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J. Biol. Chem., Vol. 276, Issue 14, 10641-10645, April 6, 2001
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From the
Received for publication, August 8, 2000, and in revised form, December 20, 2000
The levels of the human checkpoint gene hCHK1
were measured in human cancer cells growing in vitro after
treatment with the DNA damaging agent cis-dichlorodiammine
platinum(II) (DDP). Treatment of human cancer cell lines with
DDP induced a decrease in the hCHK1 protein levels starting 6 h
after treatment, with a further decline at 24 and 48 h. A similar
decrease in the levels of hCHK1 was found at the mRNA level by
using Northern blot analysis. By using isogenic cell systems in which
p53 was disrupted either by transfection with HPV-E6 or by targeted
homologous recombination, we found that the DNA damage-induced
down-regulation of hCHK1 was only observable in wild type
p53-expressing cells, with only a minor decline in the hCHK1 levels
observable 48 h after treatment in cells with disrupted p53.
Similarly, treatment of mutant p53-expressing human cancer cell lines
with DDP did not result in changes in the levels of hCHK1. The
p53-dependent down-regulation of hCHK1 is likely to be at
transcriptional levels, as suggested by the lack of down-regulation of
the hCHK1 when transfected under the control of a heterologous viral
promoter. In addition, p53 is able to down-regulate the luciferase
activity under the control of the 5' flanking region of the hCHK1 gene.
The data suggest a strict link between p53 and hCHK1 governing the
activation and repression of the G2 checkpoint in which
both proteins participate.
The cellular response to DNA damage includes a transient arrest of
the cell cycle either at the G1 phase, before DNA
replication (through the G1 DNA damage checkpoint), or
before mitosis (through the G2 DNA damage checkpoint),
presumably to allow time for DNA repair, minimizing the replication and
segregation of damaged DNA. The G1 checkpoint is in part
dependent on the p53-regulated transcription of p21, a potent inhibitor
of the cyclin-cdk complexes required for the G1-S
transition (1, 2). Cells lacking a functional p53 are defective in the
G1 checkpoint in response to DNA damage, still retaining
checkpoint mechanisms by which cells are arrested in G2
phase (3, 4). The G2 DNA damage checkpoint prevents the
activation of the cdc2-cyclin B1 complex, thereby inhibiting entry into
mitosis in the presence of a damaged DNA (5, 6). This is thought to be
determined by phosphorylation and inactivation of cdc25 phosphatase,
which in the phosphorylated form binds the proteins of the 14-3-3
family and is prevented from activating the cdc2 complex (7-9). The
two main kinases described to phosphorylate cdc25 are hCHK1 and hCHK2
(8-10). The mechanism by which DNA damage activates hCHK2 and/or
hCHK1, crucial enzymes in the cascade of reactions leading to
G2 arrest, is not fully elucidated yet, even if there is
evidence of an involvement of ATM (11, 12).
p53 also is an important component of the G2 checkpoint
after DNA damage, possibly through the transactivation of the p21 and
14-3-3 Moreover, a growing body of data is accumulating on a possible
cross-connection between p53 and hCHK1 and hCHK2; in particular, there is evidence that hCHK1 and hCHK2 are able to phosphorylate and
activate p53 in response to DNA damage (15-17). In the present study
we investigated the consequences of the cellular response to the
DNA-damaging agent cis-dichlorodiammine platinum(II)
on hCHK1 and in particular on its regulation by p53.
Cells and Drugs--
The human colocarcinoma cell line
HCT-116 was maintained in Iscove's modified Dulbecco's medium
supplemented with 10% fetal calf serum. The human endometrial
HEC1A cells, the human ovarian cancer cell line SKOV3, and the human
leukemic cell line Jurkat were grown in RPMI 1640 supplemented with
10% fetal calf serum. The human osteosarcoma cells U2OS, transfected
with HA-hCHK1, were kindly supplied by Dr. C. Mercurio (IEO,
Milan, Italy) and were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum and 400 µg/ml G418.
HCT-116/E6 cells were obtained and maintained as previously
described (18). HCT-116 cells with p53 gene disrupted by targeted homologous recombination (clone 392.7, p53
Cis-dichlorodiammine platinum(II)
(DDP1; Sigma) was dissolved
in medium just before use. For each cell line, DDP treatment was
performed for 24 h at the concentration approximately inhibiting the growth by 50% (IC50). Nocodazole was purchased from Sigma.
Western Blotting Analysis--
Cell extracts, obtained at the
end of treatment and at 6, 24, or 48 h after recovery in drug-free
medium, were prepared by lysing cells in 50 mM Tris-HCl (pH
7.4), 250 mM NaCl, 0.1% Nonidet P-40, 5 mM
EDTA, 50 mM NaF in the presence of aprotinin, leupeptin, and phenylmethylsulfonyl fluoride as protease inhibitors, for 30 min on
ice. Insoluble material was pelleted at 13,000 × g for 10 min at 4 °C, and the protein concentration was determined using a
Bio-Rad assay kit (Bio-Rad). Forty µg of total cellular proteins were
separated via SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose. Immunoblotting was carried out
with polyclonal anti-hCHK1 antibody (8), p53 monoclonal antibody (DO-1,
Santa Cruz Biotechnology, Heidelberg, Germany), anti-actin polyclonal
antibody (Santa Cruz Biotechnology), and anti-HA monoclonal
antibody (Roche Molecular Biochemicals). Antibody binding was
revealed by peroxidase secondary antibodies and visualized using
enhanced chemiluminescence (Amersham Pharmacia Biotech).
Northern Blotting Analysis--
Total RNA was isolated from
cells growing in culture by the guanidine-thiocyanate method according
to standard procedures (19), fractionated by electrophoresis on a
formaldehyde-agarose gel, and transferred to nylon membranes.
Filters were hybridized with cDNAs 32P-labeled
using a Rediprime kit (Amersham Pharmacia Biotech). Hybridizations were
done in 50% formamide, 10% dextran sulfate, 1% SDS, 1 M
NaCl at 42 °C for 16 h, followed by two 10-min washes at room
temperature with 2× SSC (150 mM NaCl, 15 mM
sodium citrate) and one 30-min wash at 65 °C in 2× SSC, 1% SDS.
Cell Cycle Analysis--
Cells (2 × 106) were
removed 24 h following treatment with DDP, washed twice in
ice-cold phosphate-buffered saline, fixed in ice-cold 70% ethanol,
washed in phosphate-buffered saline, resuspended in 2 ml of a solution
containing 2.5 µg/ml propidium iodide and 25 µg/ml RNase, and
stained overnight at 4 °C in the dark. Cell cycle analysis was done
on at least 10,000 cells for each sample using the FACSORT system
(Becton Dickinson). The percentage of cell cycle phase
distribution was calculated as previously described (20).
Luciferase Assays--
SKOV3 cells were cotransfected with 4 µg of pGL2-derived plasmids (Promega) containing the 5'
flanking region of the hCHK1 gene isolated from a human P1
artificial chromosome clone2
or with 4 µg of p21-Luc construct (kindly provided by Dr. C. Prives,
Columbia University, New York, NY) and with 4 µg of a plasmid
encoding for human p53. 0.05 µg of untreated pRL-SV40 were used for
internal normalization. Reporter gene activities were evaluated after
24 h using the Dual-Luciferase system (Promega). Results are
expressed as the percentage of the control luciferase reported activity
normalized by the renilla activity value. The mean ± S.D. of
three independent experiments is shown.
Treatment of human HCT-116 cells with 25 µM DDP resulted in a
decrease in the levels of hCHK1 starting 6 h after treatment, with
a further strong decrease observable at 24 and 48 h (Fig. 1A), whereas no modification
of the mobility of the protein could be observed. Treatment of the
isogenic cell line (HCT-116/E6) in which p53 was inactivated (through
HPV16/E6 transfection) with the same concentration of DDP (25 µM) did
not result in such a decrease, although at 48 h after treatment a
slight decrease in the hCHK1 levels could be found. The densitometric
analysis of the results of three independent experiments (each
performed in duplicate) is reported in Fig. 1B, where it is
shown that after an initial decline of ~25% at 6 h, by 48 h the levels of hCHK1 in the HCT-116 cell line treated with DDP are
less than 5% of the controls. At the same time point, in the
HCT-116/E6 cell line treated with DDP the levels of hCHK1 are ~65%
of the controls.
To test whether the difference in hCHK1 levels was related to a
different DDP-induced cell cycle perturbation, a time course of cell
cycle distribution induced by 25 µM DDP in HCT-116 and HCT-116/E6
cells was performed (Fig. 2A).
As expected, a G1 arrest was present only in HCT-116 wt
cells at the end of treatment and at 6 h after drug washout,
whereas in both cell lines DDP induced a G2M block
measurable at 24 and 48 h after treatment. At 48 h after drug
washout the ratio between the percentage of cells in G2M
and G1 phases was not so different between the two cell
lines (3.1 and 2.7 for HCT-116 and HCT-116/E6 cells, respectively). Treatment of both HCT-116 and HCT-116/E6 cells with 0.4 µg/ml nocodazole, causing in both cell lines an accumulation of cells in the
G2 phase of cell cycle (Fig. 2B), did not result
in any change in the levels of hCHK1 (Fig. 2C).
DNA Damage Induces p53-dependent Down-regulation of
hCHK1*
§,, and
Molecular Pharmacology Unit, Department of
Oncology, Istituto di Ricerche Farmacologiche "Mario Negri," Via
Eritrea 62, 20157 Milan, Italy and the ¶ Department of Molecular
Genetics, University of Cincinnati MSB 3005, Cincinnati, Ohio
45267
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genes (13, 14). 14-3-3 is required to sequester cdc2-cyclin B1 complexes in the cytoplasm and to prevent mitotic catastrophes, whereas p21 prevents any cdc2-cyclin B1 that enters the
nucleus from becoming activated (4).
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/) and their relative controls (clone 40-16, p53+/+) were kindly provided by Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD).
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View larger version (46K):
[in a new window]
Fig. 1.
A, Western blot analysis in HCT-116 and
HCT-116/E6 cells treated with 25 µM DDP. Extracts were obtained at
different time points after treatment. Blots were hybridized with
antibodies recognizing hCHK1, p53, and actin. B,
densitometric analysis of the levels of hCHK1 protein. Data are
expressed as percents of untreated controls and represent the mean ± S.D. of the ratio between hCHK1 and actin levels of three different
experiments, each performed in duplicate.
View larger version (49K):
[in a new window]
Fig. 2.
A and B, flow
cytometric analysis of the cell cycle phase distribution of HCT-116 and
HCT-116/E6 cells treated with 25 µM DDP (A) or 0.4 µg/ml
nocodazole (B). C, hCHK1 levels in HCT-116 and
HCT-116/E6 cells treated with nocodazole.
To check whether the DDP-induced decrease in the levels of hCHK1 was
observable also at mRNA levels, a Northern blot analysis using the
same experimental conditions reported for the Western blotting analysis
was performed (Fig. 3A). A
decrease in the levels of both hCHK1 transcripts was clearly observable
in HCT-116 cells but not in HCT-116/E6 cells. Densitometric scanning of
three independent blots (performed on the lower transcript) (Fig.
3B), in fact, resulted in an ~20% decrease at 6 h,
an 80% decrease at 24 h, and a 90% decrease at 48 h after
treatment in HCT-116 cells, whereas no appreciable changes in
HCT-116/E6 cells were found up to 24 h, with a marginal decrease
in hCHK1 mRNA levels observable at 48 h after DDP
treatment.
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To further evaluate the p53-dependent down-regulation of
hCHK1, we used a clone derived from HCT-116 cells in which the p53 gene
was disrupted by gene targeting (21). As reported in Fig. 4, 12.5 µM DDP induced hCHK1 decrease
in HCT-116 p53+/+ cells, with minor changes induced only at 48 h
in HCT-116 p53/ cells treated with the same DDP concentrations, in
agreement with the results obtained with HCT-116 cells with
HPV16-E6-inactivated p53. Similarly, when human cancer cell lines
expressing mutated p53 (HEC1A, Fig. 5;
and Jurkat, data not shown) were treated with cytotoxic concentrations
of DDP (30 µM), no decrease in hCHK1 levels was observed, again
strongly supporting the p53 dependence of hCHK1 decrease observed after
DDP treatment.
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Trying to define whether the p53-induced down-regulation of hCHK1 was
due to a specific transcriptional repression of the hCHK1 gene, we used
the p53 wild type U2OS cells transfected with the HA-CHK1 gene under
the control of a CMV promoter. Treatment of these cells with
12.5 µM DDP induced down-regulation of endogenous hCHK1 (Fig.
6), whereas no effect (if not an
increase) was found against the exogenous, HA-tagged hCHK1. The same
filter was reprobed with an anti-HA antibody, and a lack of
CMV-driven, HA-tagged hCHK1 down-regulation is clearly observed.
Importantly, the ratio between endogenous and HA-tagged hCHK1
dramatically changed after DDP treatment. In fact, by densitometric
analysis the levels of endogenous hCHK1 were 2-3-fold higher than
exogenous hCHK1 before DDP treatment, whereas at 24 and 48 h after
treatment, the ratio between endogenous and exogenous hCHK1 was
0.2.
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Finally, we analyzed luciferase constructs containing in both
orientations the 5' flanking region of the hCHK1 gene. Transfection of
an 867-base pair fragment in sense, but not in antisense, orientation induced luciferase activity (Fig. 7) in
p53 null SKOV3 cells. Cotransfection with a human p53 expression vector
reduced this activity by ~50%. Similar results were obtained when
other constructs (containing 1200 and 1600 base pairs of the 5'
flanking region of the hCHK1 gene) were used (data not shown). The same
p53-expressing vector was able to induce in the same experimental
conditions the transcription of the p53-responsive promoter p21.
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The product of the tumor suppressor gene p53 plays a central role in both G1 and G2 checkpoints (1, 2, 21). In different cellular systems, after DNA damage the cells respond by inducing an increase in the levels of p53 and consequently a transcriptional activation of genes regulated by p53 (22, 23). In addition, p53 not only activates transcription but also induces repression (through a mechanism not yet established) of different genes (23). Posttranslational modifications of p53, such as phosphorylation and acetylation, are thought to play a key role in the mechanisms of activation of p53 (24, 25). In particular, phosphorylation at the N terminus of p53 can relieve the inhibitory effect of mdm2, resulting in an increase in the levels of p53 (26, 27). The kinases possibly involved in these phosphorylations are DNA-PK, ATM, ATR, hCHK1, and hCHK2, all shown to be able, at least in vitro, to phosphorylate p53 (15-17, 24). hCHK1 is the human homologue of the yeast chk1, a protein important for the G2 DNA damage checkpoint that prevents mitosis when DNA is being repaired (8). Chk1 is phosphorylated after damage by a mechanism that required checkpoint Rad proteins, including Rad 3 and the Saccharomyces cerevisiae Mec1/Tel1 (28-30). It has been proposed that chk1 might regulate the activity of cdc2 by phosphorylating the tyrosine kinase Wee 1, which inactivates cdc2, or the protein phosphatase cdc25, which activates cdc2. Recently it has also been shown that in S. cerevisiae chk1 is required for the function of the DNA damage checkpoint maintaining the abundance of Psd1, an anaphase inhibitor (31). It seems likely that hCHK1 also operates downstream from ATM and, once activated, phosphorylates cdc25C, creating a consensus site for the binding to 14-3-3 family members and preventing the entry of cells in mitosis (5, 8, 9). As already reported by others (32), we did not observe in our experimental condition the reduction in the electrophoretic mobility of hCHK1 described in HeLa cells after UV treatment (8) and possibly related to an activation of hCHK1 through phosphorylation. These apparently contrasting results might be due to the different cellular systems used and do not exclude the possibility that other forms of hCHK1 activation do indeed occur. On the contrary, a clear-cut decrease in hChK1 protein level was observed, and this event was found to be p53-dependent. Here we show that p53, which has recently been shown to be phosphorylated and activated in vitro and in vivo by hCHK1 (15), is then able to induce a down-regulation of hCHK1. The evidence reported here, obtained in isogenic cell systems (in which p53 was inactivated by either viral transfection or targeted homologous disruption), suggests a possible regulatory loop between p53 and hCHK1 in which, after DNA damage, both p53 and hCHK1 are activated; once activated, p53 represses the transcription of the hCHK1 gene, resulting in the inactivation of this checkpoint protein at later times. This regulatory loop between hCHK1 and p53 is reminiscent of other regulatory strategies that have been described either in procaryotes or in eucaryotic systems and that underlie the fact that a specific DNA damage response (i.e. the maintenance of a sustained G2M block) might be deleterious and need to be quickly down-regulated once the cells have repaired the DNA damage (33, 34). Another negative feedback loop exists for p53 that, once activated, transcriptionally up-regulates mdm2, which is in turn a negative regulator of its transcriptional activity and stability (35).
As for the molecular mechanisms responsible for the p53-dependent hCHK1 down-regulation, the experiments performed in cells transfected with HA-tagged hCHK1 clearly show that the hCHK1 gene under the control of a viral promoter is not a target of p53-induced repression, and the initial analysis of the 5' flanking region of the hCHK1 gene strongly suggests that the action of p53 occurs, at least partially, at the level of the hCHK1 promoter. Furthermore, the use of proteosome inhibitors did not modify the DDP-induced hCHK1 down-regulation in HCT-116 cells (data not shown). The difference in cell cycle perturbations induced by the DNA damaging agent DDP in cells expressing a wt p53 compared with cells with inactivated p53 is not the reason for the drug-induced difference in the levels of hCHK1 between these cell types. In fact, in both cell systems DDP induces an accumulation of cells in G2M phases. At early times the G1 block was present, as expected, only in cells with a wt p53. Furthermore, nocodazole treatment induces a superimposable G2M arrest in both wt p53 and p53-inactivated cells without inducing changes in the levels of hCHK1 in both cell lines.
The strong p53-dependent decrease of hCHK1 protein levels
at late time points after drug treatment, together with the recent observation that cell lines with a wt p53 express lower levels of hCHK2
compared with mutant p53-expressing cells (36), might be the
way that cells tend to resume the G2 block. This phenomenon could also partially describe the stronger and persistent
G2 block induced by anticancer drug treatment in cancer
cells not expressing p53 and the effect of caffeine and other
inhibitors that was reported to occur mainly in cells without p53
(37-39).
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FOOTNOTES |
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* This work was partially supported by Project Number ICS120/RF98/73 of the Italian Ministry of Health. The generous contributions of the Italian Association for Cancer Research are gratefully acknowledged.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 and requests for reprints should be addressed. Fax: 39-2-3546277; E-mail: damia@irfmn.mnegri.it.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M007178200
2 G. Damia, L. Carrassa, and M. Broggini, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: DDP, cis-dichlorodiammine platinum(II); wt, wild type.
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 B. S. Cummings and R. G. Schnellmann Cisplatin-Induced Renal Cell Apoptosis: Caspase 3-Dependent and -Independent Pathways J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 8 - 17. [Abstract] [Full Text] [PDF]
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