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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 458-465
Early Induction of Apoptosis in Hematopoietic Cell Lines After
Exposure to Flavopiridol
By
Bernard W. Parker,
Gurmeet Kaur,
Wilberto Nieves-Neira,
Mohammed Taimi,
Glenda Kohlhagen,
Tsunehiro Shimizu,
Michael D. Losiewicz,
Yves Pommier,
Edward A. Sausville, and
Adrian M. Senderowicz
From the Laboratories of Biological Chemistry and Molecular
Pharmacology, Division of Basic Sciences, and Laboratory of Drug
Discovery Research and Development and DTP Clinical Trials Unit,
Developmental Therapeutics Program, Division of Cancer Treatment,
Diagnosis and Centers, National Cancer Institute, Bethesda, MD.
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ABSTRACT |
Flavopiridol (NSC 649890; Behringwerke L86-8275, Marburg, Germany),
is a potent inhibitor of cyclin dependent kinases (CDKs) 1, 2, and 4. It has potent antiproliferative effects in vitro and is active in tumor
models in vivo. While surveying the effect of flavopiridol on cell
cycle progression in different cell types, we discovered that
hematopoietic cell lines, including SUDHL4, SUDHL6 (B-cell lines),
Jurkat, and MOLT4 (T-cell lines), and HL60 (myeloid), displayed notable
sensitivity to flavopiridol-induced apoptosis. For example, after 100 nmol/L for 12 hours, SUDHL4 cells displayed a similar degree of DNA
fragmentation to that shown by the apoptosis-resistant PC3 prostate
carcinoma cells only after 3,000 nmol/L for 48 hours. After exposure to
1,000 nmol/L flavopiridol for 12 hours, typical apoptotic morphology was observed in SUDHL4 cells, but not in PC3 prostate carcinoma cells
despite comparable potency (SUDHL4:120 nmol/L; PC3: 203 nmol/L) in
causing growth inhibition by 50% (IC50). Flavopiridol did
not induce topoisomerase I or II cleavable complex activity. A relation
of p53, bcl2, or bax protein levels to apoptosis in SUDHL4 was not
appreciated. While flavopiridol caused cell cycle arrest with decline
in CDK1 activity in PC3 cells, apoptosis of SUDHL4 cells occurred
without evidence of cell cycle arrest. These results suggest that
antiproliferative activity of flavopiridol (manifest by cell cycle
arrest) may be separated in different cell types from a capacity to
induce apoptosis. Cells from hematopoietic neoplasms appear in this
limited sample to be very susceptible to flavopiridol-induced apoptosis
and therefore clinical trials in hematopoietic neoplasms should be of
high priority.
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INTRODUCTION |
FLAVOPIRIDOL IS A NOVEL flavonoid with
potent antiproliferative effects. Its capacity to inhibit cell growth
by 50% (IC50) is 60 and 400 times more potent than the
structurally related flavone, quercetin and the isoflavone, genistein,
respectively.1 Flavopiridol also has antitumor effects in
vivo.2 Previous studies have shown that flavopiridol is a
potent inhibitor of cyclin-dependent kinase (CDK)1,3 as
well as CDKs, 2 and 4.4 In addition, the drug can
indirectly affect CDK activity by inhibiting the normal regulatory
phosphorylation of CDKs.5 These activities can be explained
by the recent demonstration that an analog of flavopiridol can bind
directly to the adenosine triphosphate (ATP) binding site of
CDK2.6
Apoptosis is the process by which physiologic regulation of cell number
in developing organs and organisms is achieved. Apoptotic cell death is
characterized by the activation of proteases and nucleases leading to
chromatin condensation.7,8 Recent experiments have
underscored that apoptosis can be activated by many types of cancer
chemotherapeutic agents.9-11 Of great ongoing interest is
how agents of such diverse structural types can activate the apoptotic
program.
Because flavopiridol has recently entered clinical
trials,12 we have sought to acquire a basis for
prioritizing entry into Phase II trials. We report here that several
hematopoietic cell types are notably sensitive to induction of
apoptosis by flavopiridol. This sensitivity cannot be ascribed to
induction of cleavable complex activity by topoisomerases, or is it
relatable to changes in p53, bcl2, and bax levels in the
apoptosis-prone B-cell line, SUDHL4.
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MATERIALS AND METHODS |
Drugs and cell culture.
Flavopiridol (NSC 649890; Behringwerke, L86-8275 [(-)
cis-6,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1-methyl)-piperidinyl]-4h-benzopyran-4-one]) was provided by Behringwerke AG, Marburg, Germany, to the Developmental Therapeutics Program, National Cancer Institute. Flavopiridol was
dissolved in dimethyl sulfoxide as 50 mmol/L stock solutions. SUDHL4
and SUDHL6 cell lines (Southwestern University Diffuse Histiocytic
Lymphoma), histologically transformed follicular lymphoma cell lines,
were provided by Dr M. Stetler-Stevenson, Laboratory of Pathology, NCI.
MOLT4 Jurkat (T-cell, acute lymphoblastic leukemia [ALL]); HL60, K562
(myeloid); and PC3 prostate carcinoma cell lines were obtained from
(ATCC, Rockville, MD). The cells (doubling time,  24 hr) were
maintained in RPMI 1640 containing 10% (vol/vol) heat-inactivated
fetal bovine serum, 100 U/mL penicillin G, 100 µg/mL streptomycin,
and 2 mmol/L glutamine (complete medium) in an atmosphere containing
5% (vol/vol) CO2. All chemical reagents were from Sigma,
St Louis, MO, unless noted otherwise. Morphologic assessment of the
effect of flavopiridol was achieved by cytospin of 100 µL of cell
suspension, stained with Leukostat Kit (Fisher Scientific, Pittsburgh,
PA) and viewed under oil immersion microscope.
Drug effect on cell growth.
Exponentially growing hematopoietic and prostate cells were treated as
described in figure legends. In the case of PC3 cells, 2 × 103 cells per well were incubated with either drug or
vehicle. After drug exposure, cells were incubated with 10%
trichloroacetic acid and then stained with sulforhodamine B (SRB)
solution, as described in detail elsewhere.13 In the case
of hematopoietic cells, exponentially growing cells in suspension were
harvested after drug treatment, and cell numbers were counted
electronically (Coulter Electronics, Hialeah, FL) or by hemocytometer
and viability assessed by Trypan Blue exclusion.
DNA gel fractionation.
Cells grown at a density of 1 × 106 cells/mL were
exposed to flavopiridol for different concentrations and time periods
as described in the figure legends. DNA was extracted, as described by
Wang et al.14 Briefly, cells were washed once with cold
phosphate-buffered saline (PBS) and lysed with 3 mL lysis buffer (5 mmol/L Tris-HCL [pH 7.5]; 20 mmol/L EDTA; 0.5% Triton X-100) for 15 minutes at 4°C. The chromatin of the cell lysates was isolated by
centrifugation (20 minutes at 26,000g, 4°C). The
supernatants containing small DNA fragments were
extracted sequentially with
phenol, phenol:chloroform (1:1), and chloroform. Nucleic acids were
precipitated in 0.5 mol/L NaCl, 90% ethanol at -20°C overnight.
RNA was then digested by bovine RNAase A (60 µg/mL). After sequential
reextraction and reprecipitation, DNA was dissolved in 10 mmol/L
Tris-HCL (pH 7.5), 1 mmol/L EDTA, 0.5% sodium dodecyl sulfate (SDS)
before electrophoresis on 1.6% agarose gel.
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| Fig 1.
Effect of flavopiridol on cell growth. (A) SUDHL4 cell
line. Cultures were exposed to various concentrations of flavopiridol and at the indicated points counted by electronic counter at 24 hours
( ), 48 hours ( ), and 72 hours (). The data are shown as
percentage of untreated control. Total untreated control cell number/culture at 24 hours was 104,040 ± 3,750; 48 hours, 183,880 ± 6,687; and 72 hours, 371,670 ± 11,956. (B) PC3 cell line.
Exponentially growing cells were incubated with flavopiridol at the
indicated concentrations for 48 hours, and cell growth was assessed by
the colorimetric SRB assay, as described in Materials and Methods. The
data are the mean of four determinations ± standard deviation (SD)
and are representative of three experiments for each cell line.
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| Fig 2.
Morphology of SUDHL4 and PC-3 cells after exposure to
flavopiridol. (A) Untreated control SUDHL4s. (B) SUDHL4s after 12 hours exposure to flavopiridol at 1,000 nmol/L. (C) Untreated control PC-3s.
(D) PC-3s after 12 hours exposure to flavopiridol at 1,000 nmol/L.
Photography was at 1,000 ×, oil immersion microscope, after cytospin
preparation as described in Materials and Methods.
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| Fig 3.
Effect of flavopiridol on growth of SUDHL4, HL60, MOLT4,
K562, and PC3 cells. Cells (2 × 104 cells/mL) were plated
into each well of a six-well plate. After 24 hours, cells were treated
in triplicate either with vehicle or with 50, 100, and 500 nmol/L of
flavopiridol for 48 hours. Cell growth was determined by counting live
(Trypan Blue excluded) and dead (Trypan Blue stained) cells on a
hemacytometer. (A) Represents growth inhibition as percent of control
for SUDHL4 ( ), HL60 (), MOLT4 ( ), K562 ( ), and PC3 ( )
cells after 48 hours drug exposure. (B) Presents the results as percent
of total live cells (open bar) and dead cells (hatched bar) after 500 nmol/L flavopiridol exposure for 48 hours. The experiments represent
the mean of three determinations ± SD.
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DNA alkaline filter elution.
Quantitation of DNA fragmentation was performed by a modification of a
previously published procedure.15 Briefly, cells were
labeled with [14C]thymidine (0.02 µCi/mL; specific
activity, 59 mCi/mmol, from Amersham Corp, Arlington Heights, IL) for
one doubling time, followed by incubation in isotope-free medium to
allow chase of radioactivity into high molecular weight DNA. After
exposure to flavopiridol, cells were loaded onto a filter (Poretics
Corporation, Livermore, CA). The culture medium and wash fractions were
collected as "the extracellular fraction". Lysis solution (0.2%
Na Sarkosyl, 2 mol/L NaCl, 0.04 mol/L EDTA, pH 10) was added, followed
by another wash with 0.02 mol/L EDTA. This was collected as the
"lysis fraction", containing the protein-free DNA double-strand
breaks occurring as a result of induction of apoptosis. The filter was
placed into a scintillation bottle containing 0.4 mL 1 N HCL . The
filter was placed into a 65°C oven for 60 minutes, followed by
addition of 0.4 N NaOH for 60 minutes to solubilize the filter-bound
label. Radioactivity was then measured in each fraction by liquid
scintillation spectrometry, and the data plotted as the fraction of DNA
eluting from the filter.
Flavopiridol effects on topoisomerase activity.
To assess the capacity of flavopiridol to activate topoisomerase, a DNA
fragment corresponding to the 5 -end-labeled sense strand of the
c-myc proto-oncogene was used.16 The DNA was reacted with
purified topoisomerase I and II in the presence of the indicated concentrations of flavopiridol. Reactions were incubated at 30°C for 30 minutes and stopped by adding 0.5% SDS followed by proteinase K
digestion. DNA fragments were separated on a 7% denaturing
polyacrylamide gel and visualized by Phosphorimager.
DNA content and flow cytometry analysis.
Exponentially growing cells (4 × 106/20 mL) were
treated with flavopiridol as indicated in the figure legends. At each
time point, cells were washed twice with PBS, fixed in suspension in 70% ethanol and stored at -20°C. For DNA content, cells were
washed twice with PBS and resuspended in 2 mL of PBS. A total of 2 mL of phosphate-citric acid buffer (192 mL of 0.2 mol/L
Na2HPO8 and 8 mL of 0.1 mol/L citric acid, pH
7.8) was added to each sample, and cells were incubated for 15 minutes
at room temperature. Cells were washed with PBS, incubated with 50 µg/mL of propidium Iidide (Calbiochem, San Diego, CA) and 250 µg of
DNase-free Rnase A (Sigma) in the dark for 30 minutes. DNA content was
measured using a FACScan (Beckton Dickinson, San Jose, CA) flow
cytometer. Data acquisition and analysis was performed using Modfit
software (Becton Dickinson).
Western blot analysis.
Exponentially growing cells (5 × 106/25 mL) were
treated with flavopiridol as described in the figure legends. At
indicated times, cells were washed with PBS and lysed in 500 µL of
lysis buffer (50 mmol/L hepes, 150 mmol/L NaCl, 1% Triton X-100, 10% glycerol, 5 mmol/L EGTA, 15 mmol/L MgCl2, 20 mmol/L NaF, 50 mmol/L  -glycerophosphate, 2 mmol/L phenylmethylsulfonyl fluoride
[PMSF], 1 mmol/L Na3VO4, 10 µg/mL
leupeptin, and 10 µg/mL aprotinin). Cell lysates were centrifuged at
14,000 rpm for 15 minutes at 4°C. Protein content of clarified
supernatants was determined by Bradford protein assay. Cell lysates
containing equal amounts of protein (50 µg) were resolved on 12%
mini gels (Novex, San Diego, CA).
After SDS-polyacrylamide gel electrophoresis (PAGE), proteins were
transferred onto immobilon polyvinyldiene difluoride (PVDF) membranes
(Millipore, Bedford, MA) at 500 mA for 2.5 hours at 4°C using CAPS
(3-[cyclohexylamino]-1-propanesulfonic acid) buffer (10 mmol/L CAPS,
pH 11, 10% MeOH). Residual binding sites on the membrane were blocked
by incubation in TTS (20 mmol/L Tris, pH 7.4, 0.9% NaCl, and 0.05%
Tween 20) containing 3% bovine serum albumin (BSA) overnight at
4°C or for 1 hour at room temperature. Blots were probed with
either anti-bcl2 monoclonal antibody (MoAb) (Dako, Inc, Carpinteria,
CA) anti-p53 MoAb (Calbiochem) or with rabbit polyclonal bax (Santa
Cruz Biotech, Santa Cruz, CA). Immune complexes were detected using
goat antirabbit or antimouse horseradish proxidase conjugated secondary
antibodies (Amersham Corp) and were visualized using enhanced
chemilumminescence reagents (Amersham Corp).
CDK1 activity.
The activity of CDK1 was assessed, as described previously3
using CDKs1 substrate peptide.
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RESULTS |
Antiproliferative and morphologic effects of flavopiridol.
Flavopiridol inhibits the growth of SUDHL4 lymphoma cells with an
IC50 of approximately 120 nmol/L at 48 or 72 hours
(Fig 1A). For comparison, the PC3 prostate carcinoma
epithelial cell line displays comparable inhibition of growth, with
IC50 of 203 nmol/L over 48 hours (Fig 1B). A striking
finding, however, is the effect of the drug on the morphology of the
different cell types. Untreated SUDHL4 cells display homogeneous
chromatin with prominent nucleoli, similar to that expected for
lymphoid blasts (Fig 2A). After a 12-hour exposure to
flavopiridol at 1,000 nmol/L, there are typical "apoptotic"
changes, including prominent chromatin condensation, loss of normal
nuclear architecture (Fig 2B), and accumulation of nuclear debris. In
contrast, exposure of PC3 prostate carcinoma cells to flavopiridol at
the same concentrations and durations only causes loss of nucleoli
(compare Fig 2C with 2D), with no evidence of nuclear debris or
apoptotic body formation. No morphologic changes compatible with
apoptosis were observed with several other epithelial lines examined,
such as MDA 468 breast carcinoma cells and DU-145 prostate cells,
despite similar IC50s for growth inhibition over 48 hours
in these cell lines (data not shown).
To expand the variety of cell types examined, we directly contrasted
the behavior of SUDHL4 with a variety of other cell types. Figure 3A shows that after 48 hours, PC3, HL60, SUDHL4,
K562, and MOLT4 cells have comparable IC50s of 80 to 300 nmol/L. However, SUDHL4 cultures have 95% dead cells by Trypan
Blue exclusion at 500 nmol/L (Fig 3B), where growth is inhibited by
90%. Similar behavior to this is shown by HL60 and MOLT4 cells, with
90% and 60% Trypan Blue positive, respectively (Fig 3C and D). In
contrast, PC3 cells, while growth inhibited by 80% at 500 nmol/L, show
little Trypan Blue staining (Fig 3F). Interestingly, K562 chronic
myelogenous leukemia cells are more similar to PC3 cells in that they
are efficiently inhibited by flavopiridol, but show little tendency toward cell death. Qualitatively, similar conclusions were apparent after 24 hours, where at 500 nmol/L, SUDHL4, HL60, and MOLT4 showed 80%, 90%, and 40% dead cells, respectively (data not shown).
DNA fragmentation after flavopiridol treatment.
The appearance of morphologic changes consistent with induction of
apoptosis in SUDHL4 cells suggested that DNA fragmentation might be
readily evident in these cells.
Figure 4A shows that exposure
to as little as 100 nmol/L flavopiridol (approximately the
IC50; compare with Fig 1) for 14 hours, induced DNA
fragmentation with a typical "DNA ladder" in lymphoid neoplastic
cells derived from T- (MOLT4 and Jurkat) or B-cell lineage (SUDHL4 and
6). A similar effect was observed in another B-cell line, Wilson (data not shown).
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| Fig 4.
DNA fragmentation after exposure to flavopiridol.
(A) Exponentially growing SUDHL4, SUDHL6, MOLT4, and Jurkat cells
were exposed to the indicated concentrations of flavopiridol for 14 hours and genomic DNA extracted as described in Materials and Methods
before electrophoresis in a 1.6% agarose gel. (B) SUDHL4 cells were
exposed to the following concentrations of flavopiridol for the
indicated periods after prelabelling DNA with
[14C]-thymidine. The fraction of DNA eluting from filters
is indicated. Untreated control (); 100 nmol/L (); 300 nmol/L
( ); 500 nmol/L (); 1,000 nmol/L ( ). (C) PC-3 cells were
exposed to either untreated control (); 1,000 nmol/L (), or 3,000 nmol/L () flavopiridol-containing medium for 24 or 48 hours. Each
symbol represents the mean of three independent experiments.
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To quantitate the degree of DNA damage more rigorously in SUDHL4 and
PC3 cells, we used the filter elution assay described in Materials and
Methods. After exposure to 100 nmol/L flavopiridol for 6 hours, there
is clearly evidence of DNA fragmentation. By 12 hours of exposure to
300 nmol/L, there is virtually complete fragmentation of DNA (Fig 4B).
In contrast to the behavior of SUDHL4 cells, PC3 prostate carcinoma
cells are considerably more resistant to induction of DNA
fragmentation: exposure to 3,000 nmol/L of drug for 24 or 48 hours
causes only 10% or 35% of the DNA fragmentation, respectively (Fig
4C). These results, therefore, are concordant with the idea that the
SUDHL4 lymphoma cell line, similar to most of the other hematopoietic
cell lines studied here, is very sensitive to induction of apoptosis
with DNA fragmentation after exposure to flavopiridol. These data
further indicate that PC3 cells are relatively resistant to this effect
despite a similar IC50 for inhibition of cell growth in
short-term assays with continuous exposure to drug.
Activity of topoisomerase I and II after flavopiridol.
Previous studies17,18 have suggested that potential protein
kinase antagonists at relatively high concentration can modulate topoisomerase activity. The readily apparent induction of apoptosis by
flavopiridol raises the concern that flavopiridol might perturb the
activity of topoisomerases. Flavopiridol at concentrations 100-fold
greater than those associated with apoptosis in cells does not
stimulate topoisomerase I or II-induced cleavage of DNA, conditions
where, for example, VP-16 clearly causes topoisomerase II-induced DNA
cleavage of a defined target DNA sequence (data not shown).
Modulation of bcl2, p53, and cell cycle.
To begin to address the mechanisms by which flavopiridol causes
apoptosis selectively, we examined the effect of the drug on bcl2, bax,
and p53 protein levels. Figure 5A shows
essentially no change in bcl2 or bax proteins in SUDHL4 cells after
exposure to flavopiridol, and by 18 to 24 hours, there is a slight
decrease in p53. In PC3 cells, 24 hours after exposure to flavopiridol, there is a decrease in bcl2 levels (Fig 5B), without change of p53.
These changes accompanied or preceded arrest of PC3 cells with a
decrease in S and increase in G2 phase fractions
(Fig 6A), with decrease in CKD1 activity to
58% of untreated controls (data not shown), as has been described in
other cell types previously.1,4,5 In contrast, in SUDHL4
cells, CDK1 activity did not decrease, but at 3 hours after addition of
500 nmol/L flavopiridol, there was increased CDK1 activity (Fig 6B),
comparable to the threefold increases in CDK1, CDK2, and CDK4
immunoprecipitated activity induced by flavopiridol at short times
after drug addition in breast cancer cells and attributable to
decreased CDK tyrosine phosphorylation.4,5 Also in contrast
to PC3 cells, at 6, 18, and 24 hours in SUDHL4 cells, CDK1 activity
does not decrease, despite florid induction of apoptosis of SUDHL4
after 8 hours expsoure to 300 nmol/L (Fig 6C), manifest here as a
hypodiploid DNA content.
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| Fig 5.
Effect of flavopiridol on p53, bcl2, and bax proteins.
Exponentially growing SUDHL4 cells (A) and PC3 cells (B) were treated with 500 nmol/L of flavopiridol for 1, 3, 6, 18, and 24 hours. Cells
were washed with PBS, lysed, and Western blot analysis performed as
described in Materials and Methods. Proteins were visualized by
autoradiography using ECL. The arrows indicate the position of the bax
protein.
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| Fig 6.
Cell cycle distribution and effect on CDK1 activity after
exposure to flavopiridol. In (A), the fraction of PC3 cells in G1, S,
and G2/M is indicated after 12 hours exposure to 300 nmol/L flavopiridol. The experiment shown is the mean of duplicate samples with a range of < 5% and is representative of two experiments. In
(B), SUDHL4 cells were exposed to 500 nmol/L flavopiridol () or
vehicle () for the indicated time periods and CDK1 activity assayed.
The experiment shown is representative of two experiments, with each
kinase determination the average ± SD of three determinations. In
(C), SUDHL4 cells (A) were exposed to vehicle (solid line) or 300 nmol/L flavopiridol (dashed line) for 8 hours and cell cycle
distribution assayed by flow cytometry.
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DISCUSSION |
In this report, we have shown that the antiproliferative effect of
flavopiridol, a known CDK inhibitor, can be temporally linked to
induction of apoptosis in several hematopoietic cell lines. These
apoptotic events were documented by three different independent methods
and were more easily appreciated in the lymphoid cell lines, in
contrast to PC3 cells and to all epithelial cell lines studied to date
(data not shown), as well as, interestingly, the K562 myeloid cell
line. The induction of apoptosis is observed as early as 3 to 6 hours
after exposure to 100 nmol/L of flavopiridol in the SUDHL4 cell line.
This may be compared with a delayed (48 hours) and less potent effect
of the drug in the PC3 cell line in causing DNA fragmentation, despite
similar IC50 for cell growth inhibition. PC3 and K562 cells
never displayed typical apoptotic morphology after drug addition. In
contrast to VP16-213, flavopiridol did not activate cleavable complex
formation by topoisomerase II nor did it activate cleavable complex
activity by topoisomerase I at concentrations almost 1,000 times higher
than the concentration inducing apoptosis in living cells. Clear
evidence of alteration of the bcl2/bax protein ratio in SUDHL4 cells
was not obtained, or did p53 increase in SUDHL4 and PC3 cells.
Apoptosis (or programmed cell death) is a physiologic event in response
to multiple stimuli including growth factor withdrawal, radiation
therapy, and chemotherapeutic agents.19-22 Apoptotic cells
undergo shrinkage, chromatin condensation, and plasma membrane blebbing
with the activation of proteases and endonucleases. Their final
phenotype is characterized by plasma membrane-bound "apoptotic bodies".23 Several mechanisms apparently regulate this
process, such as induction of a p53-dependent-pathway after
DNA-damaging agents, modulation by the bcl2 family of proteins, and
activation of effectors including the interleukin-converting enzyme
(ICE) family of proteases and endonucleases.24-27
Interestingly, flavopiridol action appeared not to correlate with p53
status: while PC3 and K562, both p53 null,
respectively,28,29 are relatively resistant to
flavopiridol-induced apoptosis, both HL60 and Jurkat cells, also p53
null,30,31 are very sensitive to flavopiridol-induced
apoptosis. Also, in neither SUDHL4 nor PC3 cells is p53 induced after
exposure to flavopiridol.
CDKs have been implicated as modulators of apoptosis in at least two
ways. Inappropriate activation of CDKs has been correlated with
induction of apoptosis by cytotoxic lymphocytes,32 after exposure to staurosporine33,34 or the staurosporine
congener, UCN-01.14 We have also observed transient cyclin
B/cdc2 kinase activation in human leukemia HL60 treated with
topoisomerase inhibitors and DNA alkylating agents.35
Elevated expression of CDK dominant negative mutants can prevent cell
death in Hela cells.36 In other cell types, such as
proliferating PC12 cells, flavopiridol and olomucine (another CDK
inhibitor) induce apoptosis, while in differentiated PC12 cells,
flavopiridol protects from apoptosis after growth factor
withdrawal.37 These results have led Meijer38 to conclude that the influence of CDK activity on the apoptotic program
may be cell-context or cell type-dependent. However, the phase of the
cell cycle may influence the susceptibility to induction of apoptosis.
It is intriguing that despite similar IC50s for growth
inhibition of the lymphoma and prostate cell lines studied here, there is a clear difference between the epithelial and hematopoietic cells
(except K562) in the onset, concentration and apparent magnitude of the
apoptotic phenomenon. Further experiments must define whether CDK
inhibition can be related to induction of apoptosis. Consistent evidence of CDK inhibition was obtained only in the PC3 cells. SUDHL4
cells went into apoptosis so completely (Fig 6C) that evidence of cell
cycle arrest was not observed in these cells. Of great interest, CDK1
activity was maintained even after florid induction of apoptosis. The
meaning of maintained CDK activity is uncertain. One interpretation is
that SUDHL4 and other "apoptosis-prone" cell types do not become
growth arrested in the presence of stimuli that should cause cell cycle
arrest, and the ocurrence of apoptosis reflects the continued influence
of their growth-stimulating influences in the presence of the drug.
Further experiments must focus on the nature and response to drug
addition of putative CDK substrates in apoptosis-prone and
apoptosis-resistance cell types. An important caution in this regard is
that flavopiridol at concentrations > 5 µmol/L can also inhibit
several other kinases including protein kinase C, protein kinase A, and
epidermal growth factor receptor tyrosine kinase.39 Thus,
it is possible that the action of flavopiridol on other targets or in
addition to the effects of flavopiridol on CDKs contributes to
flavopiridol-induced apoptosis. Other important influences may have an
impact on susceptibility to apoptosis including altered generation of
reactive oxygen or nitric oxide intermediates or propensity for
mitrochondrial damage or protease activation. Further experiments must
clarify which pathways are preferentially activated in hematopoietic
cells34,40,41 and account for the observation that the
combination of flavopiridol with "conventional" hemotherapeutic
agents has been reported to enhance apoptosis.42 Finally,
it is also possible that the uptake or metabolism of flavopiridol is
very different in lymphoma cells in comparison to prostate carcinoma
cells, and such differential flavopiridol metabolism may also be an
explanation for the propensity to undergo apoptosis in hematopoietic
cells.
While this report was in preparation, Bible and Kaufmann43
presented evidence that high concentrations of flavopiridol (>500 nmol/L) for  24 hours was associated with cytotoxicity in several cell types. These investigators did note, however, that HL60
promyelocytic leukemia cells were exquisitely sensitive to
flavopiridol-induced apoptosis, concordant with results presented here.
In summary, flavopiridol readily induces programmed cell death in most
hematopoietic cell lines thus far examined. Irrespective of the
mechanism by which this effect occurs, these results call for early
consideration of hematopoietic neoplasms as targets for Phase II trials
with flavopiridol.
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FOOTNOTES |
Submitted January 21, 1997;
accepted September 16, 1997.
Address reprint requests to Adrian M. Senderowicz, MD, DTP
Clinical Trials Unit, Medicine Branch, National Cancer Institute, Bldg
10, Rm 6N113, NIH, Bethesda, MD 20892.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
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ACKNOWLEDGMENT |
We would like to acknowledge Carla Hemp for her valuable secretarial
assistance.
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REFERENCES |
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Sebers S,
Worland P,
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Naik R,
Sausville E:
Growth inhibition with reversible cell cycle arrest of carcinoma cells by flavone L86-8275.
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Hoffmann D,
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Antitumoral activity of flavone L86-8275.
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Carlson BA,
Kaur G,
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Potent inhibition of CDC2 kinase activity by the flavonoid L86-8275.
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M. Kim, H. Turnquist, J. Jackson, M. Sgagias, Y. Yan, M. Gong, M. Dean, J. G. Sharp, and K. Cowan
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Y. A. Elsayed and E. A. Sausville
Selected Novel Anticancer Treatments Targeting Cell Signaling Proteins
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[Abstract]
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A Phase II Trial of the Cyclin-dependent Kinase Inhibitor Flavopiridol in Patients with Previously Untreated Stage IV Non-Small Cell Lung Cancer
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B. Hagenauer, A. Salamon, T. Thalhammer, O. Kunert, E. Haslinger, P. Klingler, A. M. Senderowicz, E. A. Sausville, and W. Jäger
In Vitro Glucuronidation of the Cyclin-Dependent Kinase Inhibitor Flavopiridol by Rat and Human Liver Microsomes: Involvement of UDP-Glucuronosyltransferases 1A1 and 1A9
Drug Metab. Dispos.,
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L. Cartee, Z. Wang, R. H. Decker, S. P. Chellappan, G. Fusaro, K. G. Hirsch, H. M. Sankala, P. Dent, and S. Grant
The Cyclin-dependent Kinase Inhibitor (CDKI) Flavopiridol Disrupts Phorbol 12-Myristate 13-Acetate-induced Differentiation and CDKI Expression while Enhancing Apoptosis in Human Myeloid Leukemia Cells
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R. W. Robey, W. Y. Medina-Pérez, K. Nishiyama, T. Lahusen, K. Miyake, T. Litman, A. M. Senderowicz, D. D. Ross, and S. E. Bates
Overexpression of the ATP-binding Cassette Half-Transporter, ABCG2 (MXR/BCRP/ABCP1), in Flavopiridol-resistant Human Breast Cancer Cells
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R. J. Klasa, A. F. List, and B. D. Cheson
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T. V. Achenbach, E. P. Slater, H. Brummerhop, T. Bach, and R. Müller
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K. C. Bible, R. H. Bible Jr., T. J. Kottke, P. A. Svingen, K. Xu, Y.-P. Pang, E. Hajdu, and S. H. Kaufmann
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A. M. Senderowicz and E. A. Sausville
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K. C. Bible, S. A. Boerner, K. Kirkland, K. L. Anderl, D. Bartelt Jr., P. A. Svingen, T. J. Kottke, Y. K. Lee, S. Eckdahl, P. G. Stalboerger, et al.
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W. M. Stadler, N. J. Vogelzang, R. Amato, J. Sosman, D. Taber, D. Liebowitz, and E. E. Vokes
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G. Melillo, E. A. Sausville, K. Cloud, T. Lahusen, L. Varesio, and A. M. Senderowicz
Flavopiridol, a Protein Kinase Inhibitor, Down-Regulates Hypoxic Induction of Vascular Endothelial Growth Factor Expression in Human Monocytes
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G. I. Shapiro, D. A. Koestner, C. B. Matranga, and B. J. Rollins
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Abstract
Introduction
Abstract