | Whyte, P., Buchkovich, K. J., Horowitz, J. M., Friend, S. H., Raybuck, M., Weinberg, R. A., and Harlow, E. (1988) Nature 334, 124-129[CrossRef][Medline] [Order article via Infotrieve] |
53. | Abraham, S. E., Carter, M. C., and Moran, E. (1992) Mol. Biol. Cell 3, 655-665[Abstract] |
54. | Charles, A., Tang, X., Crouch, E., Brody, J. S., and Xiao, Z. X. (2001) J. Cell. Biochem. 83, 414-425[CrossRef][Medline] [Order article via Infotrieve] |
55. |
Chen, P. L.,
Riley, D. J.,
Chen-Kiang, S.,
and Lee, W. H.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
465-469 |
56. | Chen, P. L., Riley, D. J., Chen, Y., and Lee, W. H. (1996) Genes Dev. 10, 2794-2804[Abstract] |
57. | Fortunato, E. A., Sommer, M. H., Yoder, K., and Spector, D. H. (1997) J. Virol. 71, 8176-8185[Abstract] |
58. |
Coupland, S. E.,
Bechrakis, N.,
Schüler, A.,
Anagnostopoulos, I.,
Hummel, M.,
Bornfeld, N.,
and Stein, H.
(1998)
Br. J. Ophthalmol.
82,
961-970 |
59. | Marhin, W. W., Hei, Y. J., Chen, S., Jiang, Z., Gallie, B. L., Phillips, R. A., and Penn, L. Z. (1996) Oncogene 12, 43-52[Medline] [Order article via Infotrieve] |
60. | Nielsen, N. H., Emdin, S. O., Cajander, J., and Landberg, G. (1997) Oncogene 14, 295-304[CrossRef][Medline] [Order article via Infotrieve] |
61. | Lukas, J., Bartkova, J., Rohde, M., Strauss, M., and Bartek, J. (1995) Mol. Cell. Biol. 15, 2600-2611[Abstract] |
62. | Tam, S. W., Theodoras, A. M., Shay, J. W., Draetta, G. F., and Pagano, M. (1994) Oncogene 9, 2663-2674[Medline] [Order article via Infotrieve] |
63. |
Biswas, D. K.,
Cruz, A. P.,
Gansberger, E.,
and Pardee, A. B.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
8542-8547 |
64. |
Henry, D. O.,
Moskalenko, S. A.,
Kaur, K. J.,
Fu, M.,
Pestell, R. G.,
Jacques, H.,
Camonis, J. H.,
and White, M. A.
(2000)
Mol. Cell. Biol.
20,
8084-8092 |
65. | Ibarra-Sanchez, M. J., Wagner, J., Ong, M. T., Lampron, C., and Tremblay, M. L. (2001) Oncogene 20, 4728-4739[CrossRef][Medline] [Order article via Infotrieve] |
66. |
Johnson-Pais, T.,
Degnin, C.,
and Thayer, M. J.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
2211-2216 |
67. |
Udvadia, A. J.,
Templeton, D. J.,
and Horowitz, J. M.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
3953-3957 |
68. | Chen, L. I., Nishinaka, T., Kwan, K., Kitabayashi, I, Yokoyama, K., Fu, Y. H., Grunwald, S., and Chiu, R. (1994) Mol. Cell. Biol. 14, 4380-4389[Abstract] |
69. | Gu, W., Schneider, J. W., Condorelli, G., Kaushal, S., Mahdavi, V., and Nadal-Ginard, B. (1993) Cell 72, 309-324[Medline] [Order article via Infotrieve] |
70. |
Tamami, M.,
Lindholm, P. F.,
and Brady, J. N.
(1996)
J. Biol. Chem.
271,
24551-24556 |
71. | Hofmann, F., Martelli, F., Livingston, D. M., and Wang, Z. (1996) Genes Dev. 10, 2949-2959[Abstract] |
72. |
Sarcevic, B.,
Lilischkis, R.,
and Sutherland, R. L.
(1997)
J. Biol. Chem.
272,
33327-33337 |
73. | Leng, X., Connell-Crowley, L., Goodrich, D., and Harper, J. W. (1997) Curr. Biol. 7, 709-712[Medline] [Order article via Infotrieve] |
74. |
Sherr, C. J.,
and Roberts, J. M.
(1999)
Genes Dev.
13,
1501-1512 |
75. |
Tong, W.,
and Pollard, J. W.
(2001)
Mol. Cell. Biol.
21,
1319-1328 |
76. |
Yamamoto, H.,
Soh, J. W.,
Monden, T.,
Klein, M. G.,
Zhang, L. M.,
Shirin, H.,
Arber, N.,
Tomita, N.,
Schieren, I.,
Stein, C. A.,
and Weinstein, I. B.
(1999)
Clin. Cancer Res.
5,
1805-1815 |