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Experimental Therapeutics |
Section of Cellular and Molecular Pharmacology, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center [Z. S., A. A., D. S., Y-X. L., P. H., W. P.], and The University of Texas Graduate School of Biomedical Sciences [Z. S.], Houston, Texas 77030
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ABSTRACT |
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INTRODUCTION |
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Although ara-C has remained the mainstay of a variety of combination treatments for acute myelogenous leukemia (10, 11, 12) , resistance to ara-C-based chemotherapy is still a major problem in therapy (13, 14, 15) . Several in vitro-generated mechanisms of resistance to ara-C and other clinically active nucleoside analogues focus on their metabolism and cellular pharmacology (16, 17, 18, 19) . However, none appear prevalent in the leukemic cells of patients with resistant disease. Accordingly, no experimental models have been established for mechanisms of resistance that arise in response to nucleoside analogue incorporation into DNA. Because such incorporation appears essential to nucleoside analogue cytotoxicity (3, 4, 5, 6) , clinically relevant resistance mechanisms likely involve signaling processes that occur after the incorporation of the fraudulent nucleotides.
Most immortalized leukemic cells in S-phase die when exposed to cytotoxic concentrations of nucleoside analogues. However, when primary leukemic cells in the marrow are exposed to ara-C therapy, a substantial portion of them terminate DNA synthesis, arrest cell cycle progression, and accumulate in S-phase (20, 21, 22, 23, 24, 25, 26) . Presumably after removal of the drug, cells resume progression and population growth. Thus, the ability of cells to enact a delay in cell cycle progression that stops DNA synthesis, thereby limiting incorporation of analogue, may be a defense mechanism that spares potential toxicity. These actions are similar to mechanisms that prevent cell cycle progression of cells in G1 and G2 so that damaged DNA can be repaired (27, 28, 29) .
Several strategies have been devised to abrogate cell cycle arrest caused by therapeutic agents (30, 31, 32, 33, 34) . For example, 2-aminopurine may override multiple cell cycle checkpoints (30 , 31) . In addition, caffeine has been used to preferentially abrogate G2 arrest and enhance cytotoxicity of DNA-damaging anticancer agents, such as cisplatin and ionizing radiation (35, 36, 37, 38) . Unfortunately, the caffeine concentration required to achieve this effect in cell culture (5 mM) exceeds clinically tolerable levels by >50-fold (39) . On the other hand, the kinase inhibitor UCN-01 can abrogate the G2 checkpoint induced by cisplatin or ionizing radiation and promote cell cycle progression to mitosis at 25–100 nM (32 , 40) , a concentration of free drug that can be achieved in clinical studies (41 , 42) . Although originally developed as an inhibitor of protein kinase C (43 , 44) , UCN-01 appears to target Chk1 in G2-arrested cells. Chk1 is a cell cycle checkpoint kinase that indirectly affects the activity of Cdc2/cyclin B, which is required for exit from G2 into mitosis (45 , 46) . Although less understood, UCN-01 may also enhance cytotoxicity and abrogate a combined S-phase and G2 phase arrest induced by camptothecin in HT29 human colon carcinoma cells (47) , by cisplatin in Chinese hamster ovary cells (48) , and by mitomycin C in A431 epidermoid carcinoma cells and MCF-7 breast carcinoma cells (49) .
On the basis of these observations, we hypothesized that nucleoside analogues arrest cell cycle progression of the S-phase population to block further incorporation of analogue molecules into DNA. In the absence of nucleoside analogue, the nucleotides are cleared from the cell, and DNA damaged by incorporation is repaired. Thereafter, surviving cells are capable of proliferation. Thus, the ability of cells to enact an S-phase arrest in response to incorporation of nonlethal amounts of nucleoside analogue may serve as a mechanism of resistance to S-phase-specific agents. As a corollary, the dysregulation of S-phase arrest may contribute to analogue cytotoxicity. Here we characterize an S-phase arrest induced by the pyrimidine nucleoside analogue gemcitabine in a human myelogenous leukemia cell line and report the actions of UCN-01 on cell cycle progression and viability of this population.
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MATERIALS AND METHODS |
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Chemicals and Antibodies.
The nucleoside analogue gemcitabine was kindly provided by Dr. L. W.
Hertel (Lilly Research Laboratories, Indianapolis, IN). ara-C was
purchased from Sigma Chemical Co. (St. Louis, MO). F-ara-A was produced
by alkaline phosphatase treatment of fludarabine, which was obtained
from Berlex Laboratories (Richmond, CA). FMdC was from Hoechst Marion
Roussel (Bridgewater, NJ). UCN-01 (NSC 638850) was kindly provided by
the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment,
National Cancer Institute (Bethesda, MD). Aliquots of UCN-01 (10
mM in DMSO) were stored at -20°C and diluted in water
immediately prior to each experiment. All other chemicals were
reagent-grade.
[2-14C]Thymidine (55 mCi/mmol) was purchased
from Moravek Biochemicals (Brea, CA).
[methyl-[3
H]]Thymidine (5 Ci/mmol)
was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden).
[
-32P]ATP (4500 Ci/mmol) was purchased from
ICN Radiochemicals (Costa Mesa, CA).
Goat polyclonal antibody to Bcl-XL (sc-7122) and rabbit polyclonal antibody to Bax (sc-493) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody to Bcl-2 (1624 989) was purchased from Boehringer Mannheim Corp. (Indianapolis, IN). Mouse monoclonal antibody to PARP (66391A) and rabbit polyclonal antibody to caspase-3 were purchased from PharMingen International (San Diego, CA). Mouse monoclonal antibody to ß-actin was purchased from Sigma. Antimouse IgG horseradish peroxidase-conjugated antibody was obtained from Bio-Rad Laboratories (Hercules, CA). Antirabbit IgG horseradish peroxidase-conjugated antibody was obtained from Oncogene Research Products (Cambridge, MA).
DNA Content Analysis by PI Staining.
Flow cytometric analysis of PI-stained cells was performed to
demonstrate the effects of gemcitabine and UCN-01 on cell cycle
progression. Briefly, cells were harvested at each time point, washed
twice with ice-cold PBS, and fixed in 70% ethanol at least overnight
at 4°C. Before flow cytometry, cells were washed twice with
ice-cold PBS and stained with 1 ml of PI (15 µg/ml; Sigma)
containing 2.5 µg/ml RNase A (Boehringer Mannheim) for at least 30
min. DNA content of at least 20,000 cells was determined by a FACScan
Coulter Epics XL-MCL flow cytometer (Coulter Corp., Hialeah, FL). The
proportion of cells in a particular phase of the cell cycle was
determined by a Multicycle program (Coulter).
Cell Cycle Analysis by BrdUrd/PI Staining.
ML-1 cells were pulse-treated with 10 µM BrdUrd (Sigma)
for 30 min at 37°C and washed twice with 1% BSA/PBS to remove
unincorporated BrdUrd. Cells were then replenished with fresh,
prewarmed medium and either treated with gemcitabine or left untreated
as a control. At the indicated time, cells were washed twice with 1%
BSA/PBS and fixed with 70% cold ethanol for at least 24 h. Double
helical DNA was acid denatured with 2 N HCl/0.5% Triton
X-100 for 30 min at room temperature, and cells were neutralized with
0.1 M
Na2B4O7
(pH 8.5) for 15 min. Cells were then washed with 0.5% Tween 20/1%
BSA/PBS and resuspended in 50 µl of the same solution. They were then
incubated with 20 µl of FITC-conjugated monoclonal anti-BrdUrd
antibody (Becton Dickinson, San Jose, CA) per 1 x 106 cells for 30 min at room temperature on a
rocker. Cells were washed with 0.5% Tween 20/1% BSA/PBS and stained
with 1 ml of PI (15 µg/ml) containing 2.5 µg/ml RNase A for at
least 30 min.
Examination of Cell Morphology.
After centrifugation of 5 x 105
cells to a slide by cytospin (550 rpm for 5 min), cells were fixed with
100% methanol for 20 min, air dried, and stained by Wright’s Giemsa
stain solution (Fisher Scientific, Pittsburgh, PA) for 15 min. Cell
morphology was examined by light microscopy using a Nikon HFX-II
microscope. Apoptotic morphology was identified by the appearance of
cell shrinkage, nuclear condensation, and/or the appearance of
membrane-bound apoptotic bodies. At least 500 cells/field in a minimum
of three randomly selected fields were counted on three slides for each
sample.
Quantification of DNA Fragmentation.
DNA fragmentation was quantified as described previously
(50)
with modifications (51)
. Briefly,
exponentially growing ML-1 cells were prelabeled with
[14C]thymidine for 24 h (0.01 µCi/ml
every 12 h) at 37°C and treated with gemcitabine ± UCN-01. At harvest, 2–4 x 106 cells were pelleted, washed with
Ca2+/Mg2+-free PBS, and
lysed by incubation in 1 ml of hypotonic lysis buffer containing 10
mM Tris-Cl (pH 7.5), 1 mM EDTA, and 0.2%
Triton X-100 (Sigma) on ice for 20 min. Intact chromatin-sized DNA
(insoluble and in the pellet) was separated from fragmented DNA
(soluble at low-salt concentrations and in the supernatant) by
centrifugation at 13,000 x g for 10 min. The
pelleted chromatin was solubilized in 1 ml of Soluene-350 (Packard
Instrument Company, Meriden, CT) at 60°C for 2 h. The
radioactivities of both the pellet and supernatant fractions were then
counted in 10 ml of Liquiscint scintillation fluor (Packard) by a
liquid scintillation counter (Packard). The percentage of DNA
fragmentation was calculated as:
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Gel electrophoresis was used to visually identify DNA that had been enzymatically digested into the ladder pattern of oligonucleosome-sized DNA fragments.
DNA Synthesis Assay.
Exponentially growing ML-1 cells were prelabeled with
[14C]thymidine for 24 h (0.01 µCi/ml
every 12 h) at 37°C and incubated with 10 nM
gemcitabine ± 100 nM UCN-01 for the
indicated times in a 96-well filtration plate (Millipore Corp.,
Bedford, MA).
[methyl-[3
H]]Thymidine was added
(0.4 µCi/well) at least 30 min before incubation ended, and labeled
cells were collected on filters using a vacuum manifold (Millipore).
The filters were washed four times each with 8% trichloroacetic acid,
Millipore water, and 100% ethanol. They were then dried and punched
into a vial. Radioactivity retained on the filter was determined by a
liquid scintillation counter (Packard). 3
H values
were normalized by 14C radioactivity.
TUNEL Assay.
To identify the cell cycle position of apoptotic cells, a flow
cytometric TUNEL assay was performed using an APO-DIRECT kit (Phoenix
Flow Systems, Inc., San Diego, CA) according to the manufacturer’s
protocol. Each sample was analyzed on a FACScan Coulter Epics XL-MCL
flow cytometer (Coulter); red PI fluorescence was acquired on a linear
scale on the X axis, and green FITC staining was acquired on a log
scale on the Y axis.
Cell Lysis.
Briefly, 1–3 x 107 cells were
pelleted by centrifugation at 1500 rpm for 5 min, washed twice with
ice-cold PBS, and lysed on ice for 20 min in lysis buffer containing 25
mM HEPES (pH 7.5), 300 mM NaCl, 1.5
mM MgCl2, 0.5% sodium deoxycholate,
20 mM ß-glycerophosphate, 1% Triton X-100, 0.1% SDS,
0.2 mM EDTA (pH 8), 0.5 mM DTT, 1
mM sodium orthovanadate (pH 10), 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 20 µg/ml
leupeptin. Cells were centrifuged at 14,000 rpm at 4°C for 15 min,
and the supernatant was stored at -70°C until use. Protein content
was determined using a protein assay kit according to the
manufacturer’s instructions (Bio-Rad).
Immunoblotting Analysis.
An equal volume of 2x SDS sample-loading buffer containing 100
mM Tris-Cl (pH 6.8), 20% glycerol, 4% SDS, 0.05%
bromphenol blue, and 5% ß-mercaptoethanol was added to cell lysates.
Lysates were then heated at 95–100°C for 5 min. Ninety-µg aliquots
of total cell protein were loaded onto 8–12% SDS-polyacrylamide gels
(percentages depended on protein sizes detected), and proteins were
electrophoresed at constant voltage (70–100 V) and electrotransferred
to Immobilon-P membranes (Millipore) for 1–3 h at 250 mA. Membranes
were blocked overnight in TBS-Tween 20 containing 5% nonfat dried milk
and then incubated with primary antibodies (0.5–2 µg/ml) for 3 h, and secondary antibodies were conjugated to horseradish peroxidase
(1:2500 dilution) for 1 h. The blots were visualized by enhanced
chemiluminescence according to the manufacturer’s instructions
(Pierce, Rockford, IL).
Immunoprecipitation and Histone H1 Kinase Assay.
For each sample, 500 µg of cell protein were mixed with 3 µg of
rabbit polyclonal Cdc2 antibody on a rocker for 3 h at 4°C. The
immune complexes were then mixed with 100 µl of 20% protein
A-Sepharose beads (Amersham Pharmacia Biotech) on a rocker for another
1–2 h at 4°C, and the beads were washed twice with lysis buffer and
once in kinase buffer containing 20 mM Tris-Cl [pH 7.5,
0.1 mM EGTA (pH 7.0), 10 mM
MgCl2, and 1 mM DTT]. The Cdc2
immunoprecipitates were incubated with 30 µl of kinase buffer plus 20
µM cold ATP, 3 µg of histone H1 (Boehringer Mannheim),
and 6 µCi of [-32P]ATP for 30 min at
37°C. An equal volume of 2x SDS sample-loading buffer was added to
terminate the reaction. The mixture was then boiled for 5 min and
loaded onto a 10% SDS-polyacrylamide gel. Autoradiography was
performed, and radioactivities were quantified by a Betascope 603 blot
analyzer (Betagen, Waltham, MA).
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RESULTS |
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shows the typical cell cycle distribution of an
exponentially growing population of ML-1 cells; the S-phase fraction is
33%. In contrast, incubation of cells for 24 h (approximately
one population doubling time) with either 10 nM
gemcitabine, 5 nM FMdC, 50
nM ara-C, or 2 µM F-ara-A
resulted in an increase in the S-phase fraction to 60–70% (Fig. 1, B
-E, respectively). Treatments were minimally
toxic to the cells, because there was a <10% increase in
sub-G1 populations. Thus, the concentrations of
nucleoside analogues used were cytostatic and caused an accumulation of
cells in S-phase. Hereafter, although our data suggested that the
cellular response was similar for each of these nucleoside analogues
tested, we focused our study on gemcitabine.
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. Maximum S-phase arrest for a 24-h gemcitabine treatment
occurred with 6–15 nM gemcitabine. Lower
concentrations failed to arrest cells, whereas higher concentrations
caused accumulation at the G1-S boundary and most
likely killed S-phase cells. Thus, we chose 10 nM
gemcitabine to induce S-phase arrest in ML-1 cells for additional
experiments. S-phase accumulation reached a plateau after 16 h of
incubation and was maintained through 34 h (Fig. 2B)
.
To evaluate the stability of this arrest, cells were pulsed with BrdUrd
for 30 min, washed into fresh medium, and incubated with 10
nM gemcitabine. For cells not treated with
gemcitabine, those in S-phase at the time of BrdUrd labeling exited
S-phase by 9 h and cycled back to the subsequent S-phase by
18 h. In contrast, during continuous treatment with gemcitabine
for as long as 34 h, <25% of S-phase cells were able to transit
out of S-phase (Fig. 2C)
. The significant delay in
progression through S-phase during treatment with a cytostatic
concentration of gemcitabine was consistent with an effective cell
cycle arrest.
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. The absence of BrdUrd-positive cells with a
sub-G1 DNA content (data not shown) and the
maintenance of cell numbers (detected by particle counts) suggested
that the treatment did not cause cell lysis.
Lack of Cell Cycle Progression after UCN-01 Treatment of Cells
Arrested in S-Phase by Gemcitabine.
Prior studies have indicated that UCN-01 can abrogate cell cycle
blockage of cells arrested in G2 by ionizing
radiation (40)
, cisplatin (32)
, or DNA
topoisomerase I inhibitors (47)
. In this study, we
investigated the actions of UCN-01 on cells arrested exclusively in
S-phase. Dose-response experiments demonstrated that continuous
incubation with 500 nM UCN-01 had no effect on the
viability of ML-1 cells after 72 h; therefore, we chose 100
nM UCN-01 for further investigation, because it was clearly
nontoxic and had no effect on cell cycle distribution (data not shown).
Addition of 100 nM UCN-01 to ML-1 cells after a 24-h
incubation with 10 nM gemcitabine resulted in a rapid
decrease in the S-phase fraction (Fig. 3A)
. If this promoted cell cycle progression, we would expect
the G2 fraction to begin to expand as the S-phase
population diminished. However, no increase in the
G2 population was evident after UCN-01 addition
(Fig. 3B)
, and the G1 population
appeared stable (Fig. 3C)
. Rather, there was a concomitant
increase in cells with a sub-G1 DNA content (Fig. 3D)
. These results suggested that addition of an otherwise
nontoxic concentration of UCN-01 to S-phase-arrested cells initiated
cell death without cell cycle progression.
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. Gemcitabine treatment for 24 h inhibited DNA
synthesis by 65%, and synthesis did not increase after addition of
UCN-01. In fact, there was a small but reproducible decrease to only
7% of control values, indicating that DNA synthesis was not
reactivated by UCN-01. In addition, immunoprecipitated Cdc2 kinase did
not increase in gemcitabine-arrested cells or after addition of UCN-01
(Fig. 4B)
. Because activated Cdc2 kinase is essential for
cell cycle progression through G2 into mitosis,
these results supported the conclusion that UCN-01 did not promote the
ability of S-phase-arrested cells to pass through the cell cycle.
Finally, there was no increase in the absolute number of cells in the
culture within 10 h after UCN-01 addition (data not shown).
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Induction of Apoptosis by UCN-01 in Gemcitabine-arrested ML-1
Cells.
The marked increase in the sub-G1 population of
S-phase-arrested cells after a 10-h incubation with 100 nM
UCN-01 (Fig. 3D)
suggested that UCN-01 induced cell death.
We therefore studied the cellular morphology of these cells and found
cell membrane blebbing, nuclear condensation, and apoptotic bodies
consistent with apoptosis (data not shown). Apoptotic cells were
counted every 2 h after addition of 100 nM
UCN-01, and the proportion of cells with apoptotic morphology increased
over time during combination treatment with gemcitabine and UCN-01,
rising to 50% after 10 h (Fig. 5A)
. As an additional parameter of apoptosis, nucleosomal DNA
fragmentation assays showed that DNA fragments appeared in
gemcitabine-arrested cells 2 h after UCN-01 addition and increased
with time (data not shown). To quantitate the DNA fragmentation, we
labeled ML-1 cells with [14C]thymidine prior to
gemcitabine treatment and determined the percentage of soluble DNA
fragments relative to the whole DNA content of the cultures (Fig. 5B)
. After addition of UCN-01, the induction of DNA
fragmentation occurred at approximately the same time as the appearance
of apoptotic morphology.
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. This was associated with the cleavage of the
caspase-3 substrate, PARP, into an Mr
85,000 fragment. These results suggested that UCN-01 initiated an
apoptotic cascade in the S-phase-arrested cells. Finally, because Bcl-2
family members may play a critical role in regulating cell survival or
death, we examined whether the protein expression of some of these
family members was affected by cytostatic treatment with gemcitabine or
by addition of UCN-01. Immunoblotting analysis of Bcl-2 and
Bcl-XL, which are inhibitors of cell death, and
of Bax, which is a promoter of cell death, showed that protein levels
were not affected by incubation with cytostatic concentration of
gemcitabine or addition of UCN-01 (Fig. 5D)
.
Selective Killing of S-Phase-arrested Cells by UCN-01.
The rapid loss of cells with an S-phase DNA content (Fig. 3A)
, the absence of an increase in the
G2 population (Fig. 3B)
, and the
concomitant increase in cells with apoptotic markers after addition of
UCN-01 (Fig. 3D)
suggested that UCN-01 selectively killed
the S-phase population. To evaluate this possibility, we used the TUNEL
assay. Only a minor fraction of the population appeared to be TUNEL
positive after 24-h cytostatic exposure to gemcitabine (Fig. 6A)
. However, a time course investigation after UCN-01
addition demonstrated that the TUNEL-positive cells appeared to arise
from the portion of the population defined by PI staining to have an
S-phase DNA content. Quantitation of these data indicated that the
percentage of TUNEL-positive cells increased from 10% to >50% in the
10 h after UCN-01 addition (Fig. 6B)
. In contrast, the
number of TUNEL-positive cells did not significantly increase with
continued incubation with gemcitabine alone for as long as 34 h.
Finally, the TUNEL-negative populations in the cells with an S-phase
DNA content reciprocally decreased over time after the addition of
UCN-01 (Fig. 6C)
. Because there was no significant change in
TUNEL-negative cells in G1 and
G2 (data not shown), we concluded that UCN-01
induced a rapid onset of apoptosis specific for the cells arrested in
S-phase.
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DISCUSSION |
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Cell cycle checkpoints serve as surveillance systems to interrupt cell cycle progression when damage to the genome or spindle is detected or when cells have failed to complete a preceding event (52) . Thus far, emerging studies have emphasized DNA damage-induced G1 or G2 checkpoints. However, little is known about the molecular mediators of S-phase-arresting mechanisms in mammalian cells. Although previous investigators have used agents that are not entirely specific for S-phase cells, such as cisplatin and camptothecin, we chose to use the nucleoside analogue gemcitabine because nucleoside analogues act by incorporating into DNA during replication, and therefore more specifically target cells in S-phase.
Although cytotoxic concentrations of gemcitabine and other nucleoside analogues selectively kill S-phase cells in human leukemia cell lines (8 , 9) , substantial literature demonstrates that not all S-phase cells are killed during cytotoxic chemotherapy. One goal of early therapeutic studies was to use the S-phase-specific nucleoside ara-C to synchronize the population of leukemic blasts in S-phase. Flow cytometry has clearly demonstrated an accumulation of cells in S-phase, indicating the resistance of at least a portion of the S-phase population to potentially lethal cell cycle-specific treatment (20, 21, 22, 23, 24, 25, 26) . The high incidence of leukemic regrowth, even after intensive ara-C therapy that should have extended beyond the cell cycle time of most human leukemias (53 , 54) , suggests a mechanism of clinical resistance that is not seen in metabolic models (55 , 56) . In the clinic, it is possible that blasts fully in S-phase could encounter low-level incorporation of the analogue into DNA at the beginning of infusions, when triphosphate concentrations are low. Additionally, some cells may transit into S-phase some time after the end of nucleoside infusion, when metabolic elimination has reduced the cellular level of triphosphate analogues. Here, we have used cytostatic treatment of an acute myelogenous leukemia cell line as a model of arrest during S-phase progression to evaluate it as a defense response to nonlethal analogue incorporation.
We found relatively narrow ranges of concentrations of each nucleoside
analogue that optimized S-phase accumulation in the absence of
cytotoxicity (Fig. 1
and data not shown), as illustrated specifically
by gemcitabine (Fig. 2A)
. Presumably, the differences in
concentrations among the analogues reflect their cellular pharmacology,
leading to incorporation of a critical amount of each analogue in DNA,
and possibly indicate differences in affinity of the putative sensor
for a specific nucleotide. Because nucleotides of gemcitabine,
fludarabine, and FMdC inhibit ribonucleotide reductase, it is possible
that an imbalance in deoxynucleotide pools may also contribute to the
S-phase arrest of cells incubated with these nucleosides. After release
from S-phase arrest, populations exhibited a lag that reflected the
duration of nucleoside analogue exposure before resuming proliferation
at a rate comparable with controls (Fig. 2D)
. This suggested
that an S-phase checkpoint had been activated, in which cell cycle
progression through the S-phase at its normal rate was prevented, and
incorporation of additional analogues was subsequently blocked. It is
likely that the lag prior to continued proliferation reflects the time
needed for elimination of cytostatic concentrations of the analogue
nucleotides and repair of damage associated with incorporated drug and
stalled replication forks. Thus, the induction of cytostasis and
subsequent recovery may model a mechanism of resistance to nucleoside
analogues. Furthermore, activation of the S-phase checkpoint by
gemcitabine appeared independent of p53 status, because S-phase arrest
was also observed in the p53 mutant cell line CCRF-CEM (data not
shown).
The progression of cells through the cell cycle is controlled by core
cell cycle regulators, the most prominent of which are a family of
kinases under the direction of their regulatory subunits, the cyclins.
Although these cyclin-dependent kinases have specific substrates that
enable orderly progression through the cell cycle, a complex hierarchy
of other kinases and phosphatases provide an overlay of regulation on
these processes. In particular, the activity of the S-phase-specific
complex of cyclin-dependent kinase 2 (Cdk2) and cyclin A is enabled by
phosphorylation of Thr-160 and inhibited by phosphorylation of Thr-14
and Tyr-15 on Cdk2. The Tyr-15 phosphorylation is antagonistically
regulated by wee1 kinase and Cdc25A/Cdc25B phosphatases
(57, 58, 59)
, the direct upstream regulators of Cdk2. Recent
studies have shown that the checkpoint kinases Chk1 and Chk2 are
upstream serine/threonine protein kinases that regulate both wee1
kinase and Cdc25A/B phosphatase activity (60
, 61)
. Chk2
becomes phosphorylated and activated in response to DNA damage or
replication blockage (62, 63, 64)
, and Chk1 is also an
important effector of the DNA damage checkpoint (61
, 65
, 66)
and serves an essential function in the DNA replication
checkpoint in the absence of Chk2 (67
, 68)
. More recently,
studies from two different groups both identified the Chk1 kinase and
the Cdc25C pathway as potential targets of G2
checkpoint abrogation by UCN-01 (45
, 46)
. Thus, we
hypothesized that increased phosphorylation of Tyr-15 on Cdk2 may be
associated with a nucleoside analogue-induced, S-phase checkpoint.
Disruption of this phosphorylation may permit cells to replicate their
DNA in the presence of DNA damage, which could have lethal consequences
for the cell. In support of this postulate, we have demonstrated that
cells arrested in S-phase by gemcitabine treatment are sensitized to
otherwise nontoxic concentrations of the protein kinase inhibitor
UCN-01 (Figs. 3D
and 5
, A–C).
Furthermore, preliminary studies of ML-1 cells arrested in S-phase by
F-ara-A demonstrated an increased level of Tyr-15 phosphorylation on
Cdk2 that was associated with a decreased kinase activity. Upon
addition of UCN-01, this inhibitory phosphorylation was decreased with
the concomitant increase in Cdk2-associated kinase activity
(69)
. This suggests that a molecular regulation analogous
to that observed in G2-arrested cells, perhaps
functioning through Chk1/Chk2 and the Cdc25A/B pathway, may be involved
in S-phase arrest induced by nucleoside analogues and dysregulated by
UCN-01 addition.
Recently, several laboratories have reported that UCN-01 potently
abrogates the G2 checkpoint (32
, 40)
and possibly the S-phase checkpoint (47, 48, 49)
by allowing
arrested cells to progress into the next phase of the cell cycle.
However, for ML-1 cells arrested in S-phase by nucleoside analogues, we
demonstrated that UCN-01 addition affected the S-phase-arrested
fraction specifically, and that these cells underwent apoptosis without
cell cycle progression: (a) DNA synthesis determined by
[3
H]thymidine incorporation was not increased
but instead decreased after the addition of UCN-01 (Fig. 4A)
; (b) as the population of cells with S-phase
DNA content decreased, the fraction of G2 cells
did not increase but rather decreased, indicating that there were no
cells coming from the S-phase and that cells previously in
G2 may have moved to the G1
phase (Fig. 3B)
; (c) Cdc2 kinase activity did not
increase after the addition of UCN-01 to gemcitabine-arrested cells,
suggesting that cells were not transiting from G2
to mitosis (Fig. 4B)
; and (d) the TUNEL flow
cytometric assay, used to determine the cell cycle specificity of
apoptotic cells, demonstrated that it was the S-phase fraction that was
selectively induced to undergo apoptosis (Fig. 6)
.
The molecular mechanisms for UCN-01-induced apoptosis in the S-phase-arrested cells are not fully understood. It has been shown that in F-ara-A-arrested cells, signaling through c-Jun NH2-terminal kinase is activated shortly after UCN-01 addition but before the cells begin to show biochemical or morphological evidence of apoptosis (70) . Whether this pathway is also activated by gemcitabine needs further investigation. On the other hand, it has also been suggested that suppression of survival signaling pathways is involved in this treatment system. Recent evidence has shown that activation of the phosphatidylinositol 3-kinase-Akt kinase signaling pathway culminates in the phosphorylation of the Bcl-2 family member BAD, thereby suppressing apoptosis and promoting cell survival (71, 72, 73) . Our preliminary results indicated that UCN-01-mediated changes in the phosphorylation status inhibited the phosphatidylinositol 3-kinase-Akt-BAD survival pathway and were associated with induction of apoptosis in gemcitabine-arrested cells (74) .
In conclusion, the lack of metabolic resistance characteristics established in cell lines and in the leukemia blasts isolated from patients refractory to nucleoside analogue therapy prompted our hypothesis that S-phase arrest occurs in response to minimal damage. In our model, we demonstrate that ML-1 cells arrest cell cycle progression in S-phase in response to a variety of S-phase-specific analogues but are able to continue proliferation after removal of the drugs. Incubation with otherwise nontoxic concentrations of UCN-01 causes a rapid onset of apoptosis without cell cycle progression in nucleoside analogue-arrested cells. Thus, S-phase arrest appears to sensitize cells to actions of UCN-01 that inhibit survival pathways and/or activate cell death mechanisms. Based in part on these findings, a clinical trial of ara-C in combination with UCN-01 has been initiated in patients with relapsed acute myelogenous leukemia.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grants CA28596, CA32839,
CA55164, and Cancer Center Core Grant P30 CA16672 from the National
Cancer Institute; Grant DHP-1 from the American Cancer Society; and the
Rosalie B. Hite Fellowship in Cancer Research (to Z. S.). 
2 To whom requests for reprints should be
addressed, at Department of Experimental Therapeutics, Box 71, The
University of Texas M. D. Anderson Cancer Center, 1515 Holcombe
Boulevard, Houston, TX 77030. Phone: (713) 792-3335; Fax:
(713) 794-4316; E-mail: wplunket{at}mdanderson.org
3 The abbreviations used are: ara-C,
1-ß-D-arabinofuranosylcytosine; UCN-01,
7hydroxystaurosporine; FMdC,
(E)-2'-deoxy-2'-fluoromethylene cytidine; F-ara-A,
9-ß-D-arabinofuranosyl-2-fluoroadenine; PI, propidium
iodide; BrdUrd, bromodeoxyuridine; TUNEL, terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling; PARP,
poly(ADP-ribose)polymerase; Cdk, cyclin-dependent kinase.
Received 7/ 7/00. Accepted 11/29/00.
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) or medium containing 10 nM
gemcitabine (•). At indicated times, cells were then harvested, fixed
in 70% ethanol, stained for BrdUrd and PI, and analyzed by flow
cytometry. D, proliferation of ML-1 cells after release
from gemcitabine. Cells were either untreated (
) or treated with 10
nM gemcitabine for 24–48 h, washed into drug-free medium
at 24 h (
), 36 h (
), and 48 h (
), and incubated
for up to 288 h. As a procedural control, untreated cells were
washed at 24 h and resuspended in fresh medium. Cells were counted
with a Coulter counter at the indicated times.
