From the Division of Molecular Oncology, Institute
for Genetic Medicine, Hokkaido University, Kita-ku, Sapporo
060-0815, the § Department of Viral Oncology, Cancer
Institute, Japanese Foundation for Cancer Research, Toshima-ku, Tokyo
170-8455, and the ¶ Department of Surgery, Kitasato University
School of Medicine, Sagamihara, Kanagawa 228-8555, Japan
Received for publication, August 31, 2000, and in revised form, December 14, 2000
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ABSTRACT |
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The activity of the retinoblastoma protein pRB is
regulated by phosphorylation that is mediated by G1
cyclin-associated cyclin-dependent kinases (CDKs). Since
the pRB-related pocket proteins p107 and p130 share general structures
and biological functions with pRB, their activity is also considered to
be regulated by phosphorylation. In this work, we generated
phosphorylation-resistant p107 and p130 molecules by replacing
potential cyclin-CDK phosphorylation sites with non-phosphorylatable
alanine residues. These phosphorylation-resistant mutants retained the
ability to bind E2F and cyclin. Upon introduction into
p16INK4a-deficient U2-OS osteosarcoma cells, in which
cyclin D-CDK4/6 is dysregulated, the phosphorylation-resistant
mutants, but not wild-type p107 or p130, were capable of inhibiting
cell proliferation. Furthermore, when ectopically expressed in
pRB-deficient SAOS-2 osteosarcoma cells, the wild-type as well as the
phosphorylation-resistant pRB family proteins were capable of inducing
large flat cells. The flat cell-inducing activity of the wild-type
proteins, but not that of the phosphorylation-resistant mutants, was
abolished by coexpressing cyclin E. Our results indicate that the
elevated cyclin D- or cyclin E-associated kinase leads to systemic
inactivation of the pRB family proteins and suggest that dysregulation
of the pRB kinase provokes an aberrant cell cycle in a broader range of
cell types than those induced by genetic inactivation of the RB gene.
The retinoblastoma tumor suppressor protein pRB is a nuclear
phosphoprotein that is ubiquitously expressed in somatic cells. It
inhibits cell proliferation when ectopically expressed and is thought
to play an important role in the growth decision-making in the late
G1 phase of the cell cycle (1, 2). pRB is considered to
inhibit cell proliferation by physically interacting with cellular proteins, most notably with the E2F family of transcription factors (3-5). Upon complex formation with E2F, pRB inhibits transcriptional activation of those E2F-dependent genes whose products are
essentially required for G1-to-S phase cell cycle
transition. Furthermore, the pRB-E2F complex is capable of acting as a
repressor against promoters containing E2F-binding sites, thereby
actively repressing transcription in an E2F-dependent
manner (4-6). Sequential phosphorylation of pRB during G1
by cyclin D-associated cyclin-dependent kinase (CDK)1-4/6 and cyclin E-CDK2
abolishes the ability of pRB to form physical complexes with cellular
proteins, including E2F, and leads to G1-to-S phase cell
cycle progression and subsequent cell division.
The "p16INK4a-pRB" pathway is perturbed in most, if not
all, cancer cells (7-12). The changes include mutational inactivation
of p16INK4a, cyclin D overexpression, and production of
CDK4 mutants that cannot interact with p16INK4a. All of
these changes lead to biochemical inactivation of pRB through
phosphorylation, indicating that pRB plays a central role in preventing
cellular transformation. On the other hand, genetic inactivation of a
single copy of the RB gene predisposes only a limited set of
malignancies such as retinoblastoma and osteosarcoma. This raises an
intriguing possibility that dysregulation of the p16INK4a-pRB pathway gives rise to transformation in a
broader range of cell types than those induced by RB gene inactivation.
There are two proteins, p107 and p130, that share the so-called
"pocket domain" with pRB (13-16). They are also capable of inhibiting cell growth upon ectopic expression (17-19). Among the members of the pRB pocket protein family, p107 and p130 are more homologous to each other than to pRB and share unique properties allowing interaction with cyclins and CDKs (14, 15, 20-22).Through the
interaction, p107 or p130 is capable of inhibiting the kinase activity
of cyclin-CDK (23, 24). In addition, p107 and p130 selectively bind
E2F-4 and E2F-5, whereas pRB preferentially binds E2F-1, E2F-2, and
E2F-3 (25-30). Hence, each member of the pRB family is likely to
perform shared as well as unique cell cycle regulatory roles in a
single cell. Furthermore, we (33, 36) and others (30-32, 34, 35) have
recently shown that a different member of the pRB pocket protein family
may become a key regulator of the cell cycle in different cell types.
p107 and p130 are phosphorylated in a cell cycle-dependent
manner. Like pRB, p107 can be a substrate for cyclin D-CDK4, and p107-induced cell cycle arrest was reportedly released by cyclin D-CDK4, but not by cyclin E-CDK2 (19, 37, 38). In the case of p130,
however, both D-type cyclins and cyclin E are capable of reversing the
growth suppression mediated by p130. These observations raise the idea
that the function of the entire pRB family of proteins is collectively
regulated by phosphorylation, most likely through G1
cyclin-CDK. Although this idea has long been suspected, there is little
published information as to the functional regulation of p107 and p130
by phosphorylation.
In this work, we generated p107 and p130 mutants that are resistant to
cyclin-CDK-mediated phosphorylation. By expressing these
phosphorylation-resistant molecules, we demonstrate that the
growth-inhibitory activity as well as the cell
differentiation/senescence-promoting activity of pRB, p107, and p130
are inactivated by phosphorylation. Our results indicate that elevation
of the levels of pRB kinases through either p16INK4a loss
or cyclin E overexpression abolishes the total activities of the pRB
family proteins and, as a result, predisposes a broad spectrum of cells
to a dysregulated cell cycle.
Cells--
The human osteosarcoma line SAOS-2 was provided by
Dr. Phil Hinds (Harvard Medical School, Boston). The human osteosarcoma line U2-OS was obtained from American Type Culture Collection. The
cells were cultured in Dulbecco's modified Eagle's medium with 10%
(for U2-OS) or 15% (for SAOS-2) fetal bovine serum.
Construction of Plasmids--
The phosphorylation-resistant pRB
mutant, pRB Immunoprecipitation and Immunoblotting--
Either 20 µg of
the indicated pocket protein expression plasmids or 10 µg of the
pocket protein expression plasmids and 10 µg of E1A (either 12 S or
E1A-928 mutant) expression plasmids were transfected into 1 × 106 U2-OS cells in a 100-mm plate by the calcium phosphate
precipitation method as described previously (42). After 2 days of
culture in Dulbecco's modified Eagle's medium with 10% fetal bovine
serum, the transfected cells were harvested and lysed in E1A lysis
buffer (42). Cell lysates were first treated with anti-HA monoclonal antibody (12CA5) for 1 h, and the immune complexes were collected on protein A-Sepharose beads. After washing the beads,
immunoprecipitates were subjected to SDS-polyacrylamide gel
electrophoresis, 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 A (H-432, sc-75, Santa Cruz Biotechnology), anti-E2F-4
(C-108, sc-512, Santa Cruz Biotechnology), and anti-adenovirus E1A
(14161A, Pharmingen).
Colony Formation Assay--
20 µg of the expression plasmid
was transfected into U2-OS cells together with 2 µg of the puromycin
resistance gene (pBabe-puro) (43) by the calcium phosphate
precipitation method. At 16 h after the transfection, the cells
were split into four 100-mm plates. The transfected cells were then
selected in Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum in the presence of 2 µg/ml puromycin for 2 weeks. During
the selection, the medium was changed twice a week. After the drug
selection, the cells were stained by the May-Giemsa method, and the
number of puromycin-resistant colonies was counted.
Flat Cell Formation Assay--
The expression plasmid (20 µg)
was transfected together with 2 µg of pBabe-puro into SAOS-2 cells by
the calcium phosphate precipitation method. At 16 h after the
transfection, the cells were split into four 100-mm plates, and the
transfected cells were selected in Dulbecco's modified Eagle's medium
with 15% fetal bovine serum in the presence of 0.5 µg/ml puromycin
for 2 weeks. After the drug selection, the cells were stained with
May-Giemsa solution and crystal violet. For cyclin E coexpression, 10 µg of the pRB family expression plasmid and 2 µg of pBabe-puro were transfected together with 10 µg of pCMV or pCMV-cyclin E.
Flow Cytometric Analysis--
U2-OS cells were transfected with
20 µg of the pRB family expression plasmid together with 2 µg of
the CD20 expression plasmid (pCMV-CD20) (40) as a marker. 40 h
after the transfection, cells were harvested and treated with an
anti-CD20 antibody (fluorescein isothiocyanate-labeled CD20,
Becton Dickinson) for 20 min on ice, washed with phosphate-buffered
saline, and fixed in 70% ethanol on ice. The cells were washed again
and resuspended in 100 µl of phosphate-buffered saline containing 25 µg/ml RNase A for 20 min. Prior to flow cytometry, 700 µl of
propidium iodide solution (100 µg/ml propidium iodide and 0.1%
sodium citrate) was added to the cell suspension, and cells were
incubated for another 15 min on ice. The intensity of propidium iodide
staining was measured by a FACSCalibur (Becton Dickinson) on cell
populations that were positive for CD20 expression to determine the DNA
content. Cell cycle profiles of the CD20-positive cells were analyzed
using CELL Quest and ModFit cell cycle analysis software (Becton Dickinson).
Generation of Phosphorylation-resistant p107 and p130--
To
address the role of cyclin-associated CDK-dependent
phosphorylation in the function of p107 and p130, we generated
phosphorylation-resistant mutants as described previously in a study on
the function of pRB (33). Human p107 possesses 18 Ser-Pro or Thr-Pro
((Ser/Thr)-Pro) motifs that are potential phosphorylation targets for
cyclin-CDK. The serine and threonine residues making up all of the
(Ser/Thr)-Pro motifs were replaced by alanine to generate p107 Interaction of the Phosphorylation-resistant pRB Family Proteins
with E2F and Cyclin--
Since the phosphorylation-resistant mutants
possess multiple point mutations, we first investigated whether or not
these proteins retain sufficient structural integrity to interact with
cyclins and E2F proteins in the manner of their respective wild-type
proteins. To do so, cDNA encoding the wild-type or the
phosphorylation-resistant mutant protein was transfected into U2-OS
cells. As demonstrated in Fig. 2
(A, upper panel, lanes 2,
4, and 6; and C, upper
panel, lanes 2, 4, 6,
10, 12, and 14), the wild-type pocket
proteins were detected as broad bands, indicating that they were
variably phosphorylated. In contrast, the phosphorylation-resistant
mutants were all detected as fast migrating bands (Fig. 2,
A, upper panel, lanes 3, 5,
7, and 8; and C, upper
panel, lanes 3, 5, 7,
8, 11, 13, 15, and
16), indicating that they were resistant to phosphorylation in cells.
In sequential immunoprecipitation-immunoblotting analyses, endogenous
E2F-4 proteins were co-immunoprecipitated with all wild-type and mutant
pocket proteins, with the exception of p130
Conformation of the phosphorylation-resistant mutant as the "pocket
protein" was further examined with the use of the adenovirus E1A 12 S
product, which specifically binds the pocket domains and inactivates
the pRB family proteins (45-47). Upon transient coexpression in U2-OS
cells, the mutant pRB family molecules, except p130 G1 Cell Cycle Arrest of p16INK4a-defective
U2-OS Cells by the Phosphorylation-resistant pRB Family
Proteins--
It has been shown that U2-OS cells are highly resistant
to pRB overexpression (19). Since the U2-OS cells do not express functional p16INK4a (51), a specific inhibitor of cyclin
D-CDK4/6, the cells exhibit dysregulated pRB kinase activity (52). It
is therefore considered that, in U2-OS cells, the dysregulated pRB
kinase instantly inactivates ectopically expressed wild-type pRB
proteins. If this is the case, then the growth of U2-OS cells must be
inhibited by phosphorylation-resistant pRB. To address this, a cDNA
expression vector for wild-type pRB or the phosphorylation-resistant
pRB mutant, pRB
The U2-OS cells allowed us to examine whether the growth-suppressive
activity of p107 or p130 is also under the control of cyclin-CDK-mediated phosphorylation. To this end, we transiently introduced wild-type p107 or p107
The wild-type and two forms of phosphorylation-resistant p130
molecules, p130 Growth Suppression of U2-OS Cells by the
Phosphorylation-resistant pRB Family Proteins--
We next
investigated the long-term effects of the pRB family proteins on the
growth of U2-OS cells. To do so, we transfected cDNA expression
vectors for the pRB family proteins together with the puromycin
resistance gene. After selection of the transfected cells with
puromycin, the number of puromycin-resistant colonies was counted. As
shown in Fig. 4, expression of wild-type
pocket proteins in U2-OS cells resulted in a weak reduction of
puromycin-resistant colonies, indicating that these proteins are
growth-inhibitory.
As expected from the transient assay, the phosphorylation-resistant pRB
and p107 molecules reduced puromycin-resistant colonies quite strongly
(Fig. 4). Furthermore, in this colony formation assay, the
phosphorylation-resistant p130 mutant p130 p107- and p130-Mediated Flat Cell Formation in pRB-defective SAOS-2
Cells--
Expression of pRB in pRB-defective SAOS-2 human
osteosarcoma cells causes strong G1 cell cycle arrest that
is associated with a large flat cell formation (42, 54-56). The
pRB-induced flat cells express bone differentiation markers as well as
cell senescence markers such as
We next employed several deletion mutants of p107 (see Fig. 1) to
delineate the regions responsible for the flat cell formation. The
p107N385 mutant, which lacks the amino-terminal one-third of
p107, was capable of inducing flat cells. In contrast, deletion of the central one-third (p107DE) or truncation of the C-terminal region (p107L19) resulted in a total loss of the ability to induce flat
cells (Fig. 5). This indicates that, as in pRB, the pocket function is
involved in the flat cell induction by p107. The conclusion was further
supported by a series of p130 deletion mutants (see also Fig. 1). In
these mutants, deletion of the spacer region (p130
By employing the flat cell assay, we were also able to determine the
(Ser/Thr)-Pro motifs whose mutations lead to functional inactivation of
p130. To this end, we generated two additional mutants, p130 Effect of Cyclin E on p107/p130-mediated Flat Cell
Formation--
The ability of p107 and p130 to induce SAOS-2 flat
cells gave us another opportunity to examine whether or not the
function of these pRB homologs is regulated by cyclin-CDK-mediated
phosphorylation. In the SAOS-2 flat cell assay with pRB, coexpression
of cyclin E together with pRB has been shown to promote pRB
hyperphosphorylation by cyclin E-CDK and, as a result, to abolish the
ability of pRB to induce flat cells (42). With this observation in
mind, we wondered whether the flat cell-inducing activity of p107 or
p130 might also be controlled by the cyclin E-associated kinase. We thus cotransfected the p107 or p130 expression vector together with the
cyclin E expression vector into SAOS-2 cells and examined p107- or
p130-mediated flat cell induction in the presence or absence of ectopic
cyclin E (Fig. 6). In the case of
wild-type p107 or p130, the number of flat cells generated was severely reduced by coexpression with cyclin E. In striking contrast, both p107 In this work, we provide evidence that the activities of the
entire pRB family of proteins are collectively regulated by
phosphorylation through cyclin D- or cyclin E-associated CDK with the
use of the phosphorylation-resistant pRB family proteins. Cells lacking
p16INK4a are highly resistant to ectopic overexpression of
wild-type pRB during their growth stage (19). Since
p16INK4a acts as a specific inhibitor of cyclin D-CDK4/6,
dysregulated cyclin D-CDK4/6 is suspected to phosphorylate and
inactivate pRB in cells with p16INK4a loss (52, 58). This
idea is confirmed by our current work, in which
phosphorylation-resistant pRB inhibited G1-to-S phase cell
cycle progression of the p16INK4a-defective U2-OS cells
(51), whereas wild-type pRB failed to do so. A similar result has been
recently reported with the expression of a distinct
phosphorylation-resistant pRB mutant in which 10 out of 16 potential
CDK phosphorylation sites have been replaced by alanine residues. As is
the case of our transient expression experiment, U2-OS cells in which
expression of phosphorylation-resistant pRB was induced arrested in
G1 within 48 h. Intriguingly, however, the
G1-arrested cells gradually entered into S phase after
48 h and underwent endoreplication between 4 and 6 days (59).
We demonstrate in this work that phosphorylation-resistant p107 and
p130 are also capable of inhibiting U2-OS cell growth. Again in these
cases, the wild-type molecules had much weaker effects on the growth of
U2-OS cells. This indicates that the U2-OS cell cycle is principally
sensitive not only to pRB, but also to p107 and p130. However, like
that of pRB, the growth-inhibitory activities of wild-type p107 and
p130 are neutralized via phosphorylation in U2-OS cells. This in turn
suggests that, to initiate cell cycle progression, the cells need to
inactivate all of the pRB family pocket proteins that are functioning
as cell cycle brakes.
Since cyclin D-associated kinase is capable of phosphorylating p107
(30, 37, 38), it is logical to speculate that cyclin D-CDK4/6, which is
deregulated in p16INK4a-defective U2-OS cells, is involved
in the inactivation of p107 and p130. This conclusion is consistent
with the previous observation that cell growth inhibition by p107 or
p130 is ameliorated by ectopic coexpression of cyclin D (19, 37, 38,
60).
We further demonstrate in this work that p107 and p130 share with pRB
the biological activity of inducing flat SAOS-2 cells. Previous reports
have demonstrated that these pRB homologs, particularly p130, shows
little induction of flat cells in the SAOS-2 cell assay (19, 57). We
suspect that the difference is due to the protein expression levels. In
our system, the cDNA-directed proteins were expressed with the use
of the SR One intriguing result of this study is that the flat cell-inducing
activity of wild-type p107, p130, and pRB is abolished by cyclin E
coexpression. In contrast, cyclin E had little effect on the flat cell
induction by phosphorylation-resistant pRB, p107, or p130. Although
cyclin E is considered to be a critical downstream effector of "pRB
family-E2F interplay" for entry into the S phase (61, 62), our
observation clearly points to the notion that cyclin E acts as an
upstream regulator of the pRB family proteins and that elevated cyclin
E-CDK2 inactivates the flat cell-inducing activity of pRB, p107, or
p130 through phosphorylation. This study thus demonstrates that the two
established pRB kinases, cyclin D-CDK4/6 and cyclin E-CDK2, are capable
of phosphorylating and functionally inactivating the pRB homologs p107
and p130 as well. In other words, the total activity of the pRB family
proteins is under the control of the pRB kinases.
Although we do not know the exact molecular mechanisms underlying
differentiation by the pRB family, pocket-binding molecules other than
E2F proteins may be involved in this process since pRB mutants that
cannot interact with E2F still retain the ability to induce flat cells
(57). The idea is indeed supported by our current observation that
cyclin E cannot abolish flat cell formation by the
phosphorylation-resistant pRB family molecules because the cyclin E
gene is a critical downstream target of E2F (63, 64). Since pRB could
induce flat cells at a lower level, but p107/p130 only at higher
levels, the affinity of p107 and p130 for such a differentiation
regulator may be significantly weaker than that of pRB. In striking
contrast, induction of G1 arrest by transient expression of
pRB requires its ability to bind to E2F (65-67). Accordingly, as has
been reported by a number of groups (59, 62, 68, 69), acute
G1 arrest by phosphorylation-resistant pRB may be bypassed
by ectopic overexpression of cyclin E, a major downstream effector of E2F.
The cell cycle of U2-OS or SAOS-2 cells is sensitive to any of the
three pRB family proteins because it is halted by the wild-type or
phosphorylation-resistant mutant proteins. This results in a marked
difference in the case of certain hematopoietic cells such as Ba/F3
lymphoid cells and 32D myeloid progenitor cells (33, 36). In the latter
cases, the cells are totally resistant to pRB, even in its
phosphorylation-resistant form, but are sensitive to p130. This
indicates that inactivation of pRB is not an absolute prerequisite for
cell cycle progression in all somatic cells. The conclusion was further
supported by a recent work with another cell line, C33A (35).
Furthermore, p130 inhibits proliferation of the glioblastoma cell line
T98G, which is resistant to the growth-suppressive effects of p107 and
pRB, in colony formation assay (17). This differential sensitivity of
cells to each of the pRB family proteins appears to reflect, at least
in part, differential expression of pRB-regulated E2F proteins (E2F-1, E2F-2, and E2F-3) and p107/p130-regulated E2F proteins (E2F-4 and
E2F-5) (33, 36). In cells wherein pRB-regulated E2F proteins act as
central transcriptional regulators of the cell cycle, loss of pRB
directly leads to dysregulation of the cell cycle. Conversely, in cells
in which p107/p130-regulated E2F proteins play a major role in the cell
cycle control, pRB inactivation per se has nothing to do
with cell growth. On the other hand, elevation of cyclin D-CDK and/or
cyclin E-CDK activities gives rise to the functional inactivation of
the entire pRB protein family, promoting cell cycle progression
irrespective of the cellular sensitivity to the pRB family proteins.
Our results point to a substantial difference between dysregulation of
pRB kinase and genetic inactivation of the RB gene in the
context of cell type-specific transformation and indicate that
dysregulated pRB kinase provokes an aberrant cell cycle in a broader
range of cell types than those induced by a simple loss of pRB.
Obviously, there are cells whose cell cycle is strongly dependent on
pRB. In such cells, p16INK4a is ineffective in suppressing
proliferation in the absence of pRB (70-72). In many cell types,
however, growth decision-making may involve various combinations of the
pRB family proteins. Furthermore, the relative contribution of each pRB
family member to the decision-making may also vary among different cell
types. Hence, in those cells, inactivation of the whole pRB protein
family may be an obligatory prerequisite in proceeding to the cell
cycle. This in turn indicates that, during multistep carcinogenesis,
such a situation could be most easily provided and fixed by genetically
modifying components that constitute the p16INK4a-pRB
pathway or by cyclin E overexpression, as has been frequently shown in
breast cancer (73). Finally, given the importance of the functional
loss of the pRB family proteins in cellular transformation, the
phosphorylation-resistant mutants generated in this work will become
powerful therapeutic tools in gene therapies against a broad range of
human cancers.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S/T-P, was described previously (33, 39). Other mutant
constructs were generated by multiple rounds of
oligonucleotide-mediated mutagenesis with the use of the Chameleon
site-directed mutagenesis system (Stratagene) according to the
manufacturer's instructions. cDNAs encoding p107N385, p107DE, and
p107L19 were gifts from Dr. Liang Zhu (Albert Einstein College of
Medicine, New York) (40). The p130
846-1119 and p130
620-697 constructs were gifts of Dr. Peter Whyte (McMaster University, Ontario,
Canada) (23). Some of the constructs were tagged with a hemagglutinin
(HA) epitope at either the amino or carboxyl terminus. cDNAs
encoding the wild-type and mutant pRB family proteins were inserted
into mammalian expression vector pSP65-SR
(41). The pRc/CMV vector
was used to express human cyclin E cDNA, and the pSV vector for
adenovirus E1A (12 S and E1A-928 mutant).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S/T-P.
Similarly, all of the 27 (Ser/Thr)-Pro motifs in human p130 were
mutated to generate p130
S/T-P. In the case of p130, p130
CDK was
also made by mutating 12 serine/threonine residues constituting the canonical CDK consensus motif ((S/T)PX(L/R)) to alanine
residues. All of the pocket protein mutants used in this work are
summarized in Fig. 1.
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Fig. 1.
Schematic drawing of pRB family proteins and
their mutants used in this work. HA represents the
hemagglutinin epitope.
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Fig. 2.
E2F- and cyclin-binding abilities of the
phosphorylation-resistant pRB family proteins.
A, extracts were prepared from U2-OS cells
transfected with the indicated constructs, and lysates were
immunoprecipitated (IP) with anti-HA monoclonal antibody
(12CA5). The anti-HA immunoprecipitates were then subjected to 7%
SDS-polyacrylamide gel electrophoresis, followed by anti-HA
immunoblotting (upper panel). The same filter was also
immunoblotted with anti-E2F-4 (middle panel) or anti-cyclin
A ( -CycA; lower panel) antibody.
B, shown is a longer exposure of the anti-E2F-4
immunoblotting (lanes 1-3 of the middle panel in
A). C, U2-OS cells were cotransfected with
indicated constructs and adenovirus E1A (12 S or E1A-928 mutant)
expression plasmids, and then the lysates were subjected to
immunoprecipitation (anti-HA antibody) and immunoblotting (anti-HA
antibody (upper panel) and anti-E1A antibody (middle
panel)). The whole cell lysates were also immunoblotted with
anti-E1A antibody (lower panel). It should be noted that
pRB
S/T-P, which is derived from mouse RB cDNA,
migrated a bit faster than the hypophosphorylated form of the wild-type
pRB protein, which is derived from human RB cDNA.
SR
, empty vector as a negative control.
S/T-P (Fig. 2,
A, middle panel; and B). In each case,
E2F-4 molecules coprecipitated with the mutant pocket proteins were
detected in amounts similar to those coprecipitated with their
respective wild-type counterparts, although the expression levels of
the mutant proteins were significantly less than those of the wild-type
proteins. This indicates at least that the affinities of the mutant
pocket proteins for binding E2F-4 are not reduced despite introduction
of multiple mutations. Notably, the E2F-4-binding affinity of pRB or
pRB
S/T-P was significantly less than that of p107 or p107
S/T-P
(Fig. 2, A and B) as reported previously
(18, 44). Furthermore, wild-type p107, wild-type p130,
p107
S/T-P, and p130
CDK, but not p130
S/T-P, physically interacted with endogenous cyclin A (Fig. 2A, lower
panel). Again, in each case, coprecipitated cyclin A levels were
found to be similar to the levels coprecipitated with their respective
wild-type counterparts. In contrast, wild-type pRB and pRB
S/T-P did
not associate with cyclin A because they do not possess cyclin
A-binding spacer sequences. These observations indicate that
p107
S/T-P, p130
CDK, and pRB
S/T-P are capable of interacting
with cellular targets with affinities comparable to those of their
respective wild-type molecules.
S/T-P, formed
stable complexes with E1A to levels comparable to those associated with
their respective wild-type counterparts (Fig. 2C). Moreover,
an E1A point mutant, E1A-928, which does not bind to wild-type pRB, did
not bind pRB
S/T-P as well (Fig. 2C, middle
panel, lanes 10 and 11). The E1A mutant is
also known to bind p107 and p130 less effectively. Consistently, p107
S/T-P and p130
CDK exhibited reduced affinities for E1A-928, as is the case with wild-type p107 and p130 (Fig. 2C,
middle panel, compare lanes 4-7 with lanes
12-15) (48-50). These observations provide further evidence that
the structural conformations of the phosphorylation-resistant pocket
protein mutants pRB
S/T-P, p107
S/T-P, and p130
CDK are
indistinguishable from those of their respective wild-type molecules,
pointing to the notion that these mutants function through normal
biological pathways that involve their wild-type counterparts when
expressed in cells. On the other hand, p130
S/T-P, which has 27 point
mutations, lost its structural integrity as a pocket protein because of
the introduced mutations.
S/T-P, was cotransfected with the CD20 expression
vector, and the cell cycle distribution of the CD20-positive cells at
40 h after the transfection was examined with the use of a flow
cytometer. As previously reported (19), ectopic expression of pRB had
very little effect on the cell cycle distribution profile of U2-OS
cells (Fig. 3). On the other hand,
pRB
S/T-P caused severe G1 cell cycle arrest in U2-OS cells (Fig. 3). This indicates that, in these cells, the
growth-suppressive activity of pRB is neutralized by phosphorylation,
most likely through the dysregulated cyclin D-CDK kinase because of the
lack of p16INK4a.
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Fig. 3.
G1 cell cycle arrest of U2-OS
cells by phosphorylation-resistant pRB family proteins. An
expression vector for the wild-type pRB family protein or the
phosphorylation-resistant mutants was transiently expressed in U2-OS
cells together with the surface marker CD20. 48 h after the
transfection, cells were harvested, and DNA profiles of productively
transfected cells were evaluated by flow cytometry. Control cells were
transfected with empty vector. Percentages indicate the
proportions of cells in G1, S, and G2/M.
SR , empty vector as a negative control.
S/T-P together with CD20 into U2-OS
cells (Fig. 3). Whereas the expression of wild-type p107 did not affect
the cell cycle distribution significantly, phosphorylation-resistant p107 provoked strong accumulation of cells in G1 phase,
indicating that the mutant molecules caused G1 cell cycle
arrest in U2-OS cells. This indicates that p107 is capable of
inhibiting cell cycle progression and that the activity is under
phosphorylation control in U2-OS cells (Fig. 3).
S/T-P and p130
CDK, were next examined for their growth regulatory activity in U2-OS cells. In contrast to pRB and p107, p130, even in its phosphorylation-resistant forms, did not
significantly affect the cell cycle profile of U2-OS cells in this
transient expression assay (Fig. 3). Given the observation that
p130
S/T-P cannot bind E2F or cyclin A (Fig. 2), this finding is
consistent with the conclusion that the mutant is functionally inactive. In contrast, since p130
CDK can still bind with the target
molecules, the growth-inhibitory activity of p130, if it exists, may be
significantly weaker than that of pRB or p107 in U2-OS cells.
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Fig. 4.
Suppression of colony formation by
phosphorylation-resistant pRB family proteins. U2-OS cells were
stably cotransfected with the expression plasmids. Following
transfection, cells were selected with puromycin and, on day 14, were
fixed and stained. A, drug-resistant colonies generated
after puromycin selection; B, puromycin-resistant colony
numbers/dish. Bars represent 2 × S.D. of the
three independent experiments.
CDK suppressed U2-OS
colony formation more effectively than wild-type p130. This indicates
that p130 is also capable of inhibiting cell proliferation and that
this growth-inhibitory activity is under phosphorylation control. In
contrast, p130
S/T-P, which lacks all 27 (Ser/Thr)-Pro motifs, was
not growth-suppressive in the colony assay (Fig. 4). Accordingly, as
suggested from its inability to bind E2F-4 or cyclin A, the
introduction of mutations into one or several (Ser/Thr)-Pro sites
present in p130
CDK but absent in p130
S/T-P appears to provoke
structural inactivation of p130. Alternatively, since p130 receives
multiple phosphorylation and its function is reportedly modified
depending on phosphorylation status (53), a certain degree of basal
phosphorylation might be required for the activation of p130 as a
growth suppressor. If this is the case, then p130
S/T-P may be
functionally inactive because it cannot receive activating phosphorylation by the proline-directed kinases that target the (Ser/Thr)-Pro motifs. In any case, the failure of p130
S/T-P to inhibit cell growth argues against the idea that cell cycle-inhibitory effects of the phosphorylation-resistant mutants are due to the overexpression and accumulation of functionally inactive pocket proteins.
-galactosidase (57). Hence, pRB not
only inhibits the cell cycle, but also promotes differentiation and
senescence in SAOS-2 cells. Despite several trials, however, p107 and
p130 failed to effectively induce flat cells in SAOS-2 cells (19, 57).
Since our cDNA expression system employs the powerful SR
promoter, we wondered whether higher levels of p107 or p130 in SAOS-2
cells might induce flat cells. We therefore introduced the p107 or p130
expression vector into SAOS-2 cells together with the puromycin
resistance gene. After 2 weeks of puromycin selection, cells were
stained, and flat cell formation was examined. As demonstrated in Fig.
5, p107 caused a strong flat cell
formation comparable to that by pRB. p107
S/T-P also effectively
induced flat cells. Furthermore, wild-type p130 and p130
CDK, but not p130
S/T-P, were capable of inducing flat cells, although the efficiency was significantly lower than that of the induction by p107
or pRB. It should also be noted that the flat cells induced by p130
were more rounded than those induced by pRB or p107.
View larger version (98K):
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Fig. 5.
Flat cell production by pRB family proteins
and their mutants. SAOS-2 cells were transfected with a
puromycin resistance plasmid and the appropriate pRB family
protein-expressing construct. The transfected cells were then subjected
to selection in puromycin for 14 days and fixed and stained with
crystal violet. Photographs of the cells were made at 40-fold
magnification. SR , empty vector control.
620-697) did not
affect the flat cell-inducing activity, whereas deletion of the B box
and the C terminus (p130
846-1119), which specifically inactivates
the p130 pocket function, resulted in the loss of flat cell-inducing
activity (Fig. 5).
CDK2 and
p130
CDK3 (see Fig. 1). Like p130
CDK, p130
CDK2 was capable of
inducing flat cells when expressed in SAOS-2 cells, whereas p130
CDK3
failed to do so (Fig. 5). Taken together with the finding that
p130
620-697 (which lacks Ser-639, Ser-642, Ser-662, Ser-672,
Ser-688, and Ser-694) is still active in inducing flat cells, this
indicates that mutations within the (Ser/Thr)-Pro clusters adjacent to
the amino terminus of the A box of p130 may be involved in the
functional inactivation of p130 and suggests the importance of this
region in the structural integrity of p130.
S/T-P and p130
CDK induced flat cells irrespective of cyclin E
coexpression. We also examined the role of D-type cyclins in the flat
cell formation by p107 or p130 in SAOS-2 cells. However, in contrast to
cyclin E, ectopic expression of cyclin D was extremely toxic to SAOS-2
cells, and we were not able to successfully coexpress cyclin D (D1-D3)
together with the pocket proteins under our experimental conditions. We
concluded from the observation that the flat cell-inducing activity of
both p107 and p130 was again under the control of phosphorylation and
that cyclin E-associated kinase is capable of inactivating p107 and
p130 activities through phosphorylation.
View larger version (59K):
[in a new window]
Fig. 6.
Inhibition of pocket protein-mediated flat
cell formation by cotransfecting the cyclin E expression plasmid.
SAOS-2 cells were cotransfected with the pRB family protein and cyclin
E expression vector. Transfected SAOS-2 cells were plated, subjected to
drug selection, and stained 14 days later. After normalizing the
transfection efficiency with the drug-resistant colony numbers, the
ratio of flat cell numbers in the presence of cyclin E to flat cell
numbers in the absence of cyclin E (100%) was calculated. Two
independent experiments are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
promoter (41), which is significantly stronger than the
cytomegalovirus promoter used in the previous
works.2 Since the flat cells
exhibit differentiation phenotypes (57), all of the pRB family proteins
appear to be capable of promoting differentiation programs via the
shared pocket functions.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Liang Zhu for pCMV-CD20 and p107 mutant cDNAs and Dr. Peter Whyte for p130 mutant cDNAs. We also thank Keiko Amimoto and Yuko Shikauchi for technical help.
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FOOTNOTES |
---|
* This work was supported by a grant-in-aid for scientific research on priority areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, a research grant from the Human Frontier Science Program Organization, and a research grant from Nippon Boehringer Ingelheim Co., Ltd.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: Div. of Molecular
Oncology, Inst. for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan. Tel./Fax: 81-11-709-6482; E-mail: mhata@imm.hokudai.ac.jp.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M007992200
2 S. Ashizawa, H. Nishizawa, M. Yamada, H. Higashi, T. Kondo, H. Ozawa, A. Kakita, and M. Hatakeyama, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: CDK, cyclin-dependent kinase; HA, hemagglutinin.
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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. | Chellappan, S. P., Hiebert, S., Mudryj, M., Horowitz, J. M., and Nevins, J. R. (1991) Cell 65, 1053-1061[Medline] [Order article via Infotrieve] |
4. |
Dyson, N.
(1998)
Genes Dev.
12,
2245-2262 |
5. | Nevins, J. R. (1992) Science 258, 424-429[Medline] [Order article via Infotrieve] |
6. | 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] |
7. | Delmer, A., Tang, R., Senamaud-Beaufort, C., Paterlini, P., Brechot, C., and Zittoun, R. (1995) Leukemia (Baltimore) 9, 1240-1245[Medline] [Order article via Infotrieve] |
8. | Ruas, M., Brookes, S., McDonald, N. Q., and Peters, G. (1999) Oncogene 18, 5423-5434[CrossRef][Medline] [Order article via Infotrieve] |
9. | Ruas, M., and Peters, G. (1998) Biochim. Biophys. Acta 1378, 115-177[CrossRef] |
10. | 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] |
11. | Serrano, M. (1997) Exp. Cell Res. 237, 7-13[CrossRef][Medline] [Order article via Infotrieve] |
12. | Serrano, M., Hannon, G. J., and Beach, D. (1993) Nature 366, 704-707[CrossRef][Medline] [Order article via Infotrieve] |
13. | Ewen, M. E., Xing, Y. G., Lawrence, J. B., and Livingston, D. M. (1991) Cell 66, 1155-1164[Medline] [Order article via Infotrieve] |
14. | Hannon, G. J., Demetrick, D., and Beach, D. (1993) Genes Dev. 7, 2378-2391[Abstract] |
15. | Li, Y., Graham, C., Lacy, S., Duncan, A. M., and Whyte, P. (1993) Genes Dev. 7, 2366-2377[Abstract] |
16. | Mayol, X., Grana, X., Baldi, A., Sang, N., Hu, Q., and Giordano, A. (1993) Oncogene 8, 2561-2566[Medline] [Order article via Infotrieve] |
17. | Claudio, P. P., Howard, C. M., Baldi, A., De Luca, A., Fu, Y., Condorelli, G., Sun, Y., Colburn, N., Calabretta, B., and Giordano, A. (1994) Cancer Res. 54, 5556-5560[Abstract] |
18. | Grana, X., Garriga, J., and Mayol, X. (1998) Oncogene 17, 3365-3383[CrossRef][Medline] [Order article via Infotrieve] |
19. | Zhu, L., Heuvel, S. V. D., Helin, K., Fattaey, A., Ewen, M., Livingston, D., Dyson, N., and Harlow, E. (1993) Genes Dev. 7, 1111-1125[Abstract] |
20. | Cobrinik, D., Whyte, P., Peeper, D. S., Jacks, T., and Weinberg, R. A. (1993) Genes Dev. 7, 2392-2404[Abstract] |
21. | Ewen, M. E., Faha, B., Harlow, E., and Livingston, D. M. (1992) Science 255, 85-87[Medline] [Order article via Infotrieve] |
22. | Faha, B., Ewen, M. E., Tsai, L. H., Livingston, D. M., and Harlow, E. (1992) Science 255, 87-90[Medline] [Order article via Infotrieve] |
23. | Lacy, S., and Whyte, P. (1997) Oncogene 14, 2395-2406[CrossRef][Medline] [Order article via Infotrieve] |
24. | Zhu, L., Harlow, E., and Dynlacht, B. D. (1995) Genes Dev. 9, 1740-1752[Abstract] |
25. | Beijersbergen, R. L., Kerkhoven, R. M., Zhu, L., Carlee, L., Voorhoeve, P. M., and Bernards, R. (1994) Genes Dev. 8, 2680-2690[Abstract] |
26. | Hijmans, E. M., Voorhoeve, P. M., Beijersbergen, R. L., van't Veer, L. J., and Bernards, R. (1995) Mol. Cell. Biol. 15, 3082-3089[Abstract] |
27. |
Ikeda, M. A.,
Jakoi, L.,
and Nevins, J. R.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
3215-3220 |
28. | Moberg, K., Starz, M. A., and Lees, J. A. (1996) Mol. Cell. Biol. 16, 1436-1449[Abstract] |
29. | Sardet, C., Vidal, M., Cobrinik, D., Geng, Y., Onufryk, C., Chen, A., and Weinberg, R. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2403-2407[Abstract] |
30. | Vairo, G., Livingston, D. M., and Ginsberg, D. (1995) Genes Dev. 9, 869-881[Abstract] |
31. | Chen, P., Riley, D. J., Chen, Y., and Lee, W. H. (1996) Genes Dev. 10, 2794-2804[Abstract] |
32. | Cobrinik, D., Lee, M. H., Hannon, G., Mulligan, G., Bronson, R. T., Dyson, N., Harlow, E., Beach, D., Weinberg, R. A., and Jacks, T. (1996) Genes Dev. 10, 1633-1644[Abstract] |
33. |
Hoshikawa, Y.,
Mori, A.,
Amimoto, K.,
Iwabe, K.,
and Hatakeyama, M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8574-8579 |
34. | Hurford, R. K., Jr., Cobrinik, D., Lee, M. H., and Dyson, N. (1997) Genes Dev. 11, 1447-1463[Abstract] |
35. | Knudsen, K. E., Weber, E., Arden, K. C., Cavenee, W. K., Feramisco, J. R., and Knudsen, E. S. (1999) Oncogene 18, 5239-5245[CrossRef][Medline] [Order article via Infotrieve] |
36. | Mori, A., Higashi, H., Hoshikawa, Y., Imamura, M., Asaka, M., and Hatakeyama, M. (1999) Oncogene 18, 6209-6221[CrossRef][Medline] [Order article via Infotrieve] |
37. | Beijersbergen, R. L., Carrel, L., Kerkhoven, R. M., and Bernards, R. (1995) Genes Dev. 9, 1340-1353[Abstract] |
38. |
Xiao, Z. X.,
Ginsberg, D.,
Ewen, M.,
and Livingston, D. M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4633-4637 |
39. | Hamel, P. A., Gill, R. M., Phillips, R. A., and Gallie, B. L. (1992) Oncogene 7, 693-701[Medline] [Order article via Infotrieve] |
40. | Zhu, L., Enders, G., Lees, J. A., Beijersbergen, R. L., Bernards, R., and Harlow, E. (1995) EMBO J. 14, 1904-1913[Abstract] |
41. | 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] |
42. | 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] |
43. | Morgenstern, L. P., and Land, H. (1990) Nucleic Acids Res. 18, 3587-3596[Abstract] |
44. | Ginsberg, D., Vairo, G., Chittenden, T., Xiao, Z., Xu, G., Wydner, K. L., DeCaprio, K. L., Lawrence, J. B., and Livingston, D. M. (1994) Genes Dev. 8, 2665-2679[Abstract] |
45. | Mayol, X., Garriga, J., and Grana, X. (1996) Oncogene 13, 237-246[Medline] [Order article via Infotrieve] |
46. | Whyte, P., Williamson, N. M., and Harlow, E. (1989) Cell 56, 67-75[Medline] [Order article via Infotrieve] |
47. | Hu, Q., Dyson, N., and Harlow, E. (1990) EMBO J. 9, 1147-1155[Abstract] |
48. | Moran, E., Zerler, B., Harrison, T. M., and Mathews, M. B. (1986) Mol. Cell. Biol. 6, 3470-3480[Medline] [Order article via Infotrieve] |
49. | Wang, H. H., Rikitake, Y., Carter, M. C., Yaciuk, P., Abraham, S. E., Zerler, B., and Moran, E. (1993) J. Virol. 67, 476-488[Abstract] |
50. |
Parreno, M.,
Garriga, J.,
Limon, A.,
Mayol, X.,
Beck, G. R., Jr.,
Moran, E.,
and Grana, X.
(2000)
J. Virol.
74,
3166-3176 |
51. | 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] |
52. | Hofmann, F., Martelli, F., Livingston, D. M., and Wang, Z. (1996) Genes Dev. 10, 2949-2959[Abstract] |
53. | Mayol, X., Garriga, J., and Grana, X. (1995) Oncogene 11, 801-808[Medline] [Order article via Infotrieve] |
54. | Huang, H. J., Yee, J. K., Shew, J. Y., Chen, P. L., Bookstein, R., Friedmann, T., Lee, E. Y., and Lee, W. H. (1988) Science 242, 1563-1566[Medline] [Order article via Infotrieve] |
55. | Qin, X. Q., Chittenden, T., Livingston, D. M., and Kaelin, W. G., Jr. (1992) Genes Dev. 6, 953-964[Abstract] |
56. | Templeton, D. J., Park, S. H., Lanier, L., and Weinberg, R. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3033-3037[Abstract] |
57. |
Sellers, W. R.,
Novitch, B. G.,
Miyake, S.,
Heith, A.,
Otterson, G. A.,
Kaye, F. J.,
Lassar, A. B.,
and Kaelin, W. G., Jr.
(1998)
Genes Dev.
12,
95-106 |
58. | Otterson, G. A., Khleif, S. N., Chen, W., Coxon, A. B., and Kaye, F. J. (1995) Oncogene 11, 1211-1216[Medline] [Order article via Infotrieve] |
59. | Lukas, J., Sorenson, C. S., Lukas, C., Santoni-Rugiu, E., and Bartek, J. (1999) Oncogene 18, 3930-3935[CrossRef][Medline] [Order article via Infotrieve] |
60. | Claudio, P. P., De Luca, A., Howard, C. M., Baldi, A., Firpo, E. J., Paggi, M. G., and Giordano, A. (1996) Cancer Res. 56, 2003-2008[Abstract] |
61. | Leone, G., DeGregori, J., Sears, R., Jakoi, L., and Nevins, J. R. (1997) Nature 387, 422-426[CrossRef][Medline] [Order article via Infotrieve] |
62. | Lukas, J., Herzinger, T., Hansen, K., Moroni, M. C., Resnitzky, D., Helin, K., Reed, S. I., and Bartek, J. (1997) Genes Dev. 11, 1479-1492[Abstract] |
63. | Ohtani, K., DeGregori, J., and Nevins, J. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12146-12150[Abstract] |
64. | Luo, R. X., Postigo, A. A., and Dean, D. C. (1998) Cell 92, 463-473[Medline] [Order article via Infotrieve] |
65. | Qin, X. Q., Livingston, D. M., Ewen, M., Sellers, W. R., Arany, Z., and Kaelin, W. G., Jr. (1995) Mol. Cell. Biol. 15, 742-755[Abstract] |
66. | Sellers, W. R., Rodgers, J. W., and Kaelin, W. G., Jr. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11544-11548[Abstract] |
67. | Sellers, W. R., and Kaelin, W. G., Jr. (1996) Biochim. Biophys. Acta 1288, M1-M5[CrossRef][Medline] [Order article via Infotrieve] |
68. |
Knudsen, E. S.,
Buckmaster, C.,
Chen, T.,
Feramisco, J. R.,
and Wang, J. Y. J.
(1998)
Genes Dev.
12,
2278-2292 |
69. | Chew, Y. P., Ellis, M., Wikie, S., and Mittnacht, S. (1998) Oncogene 17, 2177-2186[CrossRef][Medline] [Order article via Infotrieve] |
70. | Lukas, J., Parry, D., Agaard, L., Mann, D. J., Bartkova, J., Strauss, M., Peters, G., and Bartek, J. (1995) Nature 8, 503-506 |
71. | Medema, R. H., Herrera, R. E., Lam, F., and Weinberg, R. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6289-6293[Abstract] |
72. | 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] |
73. | Steeg, P. S., and Zhou, Q. (1998) Breast Cancer Res. Treat. 52, 17-28[CrossRef][Medline] [Order article via Infotrieve] |