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Experimental Therapeutics |
Divisions of Medical Oncology [K. C. B.] and Oncology Research [T. J. K., P. A. S., S. H. K.], Mayo Clinic, Rochester, Minnesota 55905; Department of Pharmacology [K. C. B., S. H. K., Y-P. P.] and Tumor Biology Program [K. X.], Mayo Medical School, Rochester, Minnesota 55905; and Physical Methodology [R. H. B., E. H.], Chemical Sciences Analytical, G. D. Searle and Company, Skokie, Illinois 60076
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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max from 311 to 344 nm,
demonstrating an isosbestic point typical of changes seen with
DNA-binding compounds. Reverse-phase high-performance liquid
chromatography demonstrated that flavopiridol binds to genomic
DNA to a similar extent as ethidium bromide and Hoechst 33258. Nuclear
magnetic resonance spectroscopy revealed that DNA caused extreme
broadening of flavopiridol 1H nuclear magnetic
resonance signals that could be reversed by addition of ethidium
bromide or by DNA melting, suggesting that flavopiridol binds to (and
likely intercalates into) duplex DNA. Equilibrium dialysis demonstrated
that the equilibrium dissociation constant of the
flavopiridol-DNA complex (5.4 ± 3.4 x 10-4 M) was in the same range observed for binding of the
intercalators doxorubicin and pyrazoloacridine to DNA. Molecular
modeling confirmed the feasibility of flavopiridol intercalation into
DNA and analysis of the effects of flavopiridol in the National Cancer
Institute tumor cell line panel using the COMPARE algorithm
demonstrated that flavopiridol most closely resembles cytotoxic
antineoplastic intercalators. Collectively, these data suggest that DNA
might be a second target of flavopiridol, providing a potential
explanation for the ability of this agent to kill noncycling cancer
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INTRODUCTION |
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A variety of other antineoplastic agents that kill noncycling cells (e.g., platinating and alkylating agents; Ref. 18 ) induce cytotoxicity through interactions with DNA. On the other hand, recent studies have also shown that a number of flavones inhibit the nuclear enzyme topoisomerase I (19) . On the basis of these observations, we have investigated the possibility that flavopiridol might also interact with DNA or inhibit topoisomerases. Results of these studies do not support the view that flavopiridol inhibits topoisomerase I or II, but instead provide four independent lines of evidence that flavopiridol can interact with DNA.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1°C/min. Radiochemicals were purchased from New England Nuclear
(Boston, MA). Antihuman p53 (mouse monoclonal PAb1801) was obtained
from Cambridge Research (Wilmington, DE). All other reagents were
obtained from Sigma (St. Louis, MO) or as previously described
(7
, 20)
. To prepare sonicated DNA, calf thymus DNA was dissolved in water at a concentration of 1 mg/ml, sonicated (1000 bursts; Branson Sonifier 250, Branson, Dawbury, CT), and purified by four sequential phenol/CHCl3 extractions. Agarose gel electrophoresis indicated that the average size of resulting DNA fragments was 200–300 bp.
Cell Culture.
A549 human nonsmall cell lung carcinoma cells (American Type Culture
Collection, Manassas, VA) were cultured in RPMI 1640 containing 5%
heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100
µg/ml streptomycin, and 2 mM glutamine (medium A). Cells
were maintained under subconfluent conditions at 37°C in an
atmosphere of humidified 5% (v/v) CO2 and were
passaged twice weekly.
DNA and RNA Synthesis.
A549 cells grown to 30% confluence in 60- or 100-mm tissue culture
dishes were fed with fresh medium A, exposed to the indicated
concentrations of flavopiridol for the specified times, and treated
with either [methyl-3H]thymidine (specific
activity, 88 Ci/mmol) or ([5,6-3H]uridine
(specific activity, 41 Ci/mmol) for the last 30 or 120 min of
flavopiridol exposure. Cells were then harvested and extracted with
10% trichloroacetic acid as previously described (21)
.
Immunoblotting.
To examine the effects of drugs on p53 expression, A549 cells grown to
30% confluence in 100-mm tissue culture dishes were refed with 10 ml
of medium A and then exposed to the indicated agent as described in the
text. After four washes with PBS, samples were solubilized in
alkylation buffer [6 M guanidine hydrochloride, 250 mM
Tris-HCl (pH 8.5 at 21°C), and 10 mM EDTA supplemented
immediately before use with 150 mM ß-mercaptoethanol and
1 mM 
-phenylmethylsulfonyl fluoride] and processed for
SDS-PAGE before immunoblotting using techniques previously described in
detail (22)
.
Alkaline Elution.
The formation of DNA single-strand breaks after treatment with
flavopiridol, etoposide, or ionizing radiation was assessed by alkaline
elution (23)
using techniques previously described in
detail (24)
. Briefly, A549 cells grown to 20–30%
confluence on 100-mm tissue culture dishes were treated with
[methyl-14C]thymidine (57 mCi/mmol, 0.1
µCi/ml) for 24 h, exposed to radionuclide-free medium for 1 h, released with trypsin, resuspended in fresh medium, and incubated
with the indicated concentrations of drugs for 2 h. Alternatively,
released cells were resuspended in fresh medium, incubated at 4°C for
1 h, treated with the indicated dosages of 
-irradiation, and
maintained at 4°C until processed. After treatment, suspensions of
cells were diluted with ice-cold 75 mM NaCl-2.4
mM EDTA (pH 7.4 at 4°C) and deposited on phosphocellulose
filters (Nucleopore, Costar Scientific, Cambridge, MA; 1-µm pore
size) by gentle suction. All subsequent steps were performed as
described (24)
.
Topoisomerase Assays.
Assays of topoisomerase I and II activity were performed as previously
described (25)
.
Absorption Spectroscopy.
Absorption spectra were obtained using a Beckman DU7400 diode array
spectrophotometer (Beckman Instruments, Fullerton, CA). Unless
otherwise specified, absorption spectra were recorded at 23°C.
"Size Exclusion" HPLC.
HPLC analyses were performed using a dual-pump Beckman System Gold 125
solvent module equipped with a Beckman 507e autosampler and a Beckman
168 diode array detector and using Beckman System Gold software. DNA
"size exclusion" chromatography (26)
was performed by
mixing 20 µl of 100 µg/ml genomic DNA or oligonucleotide (final
concentrations, 50 µg/ml) with 20 µl of test compound solution and
then immediately assaying 20 µl of the mixture by C18 HPLC
(4.6-mm x 250-mm x 5-µm Beckman
Ultrasphere octadecyl silane column, Brownlee MPLC Newguard C18
precolumn; mobile phase, 20% methanol in water; flow rate, 1 ml/min).
DNA, oligonucleotides, and test compounds were all dissolved in 150
mM NaCl and maintained at 4°C until analyzed.
NMR Spectroscopy.
NMR spectra were recorded using Bruker AMX-500 and AMX-600 (500 and 600
MHz 1H) NMR spectrometers (Bruker Instruments,
Rheinstetten, Germany) equipped with temperature-regulated probes. As a
starting point, the interactions between flavopiridol and sonicated
calf thymus DNA were investigated by acquiring 1H
NMR spectra of solutions containing equivalent amounts of flavopiridol
but variable amounts of sonicated calf thymus DNA and using
D2O as an NMR solvent. As described in the text,
the interactions between genomic calf thymus DNA and flavopiridol were
also examined in PBS made with 100% H2O at
37°C. Care was taken to minimize the concentrations of flavopiridol
and DNA. In these experiments, all samples contained 57 nmoles of
flavopiridol per 0.6 ml NMR sample volume (final concentration, 95
µM). Because of low solubility of flavopiridol in aqueous
solution at neutral pH, the sample pH was adjusted to
5.4.4
In these experiments, lock was established on D2O
contained in capillary tubes internal to samples, and a presaturation
pulse sequence was used to suppress the intensity of the
1H water resonance peak.
The effects of adding ethidium bromide or melting the DNA on 1H NMR spectra of flavopiridol-DNA mixtures, as well as the effects of DNA on the 1H NMR spectra of ethidium bromide, 3-hydroxy-1-methylpiperidine, and flavone acetic acid, were all examined at 37°C in PBS made with 90% H2O and 10% D2O, adjusted to pH 4.3 with DCl vapor.4 The effects of the addition of increasing amounts of ethidium bromide on the 1H NMR spectra of flavopiridol-DNA mixtures (95 µM flavopiridol and 140 µM DNA) were also studied at 37°C in PBS made with 100% H2O (pH 5.4).4
Equilibrium Dialysis.
Equilibrium dialysis was performed in the usual manner, except that
equal concentrations of the drug of interest were added to each
dialysis chamber at the start of each experiment to achieve more rapid
equilibrium. DNA chambers were prepared by drilling 6.4-mm holes in the
caps of 1.5-ml microfuge tubes and fitting them with
Mr 12,000–14,000 cutoff cellulose
dialysis membranes (Spectra/Por, Laguna Hills, CA) that had been boiled
in sodium bicarbonate and EDTA solutions to remove impurities. DNA
solutions (0.7 ml) consisted of calf thymus DNA dissolved in PBS at pH
7.40 (0.82 mM bp concentration) supplemented with
18 µM flavopiridol, 4
µM doxorubicin, or 4 µM
pyrazoloacridine, whereas dialysates (5 ml) consisted of PBS containing
either 18 µM flavopiridol, 4
µM doxorubicin, or 4 µM
pyrazoloacridine. After equilibration of the apparatus for 96 h at
37°C, concentrations of flavopiridol, doxorubicin, and
pyrazoloacridine in the two chambers were determined by absorption
spectroscopy after precipitation of DNA with 0.5 M perchloric acid.
Kd values were calculated according to
the following formula: Kd = [(concentration of free drug) x (concentration of free
DNA)]/(concentration of drug-DNA complex). Quadruplicate
determinations were made for each drug.
COMPARE Analysis.
The COMPARE algorithm of the Developmental Therapeutics Program of the
National Cancer Institute was used as described by Boyd and Paul
(27)
. In accord with previous recommendations
(27)
, Pearson’s correlation coefficients of greater than
0.6 were used as cutoffs for assessing whether two agents were likely
to share similar mechanisms of action. Both GC50
and LC50 COMPARE protocols were used in an
attempt to independently identify database compounds sharing similar
mechanisms of growth inhibition or cytotoxicity.
Molecular Modeling.
Molecular modeling was performed using the QUANTA/CHARMm (Molecular
Simulations Inc., San Diego, CA) and Gaussian 98 (M.J. Frisch,
Pittsburgh, PA) programs. Energy minimizations were carried out by
using the CHARMm force field. A randomly selected crystal structure of
a duplex DNA was optimized by an energy minimization. Different
conformations of apigenin and flavopiridol were generated by specifying
all discrete possibilities at 180° of arc increment in a range of
0–360° for all of the rotatable bonds before an energy minimization
of each conformation with the CHARMm force field. The piperidine
nitrogen atom of flavopiridol was treated as protonated. The drug-DNA
complexes were then generated by manually placing the drug apigenin or
flavopiridol in different conformations into different regions of the
duplex DNA before energy minimizations of the resulting complexes. The
binding energy of each drug was estimated by the intermolecular
interaction energy calculated with the CHARMm force field minus the
solvation energy of the drug obtained from ab initio
calculation using the CPCM model (28)
with the
HF/g-31G(2d,2p)//HF/6-31G* method. Lower binding energy resulting from
these calculations corresponds to higher binding affinity.
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RESULTS |
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, as might be expected of an agent that arrests cells in
G1 or G2 but allows
progression through S phase (29)
. Flavopiridol also
inhibited [3H]uridine incorporation into RNA
(Fig. 1, A and B)
. This effect was readily
discernible at 100 nM flavopiridol and was
maximal at 500 nM (Fig. 1B)
.
Interestingly, [3H]uridine incorporation
decreased more rapidly than [3H]thymidine
incorporation did (Fig. 1A)
. For example, treatment of A549
cells with 500 nM flavopiridol for only 30 min
inhibited RNA synthesis by 40%, whereas it no appreciable effect on
DNA synthesis during this time period (Fig. 1A)
. The
rapidity of this decrease in [3H]uridine
incorporation was reminiscent of the RNA synthesis inhibition observed
after induction of DNA damage with irradiation (30)
or
treatment with the topoisomerase I poison camptothecin (31
, 32)
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, treatment of A549
cells with 1 µM flavopiridol resulted in a
dramatic increase in p53 levels within 3 h. This effect, which was
similar to that observed after treatment with the topoisomerase II
poison etoposide (Fig. 1D
, Lane 9), was
detectable after treatment with as little as 62.5
nM flavopiridol (Fig. 1D
, Lane
4) but was not observed after a 4-h treatment with the microtubule
inhibitor paclitaxel (Lane 8), the CDK inhibitor olomoucine
(Lane 10), or two flavones that are structurally related to
flavopiridol (flavone and apigenin, Lanes 11 and
12). These data suggest that the ability of flavopiridol to
up-regulate p53 might be unique among CDK inhibitors and flavones. In
addition, they raised the possibility that flavopiridol might damage or
interact with DNA.
Effects of Flavopiridol on DNA Integrity and Topoisomerase
Activity.
To further evaluate the possibility that flavopiridol damages DNA in a
manner analogous to etoposide, the potential formation of DNA
single-strand breaks was evaluated by alkaline elution. In contrast to
etoposide (Fig. 2A)
or ionizing radiation (Fig. 2B)
, flavopiridol
did not induce DNA strand breaks after either 2 or 24 h of
treatment (Fig. 2A
and data not shown). Consistent with
these results, flavopiridol had no effect on topoisomerase II activity
in vitro (Fig. 2C)
. In contrast, the
intercalating inhibitor pyrazoloacridine (Fig. 2C)
inhibited
topoisomerase II activity. Likewise, flavopiridol concentrations up to
40 µM had no effect on topoisomerase I activity
in vitro, whereas 10-fold lower concentrations of
pyrazoloacridine inhibited topoisomerase I activity (Fig. 2D)
. The lack of effect in these assays distinguishes
flavopiridol from several cytotoxic flavones that have recently been
reported to inhibit topoisomerase I (19)
.
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When drug-DNA interactions are examined by absorption spectroscopy,
binding of DNA to other drugs shifts drug max
values to higher wavelengths about central isosbestic points
(36)
. Addition of DNA to aqueous solutions of flavopiridol
likewise resulted in a shift of the flavopiridol
max from 311 to 344 nm, producing a clear
isosbestic point (Fig. 3A)
. These results raised the possibility that flavopiridol
might be capable of directly interacting with DNA.
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, Inset). The extent
of flavopiridol-DNA binding was comparable with that seen with the
minor groove binder Hoechst 33258 and the intercalator ethidium bromide
(Fig. 3B)
. In additional experiments, the interactions between flavopiridol and DNA were investigated using NMR spectroscopy, a method that previously has been extensively used to study drug-DNA interactions (37, 38, 39, 40, 41, 42, 43, 44, 45) . In preliminary experiments, flavopiridol 1H NMR signals observed in 100% D2O at neutral pH were broadened to baseline by the addition of increasing amounts of sonicated calf thymus DNA.5 This broadening occurred without changes in the chemical shifts of flavopiridol resonances. Because of concern over uncontrolled pH, the absence of buffering salts, the high flavopiridol concentrations used in these experiments, and the potential effects of D2O on hydrogen bonding in these early experiments, we next examined the effects of DNA on the flavopiridol spectrum under somewhat more physiological conditions as described below.
When flavopiridol solutions made with 100% H2O
(no D2O) were examined at 37°C, flavopiridol
1H NMR signals were again broadened to baseline
when the molar ratio of DNA (bp) to flavopiridol reached 0.8–1.5 (Fig. 4)
. This broadening of 1H NMR signals has been
previously described with agents that intercalate into DNA (37
, 38)
. In contrast, agents that bind in the major or minor grooves
exhibit chemical shift changes but not peak broadening (37
, 38)
. In short, NMR spectroscopy confirmed flavopiridol-DNA
binding and raised the possibility that the interaction might involve
intercalation.
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or by DNA melting.5 Interestingly, addition of
increasing amounts of ethidium bromide to flavopiridol-DNA mixtures
resulted in the appearance of flavopiridol 1H-NMR
signals (Fig. 5, b and c)
before
1H-NMR ethidium signals (Fig. 5d)
,
suggesting that ethidium caused displacement of flavopiridol from
double-stranded DNA.
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Although "size exclusion" chromatography suggested that equal
amounts of flavopiridol, ethidium, and Hoechst 33258 could bind
similar amounts of DNA (Fig. 3B)
, we sought to more
precisely define the affinity of flavopiridol for DNA in comparison to
other intercalating antineoplastic agents. Equilibrium dialysis
demonstrated that the equilibrium dissociation constant of the
flavopiridol-DNA complex (Kd = 5.4 ± 3.4 x 10-4 M) is within an order of magnitude of the
dissociation constants of doxorubicin- or pyrazoloacridine-DNA
complexes (Kd = 0.68 ± 0.40 x 10-4 and 0.76 ± 0.46 x 10-4 M, respectively).
Evaluation of Sequence Specificity of Flavopiridol-Oligonucleotide
Binding Using "Size Exclusion" HPLC.
The preceding experiments demonstrated a flavopiridol-DNA interaction
by four separate and independent techniques. In an attempt to define
the nucleotide sequence specificity of this interaction, "size
exclusion" HPLC was performed using annealed, self-complementary
8-mer oligonucleotides that contain all possible 2-bp sequences (Fig. 6
, some data not shown). This analysis revealed that the intercalating
agent pyrazoloacridine (25
, 46)
binds optimally to
GGGGCCCC duplexes, less well to GGCCGGCC duplexes, and poorly to
GCGCGCGC duplexes (Fig. 6B
, indicated with asterisks) with
an intermediate level of binding to other duplexes. In contrast,
flavopiridol was found to have an intermediate level of affinity for
all tested oligonucleotides in comparison to genomic DNA (Fig. 6A)
. Although this analysis did not define the nucleotide
sequence(s) required for optimal flavopiridol-DNA binding, it did rule
out the possibility that flavopiridol binding is specified by a
particular dinucleotide sequence. Instead, the results suggest that
either (a) strands of DNA longer than 8 mer are required to allow
optimal binding of flavopiridol, possibly as a result of a requirement
for DNA secondary structure, or (b) flavopiridol-DNA binding favors a
specific nucleotide sequence of 3 or more consecutive bp not included
in the test set of oligonucleotides.
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Molecular Modeling of Flavopiridol-DNA Interactions.
To complement the experimental data, we studied the possible
intermolecular interaction of flavopiridol with DNA at the atomic level
with computer modeling (Fig. 7)
. The structurally related but considerably less cytotoxic flavone
apigenin (7)
was used as a control compound. From these
studies, the binding energies of flavopiridol and apigenin in the most
energetically favorable DNA complexes were estimated to be -238 and +5
kcal/mol, respectively, indicating that intercalation of flavopiridol
into DNA (Fig. 7A)
is energetically feasible, whereas
intercalation of apigenin is not. In the most energetically stable
flavopiridol-DNA complex (Fig. 7B)
, the chlorophenyl
flavopiridol moiety forms off-center 
– stacking
interactions with C and A bases of the DNA, whereas the
hydroxy-substituted phenyl ring at the opposite end of flavopiridol
participates in face-to-face aromatic stacking interactions with G and
T bases of the DNA. Hydrogen bonding interactions (indicated with
yellow bonds in Fig. 7B
) are found between the piperidinyl
hydroxyl group and the amino group of the C base (bond distance, 2.2
Å), between the piperidinyl NH and the carbonyl oxygen atom of a G
base (bond distance, 2.2 Å), and between one of the phenyl hydroxyl
groups and a phosphate oxygen atom of a G base (bond distance 1.9 Å).
Additionally, the piperidinyl ring forms van der Waals interactions
with the methyl group of a base. It should be noted that three of the
six energetically favorable flavopiridol-DNA interactions involve the
piperidinyl substituent, whereas the remaining three involve the
flavone ring system. These results are consistent with the experimental
observations cited above, suggesting that both the flavone and
piperidinyl moieties of flavopiridol are necessary to permit effective
binding to DNA.
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DISCUSSION |
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Data in the present study implicate DNA as a potential second target of flavopiridol. In particular, our experiments demonstrate that flavopiridol inhibits RNA synthesis and induces up-regulation of p53. In addition, four independent lines of evidence indicate that flavopiridol binds directly to DNA. Experimental data and molecular modeling studies indicate that the flavopiridol-DNA interaction might be complex, perhaps involving groove binding in addition to intercalation. Each of these observations has potentially important implications for understanding of the mechanism(s) of action of this agent.
The current model of flavopiridol action suggests that flavopiridol
arrests actively cycling cells in the G1 and
G2 phases of the cell cycle through competitive
inhibition of CDKs. According to this model, as the length of
flavopiridol treatment increases, fewer and fewer cells would be
expected to traverse S phase, resulting in a progressive reduction in
the rate of DNA synthesis. Consistent with this model, we observed that
[3H]thymidine incorporation into DNA
progressively decreased over the first several hours of flavopiridol
treatment. However, [3H]uridine incorporation
into RNA decreased even more rapidly (Fig. 1A)
. This effect
on RNA synthesis, which is difficult to understand if flavopiridol is
acting solely as a CDK inhibitor, is reminiscent of the rapid
inhibition of RNA synthesis seen after irradiation (30)
or
trapping of topoisomerase-DNA complexes (31
, 32)
. Because
inhibition of RNA synthesis by agents such as -amanitin and
5,6-dichloro-1ß-D-ribofuranosylbenzimidazole
can kill cells, it is possible that flavopiridol-induced inhibition of
RNA synthesis may in and of itself result in some degree of
cytotoxicity.
Additional experiments indicated that p53 is rapidly and dramatically up-regulated after flavopiridol treatment. These findings are not only consistent with the possibility that DNA may be a target of flavopiridol but also provide a potential alternative explanation for the induction of cell-cycle arrest by flavopiridol in cells with wild-type p53 (51 , 52) . It is important to point out, however, that up-regulation of wild-type p53 is not required for flavopiridol-induced cytotoxicity. We and others have previously reported that HL-60 cells, which are p53 null, are killed by flavopiridol (7 , 14) .
While this work was in progress, Boege et al.
(19)
reported that a number of flavones inhibit
topoisomerase I, raising the question of whether flavopiridol-induced
cytotoxicity might be mediated by this process. However, several
observations in the present study suggest that flavopiridol does not
kill cells by inhibiting topoisomerases. First, alkaline elution
experiments failed to demonstrate induction of single-strand DNA breaks
(a hallmark of covalent topoisomerase-DNA complexes) by flavopiridol
(Fig. 2A)
. These results appear to rule out the possibility
that flavopiridol acts as a topoisomerase poison (53)
.
Second, we found no evidence that flavopiridol inhibits either
topoisomerase I or topoisomerase II in vitro (Fig. 2, C and D)
.
Although we found no evidence that flavopiridol targets topoisomerases,
multiple independent lines of evidence support the view that
flavopiridol directly interacts with DNA. First, absorption
spectroscopic studies demonstrated a red shift in the flavopiridol
max about a central isosbestic point upon
addition of DNA (Fig. 3A)
, an occurrence typically seen with
DNA-binding agents (36)
. Second, addition of flavopiridol
altered the migration of DNA on reverse-phase HPLC (Fig. 3B)
. Third, addition of DNA broadened flavopiridol
1H NMR signals (Fig. 4)
, a phenomenon
characteristic of intercalating agents (37
, 38)
. Fourth,
flavopiridol was displaced from DNA or oligonucleotides (as assessed by
either absorption spectroscopy or NMR) by ethidium (Fig. 5)
or DNA
melting. Fifth, equilibrium dialysis demonstrated that flavopiridol
bound to DNA with an affinity that was only 8-fold lower than that of
doxorubicin. Consistent with these observations, molecular modeling
studies indicated that intercalation of flavopiridol into DNA was
energetically feasible, and COMPARE analyses indicated that the
nonproprietary compounds with similar toxicity patterns in the National
Cancer Institute cell line screen were all DNA interacting agents.
Having established that flavopiridol binds to DNA, we next investigated
the sequence specificity of the interaction. NMR studies suggested that
flavopiridol can intercalate nonspecifically between each adjacent set
of DNA bp under some circumstances. On the other hand, HPLC "size
exclusion" data suggested that optimal flavopiridol-DNA interactions
may require specific sequences of three or more nucleotides (Fig. 6)
, a
phenomenon more typical of groove binders than intercalating agents.
Molecular modeling simulations provided data that reconcile these
observations. Although the chlorophenyl and heterocyclic portions of
flavopiridol can intercalate between bp, the piperidinyl moiety of
flavopiridol extends into the DNA minor groove, providing additional
hydrogen bonding and van der Waals interactions (Fig. 7)
. This raises
the possibility that optimal binding may involve elements of both
intercalation and groove binding. Further studies using nuclear
Overhauser effect techniques are required to better resolve the mode of
binding.
Because of limited sensitivities of the physical chemical assays, flavopiridol concentrations of 10–100 µM were used to demonstrate flavopiridol-DNA interactions in vitro. Although these levels are 10- to 100-fold higher than concentrations achieved in the clinical setting, the interactions should not be dismissed as nonphysiological. Techniques that are universally accepted for the study of ligand-receptor interactions (e.g., X-ray crystallography and NMR spectroscopy) rely on concentrations that are often far higher than those used in the present study. The important variable is the affinity constant, not the absolute levels of the agents used to study the interactions. Equilibrium dialysis indicated that flavopiridol had an affinity for DNA that approached that of known intercalating agents. Consistent with these results, flavopiridol caused up-regulation of p53 at concentrations of <100 nM.
Collectively, the present results suggest that DNA represents a second target of flavopiridol. The recognition of this second target may help to explain the complex cellular effects of the drug. Like doxorubicin and other anthracyclines, flavopiridol may ultimately prove of therapeutic utility because of its effects on several cellular targets rather than just one. Although it is tempting to speculate that flavopiridol-DNA interactions may account for the ability of flavopiridol to kill noncycling cells, the precise contributions of DNA binding and CDK inhibition to the cytotoxicity of the drug remain to be more fully elucidated.
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ACKNOWLEDGMENTS |
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, the Mayo Clinic
Molecular Biology Core Laboratory for preparation of oligonucleotides,
and Deb Strauss for secretarial assistance. |
FOOTNOTES |
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1 Supported in part by NIH Grants U01CA66912 and
P30CA15083, American Cancer Society Grant RPG CCE-98842, and a
grant from the Jack Taylor Family Foundation. 
2 To whom requests for reprints should be
addressed, at Division of Medical Oncology, Mayo Clinic, 200 First
Street S.W., Rochester, MN 55905. Phone: (507) 284-8950; Fax:
(507) 284-3906.
3 The abbreviations used are: CDK,
cyclin-dependent kinase; HPLC, high-performance liquid chromatography;
NMR, nuclear magnetic resonance; PBS, calcium- and magnesium-free
phosphate-buffered saline; Kd, dissociation
constant.
4 At higher pH, flavopiridol sometimes
precipitated from solution at the concentrations necessary for
detection by NMR spectroscopy. Although the acidic pH used for NMR
experiments might have affected DNA tertiary structure, spectra could
not be obtained at physiological pH. It should be noted, however, that
the protonation state of flavopiridol is equivalent at pH 7.4, 5.4, and
4.3.
5 K. C. Bible and R. H. Bible, unpublished
observations.
Received 9/27/99. Accepted 3/ 3/00.
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 J. E. Karp, A. Passaniti, I. Gojo, S. Kaufmann, K. Bible, T. S. Garimella, J. Greer, J. Briel, B. D. Smith, S. D. Gore, et al. Phase I and Pharmacokinetic Study of Flavopiridol followed by 1-{beta}-D-Arabinofuranosylcytosine and Mitoxantrone in Relapsed and Refractory Adult Acute Leukemias Clin. Cancer Res., December 1, 2005; 11(23): 8403 - 8412. [Abstract] [Full Text] [PDF]
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K. C. Bible, J. L. Lensing, S. A. Nelson, Y. K. Lee, J. M. Reid, M. M. Ames, C. R. Isham, J. Piens, S. L. Rubin, J. Rubin, et al. Phase 1 Trial of Flavopiridol Combined with Cisplatin or Carboplatin in Patients with Advanced Malignancies with the Assessment of Pharmacokinetic and Pharmacodynamic End Points Clin. Cancer Res., August 15, 2005; 11(16): 5935 - 5941. [Abstract] [Full Text] [PDF]
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 B. Montaner, W. Castillo-Avila, M. Martinell, R. Ollinger, J. Aymami, E. Giralt, and R. Perez-Tomas DNA Interaction and Dual Topoisomerase I and II Inhibition Properties of the Anti-Tumor Drug Prodigiosin Toxicol. Sci., June 1, 2005; 85(2): 870 - 879. [Abstract] [Full Text] [PDF]
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A. R. Tan, X. Yang, A. Berman, S. Zhai, A. Sparreboom, A. L. Parr, C. Chow, J. S. Brahim, S. M. Steinberg, W. D. Figg, et al. Phase I Trial of the Cyclin-Dependent Kinase Inhibitor Flavopiridol in Combination with Docetaxel in Patients with Metastatic Breast Cancer Clin. Cancer Res., August 1, 2004; 10(15): 5038 - 5047. [Abstract] [Full Text] [PDF]
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X. Lu, W. E. Burgan, M. A. Cerra, E. Y. Chuang, M.-H. Tsai, P. J. Tofilon, and K. Camphausen Transcriptional signature of flavopiridol-induced tumor cell death Mol. Cancer Ther., July 1, 2004; 3(7): 861 - 872. [Abstract] [Full Text] [PDF]
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S. Grant and P. Dent Gene profiling and the cyclin-dependent kinase inhibitor flavopiridol: What's in a name? Mol. Cancer Ther., July 1, 2004; 3(7): 873 - 875. [Full Text] [PDF]
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Z. N. Demidenko and M. V. Blagosklonny Flavopiridol Induces p53 via Initial Inhibition of Mdm2 and p21 and, Independently of p53, Sensitizes Apoptosis-Reluctant Cells to Tumor Necrosis Factor Cancer Res., May 15, 2004; 64(10): 3653 - 3660. [Abstract] [Full Text] [PDF]
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K. Camphausen, K. J. Brady, W. E. Burgan, M. A. Cerra, J. S. Russell, E. E.A. Bull, and P. J. Tofilon Flavopiridol enhances human tumor cell radiosensitivity and prolongs expression of {gamma}H2AX foci Mol. Cancer Ther., April 1, 2004; 3(4): 409 - 416. [Abstract] [Full Text] [PDF]
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 R. R. Rosato, J. A. Almenara, C. Yu, and S. Grant Evidence of a Functional Role for p21WAF1/CIP1 Down-Regulation in Synergistic Antileukemic Interactions between the Histone Deacetylase Inhibitor Sodium Butyrate and Flavopiridol Mol. Pharmacol., March 1, 2004; 65(3): 571 - 581. [Abstract] [Full Text] [PDF]
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C. M. Incles, C. M. Schultes, L. R. Kelland, and S. Neidle Acquired Cellular Resistance to Flavopiridol in a Human Colon Carcinoma Cell Line Involves Up-Regulation of the Telomerase Catalytic Subunit and Telomere Elongation. Sensitivity of Resistant Cells to Combination Treatment with a Telomerase Inhibitor Mol. Pharmacol., November 1, 2003; 64(5): 1101 - 1108. [Abstract] [Full Text] [PDF]
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U. Raju, E. Nakata, K. A. Mason, K. K. Ang, and L. Milas Flavopiridol, a Cyclin-dependent Kinase Inhibitor, Enhances Radiosensitivity of Ovarian Carcinoma Cells Cancer Res., June 15, 2003; 63(12): 3263 - 3267. [Abstract] [Full Text] [PDF]
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L. Maggiorella, E. Deutsch, V. Frascogna, N. Chavaudra, L. Jeanson, F. Milliat, F. Eschwege, and J. Bourhis Enhancement of Radiation Response by Roscovitine in Human Breast Carcinoma in Vitro and in Vivo Cancer Res., May 15, 2003; 63(10): 2513 - 2517. [Abstract] [Full Text] [PDF]
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D.-M. Kim, S.-Y. Koo, K. Jeon, M. H. Kim, J. Lee, C. Y. Hong, and S. Jeong Rapid Induction of Apoptosis by Combination of Flavopiridol and Tumor Necrosis Factor (TNF)-{alpha} or TNF-related Apoptosis-inducing Ligand in Human Cancer Cell Lines Cancer Res., February 1, 2003; 63(3): 621 - 626. [Abstract] [Full Text] [PDF]
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S. Wittmann, P. Bali, S. Donapaty, R. Nimmanapalli, F. Guo, H. Yamaguchi, M. Huang, R. Jove, H. G. Wang, and K. Bhalla Flavopiridol Down-Regulates Antiapoptotic Proteins and Sensitizes Human Breast Cancer Cells to Epothilone B-induced Apoptosis Cancer Res., January 1, 2003; 63(1): 93 - 99. [Abstract] [Full Text] [PDF]
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L. Cartee, S. C. Maggio, R. Smith, H. M. Sankala, P. Dent, and S. Grant Protein Kinase C-dependent Activation of the Tumor Necrosis Factor Receptor-mediated Extrinsic Cell Death Pathway Underlies Enhanced Apoptosis in Human Myeloid Leukemia Cells Exposed to Bryostatin 1 and Flavopiridol Mol. Cancer Ther., January 1, 2003; 2(1): 83 - 93. [Abstract] [Full Text] [PDF]
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J. E. Karp, D. D. Ross, W. Yang, M. L. Tidwell, Y. Wei, J. Greer, D. L. Mann, T. Nakanishi, J. J. Wright, and A. D. Colevas Timed Sequential Therapy of Acute Leukemia with Flavopiridol: In Vitro Model for a Phase I Clinical Trial Clin. Cancer Res., January 1, 2003; 9(1): 307 - 315. [Abstract] [Full Text] [PDF]
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 L. M. Schang Cyclin-dependent kinases as cellular targets for antiviral drugs J. Antimicrob. Chemother., December 1, 2002; 50(6): 779 - 792. [Abstract] [Full Text] [PDF]
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I. Gojo, B. Zhang, and R. G. Fenton The Cyclin-dependent Kinase Inhibitor Flavopiridol Induces Apoptosis in Multiple Myeloma Cells through Transcriptional Repression and Down-Regulation of Mcl-1 Clin. Cancer Res., November 1, 2002; 8(11): 3527 - 3538. [Abstract] [Full Text] [PDF]
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 G. K. Dy and A. A. Adjei Novel Targets for Lung Cancer Therapy: Part II J. Clin. Oncol., July 1, 2002; 20(13): 3016 - 3028. [Abstract] [Full Text] [PDF]
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K. Wu, C. Wang, M. D'Amico, R. J. Lee, C. Albanese, R. G. Pestell, and S. Mani Flavopiridol and Trastuzumab Synergistically Inhibit Proliferation of Breast Cancer Cells: Association with Selective Cooperative Inhibition of Cyclin D1-dependent Kinase and Akt Signaling Pathways Mol. Cancer Ther., July 1, 2002; 1(9): 695 - 706. [Abstract] [Full Text] [PDF]
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L. Cartee, R. Smith, Y. Dai, M. Rahmani, R. Rosato, J. Almenara, P. Dent, and S. Grant Synergistic Induction of Apoptosis in Human Myeloid Leukemia Cells by Phorbol 12-Myristate 13-Acetate and Flavopiridol Proceeds via Activation of Both the Intrinsic and Tumor Necrosis Factor-Mediated Extrinsic Cell Death Pathways Mol. Pharmacol., June 1, 2002; 61(6): 1313 - 1321. [Abstract] [Full Text] [PDF]
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C. B. Matranga and G. I. Shapiro Selective Sensitization of Transformed Cells to Flavopiridol-induced Apoptosis following Recruitment to S-Phase Cancer Res., March 1, 2002; 62(6): 1707 - 1717. [Abstract] [Full Text] [PDF]
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R. R. Rosato, J. A. Almenara, L. Cartee, V. Betts, S. P. Chellappan, and S. Grant The Cyclin-dependent Kinase Inhibitor Flavopiridol Disrupts Sodium Butyrate-induced p21WAF1/CIP1 Expression and Maturation while Reciprocally Potentiating Apoptosis in Human Leukemia Cells Mol. Cancer Ther., February 1, 2002; 1(4): 253 - 266. [Abstract] [Full Text] [PDF]
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M. Motwani, C. Jung, F. M. Sirotnak, Y. She, M. A. Shah, M. Gonen, and G. K. Schwartz Augmentation of Apoptosis and Tumor Regression by Flavopiridol in the Presence of CPT-11 in Hct116 Colon Cancer Monolayers and Xenografts Clin. Cancer Res., December 1, 2001; 7(12): 4209 - 4219. [Abstract] [Full Text] [PDF]
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 Y. A. Elsayed and E. A. Sausville Selected Novel Anticancer Treatments Targeting Cell Signaling Proteins Oncologist, December 1, 2001; 6(6): 517 - 537. [Abstract] [Full Text] [PDF]
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V. Smith, F. Raynaud, P. Workman, and L. R. Kelland Characterization of a Human Colorectal Carcinoma Cell Line with Acquired Resistance to Flavopiridol Mol. Pharmacol., November 1, 2001; 60(5): 885 - 893. [Abstract] [Full Text] [PDF]
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G. I. Shapiro, J. G. Supko, A. Patterson, C. Lynch, J. Lucca, P. F. Zacarola, A. Muzikansky, J. J. Wright, T. J. Lynch Jr., and B. J. Rollins A Phase II Trial of the Cyclin-dependent Kinase Inhibitor Flavopiridol in Patients with Previously Untreated Stage IV Non-Small Cell Lung Cancer Clin. Cancer Res., June 1, 2001; 7(6): 1590 - 1599. [Abstract] [Full Text] [PDF]
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 D. W. Fry, D. C. Bedford, P. H. Harvey, A. Fritsch, P. R. Keller, Z. Wu, E. Dobrusin, W. R. Leopold, A. Fattaey, and M. D. Garrett Cell Cycle and Biochemical Effects of PD 0183812. A POTENT INHIBITOR OF THE CYCLIN D-DEPENDENT KINASES CDK4 AND CDK6 J. Biol. Chem., May 11, 2001; 276(20): 16617 - 16623. [Abstract] [Full Text] [PDF]
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