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[Cancer Research 60, 2419-2428, May 1, 2000]
© 2000 American Association for Cancer Research

Experimental Therapeutics

Flavopiridol Binds to Duplex DNA1

Keith C. Bible2, Roy H. Bible, Jr., Timothy J. Kottke, Phyllis A. Svingen, Kun Xu, Yuan-Ping Pang, Elisabeth Hajdu and Scott H. Kaufmann

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


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Flavopiridol, the first potent cyclin-dependent kinase inhibitor to enter clinical trials, was recently found to be cytotoxic to noncycling cells. The present studies were performed to examine the hypothesis that flavopiridol, like several other antineoplastic agents that kill noncycling cells, might also interact with DNA. Consistent with this possibility, treatment of A549 human lung cancer cells with clinically achievable concentrations of flavopiridol resulted in rapid elevations of the DNA damage-responsive protein p53. In further studies, the binding of flavopiridol to DNA was examined in vitro by four independent techniques. Absorption spectroscopy revealed that addition of DNA to aqueous flavopiridol solutions resulted in a red shift of the flavopiridol {lambda}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 cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Flavopiridol, the first potent CDK3 inhibitor to enter clinical trials as a potential anticancer agent (1, 2, 3) , has attracted much recent interest, not only because of its novel cellular targets but also because it inhibits the growth of a variety of human tumor cell lines in vitro and in vivo (1 , 4, 5, 6) , kills noncycling tumor cells in vitro (7 , 8) , causes apoptosis in a variety of human cancer cells and cell lines in vitro and in vivo (7, 8, 9, 10, 11, 12, 13, 14) , and induces tumor regressions in some patients (2) . Assays of CDK activity (5 , 15) and data from X-ray crystallography (16 , 17) provide strong evidence that flavopiridol can bind to the ATP site of target CDKs. Nonetheless, the toxicity of this agent in noncycling cells raises the possibility that other mechanisms might contribute to flavopiridol-induced cytotoxicity.

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.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Flavopiridol was provided by the Pharmaceuticals Resources Branch of the National Cancer Institute (Bethesda, MD). Oligonucleotides were prepared in micromole quantities by the Mayo Clinic Molecular Biology Core Laboratory (Rochester, MN) using Applied Bioscience oligonucleotide synthesizers (Applied Bioscience International, Arlington, VA). Self-complementary oligonucleotides were annealed by heating to 50°C for 60 min and then cooling to 4°C at a rate of <=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 {alpha}-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 {gamma}-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.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Flavopiridol on RNA and DNA Synthesis.
Previous studies indicated that flavopiridol kills noncycling in addition to cycling cells (7 , 8 , 13 , 14) . To investigate these unexpected findings, we examined the effects of flavopiridol on DNA and RNA synthesis in A549 nonsmall cell lung cancer cells. When the concentration of flavopiridol was fixed at 500 nM, a readily achievable concentration in the clinical setting (2) , time course studies revealed that [3H]thymidine incorporation into DNA gradually decreased over several hours (Fig. 1A)Citation , 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)Citation . This effect was readily discernible at 100 nM flavopiridol and was maximal at 500 nM (Fig. 1B)Citation . Interestingly, [3H]uridine incorporation decreased more rapidly than [3H]thymidine incorporation did (Fig. 1A)Citation . 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)Citation . 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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  		Fig. 1. Effects of flavopiridol on A549 cells. A, flavopiridol inhibits RNA synthesis more rapidly than DNA synthesis in A549 cells. Cells treated with 500 nM flavopiridol for the indicated length of time were labeled with [3H]thymidine or [3H]uridine for the last 30 min of flavopiridol treatment. Incorporation of radiolabel into macromolecules was determined as described in "Materials and Methods." Results are representative of three independent experiments. B, relationship between flavopiridol dose and inhibition of RNA synthesis. After a 15-min incubation with the indicated concentration of flavopiridol, [3H]-labeled uridine was added for 120 min. Incorporation into trichloroacetic acid-precipitable macromolecules was determined. Results are representative of four independent experiments. C, flavopiridol treatment results in increased p53 protein levels. After exposure to 1 µM flavopiridol for the indicated times, cells were prepared for immunoblotting with p53 antibody (upper panel) as indicated in "Materials and Methods." Each lane contains 50 µg of total cellular protein. To confirm equal loading, blots were reprobed with an antibody that recognizes procaspase-2 (lower panel). D, flavopiridol and etoposide cause p53 elevations, whereas olomoucine, flavone, and apigenin do not. Cells were exposed for 4 h to diluent (C, Lane 1); 15.6, 31.2, 62.5, 125, 250, or 500 nM flavopiridol (Lanes 2–7); 100 nM paclitaxel (T, Lane 8); 10 µM etoposide (E, Lane 9); 500 nM olomoucine (O, Lane 10); 500 nM flavone (F, Lane 11), or 500 nM apigenin (A, Lane 12). Samples containing 50 µg of total cellular protein were loaded in each lane. Blots were probed with antibodies that recognize p53 (upper panel) or the loading control procaspase-2 (lower panel). Results in C and D are representative of three independent experiments.

 
Effects of Flavopiridol on p53 Levels.
To examine whether flavopiridol-treated cells respond in a similar fashion to those treated with DNA-damaging agents such as ionizing radiation or etoposide (33) , we evaluated the effects of flavopiridol and several other agents on p53 levels. Previous studies have demonstrated that p53 levels increase in cells treated with the intercalating agent actinomycin D, the topoisomerase I poison camptothecin, or a variety of DNA-damaging agents (34 , 35) . These increases are thought to reflect posttranslational modification and subsequent stabilization of p53 rather than enhanced gene expression. As shown in Fig. 1CCitation , 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. 1DCitation , Lane 9), was detectable after treatment with as little as 62.5 nM flavopiridol (Fig. 1DCitation , 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)Citation or ionizing radiation (Fig. 2B)Citation , flavopiridol did not induce DNA strand breaks after either 2 or 24 h of treatment (Fig. 2ACitation and data not shown). Consistent with these results, flavopiridol had no effect on topoisomerase II activity in vitro (Fig. 2C)Citation . In contrast, the intercalating inhibitor pyrazoloacridine (Fig. 2C)Citation 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)Citation . 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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  		Fig. 2. Alkaline elution and topoisomerase assays. A and B, effects of etoposide, flavopiridol, and ionizing radiation on DNA strand breaks in A549 cells. After a 2-h treatment of A549 cells with the indicated concentrations of flavopiridol or etoposide (A) or after exposure to the indicated amounts of ionizing radiation (B), alkaline elution was performed as described in the "Materials and Methods." Data are expressed as the percentage of DNA retained on the filter. Results are representative of three independent experiments. Effects of flavopiridol on DNA topoisomerase II (C) or topoisomerase I (D) activity in vitro. C, kinetoplast network DNA (N) was incubated with the indicated concentration of pyrazoloacridine (PZA), etoposide (Etop), or flavopiridol and then was treated with nuclear extract containing 2 U of topoisomerase II. 1 U, the minimum amount of activity required to decatenate the input networks; MC, DNA minicircles. D, supercoiled plasmid DNA (SC) was incubated with the indicated concentration of PZA or flavopiridol and then was treated with nuclear extract containing 2 U of topoisomerase I activity. 1 U, the amount of activity required to relax 50% of the input supercoiled plasmid; N, nicked DNA; R, relaxed DNA. Data shown in C and D are representative of three independent experiments.

 
Evaluation of Interactions Between Flavopiridol and DNA.
Although the rapid inhibition of RNA synthesis and the up-regulation of p53 suggested that flavopiridol might be altering DNA, the preceding results provided no evidence for topoisomerase-mediated DNA effects. To evaluate the possibility that flavopiridol might be directly interacting with DNA, we next assessed flavopiridol-DNA interactions in cell-free systems using absorption spectroscopy, HPLC "size exclusion" chromatography, NMR spectroscopy, and equilibrium dialysis.

When drug-DNA interactions are examined by absorption spectroscopy, binding of DNA to other drugs shifts drug {lambda}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 {lambda}max from 311 to 344 nm, producing a clear isosbestic point (Fig. 3A)Citation . These results raised the possibility that flavopiridol might be capable of directly interacting with DNA.



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  		Fig. 3. Absorption spectroscopy and "size exclusion" HPLC. A, effects of varying concentrations of calf thymus DNA on the absorption spectrum of flavopiridol. A solution containing 50 µg/ml (114 µM) flavopiridol in aqueous solution (pH 7.4) was supplemented with calf thymus DNA at final concentrations of 0, 6.3, 12.5, 25, 50, and 500 µg/ml (0, 10.3, 20.5, 41.0, 82.1, and 821 µM with respect to bp). Similar results were obtained with 11.4 µM flavopiridol and 0.625–50 µg/ml DNA. Note that the observed effects on absorbance are maximal when DNA concentrations exceed a 1:1 ratio of bp to flavopiridol molecules. Results are representative of three independent experiments. B, effects of flavopiridol, ethidium bromide, and Hoechst 33258 on DNA as assessed by reverse-phase HPLC. DNA (50 µg/ml; 82.1 µM with respect to bp) was treated with increasing concentrations of flavopiridol, with ethidium bromide, or with Hoechst 33258. Inset, demonstrates the decline of the calf thymus DNA peak area resulting from addition of flavopiridol (due to the formation of a new flavopiridol-DNA complex that elutes at a later time). The graph quantitates this effect for varying concentrations of flavopiridol, ethidium bromide, and Hoechst 33258. Results are representative of three independent experiments.

 
DNA-flavopiridol binding was further studied by HPLC "size exclusion" chromatography (26) . For compounds that bind DNA, the area of the native DNA peak (retention time, 1.3–1.4 min) observed during reverse-phase HPLC decreases as the amount of test compound is increased. This decrease reflects the formation of a new complex (DNA bound to test compound) that elutes at a later time (26) . Similarly, when this approach was applied to the study of flavopiridol-DNA interactions, addition of flavopiridol resulted in a dose-dependent decrease in the size of the native (unbound) DNA peak (Fig. 3BCitation , 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)Citation .

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)Citation . 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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  		Fig. 4. Effects of calf thymus DNA on the 1H NMR spectrum of flavopiridol recorded in aqueous PBS (pH 5.4, no D2O, 600 MHz). Spectra a–g are those obtained from 95 µM (57 nmoles/0.6 ml) flavopiridol in the presence of increasing concentrations of DNA (2.22, 4.43, 8.86, 17.7, 35, 71, and 142 µM bp, respectively). Spectra are slightly offset to avoid peak overlap. H2O resonances have been omitted from spectra for clarity. Note that 1H NMR flavopiridol signals disappear upon addition of greater than equimolar concentration of DNA (based upon bp). Results are representative of three experiments. The acquisition time for each spectrum was 8 h, and the number indicated at the right of each spectrum represents the molar ratio of DNA bp to flavopiridol.

 
To determine whether maneuvers designed to disrupt drug-DNA interactions might reverse the broadening of NMR signals, we melted the DNA after flavopiridol binding or added ethidium bromide to the flavopiridol-DNA complex. The broadened flavopiridol signals sharpened as a result of either of these two maneuvers. In particular, flavopiridol signals were sharpened by addition of equimolar (or greater) concentrations of ethidium bromide (Fig. 5)Citation 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)Citation before 1H-NMR ethidium signals (Fig. 5d)Citation , suggesting that ethidium caused displacement of flavopiridol from double-stranded DNA.



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  		Fig. 5. Effects of ethidium bromide on the broadening of flavopiridol 1H NMR resonances by calf thymus DNA (600 MHz). Spectrum a is that of 91.6 µM flavopiridol and 137.5 µM DNA and no ethidium bromide. Spectra b–d were obtained on solutions containing the same concentrations of flavopiridol and calf thymus DNA along with 74.1, 148.3, and 296.6 µM ethidium bromide, respectively. Spectrum e was obtained on a solution containing 91.6 µM flavopiridol and 74.1 µM ethidium bromide but no DNA. Note that DNA-induced broadening of flavopiridol 1H NMR signals is reversed as a result of the addition of lower concentrations of ethidium bromide (spectra b and c) than those required for ethidium 1H-NMR signals to be seen (spectrum d). All spectra were recorded at 37°C in PBS made with 100% H2O (pH 5.4). Spectra are slightly offset to avoid peak overlap. The acquisition time for each spectrum was 8 h. F, flavopiridol peak(s); E, ethidium bromide peak(s).

 
Additional experiments revealed that neither flavone acetic acid (another cytotoxic flavone) nor 3-hydroxy-1-methylpiperidine (the piperidinyl substituent of flavopiridol) displayed similar NMR or absorption spectroscopic evidence of DNA binding, whereas ethidium bromide behaved much like flavopiridol did.5 These observations suggest that DNA binding is not a property universal to cytotoxic flavones, that flavopiridol may be unique among flavones with respect to its DNA-binding properties, and that both the flavone and piperidinyl moieties of flavopiridol may be required for its DNA binding.

Although "size exclusion" chromatography suggested that equal amounts of flavopiridol, ethidium, and Hoechst 33258 could bind similar amounts of DNA (Fig. 3B)Citation , 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. 6Citation , 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. 6BCitation , 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)Citation . 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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  		Fig. 6. Binding of flavopiridol and pyrazoloacridine to a series of annealed self-complementary oligonucleotides. "Size exclusion" HPLC was used to assess the effects of flavopiridol (A) or pyrazoloacridine (B) on native oligonucleotide HPLC peak area (see "Materials and Methods" and Fig. 3BCitation for details). Oligonucleotide concentrations were 75 µg/ml for all experiments, whereas drug concentrations were all 300 µg/ml. Reduction of oligonucleotide peak area indicates drug-oligo binding. Note that pyrazoloacridine binds optimally to GGGGCCCC duplexes, less well to GGCCGGCC duplexes, and poorly to GCGCGCGC duplexes (asterisks in B), with an intermediate level of binding to other duplexes. In contrast, flavopiridol showed an intermediate level of binding to all tested oligos, without clear specificity for any two-nucleotide sequence. Results are representative of three independent experiments.

 
COMPARE Analysis.
In an effort to determine whether the pattern of flavopiridol-induced cytotoxicity in the NCI 60 human tumor cell line screen was potentially consistent with the hypothesis that DNA might be a target of the drug, we conducted COMPARE analyses using flavopiridol as a seed compound and using LC50 data. Over 150 tested compounds had correlation coefficients of >0.6. Examination of the highly correlated test compounds with known/putative mechanisms of action revealed that most are intercalating or DNA-damaging agents. In particular, ecteinascidin 729, chromomycin A3, actinomycin D, and 3'-deamino-3'-(4-morpholinyl)-13-dihydroadriamycin were the agents with known/putative mechanisms of action and the highest correlations to flavopiridol (correlation coefficients of 0.831, 0.823, 0.820, and 0.815, respectively). All of these drugs interact with DNA: ecteinascidin 729 is a minor groove binder that forms DNA adducts at the N-2 position of guanine (47) , chromomycin A3 is a DNA-binding antitumor antibiotic that functions to inhibit transcription (48) , actinomycin D intercalates at d(GC) and inhibits transcription (49) , and 3'-deamino-3'-(4-morpholinyl)-13-dihydroadriamycin is a derivative of the anthracycline intercalating agent doxorubicin. Other highly correlated DNA interacting agents include mithramycin, saframycin, nogalamycin, cyanomorpholinoadriamycin and echinomycin.

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)Citation . 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)Citation is energetically feasible, whereas intercalation of apigenin is not. In the most energetically stable flavopiridol-DNA complex (Fig. 7B)Citation , the chlorophenyl flavopiridol moiety forms off-center {pi}–{pi} 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. 7BCitation ) 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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  		Fig. 7. Molecular modeling of the interactions between flavopiridol and DNA. A, energy-minimized flavopiridol-DNA complex. Flavopiridol is shown intercalated into duplex 5'-TCGTCAAA-3' between G and T. See "Materials and Methods" for modeling conditions. For clarity, flavopiridol is shown in green. B, expanded and simplified view of A to better illustrate hydrogen bonds (shown in yellow) and {pi}–{pi} interactions. For clarity, flavopiridol is shown by a ball and stick model, whereas DNA is represented by a stick model.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Flavopiridol is a promising new antineoplastic agent that induces arrest in the G1 and G2 phases of the cell cycle (29) , kills noncycling cancer cells (7 , 8 , 11 , 13) , induces apoptosis in cancer cells in vitro and in vivo (7, 8, 9, 10, 11, 12, 13, 14) , and produces tumor regressions in animals and humans (1 , 2 , 6 , 12) . Previously reported kinase assays and data from X-ray crystallography have unequivocally demonstrated that flavopiridol can competitively inhibit CDKs 1, 2, 4 and 7, presumably by binding to CDK ATP sites (reviewed in Refs. 1 , 5 , 15, 16, 17 , and 50 ). Although it is clear that inhibition of CDKs by flavopiridol can lead to the observed cell-cycle effects (arrest in both G1 and G2 phases of the cell cycle), it has remained unclear whether effects on these targets can account for the cytotoxicity to noncycling cells, induction of apoptosis, and tumor regressions.

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)Citation . 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 {alpha}-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)Citation . 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)Citation .

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 {lambda}max about a central isosbestic point upon addition of DNA (Fig. 3A)Citation , an occurrence typically seen with DNA-binding agents (36) . Second, addition of flavopiridol altered the migration of DNA on reverse-phase HPLC (Fig. 3B)Citation . Third, addition of DNA broadened flavopiridol 1H NMR signals (Fig. 4)Citation , 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)Citation 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)Citation , 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)Citation . 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.


   ACKNOWLEDGMENTS
 
We thank Dr. Edward Sausville for flavopiridol and helpful discussions, Scott Boerner for assistance with Fig. 1Citation , the Mayo Clinic Molecular Biology Core Laboratory for preparation of oligonucleotides, and Deb Strauss for secretarial assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grants U01CA66912 and P30CA15083, American Cancer Society Grant RPG CCE-98842, and a grant from the Jack Taylor Family Foundation. Back

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. Back

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. Back

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. Back

5 K. C. Bible and R. H. Bible, unpublished observations. Back

Received 9/27/99. Accepted 3/ 3/00.


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 ABSTRACT
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 DISCUSSION
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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]