[CANCER RESEARCH 61, 1065–1072, February 1, 2001]
S-Phase Arrest by Nucleoside Analogues and Abrogation of Survival without Cell
Cycle Progression by 7-Hydroxystaurosporine1
Zheng Shi, Atsushi Azuma, Deepa Sampath, Ye-Xiong Li, Peng Huang, and William Plunkett2
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
ABSTRACT
The mechanisms of resistance to nucleoside analogues established in
preclinical models are rarely found in primary tumors resistant to therapy
with these agents. We tested the hypothesis that cells sense sublethal
incorporation of analogues into DNA during replication and react by
arresting further DNA synthesis and cell cycle progression. After removal
of drug, cells may be able to repair damaged DNA and continue proliferation, thus escaping nucleoside analogue toxicity. As a corollary, we
evaluated whether dysregulation of this mechanism causes cell death.
Using gemcitabine as a model of S-phase-specific nucleoside analogues in
human acute myelogenous leukemia ML-1 cells, we found that DNA
synthesis decreased, cells arrested in S-phase transit, and 60 –70% of the
population accumulated in S-phase in response to cytostatic conditions.
Proliferation continued after washing the cells into drug-free medium.
S-phase-arrested cells were then treated with otherwise nontoxic concentrations of UCN-01, which caused rapid onset of apoptosis without cell
cycle progression specifically in cells with an S-phase DNA content. Thus,
S-phase arrest by nucleoside analogues sensitizes cells to UCN-01, which
appears to activate signaling for death mechanisms and/or inhibit survival
pathways. These results differ from those in cells arrested at the G2
checkpoint, in which UCN-01 abrogates cell cycle arrest, permitting cells
to progress in the cell cycle before apoptosis.
INTRODUCTION
DNA-directed nucleoside analogues, such as ara-C,3 fludarabine,
and gemcitabine, are antimetabolites effective in the treatment of a
variety of malignancies (1, 2). The major mechanism of action of
nucleoside analogues is through incorporation into DNA, and thus this
class of agents shows specificity for cells in S-phase (3–7). The
triphosphates of nucleoside analogues compete with the cellular deoxyribonucleotides for incorporation into DNA during replication.
Analogue incorporation inhibits DNA synthesis and kills the cells
either by inducing apoptosis in cells capable of this process or by
mitotic catastrophe in cells that lack the ability to conduct programmed cell death (8, 9). The precise mechanisms by which nucleoside analogues induce these forms of cell death are largely unknown.
Although ara-C has remained the mainstay of a variety of combination treatments for acute myelogenous leukemia (10 –12), resistance
to ara-C-based chemotherapy is still a major problem in therapy
(13–15). Several in vitro-generated mechanisms of resistance to ara-C
and other clinically active nucleoside analogues focus on their metabolism and cellular pharmacology (16 –19). However, none appear
Received 7/7/00; accepted 11/29/00.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
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: [email protected].
3
The abbreviations used are: ara-C, 1-␤-D-arabinofuranosylcytosine; UCN-01, 7hydroxystaurosporine; FMdC, (E)-2⬘-deoxy-2⬘-fluoromethylene cytidine; F-ara-A, 9-␤-Darabinofuranosyl-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.
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– 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 –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 (27–29).
Several strategies have been devised to abrogate cell cycle arrest
caused by therapeutic agents (30 –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 (35–38). 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
Cell Culture. ML-1, a human acute myelogenous leukemia cell line containing wild-type p53, was a gift from Dr. Michael B. Kastan (St. Jude
Children’s Research Hospital, Memphis, TN). CCRF-CEM, a human Tlymphoblastic leukemia cell line containing two mutated p53 alleles, was
obtained from the American Type Culture Collection (Rockville, MD). Cell
lines were maintained in exponential growth phase in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.,
Grand Island, NY) for ML-1 cells and 5% serum for CCRF-CEM cells. Cells
were incubated at 37°C (5% CO2) in a humidified atmosphere. Populationdoubling times were approximately 22–24 h.
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-[3H]]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 ␤-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 ␮g/ml; Sigma) containing 2.5 ␮g/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 pulsetreated with 10 ␮M 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 ␮l of the same solution. They were then incubated with 20 ␮l of
FITC-conjugated monoclonal anti-BrdUrd antibody (Becton Dickinson, San
Jose, CA) per 1 ⫻ 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 ␮g/ml) containing 2.5 ␮g/ml RNase A for at least 30 min.
Examination of Cell Morphology. After centrifugation of 5 ⫻ 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
␮Ci/ml every 12 h) at 37°C and treated with gemcitabine ⫾ UCN-01. At
harvest, 2– 4 ⫻ 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 ⫻ 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:
% DNA fragmentation ⫽
Radioactivity (dpm) of supernatant
Radioactivity (dpm) of supernatant and pellet
⫻ 100
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 ␮Ci/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-[3H]]Thymidine was
added (0.4 ␮Ci/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 ⫻ 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 ␤-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 ␮g/ml aprotinin, and 20 ␮g/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 2⫻ SDS sample-loading
buffer containing 100 mM Tris-Cl (pH 6.8), 20% glycerol, 4% SDS, 0.05%
bromphenol blue, and 5% ␤-mercaptoethanol was added to cell lysates. Lysates were then heated at 95–100°C for 5 min. Ninety-␮g 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 ␮g/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 ␮g of cell protein were mixed with 3 ␮g of rabbit polyclonal Cdc2
antibody on a rocker for 3 h at 4°C. The immune complexes were then mixed
with 100 ␮l 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 ␮l of kinase buffer plus 20 ␮M cold
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ATP, 3 ␮g of histone H1 (Boehringer Mannheim), and 6 ␮Ci of [␥-32P]ATP
for 30 min at 37°C. An equal volume of 2⫻ 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).
RESULTS
S-Phase Arrest Induced by Cytostatic Concentrations of Nucleoside Analogues in Exponentially Growing ML-1 Cells. To develop a model for S-phase arrest in exponentially growing ML-1 cells,
we first investigated the effects of cytostatic concentrations of nucleoside analogues on the cell cycle. Fig. 1A 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 ␮M 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.
To further characterize gemcitabine-induced S-phase arrest, we first
determined the dose response for S-phase accumulation in exponentially growing populations (Fig. 2A). 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
Fig. 2. Gemcitabine-induced S-phase arrest in ML-1 cells. A, concentration dependency. Cells were treated with various cytostatic concentrations of gemcitabine for 24 h. B,
time course. Cells were treated with 10 nM gemcitabine for various times, fixed in 70%
ethanol, stained with PI, and analyzed by flow cytometry. C, stability of S-phase arrest.
Cells were incubated 30 min with 10 ␮M BrdUrd, washed, and resuspended for 3–34 h in
either fresh growth medium (E) or medium containing 10 nM gemcitabine (F). 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 (f) or treated with 10 nM gemcitabine for 24 – 48
h, washed into drug-free medium at 24 h (Œ), 36 h (), and 48 h (⽧), 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.
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
Fig. 1. Induction of S-phase arrest by cytostatic concentrations of nucleoside analogues on exponentially growing ML-1 cells. ML-1 cells were untreated as control (A) or treated
for 24 h with cytostatic concentrations of nucleoside analogues: 10 nM gemcitabine (B), 5 nM FMdC (C), 50 nM ara-C (D), or 2 ␮M F-ara-A (E). Cells were fixed in 70% ethanol, stained
with PI, and analyzed by flow cytometry.
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Fig. 3. Effects of UCN-01 on gemcitabine-arrested ML-1 cells. Cells were incubated
with 10 nM gemcitabine continuously for 34 h (E), and 100 nM UCN-01 was added at 24 h
to one portion (F) when the culture was split. Cells were harvested at indicated times for
analysis by flow cytometry. Quantitative data of relative DNA content from at least three
independent experiments are shown: A, cells in S-phase; B, cells in G2-M phase; C, cells
in G1 phase; and D, cells with a sub-G1 DNA content. Bars, SD.
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.
Next, we determined whether S-phase-arrested cells could proliferate after removal of gemcitabine, as would be expected in a resistant
phenotype. Growth was quantitated after cells were treated with 10 nM
gemcitabine for 24, 36, and 48 h and washed into fresh medium.
Incubation for 24 –36 h caused a major arrest of the population in
S-phase. However, after the analogue was washed out, cells continued
proliferating at a rate comparable with controls, although proliferation
restarted after lag times that correlated with the times spent in gemcitabine. Cells treated with gemcitabine for 48 h (more than two
population doubling times) did not recover proliferative capacity ⬎10
days after the drug was washed out (Fig. 2D). 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-phasearrested cells initiated cell death without cell cycle progression.
If UCN-01 were to promote cell cycle progression of the S-phasearrested cells induced by cytostatic treatment with gemcitabine, it
would be expected that cells would reinitiate DNA synthesis. To
evaluate this possibility, we quantitated DNA synthesis in gemcitabine-arrested cells before and after UCN-01 addition (Fig. 4A). 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).
Cells arrested in S-phase by F-ara-A or ara-C showed similar
responses to UCN-01, suggesting a general nature of this response
(data not shown). In parallel studies, human T-lymphoblastic leukemia CCRF-CEM cells, which contain two mutated p53 alleles, accumulated in S-phase in response to cytostatic treatments with gemcitabine and F-ara-A. Subsequently, UCN-01 also specifically decreased
the S-phase population in these nucleoside analogue-arrested cells
(data not shown). This suggested that the ability of nucleoside analogues to arrest cells in S-phase and the effects of UCN-01 on these
populations were both independent of p53 status.
Induction of Apoptosis by UCN-01 in Gemcitabine-arrested
ML-1 Cells. The marked increase in the sub-G1 population of Sphase-arrested cells after a 10-h incubation with 100 nM UCN-01 (Fig.
Fig. 4. Effects of UCN-01 on cell cycle progression of S-phase-arrested cells. Cells
were incubated with 10 nM gemcitabine continuously for 34 h, and 100 nM UCN-01 was
added at 24 h to one portion when the culture was split. A, no reactivation of DNA
synthesis. DNA synthesis was measured by [3H]thymidine incorporation at the indicated
times; data shown are the means of two independent experiments. Control incorporation
was 81,947 ⫾ 459 dpm (n ⫽ 2). Bars, SD. B, lack of effect of gemcitabine and UCN-01
on Cdc2 kinase activities. Immunoprecipitates were collected from cell extracts with
anti-Cdc2 antibody at indicated times. Histone H1 kinase activity was determined by a
Betascope 603 blot analyzer and was expressed relative to untreated control cells. Three
independent experiments were performed; a typical image is shown.
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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.
Consistent with apoptosis, immunoblotting demonstrated activation
of the executioner caspase, caspase-3, after addition of UCN-01 to
gemcitabine-treated ML-1 cells, as illustrated by the cleavage of
caspase-3 from its Mr 32,000 proenzyme form to a Mr 17,000 fragment (Fig. 5C). 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 cyto-
static 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 TUNELpositive 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.
DISCUSSION
Fig. 5. Induction of apoptosis by UCN-01 in gemcitabine-arrested cells. Cells were
incubated with 10 nM gemcitabine continuously for 34 h, and 100 nM UCN-01 was added
at 24 h to one portion when the culture was split. A, apoptotic morphology. Samples were
collected and analyzed at the indicated times; quantitation of apoptotic morphology in
ML-1 cells treated with gemcitabine ⫾ UCN-01 is shown. Bars, SD. B, DNA fragmentation. Samples were collected, and quantitation of DNA fragmentation was performed at
indicated times. C, caspase-3 activation and PARP cleavage. Cell lysates were prepared
at the indicated times. Equal amounts of protein (90 ␮g) were subjected to immunoblot
analysis with antibodies to caspase-3 and PARP. D, Bcl-2 family members. Cell lysates
were prepared at the indicated times. Equal amounts of protein (90 ␮g) were subjected to
immunoblot analysis with antibodies to Bcl-2, Bcl-XL, and Bax. ␤-actin was used for a
loading control.
In the present study, we demonstrated that cytostatic concentrations
of nucleoside analogues arrested cell cycle progression in S-phase and
decreased DNA synthesis in human myeloblastic ML-1 cells and that
removal of the analogues permitted renewed proliferation. However,
although nontoxic concentrations of UCN-01 alone had no effect on
population growth or cell cycle distribution of ML-1 cells, UCN-01
abruptly abrogated the survival of S-phase-arrested cells, thereby
contributing to overcoming potential resistance to nucleoside analogues. Furthermore, this abrogation was not associated with cell
cycle progression to the G2-M phase. Rather, UCN-01 appeared to
directly initiate an apoptotic cascade in S-phase-arrested cells.
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 –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
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Fig. 6. Selective killing by UCN-01 of S-phase-arrested
ML-1 cells. Cells were incubated with 10 nM gemcitabine
continuously for 34 h, and 100 nM UCN-01 was added at 24 h
to one portion when the culture was split. At the indicated
times, cells were harvested, fixed in 70% ethanol, and subjected to a TUNEL assay. A, bivariate DNA histograms of
FITC-dUTP (TUNEL labeling intensity) and PI (DNA content) double-stained contour plots. B and C, quantitative data
showing TUNEL-positive cells (B) and TUNEL-negative cells
with S-phase DNA content (C).
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 Sphase 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 (57–59), 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
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(62– 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– 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 [3H]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 (71–73).
Our preliminary results indicated that UCN-01-mediated changes in
the phosphorylation status inhibited the phosphatidylinositol 3-kinaseAkt-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.
ACKNOWLEDGMENTS
We thank Karen Ramirez for the excellent technical assistance on flow
cytometric analysis. We also thank Kerry Wright for critical editorial review.
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Research.
S-Phase Arrest by Nucleoside Analogues and Abrogation of
Survival without Cell Cycle Progression by
7-Hydroxystaurosporine
Zheng Shi, Atsushi Azuma, Deepa Sampath, et al.
Cancer Res 2001;61:1065-1072.
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