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Biochemistry |
Departments of Oncology [J. R. J., A. G., J. D. W.] and Immunology [C. I., L. A. M., A. R.], SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-irradiation or the topoisomerase I
inhibitor topotecan was used to induce G2 arrest in HeLa
cells. In the presence of the Chk1 inhibitor, the cells did not arrest
following -irradiation or treatment with topotecan, but continued
into mitosis. Abrogation of the damage-arrest checkpoint also enhanced
the cytotoxicity of topoisomerase I inhibitors. These studies suggest
that Chk1 activity is required for G2 arrest following DNA
damage. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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G2 checkpoints include regulation of cdc2 by inhibitory phosphorylations at Tyr-15 and Thr-14. At the G2-M transition, cdc2 usually is dephosphorylated at Tyr-15 and Thr-14 by the dual specificity phosphatase cdc25C, allowing cdc2 to phosphorylate its mitotic substrates (11) . However, in response to DNA damage or inhibition of DNA replication, the inhibitory phosphorylation of cdc2 remains intact and the cell cycle arrests at the G2 phase. In S. pombe, the G2 checkpoint is regulated by the checkpoint kinase Chk1, which is essential for cell cycle arrest following DNA damage (12, 13, 14) . Recently, Sanchez et al. (15) described the cloning and characterization of human checkpoint kinase (Chk1), a homologue of the yeast gene. The human Chk1 was shown to bind to and phosphorylate cdc25C on Ser-216, which results in the binding of a 14-3-3 protein to cdc25C (15 , 16) . The effect on cdc25C prevents dephosphorylation of cdc2 and thus maintains the cdc2 kinase in an inactive state, preventing entry into mitosis. Overexpression of an Ser-216-Ala cdc25C mutant, which is unable to be phosphorylated by Chk1, resulted in the partial disruption of a G2 checkpoint and the inability of DNA damaged cells to arrest at the G2 phase. These results indicated that Ser-216 of cdc25C is a target of the DNA damage checkpoint. However, it is still important to determine whether human Chk1 is required for the G2 checkpoint response. In the present study, we identified and characterized a compound, SB-218078, that potently inhibited human Chk1. This compound prevented G2 arrest following DNA damage, suggesting that Chk1 activity is required for G2 checkpoint arrest. Consistent with the importance of the G2 checkpoint in the response to DNA damage, SB-218078 enhanced the cytotoxicity of DNA-damaging agents. These data are among the first to demonstrate a key regulatory role for human Chk1 in regulation of checkpoint controls and provide support that Chk1 inhibitors may be valuable adjuvant therapies.
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MATERIALS AND METHODS |
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Expression and Purification of GST-Chk1.
A GST-Chk1 expression construct was constructed that has the
GST2
gene fused to the NH2 terminus of
full-length Chk1 kinase via a linker containing a thrombin cleavage
site. This construct was cloned into the Baculovirus expression vector,
pFASTBAC, which was used to make the viral stock for the subsequent
infection. Spodoptera frugiperda cells (Sf9) were infected
with the virus expressing the GST-Chk1, after which the cells were
grown for 3 days, harvested, and then frozen until purification. To
purify Chk1, an Sf9 cell pellet expressing GST-Chk1 was resuspended on
ice in lysis buffer containing 50 mM Tris-HCl (pH
7.5), 250 mM NaCl, 1 mM
DTT, 0.1%Brij, a protease inhibitor cocktail [2 µg/ml E-64,
1 mM AEBSF, and 1 µg/ml pepstatin A
(Sigma)], and 1 mM sodium orthovanadate, and
cells were lysed by sonication and centrifuged at 100,000 x g for 30 min. The supernatant was added to
glutathione-Sepharose 4B (Pharmacia Biotech, Sweden), beads,
equilibrated in wash buffer (20 mM Tris-HCl, pH
7.0, 10 mM MgCl2, 100
mM NaCl, 1 mM DTT, protease
inhibitor cocktail, 1 mM sodium orthovanadate),
and rocked for 30 min. The resin with the bound GST-Chk1 was
centrifuged at 500 x g for 5 min and washed
with 14 ml of wash buffer. The beads were spun as above and resuspended
in another 14 ml of wash buffer. The suspension was transferred into a
column and allowed to pack; the wash buffer was then allowed to flow
through by gravity. The GST-Chk1 was eluted from the column with 10
mM glutathione in 50 mM
Tris-HCl (pH 8.0) in 500-µl fractions. Protein concentrations were
determined on the fractions using the Bio-Rad protein assay kit
according to the manufacturer’s instructions. Fractions containing the
GST-Chk1 were pooled and dialyzed at 4°C in dialysis buffer (20
mM HEPES, pH 7.0, 1 mM
MgCl2, 100 mM NaCl, 0.05%
Brij-35, 10% glycerol, 1 mM DTT, 1
mM sodium orthovanadate).
Expression and Purification of Human GST-cdc25C.
The GST-cdc25C Escherichia coli expression construct was the
generous gift of Dr. Laurent Meijer (Centre National de la Recherche
Scientifique, Roscoff, France). The construct contained the
GST gene fused to the NH2 terminus of
full-length cdc25C by a linker containing a thrombin cleavage site.
This was subcloned into the baculovirus expression vector, pFASTBAC,
which was used to generate viral stock for subsequent infection. Sf9
cells were infected with virus and grown for 3 days prior to
harvesting. Cell pellets were lysed by an Avestin cell disrupter in 20
mM Tris-HCl (pH 8.0), 137
mM NaCl, 2 mM EDTA, 10%
glycerol, 1% Triton X-100, 5 mM DTT, and
protease inhibitor cocktail. Cell lysates were clarified by
centrifugation at 28,000 x g at 4°C for
1 h. The supernatant was mixed with 10 ml of glutathione-Sepharose
4B and shaken gently at 4°C for 2 h. The resin was packed into a
Pharmacia XK26/50 column, and unbound material was washed out with 20
mM Tris-HCl (pH 7.0), 100
mM NaCl, 12 mM
MgCl2, 1 mM DTT (wash
buffer). Proteins bound to the resin were then eluted with 50
mM Tris-HCl (pH 8.0), 10 mM
glutathione, and 0.05% Brij-35, and assayed and dialyzed as described
above for Chk1.
Chk1 Kinase Assay.
Each well of a 96-well Flashplate (Amersham, Arlington Heights, VA) was
coated with 1 µg of the GST-cdc25C fusion protein diluted in PBS.
Plates were incubated overnight at 4°C, and then washed twice in PBS
and dried for 5–30 min at 37°C. DMSO vehicle or compounds are added
as 2 µl/well prior to the addition of 0.5µCi/well of
[33P]-ATP, 10 µM cold ATP, and
kinase reaction buffer containing 20 mM HEPES (pH 7.4), 50
mM KCl, 10 mM MgCl2, 1
mM EGTA, 0.5 mM DTT. The reaction was initiated
by the addition of GST-Chk1 (0.1 µg/well) and was allowed to proceed
for a time predetermined to be linear on a time versus
phosphorylation plot. Reactions were terminated with the addition of an
equal volume (50 µl) of 50 mM EDTA. Plates are
washed four times in PBS, dried for 30 min at 37°C, and quantitated
by liquid scintillation counting.
Cell Cycle Studies.
Cellular studies used HeLa S3 and HT-29 adenocarcinoma lines obtained
from ATCC. HeLa and HT-29 cells were grown in DMEM and McCoy’s medium
(Life Technologies, Inc., Gaithersburg, MD), respectively (all
supplemented with 2 mM L-glutamine and 10%
FBS) in a 37°C, 5% CO2 environment. For cell
cycle studies, cells were plated at a concentration sufficiently low
such that 24 h later they were at 10–20% confluence (typically
2 x 105 cells/15
cm3). Cells were then synchronized in S-phase by
a repeated thymidine block. Briefly, cells were treated with 2
mM thymidine for 18 h, released for 8 h by three
washes, and then treated again with thymidine. Following the second
release from thymidine, 95% of cells were in S-phase. Synchronized
cells were then returned to complete media containing 50 nM
topotecan (a dosage we have found to be sufficient to arrest cells in
early G2 phase without inducing apoptosis) alone
and in combination with SB-218078 for up to 18 h. Studies using
-irradiation were carried out in a GAMMACELL 40 irradiator with a
137Cs source. The exposure rate was 91 rad/h, and
cells were exposed for 5.5 min to achieve a dose of 500 rads, or 5 Gy.
Cell cycle profiles were performed cytometrically using a procedure for
propidium iodide staining of nuclei (19)
.
Cytotoxicity Assays.
The proliferation assay was performed with HeLa and HT-29 cells, and
used a colorimetric change resulting from reduction of the tetrazolium
reagent XTT into a formazan product by metabolically active cells
(20)
. Cells were seeded in 100-µl aliquots into
96-well plates to roughly 10% confluence (HT-29, 2.75 x 103 cells/well; HeLa, 5.5 x 103 cells/well) and grown for 24 h.
Compounds were then added with or without sufficient vehicle-containing
medium to raise the cells to a 200-µl final volume containing
chemical reagents in 0.2% DMSO. Cells received multiple concentrations
of topotecan, SB-218078, and combination treatment at 37°C, 5%
CO2. Ninety-six h later, 50 µl of an
XTT/phenazine methosulfate mixture were added to each well, and cells
were left to incubate for 90 min. Plates were read at 450 nm, and
antiproliferative effects were compared relative to maximal effect of 5
µM topotecan.
Clonogenic assays (10) were performed with HT-29 cells seeded at 5 x 103 cells/ml into 12-well plates. Twenty-four h after plating cells, the medium was removed and various dilutions of camptothecin or vehicle were placed in the wells. After 7 h, the camptothecin medium was removed, and adherent cells were washed once with warmed PBS and then dosed with dilutions of SB-218078, UCN01, or vehicle. After 18 h of drug treatment, cells were harvested by trypsinization and reseeded into 6-well plates at a consistent volume determined to give control cells a concentration of 3 x 102 cells/well. Cells were grown for 10 days, and then fixed in 100% methanol for 5 min and stained with 0.04% methylene blue to permit counting.
Chromatin condensation studies (21) were performed with thymidine-synchronized HeLa cells. Drug or vehicle was introduced to cells 4 h after release from the final thymidine arrest. Thirty min later, cells were subjected to a mock dose or a 5-Gy dose of irradiation, sufficient to induce G2 arrest. Cells were harvested beginning at 6 h post release and prepared for nuclear staining with DAPI (Calbiochem), according to the manufacturer’s protocol. Briefly, cells were swollen hypotonically for 5 min in 75 mM KCl, gently pelleted, and fixed in 0.5 ml of Carnoy’s fixative (3:1 methanol:glacial acetic acid, v/v) for 5 min. After a final wash and resuspension in fixative, cells were dropped on prewetted slides and allowed to air dry. Fixed cells were then stained in 1 µg/ml DAPI for 15 min, followed by one wash in methanol. Stained cells were visualized using an Olympus IX-70 digital camera with fluorescence filters (exciter filters, BP360-370 nm), and multiple cell field pictures were taken at random. One hundred fifty to 250 nuclei were then evaluated for condensed nuclei.
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RESULTS |
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Staurosporine was identified as a potent inhibitor of Chk1, with an
IC50 of 8 nM (Fig. 1A)
. Staurosporine is known to inhibit many kinases, and the
lack of selectivity (Table 1)
limits its utility in cellular studies. Hoping to find a more
selective Chk1 inhibitor, we examined the activities of other compounds
related to staurosporine, and identified SB-218078 (Fig. 1B)
, which inhibited Chk1 phosphorylation of cdc25C with an
IC50 of 15 nM (Fig. 1A)
. In kinase selectivity assays, SB-218078 had
IC50 values for cdc2 and PKC of 250
nM and 1000 nM,
respectively (Table 1)
. Selectivity over cdc2/cyclinB is of particular
importance because Chk1 is predicted to have a role in negative
regulation of cdc2/cyclin B. Thus, inhibition of Chk1 should allow
activation of cdc2, and this would have only a cellular effect only if
the inhibitor does not also inhibit cdc2. We also tested the related
compound UCN-01 and determined that it had an
IC50 of 7 nM for Chk1.
UCN-01 previously has been demonstrated to abrogate DNA-damage arrest
(7)
, but the target of this effect had been unknown.
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, panel 2). To determine the effect of the Chk1
inhibitor on the DNA damage-induced arrest, 50 nM
topotecan was used in combination with several concentrations of
SB-218078. As shown in Fig. 2A
, when topotecan was combined
with either 2.5 or 5 µM SB-218078, the cell
cycle profile was very similar to that of the control cells, suggesting
that they were unable to arrest in G2 in response
to the DNA damage. When the concentration of SB-218078 was 1.2
µM, the cells remained arrested in
G2. Thus, 2.5 µM was the
minimum concentration of SB-218078 capable of causing complete
abrogation of G2 arrest within 18 h. It is
important to note, however, that complete abrogation of
G2 arrest may not be necessary to enhance the
effects of DNA damage on the cells. Thus, lower concentrations of this
compound could have effects in other cellular assays.
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-irradiation, which activates only the damage checkpoint. Five
Gy of radiation caused the majority of HeLa cells to accumulate in
G2 (Fig. 2A
, panel 7).
However, in the presence of the Chk1 inhibitor, the irradiated cells
did not arrest (Fig. 2A
, panel 8).
Additional experiments were conducted to confirm that the cell cycle
profile observed when cells were treated with SB-218078 and topotecan
was due to abrogation of G2 arrest and not an
alternative effect such as a G1 delay. HeLa cells
were synchronized at the G1-S border by
double-thymidine block. Cells were released by washing out the
thymidine and were incubated for 1.5 h prior to treatment with 50
nM topotecan and 5 µM SB-218078. The cell
cycle profile was then analyzed 16 h later. At the time of
treatment, the cells had clearly progressed into S-phase (Fig. 2B
, panel 2 compared with panel 1).
Topotecan alone caused the majority of cells to arrest in
G2, whereas SB-218078 caused the
topotecan-treated cells to continue through the
G2 and M phases and return to a normal cell cycle
profile similar to untreated cells 18 h after release from the
double-thymidine block (Fig. 2B
, panels 3–5).
Staurosporine, which also inhibits Chk1 but has no selectivity over
cdc2, was also tested in this study, and we observed that 1
µM staurosporine alone caused the cells to
arrest in G2. In addition, 1
µM staurosporine was unable to abrogate
G2 arrest in the topotecan-treated cells (Fig. 2B
, panels 7 and 8). We also evaluated
a potent, selective PKC inhibitor, Ro 34-0432 (22)
, as a
control because SB-218078 does have weak PKC inhibitory activity. Ro
34-0432 was used at 1 µM, which is more than
sufficient to inhibit PKC in cells because cellular effects
attributable to PKC inhibition have been reported to have
IC50s in the 0.1 µM range
(23)
. This PKC inhibitor was unable to prevent cells from
arresting in G2 with topotecan (Fig. 2B
, panel 6). A time course analysis of cell
cycle progression in synchronized SB-218078-treated cells further
demonstrated that this compound did not significantly perturb the cell
cycle in the absence of DNA damage (Fig. 3)
. Control cells reached G2 6–8 h after release
from a double-thymidine block, progressed through mitosis by 10 h,
and had returned to a normal cell cycle profile by 20 h. Cells
treated with 2.5 µM SB-218078 reached
G2 with a slight delay, 8–10 h after release,
progressed through mitosis by 12–16 h post release, and returned to a
normal cell cycle profile by 20 h. The minor
G2-M delay could be due to partial inhibition of
cdc2/cyclin B at the concentration used. Additional studies
demonstrated that concentrations of SB-218078 at 10
µM or higher caused significant
G2 arrest (data not shown). Thus, the activity of
SB-218078 as judged by complete abrogation of G2
arrest was limited to a range between 1.2 and 10
µM.
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. This was followed by decondensation between 8 and 10 h as cells
exited from mitosis. Cells that were -irradiated 4.5 h after
release from double-thymidine block were at 50% condensation at 6 h. This increase over the control cells is likely due to some
spontaneous premature chromatin condensation caused by the radiation as
reported previously (24)
. However, the irradiated cells
did not have any significant progression in chromatin condensation
between 6 and 10 h. In contrast, when irradiated cells were also
treated with SB-218078, there was a transient peak in chromatin
condensation 
1 h behind the control cells, which suggests only a
slight delay in prophase entry, and normal progression through mitosis.
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. However, when these treatments were combined, survival was decreased
to 50%. Similar results were also seen with the HT-29 colon carcinoma
cell line, suggesting that the Chk1 inhibitor can enhance the
cytotoxicity of very low amounts of DNA damage.
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. The effect was very similar to that of an equivalent concentration of
UCN-01.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-irradiation or a topoisomerase I inhibitor as reported
previously for UCN-01 (7
, 10)
. Consistent with the
pharmacology of other agents that abrogate the G2
checkpoint, SB-218078 enhanced the cytotoxicity of both topotecan and
camptothecin in either HeLa or HT-29 cell lines.
SB-218078 is related to UCN-01 and staurosporine, and we observed that
these compounds are all potent inhibitors of Chk1 kinase (Table 1)
,
although SB-218078 appears to be more selective versus PKC.
Thus, it seems likely that the checkpoint abrogation of UCN-01 could be
also attributed to Chk1 inhibition. Staurosporine has been reported to
abrogate DNA-damage checkpoints (29)
at very low
concentrations, but it has also been reported to cause
G2 arrest at slightly higher concentrations
(30)
. This can be explained by the observation that
staurosporine inhibits both Chk1 and cdc2 with similar potency, and
illustrates why there are few reports of staurosporine overriding
DNA-damage arrest. SB-218078 has significantly better selectivity
versus cdc2, and is therefore a more useful pharmacological
tool for checkpoint modulation. Another recent report identified
isogranulatimide as a G2 checkpoint
inhibitor by use of a specific cell-based screening assay
(31)
. This compound is a substructural component of
SB-218078 and also quite possibly is a Chk1 kinase inhibitor.
We observed that SB-218078 was able to significantly enhance the
cytotoxicity of topotecan at concentrations where either agent alone
had no significant effect (Fig. 5)
. The concentration of SB-218078 that
caused this effect was 500–625 nM, which is 3–4-fold
lower than that required for complete abrogation of
G2 arrest by topotecan in a 1-day assay. Similar
results have been reported for UCN-01, which required a concentration
of at least 0.3 µM to completely abrogate
G2 arrest yet was able to significantly enhance
the lethal effects of -irradiation at a 3-fold lower concentration
of 0.1 µM (7)
. This is an interesting
observation and may reflect the difference in the duration of the
assays. The survival assays are several days in length and are
sensitive to lower concentrations of Chk1 inhibitor. Indeed, we also
observed that higher concentrations of SB-218078, above 1
µM, did begin to inhibit cell proliferation in these
longer-term assays. It is not clear if this was due to inhibition of
Chk1 or another effect of the compound, for example, possible weak
inhibition of cdc2. Nevertheless, we specifically chose subcytotoxic
concentrations of SB-218078 and topotecan in these assays to maximally
demonstrate their combined effects. These data suggest that it may not
be necessary to completely abrogate G2 arrest to
have a significant impact on the ability of cells to handle DNA damage
and survive over several days. In the clonogenic assay (Fig. 6)
, we
sought to compare SB-218078 and UCN-01 directly, using conditions
reported previously for UCN-01 (10)
. This study, which is
10–14 days in length, used a different treatment schedule in which the
Chk1 inhibitor is added after transient treatment with camptothecin.
Both SB-218078 and UCN-01, at 100 nM, enhanced the
cytotoxicity of camptothecin, and the maximal enhancement was apparent
at the lowest doses of camptothecin.
A second checkpoint kinase has been identified very recently. This kinase, known as Cds1 in S. pombe and Chk2 in humans, is both structurally and functionally distinct from Chk1 (32, 33, 34, 35, 36) . Both Chk1 and Chk2/Cds1 phosphorylate cdc25C on Ser-216, and this phosphorylation has been proposed to be a critical event in preventing progression into mitosis (16) . Nevertheless, it is unlikely that these two kinases are completely redundant. In S. pombe, both Cds1 and Chk1 must be mutated to override a replication block (32) , but mutation of Chk1 alone is sufficient to override DNA-damage arrest (12) . Moreover, these kinases may have different additional substrates other than cdc25C. For example, Cds1 can phosphorylate Wee1, whereas Chk1 does not (32) . In humans, the extent of overlapping functions for Chk1 and Chk2 remains to be determined. We have demonstrated that the Chk1 inhibitor SB-218078 can inhibit the DNA-damage checkpoint, but the requirement for Chk2 activity in the checkpoint remains to be determined. On the basis of the structural divergence of Chk1 and Chk2, including the kinase domain, it is quite possible that SB-218078 would not be a potent inhibitor of Chk2, but this has not been tested.
The current model of checkpoint regulation mediated via phosphorylation of cdc25C on Ser-216 makes several predictions. For example, phosphorylation of Ser-216 and 14-3-3 binding should be inducible by DNA damage. If this were true, then one could test whether a Chk1 inhibitor such as SB-218078 could block Ser-216 phosphorylation in DNA-damaged cells. However, we and others have observed that Ser-216 on cdc25C is constitutively phosphorylated throughout interphase in both humans and yeast (data not shown; Refs. 16 , 34 , 37 ) and thus, DNA-damage-inducible phosphorylation on this residue cannot be observed during the S or G2 phases. It was interesting that the constitutive phosphorylation of Ser-216 was seen in a S. pombe strain lacking both Chk1 and Cds1 (37) , suggesting that additional kinase(s) also target this site. Indeed, there is a third human kinase, C-TAK1, that can phosphorylate cdc25C on Ser-216 (38) . It is possible that cdc25C falls under the regulation of Chk1 or Chk2 only during mitosis, or that there are specific subcellular pools of cdc25C that are subject to Chk1 and/or Chk2 regulation. In any event, the regulation of DNA-damage and replication checkpoints by the Chk1 and Chk2 kinases is quite possibly more complicated than the simple model involving Ser-216 phosphorylation and 14-3-3 binding on cdc25C. Nevertheless, our data suggest a key role for Chk1 in regulating the DNA-damage checkpoint and suggest that Chk1 inhibitors may have therapeutic utility in cancer by enhancing the efficacy of some chemotherapeutics and preventing acquired resistance via repair of DNA damage.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at SmithKline Beecham Pharmaceuticals, Oncology Research,
UW2532, 709 Swedeland Road, King of Prussia, PA 19406. Phone:
(610) 270-5742; Fax: (610) 270-5381; E-mail: Jeffrey_R_Jackson{at}SBPHRD.com 
2 The abbreviations used are: GST,
glutathione-S-transferase; FBS, fetal bovine serum;
DAPI, 4',6'-diamidino-2-phenylindole; PKC, protein kinase C.
Received 7/19/99. Accepted 11/29/99.
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B. Zhao, M. J. Bower, P. J. McDevitt, H. Zhao, S. T. Davis, K. O. Johanson, S. M. Green, N. O. Concha, and B.-B. S. Zhou Structural Basis for Chk1 Inhibition by UCN-01 J. Biol. Chem., November 22, 2002; 277(48): 46609 - 46615. [Abstract] [Full Text] [PDF]
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 H. Zhao, J. L. Watkins, and H. Piwnica-Worms Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints PNAS, November 12, 2002; 99(23): 14795 - 14800. [Abstract] [Full Text] [PDF]
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