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Experimental Therapeutics, Preclinical Pharmacology |
Departments of Medicine [S. H., R. D., Y. D., L. K., G. S., S. G.], Pharmacology [S. G.], Microbiology [L. T., S. G.], Biochemistry [S. G.], and Radiation Oncology [P. D.], Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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m), cleavage/activation of procaspase-9 and
procaspase-3, DNA fragmentation, and degradation of poly-ADP(ribosyl)
polymerase. Coadministration of UCN-01 with F-ara-A was also associated
with diminished phosphorylation of the cdc25 phosphatase. In contrast,
exposure of cells to the sequence UCN-01, followed by F-ara-A, resulted
in only a modest increase in apoptotic cells. The ability of UCN-01 to
potentiate F-ara-A-mediated lethality was not mimicked by the selective
PKC inhibitor bisindolylmaleimide, nor did treatment of cells with
UCN-01 enhance formation of F-ara-ATP or increase incorporation of
[3H]F-ara-A into DNA. Enhanced apoptosis in cells exposed
sequentially or simultaneously to F-ara-A and UCN-01 was accompanied by
a substantial reduction in colony formation (e.g., to
0.01% of control values). Cotreatment with UCN-01 also increased
F-ara-A-mediated apoptosis and loss of m in U937
cells ectopically expressing Bcl-2, although not to the same extent as
that observed in empty-vector controls. Finally, simultaneous exposure
(24 h) of malignant B lymphocytes from the pleural effusion of a
patient with indolent non-Hodgkin’s lymphoma to F-ara-A and UCN-01
ex vivo resulted in a striking increase in
apoptosis, as determined by terminal
deoxynucleotidyltransferase-mediated nick end labeling assay.
These findings indicate that UCN-01 increases F-ara-A-induced
mitochondrial damage and apoptosis in human leukemia cells in a
sequence-dependent manner, and that these events occur in at least some
primary human lymphoma cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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UCN-01 (7-OH staurosporine) was developed as an inhibitor of protein
kinase C, a serine/threonine kinase involved in diverse cellular
processes, including mitogenesis, differentiation, and
stimulus-response coupling (8)
. However, UCN-01 also
functions as a checkpoint abrogator and has been shown recently to
inhibit Chk 1, an enzyme intimately involved in cell cycle arrest after
DNA damage (9)
. The ability of UCN-01 to abrogate cell
cycle checkpoints appears to depend upon p53 status in some, but not
all, cell types (10)
. UCN-01 has been shown to potentiate
the antitumor activity of agents that induce G2-M
arrest, including cisplatin, mitomycin, and ionizing radiation
(10, 11, 12)
, although enhancement of the toxicity of
S-phase-specific agents such as camptothecin has also been observed
(13)
. In human leukemia cells, UCN-01 induces apoptosis in
a dose- and time-dependent manner, a phenomenon associated with
dephosphorylation of CDK1 and CDK2 (14)
. Unlike
staurosporine, UCN-01 exhibits in vivo antitumor activity in
animals (15)
. However, free plasma UCN-01 levels in humans
appear to be limited by extensive binding of the parent compound to
1 acidic glycoprotein (16)
.
Nevertheless, interest in UCN-01 as a antitumor agent and signal
transduction modulator persists, and multiple clinical trials involving
this compound are under way (17)
.
Although the activity of fludarabine in hematological malignancies is well established (1) , recent preclinical studies suggest that UCN-01 may also play a role in these disorders (18) . In a recent communication, we demonstrated that the PKC activator and down-regulator bryostatin 1 interacted synergistically with F-ara-A in human leukemia cells, a phenomenon that appeared to involve both induction of leukemic cell apoptosis and differentiation (19) . In addition, UCN-01 has been shown to mimic the actions of bryostatin 1 in circumventing, at least in part, resistance of Bcl-2-overexpressing leukemic cells to apoptosis induced by the pyrimidine analogue ara-C (20) . Currently, little information is available concerning interactions between UCN-01 and purine analogues such as F-ara-A in malignant hematopoietic cells. The purpose of the present studies was to determine whether, and to what extent, UCN-01 might enhance the lethal actions of F-ara-A in human leukemia cells. Our results indicate that UCN-01 substantially increases F-ara-A-mediated mitochondrial injury, caspase activation, and apoptosis in these cells, and that this effect is schedule dependent but unrelated to enhanced F-ara-A metabolism. Furthermore, enhanced lethality for this drug combination can also be demonstrated in at least some primary patient-derived lymphoma cells.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transfectant U937 cells were generated by electroporation, as described previously in detail (20) , with plasmids containing a full-length Bcl-2 cDNA (provided by Dr. M. Cleary, Stanford University, Palo Alto, CA), along with a hygromycin selection marker. These transfected cell lines, designated U937/Bcl-2, and their empty vector counterparts, designated U937/pCEP4, were maintained under selection pressure in 400 µg/ml hygromycin B (Boehringer Mannheim, Mannheim, Germany). U937/Bcl-2 express approximately a 7-fold increase in Bcl-2 protein compared to their empty vector counterparts U937/PCEP4.
Drugs.
UCN-01 was kindly provided by Dr. Edward Sausville (Division of
Clinical Sciences, National Cancer Institute, Bethesda, MD). It was
stored frozen as a 1 mM stock solution in DMSO in
light-protected microcentrifuge tubes at -20°C and was subsequently
diluted 1:10 in sterile PBS (1x, 137 mM NaCl, 2.7
mM KCl, 10.2 mM
Na2HPO4, and 1.76
mM KH2PO4) to a
concentration of 100 µM prior to each experiment. The
diluted UCN-01 solution was directly added to each flask to achieve the
desired final concentration. F-ara-A was purchased from Sigma Chemical
Co. (St. Louis, MO) and stored in powder form under light protected
conditions at 4°C and formulated in sterile PBS prior to use.
F-ara-AMP was provided by Berlex Pharmaceuticals (Richmond, CA).
Although F-ara-AMP is rapidly converted to its nucleoside derivative by
nucleotidases present in the plasma, this process occurs to varying
degrees in cells in tissue culture. For this reason, F-ara-A was used
in the large majority of studies.
[3
H]Fludarabine (10 Ci/mmol) was purchased from
Moravek Biochemicals (Brea, CA). F-ara-ATP was kindly provided by Dr.
W. Plunkett (M. D. Anderson Cancer Center, Houston, TX). GFX (GF
109203X) was purchased from Sigma, formulated in DMSO, and stored
frozen in light-protected vials prior to use. Boc-fmk was purchased
from Enzyme Systems Products (Livermore, CA), formulated in DMSO, and
stored frozen until use.
Experimental Format.
Logarithmically growing cells (approximately 2 x
105 cells/ml) were placed in 25 or 75
cm2 T-flasks (Greiner Labortechnik,
Frickenhausen, Germany) and incubated simultaneously with 2.0
µM F-ara-A and 100 nM UCN-01 for 24 h.
Alternatively, cells at 4 x 105/ml were
incubated in the presence of 2.0 µM F-ara-A for 6 h,
after which they were washed three times with serum-free medium and
resuspended in fresh medium containing 10% FCS at 2 x
105 cells/ml in the presence or absence of 100
nM UCN-01. In some experiments, cells were exposed to the
opposite sequence, e.g., 18 h of UCN-01
6 h of
F-ara-A. At the end of the incubation period, cells were harvested and
prepared for analysis as described below.
Assessment of Apoptosis.
After the indicated drug exposure, morphological evidence of apoptosis
was monitored by evaluating cytocentrifuge preparations stained with
the Diff-Quik stain (Dade Diagnostics, Aguada, Puerto Rico), set, and
viewed under light microscopy. The percentage of apoptotic cells was
determined by scoring the number of cells exhibiting classic features
of apoptosis (e.g., cell shrinkage, nuclear condensation,
extensive formation of membrane blebs, and apoptotic bodies, and
others) as described previously (20)
. Five different
fields were randomly selected, and at least 500 cells were scored for
each drug treatment. In some cases, results were confirmed with TUNEL
assay using terminal transferase and fluorescein-12-dUTP as per the
manufacturer’s instructions (Boehringer Mannheim, Indianapolis, IN).
After fixation in 4% formaldehyde/PBS for 10 min, slides were washed
in PBS and incubated in a 1:2 acetic acid:ethanol solution at -20°C
for 5 min. Slides were washed as before and blocked in 1 mg/ml BSA/PBS
for 1 h prior to treatment with terminal transferase and
fluorescein-12-dUTP. Slides were treated with Vectashield containing
propidium iodide (3:1) and viewed under a fluorescence microscope.
Clonogenic Assays.
A modification of a previously described method was used
(21)
. Briefly, after drug treatment, cells were washed
three times with serum-free medium, cell numbers were normalized, and
500 or 5000 cells/well were plated in 12-well plates (Costar,
Cambridge, MA) containing RPMI 1640, 20% FCS, and 0.3% Agar (Sigma).
The plates were incubated in a 37°C, 5% CO2,
fully humidified incubator for 10–12 days, after which colonies
consisting of groups of 
50 cells were scored using an Olympus Model
CK inverted microscope (Olympus, Lake Success, NY).
F-ara-ATP Formation.
After drug treatment, approximately 20 x
106 cells/condition were washed in cold PBS,
pelleted, lysed in 0.6 N trichloroacetic acid, and
extracted in 1:3.5 trioctylamine:1,1,2-trichlorotrifluoroethane
(SigmaAldrich). The aqueous phase was stored at -80°C until
analysis. Immediately prior to column addition, samples were thawed and
extracted in succession with 0.5 M sodium periodate, 4.0
M methylamine, and 1.0 M rhamnose to convert
the nucleotide triphosphates to their respective bases
(22)
. Extracts were separated on a Waters Radial-Pak 10
µSAX cartridge (Waters, Millipore, Bedford, MA), and absorbance was
monitored at 254 nm using a Beckman 160 detector (Beckman, Fullerton,
CA). Samples were separated using a flow rate 3 ml/min for 22 min in
25% ammonium phosphate (0.75 M, pH 3.7)/75% ammonium
phosphate (5 mM, pH 2.8), which was then ramped up to 100%
of the 0.75 M ammonium phosphate over the next 40 min.
Peaks were identified by relative retention times compared with
authentic F-ara-ATP, as described previously (19)
.
[3
H]F-ara-A DNA Incorporation.
Cells were incubated with 2.0 µM
[3
H]F-ara-A (final specific activity, 10
µCi/mmol) for 24 h as we have described previously
(19)
in the presence or absence of 100 nM
UCN-01. At the end of the incubation period, 5 x
106 cells were centrifuged at 300 x
g for 5 min and resuspended in 200 µl of PBS. After
incubation for 2 min in 100 mg/ml RNase A at room temperature (to
obtain RNA-free genomic DNA), 20 µl of Proteinase K and 200 µl of
Buffer AL provided in the Qiagen DNeasy Tissue kit (Qiagen, Inc.,
Chatsworth, CA), were added, vortexed, and incubated at 70°C for
10 min. After the incubation period, 200 µl of 100% ethanol were
added and vortexed, and the mixture was transferred to a DNeasy spin
column and collecting tube (provided) and centrifuged at 8000 rpm for 1
min. The centrifugation step was repeated with the addition of 500 µl
buffer AW1, buffer AW2 (centrifuged 3 min), and 2 x 200 µl of
buffer AE after a 1-min incubation (all provided) to yield 400 µl of
eluate containing total cellular DNA. Spectrophotometry was used to
determine total DNA from cells, and the radioactivity was quantified by
scintigraphy. The quantity of [3
H]F-ara-A
incorporated into U937 cell DNA was calculated and expressed as fmol
[3
H]F-ara-A/µg DNA.
Western Analysis.
After drug treatment, whole-cell pellets (5 x
105 cells/condition) were washed twice in PBS,
resuspended in 100 µl of PBS, and lysed by the addition of 100 µl
of 2x sample buffer [1x, 30 mM Tris (pH 6.8), 2% SDS,
2.88 mM ß-mercaptoethanol, and 10% glycerol]. The
lysates were sonicated, boiled for 5 min, and centrifuged at
12,8000 x g for 5 min, and protein was quantified
using Coomassie protein assay reagent (Pierce, Rockford, IL). Equal
amount of protein (20 µg) were separated by SDS-PAGE, transferred
electrophoretically to Optitran nitrocellulose filters (Schleicher and
Schleicher, Keene, NH), and blocked in PBS-Tween (PBS-T; 0.05%)/5%
dry milk for 1 h at 22°C. The blots were then probed for 4 h at 22°C or overnight at 4°C with primary antibodies:CPP32
(1:1000; Transduction Laboratories, Lexington, KY), PARP (1:2000;
Biomol Research Laboratories, Plymouth Meeting, PA), caspase 9 (1:2000;
PharMingen International, San Diego, CA), Bcl-XL
(1:1000; Santa Cruz, Santa Cruz, CA), Bax (1:2000; PharMingen, San
Diego, CA), Mcl-1 (1:1000; PharMingen, San Diego, CA), Bcl-2 (1:2000;
Dako, Carpinteria, CA), XIAP (1:2000; R&D Systems, Minneapolis, MN),
phosph-ERK1/2 (1:1000; Cell Signaling Technology, Beverly, MA), or
serine 216 phospho-cdc25 (1:500; Cell Signaling Technology, Beverly,
MA) as per the manufacturer’s instructions. Blots were subsequently
washed 3 x 5 min each in PBS-T and incubated with horseradish
peroxidase-conjugated secondary antibody (Kirkegaard and Perry
Laboratories, Gaithersburg, MD) in PBS-T for 1 h at 22°C. The
blots were again washed 3 x 5 min each in PBS-T and developed
with the enhanced chemiluminescence method (Amersham, Arlington
Heights, IL). Equivalent loading and protein transfer were documented
by reprobing blots with anti-actin antibody (1:2000; Sigma).
Assessment of MMP (
m).
MMP was monitored using DiOC6 as we have
described previously (21)
. For each condition, 4 x
105 cells were incubated for 15 min at 37°C in
1 ml of 40 nM DiOC6 and subsequently
analyzed using a Becton Dickinson FACScan cytofluorometer with
excitation and emission settings of 488 and 525 nm, respectively.
Values were expressed as the increase in the percentage of cells
displaying reduced levels of DiOC6 uptake
relative to untreated controls.
Primary Patient Samples.
Malignant B lymphocytes were obtained with informed consent under
sterile conditions from the malignant pleural effusion of a patient
with indolent non-Hodgkin’s lymphoma undergoing a therapeutic
thoracentesis. These studies have been approved by the Investigational
Review Board of the Medical College of Virginia/Virginia Commonwealth
University. Cells were pelleted by centrifugation at 400 x
g for 6 min at room temperature, washed three times in fresh
medium, and resuspended in sterile tissue culture flasks at a cell
density of 5 x 105 cells/ml in RPMI 1640
containing 10% FCS. F-ara-A and UCN-01 were added to the medium as
described above, and the flasks were placed in a 37°C, 5%
CO2 incubator for 24 h. At the end of the
incubation period, cytospin preparations were obtained, and apoptosis
was monitored by TUNEL assay as outlined above.
Statistical Analysis.
The significance of differences between experimental values was
determined using Student’s t test for unpaired observations
or Mann-Whitney test for nonparametric data. Drug interactions were
characterized using median dose effect analysis in conjunction with a
commercially available software program (Calcusyn; Biosoft, Cambridge,
United Kingdom; Ref. 23
).
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RESULTS |
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. This concentration of UCN-01 was
selected because it exerted minimal toxicity when administered alone
(e.g., < 10% apoptotic cells). Alternatively, cells
were simultaneously exposed to F-ara-A and UCN-01 for 24 h to make
comparisons with the sequential schedules (Fig. 1
C). It can
be seen that whereas 2 µM F-ara-A alone for
6 h also induced apoptosis to a very limited extent in these cells
(e.g., 
10%), subsequent exposure to a minimally toxic
concentration of UCN-01 substantially increased apoptosis
(e.g., to 40%; Fig. 1
A). Similar results were
obtained when the extent of apoptosis was monitored by the TUNEL assay
(data not shown). In contrast, pretreatment of cells with 100
nM UCN-01, followed by F-ara-A (2
µM; 6 h) resulted in a more modest
increase in the percentage of apoptotic cells (e.g., to
20%; Fig. 1B), the extent of which was significantly
less than that observed for the sequence F-ara-A UCN-01
(P < 0.02). In addition, when cells were
simultaneously exposed to UCN-01 (100 nM) and 2
µM F-ara-A (24 h each), the large majority of
cells (e.g., 85%) were apoptotic (Fig. 1
C).
Finally, when cells were exposed to F-ara-A (2
µM; 24 h), washed free of drug, and
subsequently incubated with UCN-01 (100 nM) for
an additional 24 h, essentially all cells (e.g.,
>98%) exhibited apoptotic features; in contrast, the extent of
apoptosis in cells exposed to the opposite sequence [e.g.,
UCN-01 (24 h) F-ara-A (24 h)] was only modestly greater than that
observed in cells treated with F-ara-A alone (data not shown). These
findings indicate that simultaneous or subsequent administration of
UCN-01 substantially potentiates F-ara-A-induced apoptosis in U937
cells, whereas prior exposure of cells to UCN-01 is less effective in
this regard. It should be noted that although the sequence-dependent
interactions between F-ara-A and UCN-01 in U937 cells are qualitatively
similar to those we have observed previously in the case of bryostatin
1 (19)
, the combination of F-ara-A and UCN-01 is
significantly more lethal in this cell line than the latter
combination.
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m) was monitored (Fig. 1)
. Interestingly,
UCN-01 was as effective or slightly more potent than F-ara-A in
disrupting the MMP. As noted above, simultaneous (Fig. 1
C)
or sequential (Fig. 1
A) exposure of cells to F-ara-A,
followed by UCN-01, resulted in a very marked increase in the
percentage of cells displaying mitochondrial dysfunction compared with
cells exposed to either drug alone, whereas this effect was less
pronounced in cells exposed to the sequence UCN-01F-ara-A (Fig. 1
B).
To determine whether the ability of UCN-01 to potentiate
F-ara-A-mediated apoptosis might be related to inhibition of PKC,
parallel studies were used using the selective PKC inhibitor GFX (1
µM). In marked contrast to findings obtained with UCN-01,
simultaneous exposure to GFX failed to potentiate F-ara-A-induced
apoptosis (P 0.05 versus F-ara-A alone;
Fig. 2
). In separate studies, this concentration of GFX was found to attenuate
several PKC-dependent events, including phorbol myristate
acetate-mediated G1 arrest,
p21CIP1 induction (data not shown), and
potentiation of phosphorylation/activation of p42/44 mitogen-activated
protein kinase (Fig. 2
, inset). These findings suggest that
the capacity of UCN-01 to enhance F-ara-A-mediated apoptosis involves
factors other than, or in addition to, interruption of the PKC pathway.
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A). Combined drug exposure was also associated with enhanced
activation of the apoptotic caspase cascade, manifested by more
pronounced cleavage of procaspase-9 and procaspase-3 (reflected by the
appearance of a Mr 17,000
cleavage product), as well as degradation of the major caspase-3
substrate, PARP (Fig. 3
B). Similar results were noted in
cells exposed sequentially to F-ara-A, followed by UCN-01 (data not
shown). Treatment of cells with UCN-01 or the combination of UCN-01 and
F-ara-A also resulted in a reduction in cdc25 phosphorylation on serine
216 (Fig. 3
C), consistent with the known inhibitory effects
of UCN-01 on Chk1 activity (9)
. Finally, median dose
effect analysis was used to characterize the interaction between
F-ara-A and UCN-01 over a range of drug concentrations with respect to
induction of apoptosis (Fig. 3
D). Combination index values
<1 were consistently obtained, corresponding to a synergistic
interaction.
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. Exposure of cells to F-ara-A (5 µM; 6 h) or
UCN-01 (300 nM; 24 h) individually resulted in a very
modest degree of apoptosis, reflected by TUNEL assay. However,
simultaneous exposure of cells to F-ara-A, followed by UCN-01, led to a
substantial increase in the percentage of TUNEL-positive cells
(e.g., 60%). A marked potentiation of apoptosis was also
noted in HL-60 cells exposed sequentially to F-ara-A, followed by
UCN-01 (data not shown).
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. Coexposure to UCN-01 exerted no effect on F-ara-ATP formation at
6 h and resulted in a slight reduction in F-ara-ATP levels after
24-h exposure (Fig. 5
A). UCN-01 also did not lead to a
discernible increase in F-ara-A (DNA) incorporation at 6 h (not
shown) or at 24 h (Fig. 5
B). Therefore, enhanced
apoptosis in cells exposed to the combination of UCN-01 and F-ara-A
could not be attributed to potentiation of F-ara-A metabolism.
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. A 24-h exposure to 2 µM F-ara-A reduced colony
formation by 95%, whereas treatment with 100 nM UCN-01
exerted only modest inhibitory effects (Fig. 6
A). The
reduction in clonogenicity by F-ara-A was considerably greater than the
extent of apoptosis observed at the 24-h interval, as we have reported
previously (19)
. However, simultaneous exposure to these
agents resulted in an additional 1-log reduction in clonogenic survival
(e.g., to 0.5% of control values; Fig. 6
A).
Moreover, sequential treatment of cells with F-ara-A (2
µM; 24 h), followed by UCN-01 (100
nM; 24 h), an exposure that induced
apoptosis in the large majority of cells, led to a very substantial
(e.g., 4 log) reduction in clonogenic capacity (Fig. 6
B). Similarly, simultaneous (Fig. 6
C) or
sequential (Fig. 6
D) exposure of cells to a marginally toxic
concentration of F-ara-A (e.g., 0.05
µM; 24 h) in conjunction with 100
nM UCN-01 resulted in a significant reduction in
colony formation (e.g., 50%). These findings demonstrate
that combined exposure to F-ara-A and UCN-01 is highly inhibitory to
U937 cell self-renewal capacity.
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. These cells exhibit a marked increase in Bcl-2 protein expression
compared with empty-vector controls or untransfected cells but
equivalent levels of Bcl-xL and Bax (Fig. 7
A). The extent of
apoptosis in U937/Bcl-2 cells exposed for 24 h to F-ara-A or
UCN-01 individually was modestly but significantly reduced compared
with controls (Fig. 7
B). Ectopic expression of Bcl-2 clearly
protected cells from apoptosis induced by simultaneous exposure to
UCN-01 and F-ara-A. Nevertheless, treatment of U937/Bcl-2 cells with
the combination of F-ara-A and UCN-01 induced apoptosis to an extent
that was at least as great, if not greater than, that observed in
control cells exposed to F-ara-A alone (P 0.05).
Parallel results were observed when loss of
m was monitored (data not shown).
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. It can be seen that expression of MCL-1, Bax, and Bcl-xL, and Bcl-2
did not appreciably change after drug exposure. As noted in the case of
CLL cells, a modest decline in expression of XIAP was observed in cells
treated with UCN-01, although this effect was at least partially
blocked by the caspase inhibitor Boc-fmk. A 12-h interval was used in
the latter studies because we have found the actions of caspase
inhibitors are not well sustained in these cells beyond this point. The
ability of caspase inhibitors to oppose XIAP down-regulation is
comparable with results described previously in the case of CLL cells
(24)
.
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. It can be seen that exposure of these primary cells to F-ara-A or
UCN-01 individually resulted in minimal evidence of apoptosis. However,
combined treatment induced TUNEL positivity in a substantial fraction
of these cells. This finding indicates that combined ex vivo
exposure to pharmacologically achievable concentrations of UCN-01 and
F-ara-A can effectively induce apoptosis in at least some primary
malignant B-cell specimens.
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DISCUSSION |
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C). In this context, it has been suggested that
UCN-01-mediated apoptosis in lymphoblastic leukemia cells (CCRF-CEM)
correlates more closely with dephosphorylation of CDK1 and CDK2 than
with PKC inhibition, based in part upon evidence that UCN-01-mediated
apoptosis could not be mimicked by the selective PKC inhibitor GFX
(14)
. In accord with these findings, GFX, in contrast to
UCN-01, was unable to potentiate F-ara-A-induced lethality in U937
cells. Together, these observations suggest that the modulatory effect
of UCN-01 on F-ara-A-mediated cell death, analogous to its direct
apoptotic actions (14)
, involves factors other than or in
addition to PKC inhibition. e.g., disruption of checkpoint
regulation.
For both U937 and HL-60 cells, simultaneous exposure to UCN-01 and
F-ara-A, and to a comparable extent, sequential treatment with F-ara-A
UCN-01 resulted in a marked potentiation of apoptosis. In contrast,
exposure of cells to UCN-01, followed by F-ara-A, led to a more modest
increase in cell death. The sequence-dependent nature of this
interaction is compatible with the notion that UCN-01 interferes with
certain events (e.g., inhibition of cell cycle progression)
that would ordinarily limit the extent of F-ara-A-related damage. The
finding that pretreatment of cells with UCN-01 only marginally lowered
the threshold for F-ara-A-mediated lethality suggests that the actions
of the former agent are relatively short-lived, and that ongoing or
preexisting DNA damage is required for substantial augmentation of cell
death to occur. Our results are also consistent with those of previous
reports in which simultaneous exposure of A431 cells to UCN-01 and
mitomycin C (27)
, sequential exposure of HCT29 cells to
camptothecin followed by UCN-01 (13)
, or sequential
exposure of CHO/AA8 cells to UCN-01 followed by cisplatin
(28)
resulted in a significant potentiation of lethality.
Although it is possible that sequence-dependent interactions between
UCN-01 and F-ara-A may prove to be cell type specific, the present as
well as the bulk of earlier evidence suggests that simultaneous or
subsequent exposure of DNA-damaging, agent-treated cells to UCN-01
appear to be associated with the greatest extent of apoptosis.
Although the mechanism(s) by which F-ara-A (or other nucleoside analogues) trigger the apoptotic cascade remain unknown, there is abundant evidence that cytotoxic agents induce mitochondrial dysfunction (29) , leading in turn to activation of initiator caspases and ultimately their downstream effector counterparts (30) . Consistent with this model, cotreatment of cells with UCN-01 and F-ara-A resulted in a pronounced loss of MMP, cleavage and activation of procaspase-9 and procaspase-3, and degradation of PARP. Of the various schedules examined, sequential exposure of cells to 2 µM F-ara-A (for 24), followed by 100 nM UCN-01 for an additional 24 h, was particularly effective in this regard, inducing apoptosis in the large majority of treated cells. It is noteworthy that combined exposure to UCN-01 and F-ara-A was also able to enhance mitochondrial dysfunction and cell death in Bcl-2-overexpressing U937 cells, at least to levels noted in wild-type controls treated with F-ara-A alone. However, ectopic expression of Bcl-2 continued to afford some protection to cells exposed to the combination of these agents. Such findings are analogous to those that we have described previously in HL-60 cells treated with the pyrimidine analogue ara-C and the PKC activator and down-regulator bryostatin 1 (20) . Collectively, these observations indicate that co- or subsequent exposure of F-ara-A-treated cells to UCN-01 can oppose, albeit partially, the inhibition of mitochondrial damage and apoptosis conferred by Bcl-2 overexpression.
Previous studies have demonstrated that exposure of primary CLL cells to kinase inhibitors, such as UCN-01- or flavopiridol, induced apoptosis in association with reduction in levels of certain antiapoptotic proteins (24) . In contrast to these results, we did not detect reduced Bcl-2 expression in U937 cells exposed to UCN-01, at least over a 24-h time interval. UCN-01 exposure was, however, associated with a reduction in expression of the inhibitor of apoptosis protein XIAP, as noted in some but not all CLL samples (24) . As observed in the earlier study, this effect was partially blocked by a caspase inhibitor, suggesting that down-regulation of XIAP by UCN-01 may represent, at least in part, a secondary phenomenon. It should be noted that disparities between this and the earlier report may reflect cell type-specific differences (e.g., primary CLL versus U937 monocytic leukemia cells) and/or the use of significantly lower UCN-01 concentrations in our study (e.g., 100 nM versus 1 µM).
It is widely recognized that the extent of apoptosis, particularly at early time intervals, does not necessarily correlate with loss of clonogenic survival (31) . For example, nonapoptotic forms of cell death (e.g., necrosis and giant cell formation) can also limit the self-renewal capacity of neoplastic cells (32) . Nevertheless, potentiation of apoptosis after sequential or simultaneous exposure of leukemic cells to UCN-01 and F-ara-A was accompanied by a very substantial loss of clonogenicity. Notably, sequential 24-h exposure of cells to F-ara-A, followed by UCN-01 (24 h each), led to an almost 4-log reduction in colony formation. Although it cannot be concluded that enhanced apoptosis represents the sole mechanism responsible for the marked reduction in clonogenic survival, such findings raise the possibility that leukemic cells with self-renewal capacity are particularly susceptible to combined exposure to F-ara-A and UCN-01. It is also important to note that UCN-01 enhanced F-ara-A-mediated lethality toward clonogenic cells when the latter was administered at lower, marginally toxic levels, indicating the enhanced antiproliferative effects of this drug combination is not restricted to relatively high F-ara-A concentrations.
The lethal actions of nucleoside analogues have been correlated with various pharmacodynamic determinants, particularly formation of the lethal triphosphate derivative and subsequent incorporation of this metabolite into DNA (5 , 33) . Moreover, in the case of ara-C, the PKC activator and down-regulator bryostatin 1 has been shown, under some circumstances, to enhance nucleoside analogue phosphorylation and triphosphate generation (34) . Consequently, enhancement of F-ara-A-mediated cytotoxicity by UCN-01 could potentially reflect perturbations in F-ara-A anabolism. However, we were unable to detect an increase in F-ara-ATP formation or [3 H]F-ara-A (DNA) incorporation in leukemic cells coexposed to UCN-01, rendering this possibility unlikely. Instead, such findings suggest that UCN-01 lowers the threshold for F-ara-A-mediated activation of apoptotic caspases, allowing cells that might otherwise remain unaffected to engage the cell death program.
In summary, the present findings indicate that UCN-01 augments
F-ara-A-mediated mitochondrial damage, caspase activation, and
apoptosis in human leukemia cells (U937) in a schedule-dependent
manner, that this phenomenon does not stem from enhanced F-ara-A
metabolism, and that it is associated with a substantial loss of
clonogenic capacity. Moreover, the data obtained using GFX suggest that
potentiation of F-ara-A-related lethality by UCN-01 involves factors
other than, or in addition to, inhibition of PKC activity. It is
important to note that the extent of apoptosis that occurred in cells
exposed to F-ara-A and UCN-01 was significantly greater than that which
we have previously observed in studies combining F-ara-A and the PKC
activator and down-regulator bryostatin 1 (19)
. In this
context, early clinical results of a Phase I trial combining the latter
agents in patients with CLL and refractory indolent non-Hodgkin’s
lymphoma appear encouraging (35)
. It is also worth noting
that the combination of UCN-01 and F-ara-A potently induced apoptosis
ex vivo in primary malignant B cells obtained from a patient
with indolent non-Hodgkin’s lymphoma (Fig. 8)
. Although avid binding
of UCN-01 to acidic -glycoprotein appears to limit free plasma
levels (16)
, the low concentrations used in the present
study (e.g., 100 nM) should be
achievable, as should be plasma F-ara-A levels of 2
µM (36)
. On the basis of
these considerations, as well as: (a) the known activity of
F-ara-A in chronic lymphocytic leukemia and some forms of
non-Hodgkin’s lymphoma (1
, 3 , 37)
; and (b)
very recent evidence suggesting that UCN-01 may increase the efficacy
of cytotoxic drugs in patients with lymphoma (38)
, efforts
to develop combination regimens incorporating these agents appear
warranted.
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FOOTNOTES |
|---|
1 This work was supported by Awards CA 63753 and
77141 from the NIH, Grant BC98-0148 from the United States Department
of Defense, and Grant 6630-01 from the Leukemia Society of America. 
2 To whom requests for reprints should be
addressed, at Division of Hematology/Oncology, Medical College of
Virginia/Virginia Commonwealth University, MCV Station Box 230,
Richmond, VA 23298. Phone: (804) 828-5211; Fax: (804) 828-8079;
E-mail: stgrant{at}hsc.vcu.edu
3 The abbreviations used are: F-ara-AMP,
fludarabine; F-ara-A, 2-fluoroadenine
9-ß-D-arabinofuranoside; ara-C,
1-ß-D-arabinofuranosylcytosine; GFX, bisindolylmaleimide;
PARP, poly-ADP(ribosyl)polymerase; TUNEL, terminal
deoxynucleotidyltransferase-mediated nick end labeling; CLL, chronic
lymphocytic leukemia; CDK, cyclindependent kinase; PKC, protein
kinase C; DiOC6, 3,3-dihexyloxacarbocyanine iodide; MMP,
mitochondrial membrane potential; XIAP, X inhibitor of apoptosis
protein.
Received 6/ 5/00; revised 11/ 7/00; accepted 11/ 7/00.
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) or loss of MMP (
). Values represent the means for three separate experiments
performed in triplicate; bars, SD. *, significantly
greater than values for either drug alone (P 
0.05).
) and U937/Bcl-2 cells (
