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INTRODUCTION |
In the vertebrate central nervous system, neurons withdraw from
the cell cycle immediately after differentiation from their proliferative precursors, termed neuroepithelial stem cells.
Differentiated neurons are absolutely incompetent to divide even in the
presence of extracellular stimuli that promote cell cycle progression
of proliferative cells. Therefore, the permanent arrest of the cell cycle is the most fundamental feature displayed by differentiated neurons. However, little is known about the molecular mechanism whereby
neurons exit from the cell cycle and remain quiescent all of their
lives. In proliferative cells, the cell cycle is strictly controlled by
various regulatory proteins. Among them, E2F1 is a principal
transcription factor that controls cell cycle progression of dividing
cells (reviewed in Ref. 1). In the G1 phase, E2F1 is
inactivated by interacting with retinoblastoma protein
(Rb).1 During
G1-S transition, this interaction disappears by
phosphorylation of Rb, and released E2F1 activates transcription of its
target genes that are indispensable for DNA replication. Thus, the
Rb-E2F1 system is thought to govern exit from or passage through the
cell cycle (reviewed in Refs. 2 and 3). Several previous studies have
suggested that the Rb-E2F1 system is also involved in the growth arrest
associated with neuronal differentiation (4-6).
Necdin is a 325-amino acid residue protein encoded in a cDNA
sequence isolated from the library of neurally differentiated murine
embryonal carcinoma P19 cells (7). The necdin gene is expressed in
postmitotic neurons derived from embryonal carcinoma cells but not in
transformed cell lines originating from neuroblastomas and
pheochromocytomas even after they are induced to differentiate (8). In
developing mouse brain, the necdin gene is constitutively expressed in
neurons from early embryonic stages (e.g. embryonic day 10 at the forebrain) until late adult periods, whereas necdin mRNA is
undetectable in neuroepithelial stem cells in the neural tube (8, 9).
These observations suggest that necdin is expressed in postmitotic
neurons that are differentiated from their precursor cells in an
irreversible manner. Ectopic expression of necdin in NIH 3T3 cells
suppresses the cell growth without affecting cell viability (10).
Intriguingly, necdin binds to viral oncoproteins such as SV40 large T
antigen and adenovirus E1A (11). Moreover, necdin interacts with the
transcription factor E2F1 and suppresses E2F1-dependent
transcription (11). These characteristics of necdin resemble those of
Rb, although these proteins are structurally dissimilar.
Both necdin and Rb bind to the COOH-terminal transactivation domain of
E2F1 (11). This transactivation domain is also a target of MDM2, a
cellular oncogene product that binds to SV40 large T antigen (12). The
necdin-binding site on E2F1 is located in a close proximity to the
MDM2-binding site (11, 13), although MDM2, unlike necdin, enhances
E2F1-mediated transcriptional activation (13). Furthermore, MDM2
interacts with the transactivation domain of the tumor suppressor p53
and represses the p53-driven transcriptional activity (14, 15). There
is a substantial degree of homology between the E2F1 and p53 activation
domains (13, 14), suggesting a conservation of binding sites for
specific proteins. These findings prompted us to examine whether necdin
also interacts with p53.
In this study, we demonstrate that necdin does form a specific complex
with p53 to modulate p53-mediated biological functions. We have used
the yeast two-hybrid system and in vitro binding analyses to
map the necdin-binding region on p53. Using cultured cells that are
often adopted for functional analyses of p53, we have examined the
effects of necdin on p53-mediated transcriptional activation, growth
arrest, and apoptosis. The present results implicate that necdin is a
novel type of growth suppressor that targets both p53 and E2F1.
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EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Assay--
GAL4 DNA-binding domain vector
(pGBT9) and GAL4 activation domain vector (pGAD424) were purchased from
CLONTECH. cDNAs encoding various p53
subsequences were generated from full-length p53 cDNA (a gift from
Dr. T. Akiyama, University of Tokyo) by polymerase chain reaction using
specific primers and inserted into pGAD424. These vectors were
introduced along with pGBT9 harboring necdin cDNAs (11) into
Saccharomyces cerevisiae SFY526. Transformants were grown on
leucine- and tryptophan-deficient synthetic dropout medium plates, and
the colony lift filter assay for
-galactosidase activity was carried
out as recommended by CLONTECH. The reaction was
evaluated, and the results were separated into four ranks with the time
for the appearance of blue colonies at 30 °C: +++, less than 2 h; ++, 2-6 h; +, 6-12 h;
, remaining white over 12 h.
In Vitro Binding Assay--
Various fragments of p53 cDNA
were subcloned into pMALC2 (New England Biolabs) to make
maltose-binding protein (MBP) fusion proteins. The MBP fusion proteins
were affinity-purified with amylose resin as recommended by New England
Biolabs. Full-length necdin cDNA was subcloned into a baculovirus
transfer vector (pBlueBacHis2/B) (Invitrogen) for expression as a
His-tagged fusion protein. The transfer vector and a wild-type
baculovirus DNA (Bac-N-Blue AcMNPV) were introduced to Sf21
insect cells to obtain AcMNPV-Ndn. The recombinant His-tagged necdin
expressed in AcMNPV-Ndn-infected Sf9 insect cells was purified
using Probond metal-binding resin (Invitrogen). MBP-p53 fusion proteins
(1 µg) bound to amylose resin (5 µl) was incubated with purified
His-tagged necdin (100 ng) at 4 °C for 30 min in 0.5 ml of binding
buffer (20 mM Tris-HCl (pH 7.5), 200 mM NaCl,
and 1 mM EDTA). The resin was washed with the binding
buffer, and bound proteins were eluted with 20 mM maltose.
His-tagged necdin was separated by 10% SDS-polyacrylamide gel
electrophoresis, transferred to Immobilon membrane (Millipore) by
electroblotting, and detected with an anti-necdin antibody (C2)
(1:1000) (7) and peroxidase-conjugated anti-rabbit IgG (Cappel) using
chemiluminescence method (Renaissance, NEN Life Science Products).
Protein concentrations were determined by the Bradford method
(Bio-Rad).
Electrophoretic Mobility Shift Assay--
The oligonucleotide
probe containing the p53-binding site
(5'-TACAGAACATGTCTAAGCATGCTGGGG-3') was labeled with
[
-32P]ATP (Amersham Pharmacia Biotech) using T4
polynucleotide kinase. cDNAs encoding Myc tag p53 proteins were
cloned into pRc/CMV (Invitrogen) for expression of Myc-tagged p53
(amino acids 1-393) (pRc-Myc-p53), its NH2-terminal
deletion mutants p53 (amino acids 55-393) (pRc-Myc-p53 (55-393)), and
p53 (amino acids 75-393) (pRc-Myc-p53 (75-393)). The Myc tag was
added to the p53 subsequences using a 6 × Myc tag plasmid (a gift
from Dr. M. W. McBurney, University of Ottawa). The expression
vectors were transfected into SAOS-2 cells by the calcium phosphate
method (16). Nuclear extracts (2-4 µg of protein) were prepared by
the small scale extraction method (17) and incubated in a reaction
mixture (20 µl) containing 15 mM Hepes (pH 7.9), 1 mM EDTA, 4% Ficoll 400, 4 mg/ml bovine serum albumin, 50 mM KCl, 1 mM dithiothreitol, 1 µg of
sonicated salmon sperm DNA, and the 32P-labeled
oligonucleotide probe. For supershift analysis, the reaction mixture
was incubated at 4 °C for 60 min with hybridoma culture media (1:10)
containing anti-Myc (9E10) and anti-E1A (M73) antibodies or with
purified His-tagged necdin (100 ng). DNA binding activities were
analyzed by electrophoresis in a 4% nondenaturing polyacrylamide gel
run in a buffer (25 mM Tris borate (pH 8.2), 50 mM EDTA) at 4 °C.
Immunoprecipitation--
Combinations of expression vectors of
pRc-necdin, pRc-Myc-p53, and pRc-Myc-p53 (75-393) were transfected
into SAOS-2 cells, and nuclear extracts were prepared 48 h after
transfection. Aliquots (40 µg of protein) of extracts were incubated
for 2 h at 4 °C with the anti-Myc antibody (1:5) or with an
anti-necdin antibody C2 (1:125) in 250 µl of a buffer containing 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 0.1% Triton
X-100, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride. The antibody-protein complexes were pelleted with protein A-Sepharose (Amersham Pharmacia Biotech), separated by
SDS-polyacrylamide gel electrophoresis, and analyzed by immunoblotting
as described above.
Reporter Assay for p53-driven Transactivation--
A
2.4-kilobase pair fragment of human p21/WAF1 promoter (18) (a gift from
Dr. T. Akiyama, University of Tokyo) was subcloned into the
HindIII site of the luciferase reporter vector pGL2-basic (Promega). To construct the expression vectors for Myc-p53 (amino acids
1-37) (pRc-Myc-p53 (1-37)) and Myc-p53 (amino acids 35-62) (pRc-Myc-p53 (35-62)), respective p53 cDNA inserts in pMALC2 were subcloned into the Myc tag plasmid and then into pRc/CMV. The p53
expression vectors in combination with the expression vector for necdin
(amino acids 1-325) (pRc-necdin) (11) or necdin (amino acids 110-325)
(pRc-necdin
N) (11) were transfected into ~70% confluent SAOS-2
cells in 35-mm dishes by the calcium phosphate method (16).
Transfectants were harvested 36 h after transfection, and
luciferase activities were measured with a luminometer (Lumat LB9501,
Berthold) using a reagent kit (Toyo Ink, Tokyo). Transfection efficiency was normalized with co-transfected LacZ reporter plasmid (pRc-LacZ) (11).
Cell Growth Assay--
The colony formation assay using SAOS-2
cells was carried out as described previously (11). SAOS-2 cells grown
in 60-mm dishes were transfected with pRc-Myc-p53 (5 µg), pRc-necdin
(5 µg), or both (5 µg each) by the calcium phosphate method (16). G418 (500 µg/ml) was added to the culture medium 48 h after
transfection. The cells were incubated for 14 days, fixed with 10%
acetate/10% methanol for 15 min, and stained with 0.4% crystal violet
in 20% ethanol for 15 min to visualize the colonies. For
bromodeoxyuridine (BrdUrd) labeling, the 293 cells grown on coverslips
in 35-mm dishes were transfected with pRc-LacZ (0.5 µg) and
combinations of pRc-Myc-p53 (0.8 µg), pRc-necdin (1.6 µg), and
pRc-necdin
N (1.6 µg). The empty vector pRc/CMV was added to
equalize the amounts of transfected DNA (4 µg/assay). BrdUrd was
added to the medium (final concentration, 10 µM) 36 h after transfection, and the cells were fixed 2 h later with 70%
ethanol containing 20 mM glycine-HCl (pH 2.0) for 25 min at
20 °C. BrdUrd and
-galactosidase were detected by fluorescence
immunocytochemistry with an anti-BrdUrd monoclonal antibody (1:200)
(Roche Molecular Biochemicals) and an anti-
-galactosidase polyclonal
antibody (1:2000) (Chemicon). BrdUrd and
-galactosidase were
visualized with fluorescein isothiocyanate-conjugated goat anti-mouse
immunoglobulin (1:500) (Cappel) and rhodamine B-conjugated goat
anti-rabbit immunoglobulin (1:500) (Cappel), respectively, using a
fluorescence microscope (BX 50-34-FLAD 1, Olympus). BrdUrd-positive
cells among 100
-galactosidase-positive cells were counted.
Assay for p53-dependent Apoptosis--
U2OS cells
(~70% confluence) grown on coverslips in 35-mm dishes and
transfected with pRc-Myc-p53 and/or pRc-necdin. The cells were fixed at
48, 60, and 72 h with 4% formaldehyde (pH 7.4) for 25 min at
4 °C and treated with methanol for 20 min at 20 °C and incubated
with the anti-Myc antibody (1:10) and the anti-necdin antibody C2
(1:1000) for 1 h at 20 °C. Myc-tagged p53 and necdin were
visualized with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin and rhodamine B-conjugated anti-rabbit immunoglobulin, respectively. For Hoechst dye staining, the fixed cells were treated with 3.3 µM Hoechst 33342 for 15 min at 20 °C and
observed with the fluorescence microscope. p53-immunopositive cells in
10 nonadjacent fields (total area, 10 mm2) were counted.
Statistical significance was tested using Student's t test.
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RESULTS |
Necdin Interacts with the Transactivation Domain of p53--
We
first examined whether necdin interacts with p53 by the yeast
two-hybrid assay. Because the NH2-terminal sequences of
necdin fused to GAL4 DNA-binding protein stimulate the transcription even in the absence of GAL4 activation fusion proteins (11), we used an
NH2-terminally truncated form of necdin (amino acids 83-325) as a DNA-binding fusion protein in this assay. As shown in
Fig. 1, full-length p53 (amino acids
1-393) strongly interacted with necdin (amino acids 83-325). Because
the transactivation domain of p53 is located at the NH2
terminus, NH2-terminally truncated p53 mutants were tested
for necdin binding activities. An MDM2-binding site-deleted mutant
(amino acids 35-393) still retained the ability to bind to necdin, but
further deletions (amino acids 55-393 and 75-393) failed to bind to
necdin. These results suggest that necdin binds to the transactivation
domain, in which a region (amino acids 35-55) is indispensable.

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Fig. 1.
Yeast two-hybrid assay for interaction
between necdin and p53. cDNAs for full-length p53 and its
NH2-terminally truncated mutants were inserted in GAL4
activation domain fusion vector pGAD424 and introduced into yeast cells
along with NH2-terminally truncated necdin (amino acids
83-325) cDNA in GAL4 DNA-binding domain fusion vector pGBT9. The
-galactosidase activities were semi-quantified as described under
"Experimental Procedures." The domain structure of human p53 is
shown on top. Transactivation, transcriptional
activation domain; DNA binding, sequence-specific
DNA-binding region; MDM2, MDM2-binding region;
Pro, proline-rich domain; Oligo, oligomerization
region.
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We then tested the in vitro binding between necdin and p53
(Fig. 2). We prepared a series of p53
deletion mutants as MBP fusion proteins and confirmed that purified
MBP-p53 fusion proteins had predicted sizes of polypeptides (Fig.
2A). These MBP-p53 fusion proteins were incubated with
His-tagged necdin, and formation of p53-necdin complexes in
vitro was examined. As shown in Fig. 2B, both p53
(amino acids 1-393) and p53 (amino acids 35-393) bound to necdin,
whereas neither p53 (amino acids 55-393) nor p53 (amino acids 75-393)
had necdin binding activities. An NH2-terminal region of
p53 (amino acids 1-83), which encompasses the transactivation domain,
was competent to interact with necdin. An NH2-terminal subsequence of p53 (amino acids 1-37), which contains the entire MDM2-binding region, failed to interact with necdin, whereas a region
(amino acids 35-62) located between the MDM2-binding site and the
proline-rich region retained the necdin binding activity. These data
are schematically shown in Fig. 2C.

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Fig. 2.
In vitro binding assay for
interaction between necdin and p53. A, expression of
MBP-p53 fusion proteins. Purified MBP fusion proteins of p53 deletion
mutants were electrophoresed on a 10% SDS-polyacrylamide gel and
visualized by Coomassie Brilliant Blue staining. Molecular size markers
(in kDa) are at the left. B, in vitro
binding assay. Recombinant His-tagged necdin was prepared from
AcMNPV-Ndn-infected Sf9 insect cells. Purified MBP fusion
proteins were immobilized on amylose resin and incubated with
His-tagged necdin. Bound His-tagged necdin was detected by
immunoblotting using an anti-necdin antibody. Necdin,
His-tagged necdin (53 kDa). C, summary of interactions of
p53 deletion mutants with necdin. The results shown in B are
schematically presented.
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We have previously reported that the large T antigen, E1A, and E2F1
bind to the central domain of necdin (amino acids 83-292) (11). The
p53-binding region on necdin was determined using various necdin
deletion mutants in the two-hybrid system (Fig. 3). Both necdin (amino acids 83-325) and
necdin (amino acids 102-325) strongly bound to p53, but further
NH2-terminal truncations of necdin (amino acids 110-325,
167-325) failed to interact with p53. Although a COOH-terminally
truncated form of necdin (amino acids 83-292) retained the ability to
bind to p53, a further truncated form of necdin (amino acids 83-279)
had no p53 binding activity. These results suggest that the central
region of necdin is indispensable for the interaction with p53. This
region coincided with the region required for the interactions with the
large T antigen (11). Although p53 and E2F1 share the binding domain on
necdin, p53 showed a stronger necdin binding activity than E2F1 (Fig.
3).

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Fig. 3.
Determination of p53-binding domain of
necdin. cDNAs for necdin deletion mutants were inserted in
pGBT9 and introduced into yeast cells along with pGAD424 carrying p53
(amino acids 1-393) cDNA or mouse E2F1 (amino acids 55-430)
cDNA. E2F1 was used as a control. The -galactosidase activities
were semi-quantified as described under "Experimental Procedures."
PA, a domain rich in proline and acidic amino acids;
LT, the large T antigen-binding domain.
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Necdin Interacts with p53-DNA Complex--
We carried out the
electrophoretic mobility shift assay to examine whether necdin affects
the ability of p53 to bind to its specific DNA sequence (Fig.
4). SAOS-2 cells transfected with cDNAs for Myc-tagged fusion proteins of p53 (amino acids 1-393), p53 (amino acids 55-393), and p53 (amino acids 75-393) expressed the
products of predicted sizes (Fig. 4A). These three p53
species, all of which contain the sequence-specific DNA-binding region, formed complexes with p53 site-carrying DNA (Fig. 4B,
lanes 3, 6, and 9). The signals of
these p53-containing complexes were competed with excess amounts of the
oligonucleotide (data not shown). These major bands were supershifted
or diminished in density by the anti-Myc antibody (Fig. 4B,
lanes 5, 8, and 11) but not by the
anti-E1A antibody used as a negative control (Fig. 4B, lanes 4, 7, and 10), indicating that
shifted complexes contain Myc-tagged p53 proteins. Addition of purified
His-tagged necdin protein to the reaction mixture supershifted the
complexes containing p53 (amino acids 1-393) and p53 (amino acids
55-393) (Fig. 4C, lanes 2 and 4) but
not the complex containing p53 (amino acids 75-393) (Fig.
4C, lane 6). These results imply that the
necdin-p53 complexes are competent for DNA binding. In this analysis,
the NH2-terminally truncated mutant p53 (amino acids
55-393), which failed to interact with necdin in the two-hybrid and
in vitro binding assays (Figs. 1 and 2), bound to necdin. It
seems likely that a potential binding site present in p53 (amino acids
55-74) is manifested by a conformational change due to p53-DNA complex formation in the mobility shift assay. We were unable to reconstitute the necdin-p53 complex competent for DNA binding by using purified MBP-p53 fusion protein instead of nuclear extracts of p53
cDNA-transfected cells (data not shown), suggesting that additional
nuclear factors are required for the complex formation.

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Fig. 4.
Electrophoretic mobility shift assay for
interaction between necdin and p53. A, Myc-tagged p53
expressed in SAOS-2 cells. Nuclear extracts were prepared from SAOS-2
cells transfected with pRc-Myc-p53 (amino acids 1-393), pRc-Myc-p53
(amino acids 55-393), or pRc-Myc-p53 (amino acids 75-393). Expressed
Myc-tagged p53 proteins were detected by immunoblotting with an
anti-Myc antibody. B, electrophoretic mobility shift assay
for p53-DNA complexes. Nuclear extracts were prepared from SAOS-2 cells
transfected with above three vectors, and DNA binding activities of the
extracts were analyzed by the mobility shift assay using
32P-labeled p53 site oligonucleotide probe. Supershifts
with the complexes were examined by adding antibodies against Myc
( Myc) and E1A ( E1A) (a negative control) to
the reaction mixture. Protein-DNA complexes were electrophoresed on a
4% nondenaturing polyacrylamide gel. C, interactions of
necdin with p53-DNA complex. Recombinant His-tagged necdin protein was
added to the DNA-binding reaction mixture. Note the supershifted bands
of necdin-p53 (amino acids 1-393) and necdin-p53 (amino acids
55-393). S, specific shifts; NS, nonspecific
shifts; F, free probe (in B and
C).
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Necdin Represses p53-driven Transcription--
We examined the
effect of necdin on p53-driven transactivation using SAOS-2 cells. As
shown in Fig. 5A, necdin
formed a nuclear complex with Myc-tagged p53 in SAOS-2 cells. Necdin
bound to full-length p53 (amino acids 1-393) but not to the
NH2-terminally truncated p53 (amino acids 75-393). We then
transfected full-length necdin and p53 into SAOS-2 cells with a
luciferase reporter vector driven by the p21/WAF1 promoter (18), which
contains the p53-binding site. As shown in Fig. 5B, necdin
had no appreciable effect on the reporter activity in the absence of
co-transfected p53, whereas the p53-stimulated activity (6-fold of the
basal activity) was suppressed by full-length necdin in a
dose-dependent manner. Necdin (amino acids 110-393), which
lacks the p53 binding activity, had no suppressive effect. The
suppression of p53-dependent transcriptional activity by
necdin was not a result of reduced quantities of p53 protein because
co-expressed necdin did not affect the p53 protein level in SAOS-2
cells (data not shown). As shown in Fig. 5C, necdin-induced suppression was partially restored by Myc-tagged p53 (amino acids 35-62), which contains the minimal necdin-binding region but not by
Myc-tagged p53 (amino acids 1-37) lacking the necdin-binding region.
These results suggest that necdin-induced suppression of p53-driven
transcription is mediated through the specific sequence of p53 (amino
acids 35-62).

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Fig. 5.
Necdin represses p53-dependent
transcriptional activation. A, detection of necdin-p53
complex by immunoprecipitation. Nuclear extracts were prepared from
SAOS-2 cells transfected with pRc-necdin (Necdin) in
combination with pRc-Myc-p53 (Myc-p53) or pRc-Myc-p53
(75-393) (Myc-p53 N). Left, immunoprecipitated
with anti-Myc antibody and detected with anti-necdin antibody;
right, immunoprecipitated with anti-necdin antibody and
detected with anti-Myc antibody. B, suppression of
p53-driven transcriptional activity by necdin. A luciferase reporter
vector carrying the p21/WAF1 promoter was transfected into SAOS-2 cells
with varying amounts (in µg) of pRc-necdin (Necdin) or
pRc-necdin N (Necdin N) in combination with pRc-Myc-p53
(Myc-p53). The luciferase activities were measured with a
luminometer. C, effects of p53 NH2-terminal
subsequences on the necdin-induced transcriptional suppression.
Combinations of pRc-Myc-p53 (Myc-p53), pRc-necdin
(Necdin), pRc-Myc-p53 (35-62) (Myc-p53(35-62)),
and pRc-Myc-p53 (1-37) (Myc-p53(1-37)) were transfected
into SAOS-2 cells along with the reporter vector. The amount of each
plasmid is in µg/assay. The total amount of plasmids was adjusted to
4 µg/assay by adding pRc/CMV. The mean values are presented
(n = 3).
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Necdin Has No Inhibitory Effect on p53-induced Growth
Suppression--
Both necdin and p53 induce growth suppression of
SAOS-2 cells (11, 19). The p21/WAF1 protein is a major effector that mediates p53-induced growth suppression (20), and transcription of the
p21/WAF1 gene was suppressed by co-expressed necdin as shown in Fig. 5.
Thus, we examined whether the growth suppressive effect of p53 is
inhibited by co-expressed necdin in SAOS-2 cells. Both p53 and necdin
suppressed the colony formation, and these two proteins in combination
reduced the number of colonies in an additive manner (Fig.
6A). In SAOS-2 cells,
p53-mediated suppression of colony formation can occur as a result of
cell cycle arrest, apoptosis, or both, although it is difficult to
differentiate these two phenomena in this system (21). On the other
hand, growth of embryonic kidney 293 cells is markedly reduced without inducing apoptosis by overexpressed p53 (22). We thus tested the effect
of necdin on p53-induced growth suppression of the 293 cells. The 293 cells were transiently transfected with
-galactosidase-expressing vector and combinations of expression vectors for Myc-tagged
full-length p53, full-length necdin, and necdin (amino acids 110-325).
The BrdUrd incorporation was analyzed by immunocytochemistry to
estimate the cell population in S phase. Both necdin and Myc-p53
reduced the number of BrdUrd-positive cells among
-galactosidase-positive cells, whereas necdin and p53 in combination
caused a larger reduction in the number of BrdUrd-positive cells than
either protein (Fig. 6B). In contrast, the
NH2-terminally truncated necdin (amino acids 110-325),
which is incapable of interacting with E2F1, p53, the large T antigen,
or E1A (Ref. 11 and Fig. 3), had little or no effect on the cell number
in S phase. These results suggest that necdin exerts no inhibitory
effect on p53-induced growth arrest, although it suppresses p53-driven
transcription.

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Fig. 6.
Effects of necdin on p53-mediated growth
suppression. A, colony formation analysis. SAOS-2 cells
grown in 60-mm dishes were transfected with empty pRc/CMV
(Ctl), pRc-necdin (Necdin) (5 µg), pRc-Myc-p53
(p53) (5 µg), or both (Necdin/p53) (5 µg
each). The total amount of plasmids was adjusted to 10 µg/assay by
adding pRc/CMV. Cells were cultured in the presence of G418 for 14 days, and the drug-resistant colonies were visualized by crystal violet
staining. B, BrdUrd incorporation analysis. The 293 cells
were transfected with pRc-LacZ in combination with pRc-Myc-p53
(Myc-p53), pRc-necdin (Necdin), and pRc-necdin
(110-325) (Necdin N). The amount of each plasmid is in
µg/assay. The total amount of plasmids was adjusted to 4 µg/assay
by adding pRc/CMV. Cells were incubated for 36 h, treated with
BrdUrd for 2 h, and double stained for BrdUrd and
-galactosidase. BrdUrd-positive cells among 100 -galactosidase-positive cells were counted (BrdUrd-positive
cells (%)). Each value represents the mean ± S.E.
(n = 3). The asterisk indicates a value
significantly different (p < 0.02) from the value of
p53 alone or necdin alone.
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Necdin Blocks p53-induced Apoptosis--
Overexpression of p53 in
U2OS osteosarcoma cells induces transient cell cycle arrest followed by
rapid apoptotic cell death (23). We used this cell line to examine the
effect of necdin on p53-induced apoptosis (Fig.
7). U2OS cells were transfected with
Myc-p53 alone or in combination with necdin, and morphological changes
of p53-expressing cells were examined by fluorescent
immunocytochemistry. p53-positive cells were morphologically intact
48 h after transfection, and immunoreactive p53 was localized to
the nuclei (Fig. 7, A and B). Co-expression of
necdin had no appreciable effects on the p53-expressing cells. After
60 h, the p53-accumulating cells had abnormal nuclei exhibiting
condensation and fragmentation, which are characteristic of apoptosis
(Fig. 7, C and D). However, co-expression of
necdin blocked the apoptotic changes of p53-positive cells even 72 h after transfection. In these transfectants, both p53 and necdin were
present in the nuclei that appeared morphologically intact (Fig. 7,
E and F). The protective effect of necdin on
p53-inducible apoptosis was quantified by counting the transfectants
having p53-accumulating nuclei (Fig. 7G). The number of
p53-positive cells was markedly reduced at 60 and 72 h, but the
reduction was significantly restored by co-expressed necdin. These
results suggest that necdin inhibits p53-induced apoptosis of U2OS
cells.

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Fig. 7.
Inhibition of p53-induced apoptosis by necdin
in U2OS cells. A-F, morphological changes of p53
cDNA transfectants. U2OS cells were transfected with pRc-Myc-p53
alone (A, C, and D) or in combination
with pRc-necdin (B, E, and F). Cells
were fixed at each time point and stained for Myc-p53 (A,
B, C, and E). Morphological changes of
the nuclei were examined by Hoechst dye staining (D).
A and B, p53 in Myc-p53 cDNA-transfected
cells without (A) or with (B) co-transfection of
necdin cDNA at 48 h; C and D,
double-staining for p53 (C) and Hoechst dye-reactive DNA
(D) in the cells transfected with Myc-p53 cDNA alone at
60 h. E and F, double-staining for p53
(E) and necdin (F) in the cells transfected with
both p53 and necdin cDNAs at 72 h. Note that p53-expressing
cells transfected with p53 cDNA alone have condensed and fragmented
nuclei (arrows in C and D) at 60 h, whereas the cells co-transfected with necdin cDNA have
morphologically intact nuclei (arrowheads in E
and F). Scale bar, 40 µm (in A).
G, quantification of p53 immunoreactive cells. U2OS cells
were transfected with pRc-Myc-p53 alone (p53) or in
combination with pRc-necdin (p53/Necdin), and the
p53-positive cells in 10 nonadjacent fields (total area, 10 mm2) were counted. Each value represents the mean ± S.E. (n = 3). The asterisk indicates a value
significantly different (p < 0.01) from the values of
p53 alone.
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DISCUSSION |
The present study has demonstrated that necdin is a novel type of
growth suppressor that interacts with the transactivation domain of
p53. Necdin can be placed into a group of p53-interacting proteins such
as MDM2 (14, 15), WT-1 (24), Ref-1 (25), p300 (26), and
p33ING1 (27), all of which modify p53-mediated biological
functions. We found that p53-driven transactivation of the p21/WAF1
promoter was greatly suppressed by necdin (Fig. 6). Furthermore, the
necdin-p53 complex was still competent for DNA binding (Fig. 5). These
features resemble those of the Rb-E2F1 system. The Rb-E2F1 complex,
which is competent for DNA binding, inhibits transcription of various E2F1-responsive genes that are required for DNA replication. By analogy
with the Rb-E2F1 system, we infer that synchronized expression of a
battery of genes possessing the p53 site-carrying promoters is
controlled by the necdin-p53 complex.
p21/WAF1 is considered as a major mediator through which p53 suppresses
cell proliferation (20). Although necdin suppressed p53-dependent stimulation of the p21/WAF1 promoter, necdin
had no inhibitory effects on p53-mediated growth suppression of SAOS-2 cells (Figs. 5 and 6). This may be because p53 exerts its growth suppression through a p21/WAF1-independent process in the presence of
necdin. However, we cannot rule out the possibility that necdin inhibits growth suppression mediated by p53 that conversely potentiates the growth suppressive effect of necdin. During mouse embryogenesis, the p21/WAF1 gene is expressed during terminal differentiation of
skeletal muscles, cartilage, skin, and nasal epithelium, whereas p21/WAF1 mRNA is absent from the embryonic brain and spinal cord (28). Because necdin is highly expressed in postmitotic neurons in the
embryonic brain (9), it is possible that necdin substantially suppresses p53-dependent p21/WAF1 expression during
neuronal differentiation.
The present study also demonstrated that necdin blocked p53-induced
apoptosis (Fig. 7). These characteristics resemble those of the Wilms'
tumor suppressor WT-1. WT-1 binds to p53 and inhibits p53-induced
apoptosis without affecting the p53-mediated cell cycle arrest (23).
However, WT-1, unlike necdin, shows enhancement of
p53-dependent transactivation. On the other hand, apoptosis of HeLa cells mediated by p53 is significantly suppressed by
co-expression of Rb, which does not interfere with the transcriptional
activity of p53 (29). Thus, necdin may be a novel type of growth
suppressor that inhibits both p53-dependent transactivation
and apoptosis. The necdin-binding region of p53 was mapped to amino
acids 35-55 and 35-62 as determined by the two-hybrid assay and by
the in vitro binding assay, respectively. This region
resides between the binding site for TATA box-binding
protein-associated factor (30, 31) and the proline-rich domain. Recent
studies have shown that p53 mutants devoid of the proline-rich domain
lack apoptotic activities but can still mediate growth arrest (21, 32),
indicating that the proline-rich domain is responsible for p53-mediated
apoptosis. Thus, the necdin-binding domain of p53 is appropriately
situated to exert negative effects on both transactivation (through the
TATA box-binding protein-associated factor-binding site) and apoptosis
(through the proline-rich domain).
The necdin gene is expressed in postmitotic neurons but not in
proliferative neuroepithelial stem cells (8, 9). Thus, necdin may not
modulate p53-mediated cellular responses in the same manner as it does
in replicating cells. Although p53 has been implicated in the induction
of cell cycle arrest and apoptosis of dividing cells, several lines of
evidence suggest that p53 is also involved in differentiation and
apoptosis of postmitotic neurons. Infection of cultured cells with a
recombinant retrovirus encoding a dominant negative inhibitor of
endogenous p53 inhibits neuronal differentiation of PC12
pheochromocytoma cells and protects cultured primary neurons from
spontaneous apoptotic death (33). Mice lacking the functional p53 gene
have an unusually large number of birth defects such as exencephaly and
impaired neural tube closure (34, 35). These findings suggest that p53
plays a regulatory role in directing neurons toward differentiation and death at early stages of neuronal development. Necdin may prevent nascent neurons from p53-mediated apoptosis and keep them viable during
neurogenesis. On the other hand, p53 mediates or induces apoptosis of
fully differentiated neurons. For example, p53 overexpression by
adenovirus-mediated gene delivery induces neuronal death with features
characteristic of apoptosis (36). Wild-type (p53+/+)
neurons are severely damaged by exposure to excitotoxins such as
glutamate and kainate as well as by treatment with the topoisomerase I
inhibitor camptothesin, whereas p53-deficient (p53
/
)
neurons are resistant to these compounds (37, 38). Because neurons
continue to express necdin until late adult stages of mice (8), necdin
potentially blocks p53-mediated apoptosis of fully differentiated
neurons throughout their lives. We are now studying the functional
interactions between necdin and p53 in postmitotic neurons using
adenovirus-mediated gene transfer and cultured neurons derived from
necdin-deficient mice.
The human necdin gene is localized to chromosome 15q11.2-q12 within the
Prader-Willi syndrome (PWS) deletion region and contains CpG-rich
regulatory sequences in the 5'-end region (39). Recent studies have
demonstrated that this gene is maternally imprinted and deleted in PWS
(40, 41). Major symptoms of PWS (e.g. hypogonadism, feeding
problems, and gross obesity) have been suggested to be hypothalamic in
origin. Among discrete brain regions of mice, necdin mRNA is most
abundant in the hypothalamus (9). Therefore, it is tempting to
speculate that deficiency of necdin gene expression promotes
p53-dependent neuronal death in the brain, especially in
the hypothalamus, to cause various types of abnormalities seen in PWS.
Studies using various neuronal cell lines and necdin-deficient mice are
currently in progress in our laboratory to clarify physiological and
pathological implications of necdin-p53 interactions in neuronal functions.