From the Laboratorio Oncogenesi Molecolare, Istituto
Regina Elena, Rome 00158, the
Dipartimento di Biologia Animale
Università di Modena e Reggio, Modena 41100, Italy, and
the ** Medizinische Klinik II, Max-Burger-Forschungszentrum, Universitat
Leipzig, Leipzig D-04103, Germany
Received for publication, July 10, 2000, and in revised form, October 31, 2000
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ABSTRACT |
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During normal cell cycles, the function of
mitotic cyclin-cdk1 complexes, as well as of cdc25C phosphatase,
is required for G2 phase progression. Accordingly,
the G2 arrest induced by DNA damage is associated with a
down-regulation of mitotic cyclins, cdk1, and cdc25C phosphatase
expression. We found that the promoter activity of these genes is
repressed in the G2 arrest induced by DNA damage. We asked
whether the CCAAT-binding NF-Y modulates mitotic
cyclins, cdk1, and cdc25C gene
transcription during this type of G2 arrest. In our
experimental conditions, the integrity of the CCAAT boxes of
cyclin B1, cyclin B2, and cdc25C
promoters, as well as the presence of a functional NF-Y complex, is
strictly required for the transcriptional inhibition of these
promoters. Furthermore, a dominant-negative p53 protein, impairing
doxorubicin-induced G2 arrest, prevents transcriptional
down-regulation of the mitotic cyclins, cdk1,
and cdc25C genes. We conclude that, as already demonstrated
for cdk1, NF-Y mediates the transcriptional inhibition of
the mitotic cyclins and the cdc25C genes during
p53-dependent G2 arrest induced by DNA damage.
These data suggest a transcriptional regulatory role of NF-Y in the
G2 checkpoint after DNA damage.
In mammalian cells, progression through the G2 phase
of the cell cycle is mediated by the activity of a specific set of
proteins, which includes mitotic cyclins A, B1, and B2, mitotic kinase
cdk1 (alias p34cdc2), and mitotic phosphatase
cdc25C. The kinase activity of cdk1 during the G2 phase is
dependent on its dephosphorylated status of specific residues triggered
by the phosphatase activity of cdc25C protein (1-6), as well as on the
levels of cyclins A and Bs. In proliferating cells, oscillations of
mitotic cyclin amounts are tightly regulated at the transcriptional
level (7-12). In particular, activation of cdk1 does not occur until
sufficient cyclin B1 protein is synthesized (13). The accumulation of
mitotic cyclins and cdk1 correlates with nascent gene expression, and their mRNAs can only be detected in particular phases of the cell cycle (2, 7, 10, 14).
Cell cycle progression through the G2 phase is controlled
by the G2 checkpoint. This checkpoint ensures correct DNA
synthesis during cell proliferation and genome integrity after DNA
damage. In the latter condition, cells can arrest at the G1
and/or the G2 checkpoint, depending on cell type, cell
cycle phase, and checkpoint integrity (15-17). The
G1/G2 arrest after DNA damage is regulated, at
least in part, by the activities of the tumor suppressor gene p53. Indeed, The CCAAT motif, G/AG/ACCAATC/GA/GC/G, is present in 30% of the
eukaryotic promoters of tissue-specific, housekeeping, and cell
cycle-regulatory classes of genes (25).
NF-Y1 has been shown to bind
to more than 120 CCAAT-containing promoters (26). It is composed of
three subunits, NF-YA, -B, and -C, whose highly conserved genes have
been cloned in mammals, yeast, plants, and parasites (27-31). All
three subunits are required for CCAAT binding (32, 33). The promoters
of cyclin A, cyclin B1, cyclin B2,
cdk1, and cdc25C genes all contain CCAAT boxes,
and it has been demonstrated that NF-Y modulates, at least in part,
their activity during the cell cycle (11, 33, 34). Thus, we asked whether NF-Y is involved in the modulation of these promoter activities during the G2 arrest induced by DNA damage.
For this purpose, we induced a G2 arrest by doxorubicin
(Adriamycin; ADR)-mediated DNA damage in C2C12 nontransformed skeletal muscle cells, which possess a wild type p53 (35). We found that ADR
treatment down-regulates the promoter activities of the mitotic cyclins, cdk1, and cdc25C genes. This
down-regulation involves a molecular mechanism requiring, at least in
part, the CCAAT boxes and the transcription factor NF-Y. By using a
dominant-negative p53 protein (DD-p53), we also show that the
NF-Y-dependent down-regulation of mitotic
cyclins, cdk1, and cdc25C occurs only
in the presence of a functional p53 protein.
Cell Cycle Analysis--
DNA distribution analysis of propidium
iodide-stained cells was performed according to standard procedures.
For each sample 104 events were analyzed by an Epics
cytofluorometer (Coulter). DNA content and cell cycle distribution were
determined using computer-assisted analysis.
Mitotic Index--
The cells were washed twice with phosphate
buffered saline (PBS) and fixed with 2% formaldehyde in PBS for 10 min
at room temperature. After permeabilization with 0.25% Triton X-100,
nuclei were stained with Hoechst 33342.
Terminal Nucleotidyl Transferase
Assays--
Approximately 2 × 104 cells were
cytocentrifuged onto glass slides, and cytospin preparations were air
dried, fixed with paraformaldehyde solution (4% in PBS, pH 7.4) for 30 min at room temperature, rehydrated with PBS, incubated in
permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate)
for 2 min on ice (4 °C), and preblocked for 30 min at room
temperature. Cells were incubated with fluorescein-conjugated dUTP
terminal deoxynucleotide transferase mixture for the assay (Roche
Diagnostics S.p.A, Monza, Italy) following the manufacturer's conditions, counterstained with 0.1 µg/ml Hoechst 33258 dye for 2 min, and mounted with coverslips in 25% glycerol in PBS. Apoptosis was
quantified by determining the percentage of stained terminal nucleotidyl transferase-positive cells on a total of 400 cells.
Reporter Plasmid Constructs--
p332B1CAT, p240B1CAT (10),
pmtupCCAAT, pmtdownCCAAT, pmtup/downCCAAT (33), pcycA LUC (36),
B2-Luci, Y1,2 m, Y1,2,3 m (34), pcdk1CAT (14), pcycD1 (37), and pE Cell Culture, DNA Transfections, CAT and LUC Assay--
Cells
were cultured in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum and antibiotics. Transient DNA transfections were
performed using the calcium phosphate precipitation technique (38)
modified by Chen and Okayama (39). In each 60-mm plate, 1.5 × 105 cells were transfected with precipitates containing
10 µg of each reporter construct and 1 µg of CMV
Western Blot Analysis--
Total cell lysates were obtained by
freeze and thaw in 0.4 M NaCl buffer containing 50 mM Tris-HCl, pH 8, 25% glycerol, 1 mM EDTA,
2.5 mM dithiothreitol, 10 µg/ml leupeptin, 4 µg/ml
pepstatin, 5 µg/ml aprotinin, 50 mM NaF, and 1 mM orthovanadate. 50 µg of protein/sample was separated
on SDS-polyacrylamide gel electrophoresis (12.5%) and electroblotted
onto nitrocellulose. After blocking in 5% nonfat dried milk, filters
were immunoreacted with 0.1 µg/ml anti-cyclin B1 rabbit polyclonal
antibody, 0.1 µg/ml anti-cdc2p34 rabbit
polyclonal antibody, 0.1 µg/ml anti-cyclin A rabbit polyclonal antibody, 1 µg/ml anti-cdk7 rabbit polyclonal antibody, 2 µg/ml anti-cdc25C rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.), 0.2 µg/ml anti-cdk9 rabbit polyclonal antibody (kindly provided by Antonio Giordano and Antonio DeLuca), 1 µg/ml anti-p53 sheep polyclonal antibody (Calbiochem, Inalco SPA, Milano Italy), and
0.25 µg/ml anti-Hsp70 mouse monoclonal antibody (Stress Gen, Biotechnologies Corp., Victoria Canada), following the manufacturer's directions. Peroxidase activity of the appropriate secondary antibodies was visualized by enhanced chemiluminescence detection system (Amersham, Little Chalfont, U. K.).
Viruses and Infections--
LXSN-producing cells (GP+E LXSN)
(42) and LDDSN producing cells (GP+E LDDSN) (kindly provided by Prof.
M. Oren) were employed. Approximately 2.5 × 105
Polybrene-treated C2C12 cells were incubated for 12 h with
virus-containing supernatants. Infected cells were selected in the
presence of 1.5 mg/ml G418.
ADR-mediated DNA Damage Induces G2 Arrest in C2C12
Cells--
It has been reported that in cells expressing wild type p53
protein, ADR-mediated DNA damage induces G2 arrest (43). To identify experimental conditions that efficiently induce an
accumulation of the cells in the G2 phase, dose-response
curves to ADR were performed on C2C12 cells. These conditions were
reached by a 22-h treatment with 0.5 µg/ml ADR (data not shown). The
following experiments were all performed under these conditions.
The typical DNA content analysis of untreated (panel A) and
ADR-treated C2C12 cells (panel B) is reported in Fig.
1. Accumulation of ADR-treated cells in
G2/M phases is accompanied by a decrease in the fraction of
the cells in the S phase of the cell cycle. To evaluate the amount of
apoptosis, the terminal nucleotidyl transferase assay was performed. As
shown in Table I, no significant amount
of apoptotic cells was found after ADR treatment. Finally, to
distinguish between G2 and M phases, the mitotic index was evaluated. Fewer than 2% of the ADR-treated C2C12 cells presented mitotic figures (Table I). We conclude that in our experimental conditions, ADR treatment generates significant accumulation of C2C12
cells in the G2 phase of the cell cycle (19%
versus 50% approximately).
Mitotic Cyclins, cdk1, and cdc25C Promoter Activities Are
Down-regulated in ADR-treated C2C12 Cells--
It has been
demonstrated recently that during the G2 arrest induced by
ADR-mediated DNA damage, the expression of mitotic cyclins and cdk1 is
down-regulated (22, 43). Accordingly, we found that ADR-treated C2C12
cells express lower levels of mitotic cyclin-cdk complexes, as well as
cdc25C phosphatase, both at protein and mRNA levels (data not
shown). To investigate whether the down-regulation of the mitotic
cyclins, cdk1, and cdc25C expression after ADR treatment involves
mechanisms acting at the transcriptional level, we evaluated the
promoter activities of these genes. For this purpose different promoter
constructs were transfected in C2C12 cells, and promoter activities
were evaluated before and after ADR treatment. Both transient and
stable transfectants were produced. Similar results were obtained
independently of the type of transfection. The following promoter
constructs were employed: (i) pcycA LUC, containing human cyclin
A promoter fragment from Mutations of the CCAAT Boxes Impair the ADR-mediated
Down-regulation of the cyclin B1, cyclin B2, and cdc25C Promoter
Activities--
All of the above tested promoters contain a common DNA
sequence, the CCAAT box. It has been demonstrated previously that the CCAAT boxes are key sequences for the promoter activities of these genes (11, 12, 33, 34). Thus, we investigated the functional contribution of these elements to the down-regulation of the promoter activities upon ADR treatment. For this purpose, constructs with mutations of CCAAT to CTCGA in the cyclin B1 promoter, CCAAT
to TTACT in the cyclin B2 promoter, and deletion of the
CCAAT boxes in the cdc25C promoter were employed. In the
context of p240B1CAT vector, three different CAT reporter constructs
carrying single or double mutations were employed. The pmtupCCAAT
construct carries a single mutation in the upstream CCAAT box, whereas
the pmtdownCCAAT construct carries a single mutation in the downstream
element. The pmtup/downCCAAT construct carries a mutation in both CCAAT boxes (33). We used, in the context of the cyclin B2
promoter, two luciferase reporter constructs carrying two mutated
elements (Y1,2) or all (three) mutated CCAAT boxes (Y1,2,3 m) (34). We also used, in the context of the cdc25C promoter, a
luciferase reporter construct carrying a deletion spanning a region
from Dominant-negative NF-Y Protein Abrogates the Down-regulation of the
cyclin B1, cyclin B2, cdc25C, and cdk1 Promoters after the ADR
Treatment--
The CCAAT motif is recognized by the NF-Y transcription
factor (26). To evaluate the role of the NF-Y complex on the
ADR-mediated down-regulation of the cyclin B1, cyclin
B2, cdc25C, and cdk1 promoters we employed a
dominant-negative NF-Y vector, YA13 m29 (44). Upon transfection in
mammalian cells, this vector expresses a mutant protein containing a
triple amino acids substitution in the NF-YA DNA binding subdomain
enabling the subunit to interact with the NF-YB·NF-YC dimer. The
resulting trimer is inactive in terms of CCAAT recognition. The
dominant-negative NF-Y was cotransfected with p332B1CAT, p240B1CAT,
B2-Luci, pcdc25C-wt-luci, and pcdk1CAT constructs in C2C12 cells, and
the promoter activities were determined in the presence or absence of
ADR. As demonstrated previously (33, 34), YA13 m29 decreased the
activities of all tested promoters. However, the dominant-negative YA13
m29 completely impairs the ADR-mediated down-regulation of each
promoter (Fig. 6). These results indicate
that a functional NF-Y complex is required for the down-regulation of
cyclin B1, cyclin B2, cdc25C,
and cdk1 and suggest that NF-Y has a role in the modulation
of the transcription of these genes during induced G2
arrest.
C2C12 Cells Expressing Dominant-negative p53 Protein Escape the
G2 Arrest Induced by ADR Treatment and Enter
Mitosis--
It has been reported previously that p53 is essential to
sustain G2 arrest after
Dominant-negative p53 Protein Abrogates the ADR-mediated
Down-regulation of Mitotic Cyclin Complexes--
It has been reported
that exogenous wild type p53 overexpression down-regulates cyclin B1,
cyclin A, and cdk1 expression both at transcriptional and translational
levels (19, 20, 23, 24, 47). To assess whether the down-regulation of
these genes after ADR treatment in C2C12 cells requires the presence of
wild type p53, Western blot analysis was performed on the C2C12 LDDSN cells and, as control, on C2C12 cells infected with the insertless retroviral vector, LXSN (C2C12 LXSN). As shown in Fig.
8A, after 22 h of ADR
treatment, there is a substantial decrease of cyclin A, cyclin B1, and
cdc25C proteins in these cells. Although to a lesser extent, there is
also a decrease of cdk1 expression. As expected, p53 levels are
increased because of a stabilization of the protein which occurs when
the DD-p53 miniprotein is expressed (46). DD-p53 expression
significantly reduced the ADR-mediated down-regulation of cyclin A,
cyclin B1, cdk1, and cdc25C. Indeed, after DNA damage, cdk1 expression
is completely rescued by the presence of DD-p53, whereas 74% of cyclin
A, 50% of cyclin B1 and cdc25C expression was rescued in the same
conditions, as measured by densitometric analysis. We also tested, in
the same experiments, the expression of cdk7 and cdk9 kinases. Both of
these kinases are involved in transcriptional control through the
phosphorylation of the RNA polymerase II C-terminal domain (48). As
shown in Fig. 8A, after 22 h of ADR treatment, there is
a substantial decrease of cdk7 protein in C2C12 LXSN cells, and DD-p53
expression significantly reduced this down-regulation. The expression
of cdk9 is not affected at all by the treatment irrespective of the
presence or absence of a functional p53 protein. These data confirm
that mitotic cyclin-cdk complexes are modulated by wild type p53
protein upon ADR-mediated DNA damage and demonstrate also that cdc25C
and cdk7 expression is modulated by wild type p53 in these experimental
conditions.
Thereafter, the activity of mitotic cyclins,
cdk1, and cdc25C promoters was evaluated in
ADR-treated and untreated C2C12 LDDSN cells. As shown in Fig.
8B, the down-regulation of promoter activities induced by
ADR treatment was inhibited substantially in C2C12 cells expressing a
dominant-negative p53 protein. The cyclins and
cdc25C promoters were more sensitive to the presence of a functional p53 protein than the cdk1 promoter. Altogether
these data demonstrate that the down-regulation of transcription of mitotic cyclins, cdk1, and cdc25C
genes mediated by ADR-induced DNA damage depends on the presence of a
functional p53 protein.
The progression through the G2 phase of the cell cycle
is regulated in part by the cyclin A-cdk1 and cyclin B-cdk1 mitotic complexes (49, 50). Although it has been described that after DNA
damage the mitotic entry is inhibited (18, 43, 51), the molecular
mechanism sustaining the G2 arrest is not completely elucidated. In this report, we show that during the ADR-mediated G2 arrest, a decrease of protein expression levels of
cyclin A, cyclin B1, cdk1, and cdc25C caused, at least in part, by a
transcriptional level of regulation, is observed. Indeed, we
demonstrate that after ADR treatment the promoter activities of the
cyclin A, cyclin B1, cyclin B2,
cdk1, and cdc25C genes are down-regulated. These results indicate that key molecules that control G2/M
transition in the normal cell cycle are transcriptionally modulated
during the G2 checkpoint induced by DNA damage.
The repression of cyclin B1, cyclin B2, and
cdc25C promoters, occurring in the G2 arrest,
requires the integrity of the CCAAT boxes present in the 5' region of
these genes, suggesting a role for the transcription factor NF-Y (Figs.
3 and 4). It has been demonstrated previously that NF-Y binds the CCAAT
boxes of cyclin B1, cyclin B2, and
cdc25C promoters (11, 33, 34). Here we show that a
functional NF-Y complex is required for the inhibition of cyclin
B1, cyclin B2, and cdc25C promoters during
G2 arrest. Indeed, the expression of a dominant-negative
NF-YA abolishes the ADR-mediated repression of these promoters.
Moreover, as already demonstrated, this is true also for the
cdk1 promoter (24).
The down-regulation of expression and transcription of mitotic
cyclin-cdk1 complexes, as well as of cdc25C phosphatase, after DNA
damage requires the presence of a functional p53 protein (Fig. 8). In
agreement with this result it has been reported that overexpression of
p53 in p53-null cells suppresses the transcriptional
activity of cyclin A, cyclin B1, cyclin
B2, and cdk1 promoters (19, 21-24, 52). Nevertheless,
this is the first evidence that the cdc25C promoter activity
is down-regulated also.
None of the tested promoters contains a canonical DNA binding site for
p53, thus our results lead to the speculation that p53 could interfere
with the function of NF-Y. Indeed, it has been described that p53
protein can repress transcription by binding to and preventing the
function of specific transcription factors (53, 54). In shift
experiments performed with anti NF-Y we observed that NF-Y is present
on the CCAAT boxes of cyclin B1 and cyclin B2
promoters before and after
ADR-treatment.2 Thus, p53
does not interfere with the DNA binding of NF-Y. Another possibility is
that p53 interferes with the NF-Y recruitment of coactivators and/or
general transcription factors. Indeed, it has been shown that p53 and
NF-Y bind to overlapping domains of the p300 coactivator (55, 56), and
both p53 and NF-Y bind to TATA-binding protein (57, 58).
Interestingly, NF-Y was shown also by other groups to be required for
the p53-mediated inhibition of cdk1 transcription (23, 24).
Furthermore, NF-Y binds the CCAAT box contained in the cyclin
A promoter (11). These findings lead to the speculation that NF-Y
modulates cdk1 and cyclin A transcription after
ADR-mediated G2 arrest by the same mechanism described here
for the cyclin B1, cyclin B2, and
cdc25C genes.
It has been shown recently that an endogenous p53 protein sustains a
G2 arrest induced by ADR through a
pRb-dependent decrease of cyclin B1 and cdk1 expression
(43). However, the pRb-dependent inhibition of cyclin B1
expression does not seem to be caused by direct binding of pRB/E2F
family proteins to the cyclin B1 promoter. Indeed, as
assessed by chromatin cross-linked immunoprecipitation, an anti-E2F1
antibody does not immunoprecipitate, from cycling cells, chromatin
containing the cyclin B1 promoter, whereas an anti-NF-Y
antibody does.3
The transcriptional regulation of the expression of mitotic kinase
complexes is not the only mechanism that sustains a G2 block. Indeed, it has been demonstrated recently that in a human colorectal cancer cell line, the p53-dependent
G2 arrest after In summary, this work provides evidences that in muscle cells, the
molecular mechanism responsible for the G2 checkpoint
induced by ADR-mediated DNA damage involves the ability of the NF-Y
transcription factor to prevent the transcription of key regulatory
molecules essential for the progression through the G2
phase of the cell cycle. This finding opens the question of whether
other genes, controlled by NF-Y during the cell cycle (26), are targets
of its activity in the cell cycle checkpoints.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-irradiated cells, knockout for the
p53 gene, progress from the G2 to the M phase
and maintain DNA content of 4n because of cytokinesis failure (18).
Overexpression of an exogenous wild type p53 induces G2
arrest associated with down-regulation of cyclin B1, cyclin A, and cdk1
expression (19, 20). In agreement with these observations, it has been
reported that wild type p53 overexpression in p53-null cells
can suppress the transcriptional activity of cyclin A,
cyclin B1, and cdk1 promoters (19, 21-24). However, the specific molecular mechanisms responsible for the down-regulation of the transcription of these genes in the
G2 arrest are still unknown.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
have been described previously. The cdc25C-wt-luci construct was
obtained by polymerase chain reaction-amplifying the human promoter
region with the primers 696 5'-CGA GCC CAA CAG CTT AGA GGC GAG CGG
GG-3' and 743R 5' CCT CTA AGC TGT TGG GCT CGC AGA TCA CC-3' and cloning
the
1433 to
1 (numbering relative to the translational start)
promoter fragment into the BglII/NcoI sites of
the pGL3-Basic plasmid (Promega Mannheim Germany). The cdc25C deletion
mutant was created by polymerase chain reaction with the primers
25C-3'-mut 5'-CGT AGC CAT GGT CTT CGA ATT CTC-3'.
-galactosidase plasmid as an internal control for transfection
efficiency. 16 h later, precipitates were removed, and cells were
treated with ADR (0.5 µg/ml) for 22 h. Cells were harvested, and
LUC or CAT activity was assayed on whole cell extract, as described
(40, 41). The values were normalized for
-galactosidase and protein contents.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ADR-mediated DNA damage induces a
G2 arrest in C2C12 cells. Panel A,
DNA distribution analysis of C2C12 cells. Panel B, after
ADR-mediated DNA damage C2C12 cells accumulate in G2/M. The
percentages of cells in G0/G1, S, and
G2/M phases are indicated.
Analysis of nuclei by Hoechst and terminal nucleotidyl transferase
staining
89 to +11 base pairs (36); (ii)
p332B1CAT, containing human cyclin B1 promoter fragment from
154 to +182 base pairs; (iii) p240B1CAT, containing human
cyclin B1 promoter fragment from
57 to +182 base pairs
(10); (iv) B2-Luci, containing 1.1 kilobase of murine
cyclin B2 promoter (34); (v) pcdk1CAT, containing 2 kilobases of human cdk1 promoter (14); (vi)
pcdc25C-wt-luci, containing 1,433 base pairs of human cdc25C
promoter. The activities of these promoters in untreated cells were
referred to as 100%, and the relative activities after 22 h of
ADR treatment were calculated. As shown in Fig.
2, the mitotic cyclins, the
cdk1, and the cdc25C promoter activities were
dramatically down-regulated after ADR treatment. In contrast, ADR
treatment did not down-regulate the activity of the cyclin
D1 promoter, whose activity is high in the G1 phase of
the cell cycle (37), as well as the activity of E
promoter, whose activity is tissue-specific. These results indicate
that the observed down-regulation cannot be attributed to a nonspecific
silencing of the transcription determined by toxicity of ADR treatment.
Taken together, these data demonstrate that cyclin A,
cyclin B1, cyclin B2, cdk1, and
cdc25C promoter activities are down-regulated in the
G2 arrest generated by ADR treatment.
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Fig. 2.
Mitotic cyclins,
cdk1, and cdc25c promoter activities
are down-regulated in ADR-treated C2C12 cells. Reporter constructs
carrying the cyclin A (pcycA LUC), cyclin B1
(p332B1CAT; p240B1CAT), cyclin B2 (pB2-Luci),
cdk1 (pcdk1CAT), cdc25C (pcdc25C-wt-luci),
E (pEa-luc), and cyclin D1 (pcyclinD1-luc)
promoters were transiently transfected by the calcium phosphate method
in C2C12 cells among with the CMV
gal reporter construct. The unit
activities of each promoter were made 100% (white bars),
and the relative activities after ADR treatment are indicated
(black bars). The results represent the means ± standard deviations of four independent experiments, each performed in
duplicate.
742 to
697, relative to the ATG, containing two of the three CCAAT boxes present in this promoter (cdc25C-
742/697-luci). Mutated and wild type constructs were transfected in C2C12 cells. As shown in
Fig. 3D, the mutation of all
CCAAT boxes impairs the down-regulation of the cyclin B1
promoter activity occurring after the ADR treatment. In the same
experimental conditions, the single mutation of the upstream (Fig.
3B) or downstream (Fig. 3C) cyclin
B1 CCAAT box, partially impairs the promoter activity
down-regulation. These data demonstrate that the two CCAAT boxes of the
cyclin B1 promoter act in a synergistic manner, and,
although to a different extent, each CCAAT box on its own mediates the
effect of the ADR treatment. In the context of the cyclin B2
promoter, the mutations of all (Fig.
4C), or only two (Fig.
4B) CCAAT boxes completely abolished the down-regulation
occurring after the ADR treatment. These results demonstrate that the
CCAAT boxes 1 and 2 of the cyclin B2 promoter both mediate
the effect of ADR treatment. In the context of the cdc25C
promoter, the deletion of two CCAAT boxes leads to about 20% of
down-regulation before ADR and completely abolished the down-regulation
occurring after the ADR treatment (Fig.
5). These results demonstrate that the
cdc25C promoter region from
742 to
697 with respect to
the ATG, containing two of the three CCAAT boxes present on the
promoter (11), mediates the effect of the ADR treatment. Altogether,
these data show that the CCAAT boxes are key sequences in the
down-regulation of the cyclin B1, cyclin B2, and
cdc25C promoter activities after the ADR treatment.
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Fig. 3.
Mutations of the CCAAT boxes impair the
ADR-mediated down-regulation of the cyclin B1 and
promoter activity. 10 µg of p240B1CAT vector carrying the wild
type cyclin B1 promoter (panel A), or pmtupCCAAT
vector carrying mutations in the upstream CCAAT box (panel
B), or pmtdownCCAAT vector carrying mutations in the downstream
CCAAT box (panel C), or pmtup/downCCAAT vector carrying
mutations in both boxes (panel D) was transiently
cotransfected in C2C12 cells. CAT activities were measured in untreated
(white bars) and ADR-treated cells (black bars).
Values are the means ± standard deviations of four independent
experiments. A schematic representation of the constructs employed is
shown.
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Fig. 4.
Mutations of the CCAAT boxes impair the
ADR-mediated down-regulation of the cyclin B2 promoter
activity. 10 µg of pB2-Luci vector carrying the wild type
cyclin B2 promoter (panel A), or Y1,2 m vector
carrying mutation in two CCAAT boxes (panel B) , or Y1,2,3 m
vector carrying mutation in all CCAAT boxes (panel C) was
transiently cotransfected in C2C12 cells. Luciferase activities were
measured in untreated (white bars) and ADR-treated cells
(black bars). Values are the means ± standard
deviations of four independent experiments. A schematic representation
of the constructs employed is shown.
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Fig. 5.
Mutations of the CCAAT boxes impair the
ADR-mediated down-regulation of the cdc25C promoter
activity. 10 µg of pcdc25c-wt-luci vector carrying the wild type
cdc25C promoter (panel A) or
cdc25C- 742/697-luci vector containing two of the three CCAAT boxes
present on this promoter (panel B) was transiently
cotransfected in C2C12 cells. Luciferase activities were measured in
untreated (white bars) and ADR-treated cells (black
bars). Values are the means ± standard deviations of four
independent experiments. A schematic representation of the constructs
employed is shown.
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Fig. 6.
Dominant-negative NF-Y protein abrogates the
down-regulation of the cyclin B1, cyclin
B2, cdc25C, and cdk1
promoters after ADR treatment. C2C12 cells were transfected
with 5 µg of p332B1CAT (panel A), or p240B1CAT vectors
carrying the wild type cyclin B1 promoter (panel
B), or pB2-Luci vector carrying the wild type cyclin B2
promoter (panel C), or pcdc25C-wt-luci carrying the wild
type cdc25C promoter (panel D), or pcdk1CAT
carrying the wild type cdk1 promoter (panel E).
These promoter fragments were cotransfected with 5 µg of eukaryotic
vector expressing dominant-negative NF-YA protein YA13 m29. CAT and
luciferase activities were measured in untreated (white
bars) and ADR-treated cells (black bars). Values are
the means ± standard deviations of four independent
experiments.
-irradiation (18, 45). To assess the specific
effect of p53 on cell cycle progression after ADR treatment in C2C12
cells we interfered with the activity of endogenous wild type p53 by
expressing a dominant-negative p53 (DD-p53) protein. For this purpose,
C2C12 myoblasts were infected with the LDDSN retrovirus carrying murine
DD-p53 miniprotein and, as control, with the insertless retroviral
vector, LXSN (C2C12 LXSN) (46). The dominant-negative p53 protein
(DDp53) is a miniprotein consisting of the last 89 residues of murine
wild type p53, including the oligodimerization domain, and lacking the
DNA binding and trans-acting domains. An antagonist effect
of DD-p53 toward a wild type p53 protein has been reported, at least
partially the result of the formation of functionally defective, mixed
oligomers between the two proteins (46). As expected, the p53 protein
present in the mixed oligomers has a half-life longer than that present
in the wild type oligomers, irrespective of the ADR treatment (see Fig. 8A) (46). Infected cells were maintained as polyclonal
populations after G418 selection. Western blot analysis assessed the
stable expression of DD-p53 protein in C2C12 cells (see Fig.
8A). Flow cytometric analysis of C2C12 LXSN cells (data not
shown) indicates that the insertless retroviral vector does not
influence the cell cycle distribution of these cells before and after
the ADR treatment compared with the noninfected cells showed in Fig. 1.
The cytofluorometric analysis of DDp53-expressing cells (C2C12 LDDSN)
shows that this population does not present significant alterations in
cell cycle phase distribution in normal culture conditions (compare
Fig. 1A with Fig.
7A). However, after the ADR
treatment the same cells lose the G1 checkpoint and
accumulate mainly in the G2/M phases of the cell cycle
(79%, approximately versus 21% of untreated cells) (Fig.
7B). This result indicates that DD-p53 protein, interfering with p53 activity, impairs the checkpoints of the cell cycle. To
distinguish between G2 and M phases, the mitotic index was evaluated. As shown in Table II, after
ADR treatment ~35% of C2C12 LDDSN cells entered mitosis and formed
micronuclei. These results indicate that C2C12 cells expressing a
dominant-negative p53 protein overcome the G2 checkpoint
and accumulate at the end of the M phase. We, therefore, conclude that
as reported previously for
-irradiation in other cell types (18, 45)
in C2C12 cells the G2 arrest generated by ADR-mediated DNA
damage is p53-dependent.
View larger version (18K):
[in a new window]
Fig. 7.
C2C12 cells expressing dominant-negative p53
protein escape the G2 arrest induced by ADR
treatment and enter mitosis. Panel A, DNA
distribution analysis of C2C12 cells infected with the LDDSN
retrovirus, carrying a dominant-negative p53 protein (C2C12 LDDSN).
Panel B, after ADR-mediated DNA damage, dominant-negative
p53-expressing cells escape the G1 arrest and mostly
accumulate in G2/M phases. The percentages of cells in
G0/G1, S, and G2/M phases are
indicated.
Analysis of nuclei by Hoechst staining
View larger version (22K):
[in a new window]
Fig. 8.
Dominant-negative p53 protein abrogates the
ADR-mediated down-regulation of mitotic cyclin complexes. Western
blot analysis was performed on total cell lysates from control C2C12
LXSN cells and C2C12 LDDSN cells (panel A). The extracts
were reacted with antibodies against cyclin A, cyclin B1, cdk1, cdc25C,
cdk7, cdk9, and p53 (for details, see "Experimental Procedures").
Protein loading was normalized with an anti-Hsp70 antibody. Panel
B, reporter constructs carrying the cyclin A (pcycA
LUC), cyclin B1 (p332B1CAT, p240B1CAT), cyclin B2
(pB2-Luci), cdk1 (pcdk1CAT), and cdc25C
(pcdc25C-wt-luci) promoters were transiently transfected in C2C12 LDDSN
cells along with the CMV gal reporter construct. The unit activities
of each promoter were made 100% (white bars), and the
relative activities after ADR treatment are indicated (black
bars). Values are the means ± standard deviations of four
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-irradiation is the result of induction
of 14-3-3
expression (59). Once overexpressed, 14-3-3
blocks
the cell cycle contributing to the nuclear exclusion of cdk1-cyclin B1
complexes and cdc25C phosphatase (60). Thus, after DNA damage,
transcriptional down-regulation of mitotic kinase complexes (this
paper) and their cytoplasmic segregation (56) might concomitantly
sustain the G2 checkpoint.
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ACKNOWLEDGEMENTS |
---|
We thank Stephen Dalton for human cdk1 promoter, Mark Wasner for cyclin B2 promoter, and Pidder Yansen-Durr for cyclin A promoter; Moshe Oren for LDDSN packaging cells; Marco Crescenzi for helpful discussion; Giulio Tibursi for technical advice; Antonio Giordano and Antonio De Luca for anti-cdk9 antibody; and Daniela Bona for computing assistance.
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FOOTNOTES |
---|
* This work was supported in part by Telethon-Italy Grant 1035 and the Associazione Italiana per la Ricerca sul Cancro.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.
§ Recipients of an Fondazione Italiana per la Ricerca sul Cancro fellowship.
¶ Recipient of an Italy-United States of America project fellowship.
To whom correspondence should be addressed: Laboratorio
Oncogenesi Molecolare, Istituto Regina Elena, CRS, Via delle
Messi D'Oro, 156, Rome 00158, Italy. Tel.: 39-06-4985-2531; Fax
39-06-4985-2505; E-mail: piaggio@ifo.it.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M006052200
2 I. Manni, unpublished observations
3 S. Sciortino, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: NF-Y, nuclear factor Y; ADR, Adriamycin; PBS, phosphate-buffered saline; CAT, chloramphenicol acetyltransferase; LUC, luciferase; CMV, cytomegalovirus.
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