Molecular Cancer Therapeutics
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Mol Cancer Ther. 2006;5:138-148
© 2006 American Association for Cancer Research

Flavopiridol disrupts STAT3/DNA interactions, attenuates STAT3-directed transcription, and combines with the Jak kinase inhibitor AG490 to achieve cytotoxic synergy

Yean Kit Lee1, Crescent R. Isham1, Scott H. Kaufman1 and Keith C. Bible2

Divisions of 1 Developmental Oncology Research and 2 Medical Oncology, Mayo Graduate School of Medicine, Rochester, Minnesota

Requests for reprints: Keith C. Bible, Division of Medical Oncology, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905. Phone: 507-284-8950; Fax: 507-284-3906. E-mail: bible.keith{at}mayo.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Up-regulated signal transducers and activators of transcription (STAT)–mediated signaling is believed to contribute to the pathogenesis of a variety of solid and hematologic cancers. Consequently, inhibition of STAT-mediated signaling has recently been proposed as a potential new therapeutic approach to the treatment of cancers. Having shown previously that the pan–cyclin-dependent kinase inhibitor flavopiridol binds to DNA and seems to kill cancer cells via that process in some circumstances, we evaluated the hypothesis that flavopiridol might consequently disrupt STAT3/DNA interactions, attenuate STAT3-directed transcription, and down-regulate STAT3 downstream polypeptides, including the antiapoptotic polypeptide Mcl-1. SDS-PAGE/immunoblotting and reverse transcription-PCR were used to assess RNA and polypeptide levels, respectively. DNA cellulose affinity chromatography and a nuclear elution assay were used to evaluate the ability of flavopiridol to disrupt STAT3/DNA interactions. A STAT3 luciferase reporter assay was used to examine the ability of flavopiridol to attenuate STAT3-directed transcription. Colony-forming assays were used to assess cytotoxic synergy between flavopiridol and AG490. Flavopiridol was found to (a) disrupt STAT3/DNA interactions (DNA cellulose affinity chromatography and nuclear elution assay), (b) attenuate STAT3-directed transcription (STAT3 luciferase reporter assay), and (c) down-regulate the STAT3 downstream antiapoptotic polypeptide Mcl-1 at the transcriptional level (reverse transcription-PCR and SDS-PAGE/immunoblotting). Furthermore, flavopiridol, but not the microtubule inhibitor paclitaxel, could be combined with the STAT3 pathway inhibitor AG490 to achieve cytotoxic synergy in A549 human non–small cell lung cancer cells. Collectively, these data suggest that flavopiridol can attenuate STAT3-directed transcription in a targeted fashion and may therefore be exploitable clinically in the development of chemotherapy regimens combining flavopiridol and other inhibitors of STAT3 signaling pathways. [Mol Cancer Ther 2006;5(1):138–48]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Signal transducers and activators of transcription (STAT) are transcription factors that shuttle from the cytoplasm to the nucleus in response to certain extracellular signals (1, 2). In particular, phosphorylation on specific tyrosine and serine residues induces monomeric cytoplasmic STAT proteins to dimerize and translocate to the nucleus, where they bind specific DNA sequences and activate transcription of several genes, including those encoding the antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 (3, 4). Constitutive activation of STAT3 signaling has been observed in several neoplasms, including acute leukemias (5), breast cancer (6), and squamous cell carcinoma of the head and neck (7), mycosis fungoides (8), and large granular lymphocytic leukemias (9). Consequently, there is considerable interest in developing inhibitors of STAT3-mediated signaling (10–12).

Flavopiridol, a cytotoxic small-molecule antineoplastic agent being evaluated in phase I and II clinical trials (13–15), induces apoptosis in vitro in human cancer cell lines (16–25) and human chronic lymphocytic leukemia cells (23) and in vivo in human leukemia and lymphoma xenografts grown in nude mice (17). Consistent with these results, tumor regressions have been seen in human squamous cell and prostate carcinoma xenograft models (24, 26) and in human clinical trials (13–15). Although flavopiridol was initially identified as a cytostatic inhibitor of cyclin-dependent kinases (CDK; refs. 27–30), its ability to kill noncycling cells (14, 16, 18, 23) raises the possibility that molecular targets in addition to CDKs may be at least partially responsible for its cytotoxicity.

To date, four targets that might contribute to flavopiridol-induced cytotoxicity have been identified: CDKs (28–30), glycogen phosphorylase (31, 32), P-TEFb (a component of the complex regulating activation of RNA polymerase II via phosphorylation; refs. 33, 34), and DNA (35). Although CDK inhibition has been most extensively studied, it remains to be shown whether CDK inhibition alone is sufficient to account for flavopiridol-induced cytotoxicity. Glycogen phosphorylase is unlikely to be of clinical relevance because of the high flavopiridol concentrations necessary to inhibit the enzyme in cancer cells (31, 32). Potentially consistent with its ability to inhibit P-TEFb-regulated activation of RNA polymerase II (33, 34), flavopiridol has been found to down-regulate several cellular proteins (20, 36, 37). On the other hand, treatment of yeast with flavopiridol up-regulates 80% of transcripts that are affected (38), raising the possibility that effects on P-TEFb might not explain all of the transcriptional effects of flavopiridol.

Recent work from our laboratory suggests that DNA might also be an important target of flavopiridol (35). In particular, flavopiridol binds to DNA with an affinity approaching that of the intercalator doxorubicin (35). Analysis using the National Cancer Institute COMPARE program (an analytic tool for comparing profiles of the effects of various anticancer agents on 60 tumor cell lines in vitro) also indicates that the cytotoxic effects of flavopiridol strongly correlate with those of several additional DNA-interacting agents (35). Nonetheless, flavopiridol does not induce the types of DNA damage seen with many DNA-interacting antineoplastic agents (35). In particular, flavopiridol does not induce DNA strand breaks, inhibit topoisomerase I or II, or cause DNA-DNA or DNA-protein cross-links (35). These observations suggest that flavopiridol may have a unique mechanism of action but leave unresolved the issue of how drug-DNA interactions might contribute to the cytotoxicity of flavopiridol.

We now report that flavopiridol disrupts STAT3/DNA interactions, attenuates STAT3-directed transcription, and down-regulates the antiapoptotic STAT3 transcriptional target Mcl-1 at the mRNA and protein levels in a variety of neoplastic cell lines. In conjunction with recent results showing that Mcl-1 overexpression induces a high incidence of neoplastic transformation in vivo (39) and that Mcl-1 down-regulation is sufficient to induce apoptosis in Mcl-1-expressing cells (40, 41), the present observations identify a potential mechanistic explanation for flavopiridol-induced cytotoxicity. Equally important, these results suggest that flavopiridol may represent the first cytotoxic small molecule to reach human antineoplastic clinical trials that inflicts cytotoxicity via disruption of transcription factor/DNA interactions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Flavopiridol was kindly provided by the Pharmaceutical Resources Branch of the National Cancer Institute, whereas paclitaxel and AG490 were purchased from Sigma (St. Louis, MO).

Cell Culture
Cells were cultured in the following media: A549 cells in RPMI 1640 containing 5% (v/v) fetal bovine serum; ML-1, K562, Jurkat, My-5, and HCT116 cells in RPMI 1640 containing 10% fetal bovine serum; OV17R cells in MEM containing 20% fetal bovine serum; PC-3 cells in F-12 medium containing 10% fetal bovine serum; and MDA-MB-468 cells in Iscove's modified essential medium containing 5% fetal bovine serum. All media contained 100 units/mL penicillin G, 100 µg/mL streptomycin, and 2 mmol/L glutamine. Cells were passaged once or twice weekly and maintained at 37°C in an atmosphere containing 95% air-5% CO2 (v/v).

Immunoblotting
Cells grown to 50% confluence in triplicate 100-mm tissue culture dishes were washed four times in situ with 10 mL PBS and solubilized in alkylation buffer [6 mol/L guanidine HCl, 250 mmol/L Tris-HCl (pH 8.5 at 21°C), and 10 mmol/L EDTA supplemented immediately before use with 150 mmol/L ß-mercaptoethanol and 1 mmol/L phenylmethylsulfonyl fluoride]. Samples were processed for SDS-PAGE and subsequent immunoblotting using techniques described previously (42). Antibodies were obtained as follows: anti-phospho-STAT1(Tyr701), anti-phospho-STAT3(Tyr705), anti-phospho-STAT3(Ser727), anti-phospho-STAT5(Tyr694), anti-phospho–extracellular signal-regulated kinase (ERK) 1/2(Thr202/Tyr204), anti-phospho-ELK-1(Ser383), and anti-phospho-AKT(Ser473) from Cell Signaling (Beverly, MA); anti–cyclin D1 and anti-STAT5b from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Bcl-2 and anti-Bcl-xL from BD Transduction Laboratories (Lexington, KY); anti–cyclin A, anti-phospho–cyclic AMP–responsive element binding protein (CREB) (Ser133), and anti-STAT1 from Upstate Biotechnology (Lake Placid, NY); anti-p21 and anti-p53 from Neomarkers (Union City, CA); anti-phospho–RNA polymerase II (antibody H5) and anti–RNA polymerase II (antibody 8WG16) from Convance (Richmond, CA); and anti-Mcl-1 from Dr. John Reed (Burnham Institute, La Jolla, CA).

Reverse Transcription-PCR
Cellular RNA was isolated using Trizol reagent (Life Technologies, Rockville, MD) according to established procedures (43). Cellular RNA (2 µg), random primers (1 µL), and DEPC water (14 µL) were heated to 70°C for 5 minutes and cooled to 23°C. RNasin (1 µL), 5x reverse transcription buffer (5 µL; Promega, Madison, WI), 10 mmol/L deoxynucleotide triphosphates (1 µL), and 20 units avian myeloblastosis virus reverse transcriptase (1 µL; Promega) were then added. An aliquot of each resulting mixture was incubated for 10 minutes at 23°C followed by 60 minutes at 42°C. Aliquots (2 µL) were then supplemented with 10x PCR buffer (5 µL) containing 1.5 mmol/L Mg2+, 10 mmol/L deoxynucleotide triphosphates (2 µL), respective forward and reverse primers (5 µL), distilled H2O (36 µL), and 5 units Taq polymerase (1 µL). PCR was done for 30 cycles with extension at 72°C (2 minutes), denaturation at 94°C (1 minute), and annealing 55°C (1 minute) using a Perkin-Elmer Cetus thermocycler (Wellesley, MA). After amplification, samples were subjected to electrophoresis on 1% agarose gels containing ethidium bromide, visualized under UV light, and photographed using a ChemiDoc Documentation System (Bio-Rad, Hercules, CA).

The following primers were used: ß-actin, 5'-CTTAATGTCACGCACGATTTC-3' (reverse) and 5'-ACGTTATGGTGATGATATCG-3' (forward), 654-bp product; Mcl-1, 5'-CCGTCCAGCTCCTCTTCG-3' (reverse), 5'-CGGACTCAACCTCTACTGTGG-3' (forward), 354-bp product; topoisomerase I, 5'-TCTTGGGGTGGTTGCCGCG-3' (reverse), 5'-CCGGATCCATGAGTGGGGACCACCTCCACAA-3' (forward), 185-bp product; caspase-3, 5'-AAAGAATTCTTAGTGATAAAAATAGAGTTCTTTTGTGAG-3' (reverse), 5'-AAAGGATCCTTAATAAAGGTATCCATGGAGAACACT-3' (forward), 975-bp product; cyclin A, 5'-GAAGCAATGCACTGCAGC-3' (forward) 5'-CAGATACAGGGTCTCTGC-3' (reverse), 644-bp product; and cyclin D1, 5'-CATGCGCAAGATCGTCGC-3' (forward) 5'-TCACTCTGGAGAGGAAGC-3' (reverse), 544-bp product.

DNA Cellulose Affinity Chromatography
Two hundred fifty to five hundred 100-mm dishes of subconfluent A549 cells were harvested by trypsinization. All subsequent steps were at 4°C. After three washes with ice-cold PBS, cells were suspended in 5 to 10 mL extraction buffer [1.7 mol/L NaCl, 1 mmol/L Na2EDTA, 1 mmol/L ß-mercaptoethanol, 20 mmol/L Tris-HCl, 100 mmol/L phenylmethylsulfonyl fluoride (pH 7.4)], disrupted by sonication, and sedimented. After polyethylene glycol (~6,000 molecular weight) was added to a final concentration of 10%, the sample was incubated for 15 minutes, sedimented to remove precipitated DNA, and dialyzed against buffer A [50 mmol/L NaCl, 1 mmol/L Na2EDTA, 20 mmol/L Tris-HCl (pH 7.4)] for three changes of 5 hours each. After centrifugation, the supernatant was brought to a final concentration of 10% glycerol and 0.1% DMSO and applied to a duplex DNA-cellulose column (5 g; Sigma) that had been washed extensively with buffer B [50 mmol/L NaCl, 1 mmol/L Na2EDTA, 1 mmol/L ß-mercaptoethanol, 20 mmol/L Tris-HCl, 100 µmol/L phenylmethylsulfonyl fluoride, 0.1% DMSO (pH 7.4)] after initial treatment with buffer B containing 100 µg/mL bovine serum albumin to block nonspecific binding sites. After flow-through and wash fractions were collected (20-mL fractions), the column was eluted with buffer B containing increasing concentrations of flavopiridol followed by buffer B supplemented to 2 mol/L NaCl. The resulting fractions were dialyzed against buffer A, lyophilized, resuspended in SDS sample buffer containing 10% ß-mercaptoethanol, subjected to SDS-PAGE (5–15% gradient) as described previously (42), transferred to nitrocellulose, and probed with antibodies as described in subsequent text.

Nuclear Extraction Assay
Fifty 100-mm tissue culture plates of subconfluent A549 cells were harvested by trypsinization. All subsequent steps were at 4°C. Cells were sedimented, washed thrice with PBS, swelled for 20 minutes with nuclear isolation buffer [10 mmol/L Tris-HCl, 10 mmol/L NaCl, 3 mmol/L MgSO4, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride, 100 units/mL Trasylol, 0.5 mmol/L EDTA (pH 7.4)], and homogenized in a Dounce homogenizer until cells were disrupted and nuclei were released (usually ~200 strokes). Nuclei were sedimented and extracted with 1-mL aliquots of nuclear extraction buffer [20 mmol/L HEPES, 0.2 mmol/L EDTA, 20% glycerol, 1 mmol/L DTT, 0.4 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL antipain, and leupeptin (pH 7.4)] containing increasing concentrations of flavopiridol followed by nuclear extraction buffer containing 400 mmol/L NaCl. The resulting extracts were dialyzed, lyophilized, resuspended SDS sample buffer, and subjected to SDS-PAGE followed by immunoblotting as described above.

STAT3 Luciferase Reporter Assay
Human K562 cells were cotransfected by electroporation with 10 µg pLuc-TK/STAT3 (kindly provided by Dr. Jacqueline Bromberg, Memorial Sloan-Kettering Cancer Center) and 2.2 µg pRL-TK (Promega) as a transfection efficiency control. Five hours after electroporation, cells were transferred to serum-free medium, and 250 nmol/L of the indicated drugs or DMSO diluent were added for a total exposure time of 22 hours. Human interleukin-6 (10 ng/mL) was added to the medium during the last 6 hours of drug exposure to induce STAT3 expression to increase the sensitivity of the assay. Cell lysates were collected according to the dual luciferase assay protocol (Promega). Sample light output was analyzed using a luminometer and resulting data were corrected for pRL-TK values and then normalized relative to diluent (DMSO) control. A value of 1 in this assay indicates no change from control, whereas values < 1 indicate attenuation of STAT3-directed transcription as assessed by this assay. The used drug concentrations in the displayed results were selected/optimized based on the ability of this concentration of flavopiridol to attenuate Mcl-1 levels without inducing appreciable apoptosis/cell death at the 22-hour time point. Both higher and lower concentrations of all tested agents were evaluated/explored in this assay, with the results seen at differing drug concentrations consistent with the displayed results.

Evaluation of Cytotoxic Synergy
Evaluation of cytotoxic synergy was done as described previously (16). Briefly, 750 suspended A549 human non–small cell lung cancer cells obtained from trypsinization from stock flasks of subconfluent cell cultures were deposited into each of triplicate sets of 35-mm tissue culture plates and allowed to adhere overnight. Cells were then treated for 24 hours with diluent, flavopiridol, paclitaxel, or AG490 at the concentrations indicated in the text alone and in combination. After drug removal and washing, cells were allowed to proliferate in drug-free medium for 7 to 10 days and thereafter washed twice with serum-free PBS, stained with Coomassie blue, and then manually counted. Synergy was determined by median effect analysis using the CalcuSyn software program (Biosoft, Cambridge, United Kingdom).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Flavopiridol Disrupts Transcription Factor/DNA Binding
Previous data have implicated DNA as a potential flavopiridol target but have also indicated that flavopiridol does not induce DNA cross-links/strand breaks or inhibit topoisomerase I or II (see ref. 35; see Introduction). In view of these results, we assessed the possibility that flavopiridol might instead disrupt protein/DNA interactions. Initial experiments used DNA cellulose affinity chromatography (ref. 44; Fig. 1A ). In these experiments, DNA/cellulose columns were preloaded with cellular lysate proteins and washed sequentially with flavopiridol-free mobile phase, increasing concentrations of flavopiridol, and high-salt buffer. As little as 500 nmol/L flavopiridol (a clinically achievable flavopiridol concentration; refs. 13, 14, 45) eluted STAT3 from the column. In contrast, several other polypeptides, including the transcription p53 as well as histone H1 and topoisomerase I, were not eluted by flavopiridol (Fig. 1A), thus indicating that STAT3 was eluted in this assay by flavopiridol with some degree of selectivity in comparison with several other DNA-binding proteins.


Figure 1
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  		Figure 1. Flavopiridol disrupts STAT3/DNA interactions and attenuates STAT3-directed transcription. A, flavopiridol disrupts STAT3/DNA binding as assessed by DNA/cellulose affinity chromatography. A whole-cell lysate of A549 cells was loaded on a (duplex) DNA/cellulose column and eluted with progressively higher concentrations of flavopiridol (0.5–10 µmol/L). Eluted fractions were subjected to SDS-PAGE and probed as indicated by immunoblotting. W, flavopiridol-free column wash; HS, high-salt (2 mol/L NaCl) wash; Topo I, topoisomerase I. B, flavopiridol displaces STAT3 from isolated A549 cell nuclei. Nuclei were sequentially extracted with flavopiridol-free low-salt wash followed by increasing flavopiridol concentrations (0.5–10 µmol/L) and finally by high-salt (2 mol/L NaCl) buffer as described in the text. PARP, poly(ADP-ribose) polymerase. A and B, gel lanes were loaded based on equivalent volumes of eluted fractions. C, flavopiridol attenuates STAT3-directed transcription in intact A549 cells as assessed using a STAT3 luciferase reporter assay, whereas a variety of other DNA-interacting antineoplastic agents and the microtubule-interacting agent paclitaxel do not. Results were corrected using a cotransfected control Renilla reporter plasmid and normalized to diluent-treated controls. A value of 1 indicates no effects on reporter activity, whereas values <1 indicate specific attenuation of STAT3-mediated transcription. Columns, mean of four independent experiments; bars, SD. Statistical significance indicates the probability that the attained results are significantly different from 1 (the null hypothesis, two-sided confidence intervals). NS, not significant.

 
To examine whether flavopiridol might disrupt transcription factor/DNA binding under somewhat more physiologic conditions, a nuclear extraction assay (46) was employed (Fig. 1B). Nuclei isolated from A549 cells were extracted with low-salt extraction buffer containing progressively increasing concentrations of flavopiridol. Immunoblotting showed that flavopiridol displaced STAT3 from isolated cell nuclei, confirming that flavopiridol is capable of disrupting transcription factor/DNA interactions in a second in vitro system. Under identical conditions, other nuclear-resident proteins, including p53, topoisomerase I, and poly(ADP-ribose) polymerase, remained in the nuclei until eluted with high-salt buffer, again indicating some degree of selectivity in the elution of nuclear proteins by flavopiridol.

Flavopiridol Disrupts STAT3-Directed Transcription
In view of the above data suggesting that flavopiridol might disrupt STAT/DNA interactions under cell-free conditions and in isolated cell nuclei, we next evaluated the possibility that flavopiridol might disrupt STAT3-directed transcription in intact cells using a STAT3 luciferase reporter (Fig. 1C). In these assays, a dual luciferase assay system was used to correct results for differences in transfection efficiency and nonspecific/global transcriptional effects. Whereas the microtubule-interacting agent paclitaxel and the DNA-interacting agents ethidium bromide, Hoescht 33258, pyrazoloacridine, and actinomycin D produced no selective effects on STAT3-directed transcription in this assay, flavopiridol significantly attenuated STAT3-directed transcription (Fig. 1C). It should be noted, however, that although actinomycin D was not seen to selectively attenuate STAT3-directed transcription in this assay, it decreased STAT3 and control reporter activities proportionally, presumably indicating nonspecific attenuation of transcription.

Flavopiridol Down-Regulates Mcl-1 Message Levels
We next examined whether flavopiridol decreases messages that are transcriptionally regulated by STAT3. Because flavopiridol has been reported previously to inhibit transcription based on P-TEFb inhibition (33, 34), the effects of flavopiridol (250 nmol/L) were directly compared with the classic P-TEFb inhibitor 5,6-dichloro-1-ß-D-ribobenzimidazole (DRB; 94 µmol/L). Messages that are regulated by STAT3 (Mcl-1, cyclin D1, and cyclin A) and messages that are not (topoisomerase I, caspase-3, and ß-actin) were examined using reverse transcription-PCR.

As indicated in Fig. 2 , results of these analyses showed that flavopiridol decreased levels of messages encoding Mcl-1, cyclin A, and cyclin D1. Whereas cyclin A and D1 mRNA levels declined only at later time points (most notably at 24 and 48 hours; Fig. 2), Mcl-1 mRNA levels declined dramatically after treatment with as little as 125 nmol/L flavopiridol for 6 hours, possibly reflecting the short half-life of Mcl-1 message (47).


Figure 2
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  		Figure 2. Flavopiridol rapidly attenuates levels of Mcl-1 message in A549 cells and has effects on message levels that differ from the classic P-TEFb inhibitor DRB. Although both flavopiridol and DRB down-regulate Mcl-1 message levels, DRB more dramatically attenuates cyclin A and caspase-3 message levels. C, control/diluent-treated cells; D, cells treated with 94 µmol/L DRB; F, cells treated with 250 nmol/L flavopiridol. Message levels were all assessed using reverse transcription-PCR. Representative of four independent experiments.

 
Examination of additional messages indicated that the effects of flavopiridol and DRB were distinct at assessed concentrations. In particular, although both flavopiridol and DRB decreased Mcl-1 mRNA as early as 6 hours, only DRB consistently attenuated levels of caspase-3 message at that and at later time points (Fig. 2, representative of four independent experiments), clearly indicating differential effects of the two agents on transcripts. Likewise, DRB more dramatically down-regulated cyclin A and D1 messages in comparison with flavopiridol (Fig. 2). These observations, although not definitive, suggest that the effects of flavopiridol on transcription might be distinct from and more focal than DRB, consistent with the hypothesis that flavopiridol attenuates STAT3-directed transcription with some selectivity under these assay conditions.

Flavopiridol Down-Regulates Proteins Downstream of STAT3
In view of data showing that flavopiridol can disrupt STAT3/DNA interactions (Fig. 1A and B), attenuate STAT3-directed transcription (Fig. 1C), and decrease STAT3-regulated messages (Fig. 2), we next examined whether flavopiridol might also attenuate levels of the corresponding cellular proteins. Of seven polypeptides known to be regulated by STAT3 (Mcl-1, cyclin A, cyclin D1, Bcl-xL, Bcl-2, c-Myc, and p21; refs. 3, 4, 12), three (Mcl-1, cyclin D1, and Bcl-2) have been reported previously to be down-regulated by flavopiridol in selected cell types (20, 36, 37). Experiments in A549 cells showed that Mcl-1 declined more rapidly and dramatically than the other polypeptides in response to flavopiridol (Fig. 3 ), consistent with the down-regulation of Mcl-1 message observed in Fig. 2. Examination of eight additional cell lines (Fig. 4A ), each of which is sensitive to flavopiridol-induced cytotoxicity (ref. 16; data not shown), revealed that Mcl-1 protein levels decreased in a dose-dependent manner in all of these lines, regardless of tumor type, after only 6 hours of treatment with as little as 125 nmol/L flavopiridol, a clinically achievable concentration (13, 14, 45).


Figure 3
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  		Figure 3. Effects of flavopiridol on levels of cellular proteins differ from those of the classic P-TEFb inhibitor DRB. Flavopiridol and DRB both down-regulate levels of Mcl-1 and phospho–RNA polymerase II, but only flavopiridol down-regulates c-Myc levels and up-regulates phospho-CREB (asterisks). Whole-cell extracts were subjected to SDS-PAGE (50 µg protein/lane) followed by immunoblotting as described in the text. The top pRb band corresponds to hyperphosphorylated Rb. Positive control lanes for those polypeptides found to be undetectable in assayed A549 cells showed used immunoblotting antibodies to be functional.

 

Figure 4
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  		Figure 4. Flavopiridol down-regulates Mcl-1 in flavopiridol-sensitive human cancer cell lines and abrogates 12-O-tetradecanoylphorbol-13-acetate–induced increases in Mcl-1. A, treatment with as little as 125 nmol/L flavopiridol for 6 h attenuated Mcl-1 levels in nine different flavopiridol-sensitive human cancer cell lines. B, 12-O-tetradecanoylphorbol-13-acetate treatment of A549 cells results in increased Mcl-1 levels (left), whereas pretreatment with 250 or 500 nmol/L flavopiridol (data from 500 nmol/L treatment are shown) abrogates 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced potentiation of Mcl-1. A and B, gel lanes were loaded based on equivalent amounts of protein (50 µg), and levels of polypeptides of interest were assessed by SDS-PAGE and immunoblotting. Representative of four separate experiments.

 
In addition to having the ability to attenuate levels of Mcl-1 in cancer cells cultured under ambient growth conditions, flavopiridol can also abrogate induced increases of Mcl-1. For example, the increases in levels of Mcl-1 induced by 12-O-tetradecanoylphorbol-13-acetate stimulation of A549 cells (Fig. 4B, left) were blocked by pretreatment of cells with flavopiridol (Fig. 4B, right). These data indicate that flavopiridol is capable of attenuating Mcl-1 levels not only under basal growth conditions but also under conditions of cellular stress.

Although cellular level of c-Myc and cyclin A also declined in some lines (e.g., Fig. 3), these decreases were variable and less dramatic. Levels of cyclin D1 and Bcl-xL also declined in response to flavopiridol treatment, but these decreases were not observed until 24 to 48 hours after the initiation of flavopiridol treatment (data not shown). Hence, we confirmed that the levels of five of seven polypeptides known to be transcriptionally regulated by STAT3 were attenuated in response to flavopiridol exposure.

Because flavopiridol is known under some circumstances to inhibit the activating phosphorylation of RNA polymerase II via inhibition of P-TEFb, potentially globally down-regulating transcription (33, 34), we compared the effects of flavopiridol with those produced by the classic P-TEFb inhibitor DRB in A549 cells to determine whether the effects of the two agents might be similar or, instead, distinct. Whereas flavopiridol treatment attenuated levels of c-Myc and increased levels of phospho-CREB, DRB did not (Fig. 3, asterisks). Hence, flavopiridol and the P-TEFb inhibitor DRB vary not only with respect to their effects on message levels but also with respect to their effects on various polypeptides. Collectively, these data suggest that the two agents may differ with regard to the mechanisms by which they exert their cellular effects, supporting results shown in Fig. 1, suggesting that flavopiridol may at least in some circumstances affect transcription with some selectivity.

Flavopiridol-Induced Down-Regulation of Mcl-1 Does Not Result from Effects on Phospho-STAT3, Phospho-CREB, Phospho-ELK-1, Phospho-ERK1/2, Phospho-AKT, pRb, or E2F-1
Previous studies have implicated three transcription factors in the regulation of Mcl-1 levels: phospho-STAT3 (48), phospho-CREB (49), and phospho-ELK-1 (47). To determine whether flavopiridol-induced Mcl-1 down-regulation might be attributable to alterations in one of these activated transcription factors rather than disruption of STAT3/DNA interactions, we examined the effects of flavopiridol on the cellular levels of phospho-STAT3(Tyr705), phospho-STAT3(Ser727), phospho-CREB(Ser133), and phospho-ELK-1(Ser383) (Figs. 3 and 5A ). Flavopiridol caused a (dose-dependent) increase, rather than a decrease, in phosphorylation of STAT3 on Tyr705 and Ser727 under the same conditions that down-regulated Mcl-1 (Figs. 3 and 5A). Although A549 cells had no detectable levels of phospho-ELK-1 either before or after treatment with flavopiridol, phospho-CREB was also up-regulated in response to flavopiridol treatment (Figs. 3 and 5A). Although the increased phosphorylation of STAT3 and CREB is unexplained at present (but might reflect feedback up-regulation as a consequence of altered downstream mRNA or polypeptide levels), these results clearly rule out decreased levels of the three known transcriptional regulators of Mcl-1 as a cause for flavopiridol-induced Mcl-1 down-regulation.


Figure 5
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  		Figure 5. Effects of flavopiridol on levels of cellular polypeptides were similar to those of actinomycin D but distinct from those of other DNA-interacting agents. A, both flavopiridol and actinomycin D down-regulated Mcl-1 and up-regulated phospho-STAT3 and p53 levels, whereas other DNA-interacting agents did not. B, 250 nmol/L flavopiridol treatment of A549 cells for 6 h did not up-regulate levels of nuclear E2F-1. C, in at least some cancer cell lines (OV17R shown here), flavopiridol-induced attenuation of Mcl-1 occurs in the absence of effects on polysomal RNA polymerase II levels. Concentrations of DNA-interacting agents and flavopiridol in (A, left, and C) were all 500 nmol/L. Poly(ADP-ribose) polymerase and actin are shown as loading controls, and lanes were loaded by cellular protein level (50 µg). Polypeptide levels were assessed using SDS-PAGE and immunoblotting. Nuclei for B were isolated as described in the text.

 
To determine whether flavopiridol-induced STAT3 phosphorylation occurs in other cell lines, we examined phospho-STAT3 levels in ML-1, K562, Jurkat, My-5, OV17R, PC-3, HCT116, and MDA-MB-468 cells treated with 250 nmol/L flavopiridol for 6 hours. Phospho-STAT3 levels were increased in five of nine assayed lines (A549, OV17R, PC-3, HCT116, and MDA-468; data not shown) and unaffected in the remainder, confirming that decreased levels of phosphorylated (active) STAT3 are not responsible for flavopiridol-induced Mcl-1 down-regulation in any of the cell lines. Similar experiments also failed to implicate diminished phosphorylation of CREB or ELK-1 in flavopiridol-induced Mcl-1 down-regulation in any of the cell lines (data not shown).

Several reports have also implicated signaling through the ERK and AKT pathways in the regulation of Mcl-1 (50–54). Immunoblotting with reagents that recognize phospho-ERK1/2(Thr202/Tyr204) and phospho-AKT(Ser473) suggested that flavopiridol had no effect on activation phosphorylation of AKT and that at higher concentrations flavopiridol actually increases levels of ERK1/2 phosphorylation (Figs. 3 and 5A). Hence, decreased signaling through AKT and/or ERK1/2 does not seem to be responsible for the observed flavopiridol-induced down-regulation of Mcl-1.

Finally, based on a recent report that the transcription factor E2F-1 directly represses the Mcl-1 promoter (55), we examined the possibility that flavopiridol might alter levels of hyperphosphorylated Rb (Figs. 3 and 5A) and/or nuclear levels of E2F-1 (Fig. 5B). Whereas Rb was unaffected, nuclear E2F-1 levels declined slightly (rather than increasing), hence also eliminating this pathway as a means of flavopiridol-induced down-regulation of Mcl-1. Furthermore, because Rb phosphorylation is regulated by CDK4 and CDK6, there is no indication that the ability of flavopiridol to inhibit these CDKs is playing a role in Mcl-1 down-regulation through the Rb/E2F-1 pathway.

In summary, extensive evaluation failed to provide evidence that flavopiridol altered known transcriptional regulators of Mcl-1 in a manner that could account for flavopiridol-induced down-regulation of Mcl-1 message and protein. Available evidence, therefore, suggests that flavopiridol-induced down-regulation of Mcl-1 occurs at, or downstream of, the level of phospho-STAT3-induced transcription.

Cellular Effects of Flavopiridol Are Distinct from Other DNA-Interacting Antineoplastic Agents
To determine whether other DNA-interacting agents might similarly down-regulate Mcl-1, we first compared the effects of flavopiridol, ethidium bromide, Hoechst 33258, pyrazoloacridine, topotecan, actinomycin D, and doxorubicin on Mcl-1 and phospho-STAT3 levels in A549 cells (Fig. 5A). Of all tested DNA-interacting agents, only flavopiridol and actinomycin D (indicated with asterisk) attenuated Mcl-1 levels (Fig. 5A).

Flavopiridol-Induced Mcl-1 Down-Regulation Is Independent of Altered RNA Polymerase II Phosphorylation in Some Cancer Cell Lines
Because flavopiridol has been reported previously to diminish the activating phosphorylation of RNA polymerase II by inhibiting P-TEFb (33, 34), we also examined the effects of flavopiridol on phospho–RNA polymerase II levels in a variety of cancer cell lines. In some cell lines (e.g., A549 cells), we observed that flavopiridol down-regulated RNA polymerase II phosphorylation in parallel with Mcl-1 down-regulation (Figs. 3 and 5A). In other cell lines (e.g., OV17R), however, we found that flavopiridol-induced Mcl-1 down-regulation occurred under conditions that did not produce RNA polymerase II dephosphorylation (Fig. 5C). Therefore, in at least in some cell lines, Mcl-1 down-regulation does not seem to be attributable to flavopiridol-induced decreases in RNA polymerase II phosphorylation, again consistent with the hypothesis that under at least some circumstances flavopiridol seems to affect transcription focally rather than globally.

Flavopiridol Can Be Combined with the Jak Inhibitor AG490 to Achieve Cytotoxic Synergy in A549 Cells
Having assembled much data in support of the hypothesis that flavopiridol binds to DNA (35), disrupts STAT3/DNA binding (Fig. 1A and B), attenuates STAT3-directed transcription (Fig. 1C), and reduces levels of downstream survival-critical polypeptides, including Mcl-1 (Figs. 2–5), and further that this phenomenon might under some circumstances be responsible for flavopiridol-induced cell death, we hypothesized that flavopiridol might be combined with other inhibitors of the Jak/STAT3 signaling cascade to achieve cytotoxic synergy. As depicted in Fig. 6 , we reasoned that inhibition of a single survival-critical signaling pathway at two distinct points might yield synergistic, rather than just additive, effects. To test this hypothesis, we examined cytotoxic synergy resulting between flavopiridol and the Jak inhibitor AG490 as well as between paclitaxel and AG490 (as a "negative" control, given that paclitaxel does not effect the Jak/STAT3 signaling pathway) using median effect analyses (Fig. 7 ). In these analyses, combination indices < 1 are indicative of synergy. Whereas the combination of flavopiridol and AG490 produced cytotoxic synergy, consistently yielding combination indices in the 0.2 to 0.5 range (Fig. 7A), the combination of paclitaxel and AG490 did not produce similar evidence of synergy, consistently yielding combination indices clustered around 1, indicating additivity (Fig. 7B). Furthermore, immunoblotting experiments indicated that the attenuation of phospho-STAT3 levels by AG490 closely paralleled its cytotoxicity and was achieved in the AG490 concentration ranges used in these experiments (data not shown). These data are not only consistent with the possibility that AG490 and flavopiridol may synergistically affect the same survival-critical signaling pathway (putatively Jak/STAT3) but may additionally have further implications for the future development of clinical trials combining flavopiridol with other inhibitors of the Jak/STAT3 signaling pathway.


Figure 6
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  		Figure 6. A model depicting the putative effects of flavopiridol and AG490 on STAT3-mediated transcription.

 

Figure 7
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  		Figure 7. Flavopiridol and the Jak inhibitor AG490 can be combined to produce cytotoxic synergy, whereas the microtubule-interacting agent paclitaxel (Taxol) and AG490 combine to yield only additive effects. Representative median effect, dose effect, and combination indices plots for flavopiridol combined with AG490 (A) and paclitaxel combined with AG490 (B). Colony-forming assays using A549 cells supplemented using median effect analyses served as the basis for the displayed analyses. In combination index plots, combination indices <1 indicate synergy.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results presented above support the following model of flavopiridol action (Fig. 6): (a) flavopiridol enters cancer cells and binds to dsDNA (35); (b) flavopiridol binding to DNA disrupts interactions of STAT3 with its consensus DNA-binding sequence (Fig. 1A and B); (c) consequent disruption of STAT3/DNA binding attenuates transcription of STAT3-regulated messages (Fig. 1C), including Mcl-1; and (d) resulting decreases in mRNA levels (Fig. 2) lead to down-regulation of several downstream polypeptides, including Mcl-1 (Figs. 3–5). This model can potentially explain a variety of previously seemingly disparate observations related to the action flavopiridol, including the new observation that flavopiridol can be combined with the Jak inhibitor AG490 to produce cytotoxic synergy (Fig. 7).

Previous studies have shown that flavopiridol can down-regulate cyclin D1 (36), Bcl-2 (20), and Mcl-1 (37) but leave levels of many other cellular polypeptides largely unaffected. Others have proposed that these alterations reflect the consequences of global attenuation of transcription as a consequence of flavopiridol-induced inhibition of P-TEFb, the activating kinase of RNA polymerase II (33, 34). We were intrigued, however, by the fact that cyclin D1, Bcl-2, and Mcl-1 are transcriptionally regulated by STAT3 (3, 12), leading to the present studies. In view of results presented in Figs. 1 to 5, the down-regulation of these polypeptides may reflect, at least in part, impaired STAT3-regulated transcription, providing an alternative potential mechanistic explanation for the observed transcription effects of flavopiridol. As indicated in Figs. 2 to 5, however, down-regulation of Mcl-1 message and protein levels seems to be earlier and more dramatic than down-regulation of cyclin D1. This might reflect both a shorter half-life of Mcl-1 message (47, 56) and possibly a greater reliance of Mcl-1 transcription on constitutive STAT3 activation rather than other pathways in the cell lines examined. In support of this latter possibility, we observed that constitutive ERK phosphorylation was much lower in the A549 cell line than in several other cell lines.3 Constitutive phosphorylation of the ERK substrate ELK-1 was also undetectable in these cells (Figs. 3 and 5A).

In the analysis of presented experimental results, it should be noted that the flavopiridol concentration required to disrupt STAT3/DNA binding in the DNA cellulose affinity binding assay and the nuclear extraction assay shown in Fig. 1A and B were somewhat higher (500 nmol/L) than those required to produced effects in intact cells (125–250 nmol/L). Given that the assays presented in Fig. 1A and B did not use intact cells, however, these experiments make the assumption that intracellular, and further intranuclear, flavopiridol concentrations may be roughly equivalent to applied extracellular flavopiridol concentrations, and this may or may not be the case in a particular model system. Prior analyses quantitating intracellular flavopiridol concentrations suggest that flavopiridol is not concentrated in cells (42). Because flavopiridol levels have apparently not yet been quantitated in cell nuclei, it is presently impossible to know what intranuclear flavopiridol levels might be attained in response to applied extracellular flavopiridol concentrations. It is nonetheless generally encouraging, however, that the effects seen in Fig. 1A and B were observed in the same general concentration ranges that produce STAT3 and Mcl-1 effects as well as cytotoxicity in intact cells.

To our knowledge, this is the first report of a small molecule that can attenuate STAT3-mediated transcription by disrupting the binding of STAT3 to its consensus DNA-binding sequence. STAT3 is a downstream mediator of a variety of prosurvival cytokine signaling pathways (48, 50), and up-regulation of STAT3 signaling either by constitutive activation or by up-regulated cytokine signaling has been shown in a wide variety of cancers (3–7, 12, 57, 58). Additionally, up-regulated STAT3 signaling is believed to contribute to the neoplastic phenotype of many cancers by stimulating proliferation and attenuating apoptosis via its effects on downstream polypeptides, including Mcl-1, c-Myc, cyclins, and Bcl-xL (3–7, 12, 57, 58). Previous studies have shown that inhibition of constitutively activated STAT3 signaling using antisense oligonucleotides or the Jak inhibitor AG490 can inhibit tumor growth both in vitro and in vivo, providing direct support for the contention that inhibition of STAT3-mediated signaling represents a potentially important approach for antineoplastic drug development (3–7, 12, 57, 58). The ability of a small molecule to prevent STAT3 binding to its consensus sequence provides a new means for examining the importance of this pathway in the clinical setting. This is illustrated by the observation that flavopiridol can be combined with an inhibitor of STAT3 signaling at another point in the pathway (AG490) to achieve cytotoxic synergy.

As is the case with STAT3 (see Introduction), mounting evidence indicates that Mcl-1 has an important role in cancer (59). First, Mcl-1 is often up-regulated in cancer cells (59). Second, Mcl-1 overexpression results in a high incidence of murine tumors (39). Third, down-regulation of Mcl-1 can trigger apoptosis in the absence of other proapoptotic stimuli (40). These observations suggest that the flavopiridol-induced cytotoxicity documented in previous studies (16–25) may be attributable, at least in part, to the down-regulation of antiapoptotic polypeptides, such as Mcl-1. Consistent with this possibility, we observed marked inhibition of Mcl-1 expression in a variety of flavopiridol-sensitive cancer cell lines in vitro (Fig. 4A). These results do not, of course, rule out the possibility that inhibition of STAT3/DNA binding (Fig. 1) might result in down-regulation of additional survival-critical antiapoptotic polypeptides as well.

Two reports implicate P-TEFb as a potentially important target of flavopiridol (33, 34) and further suggest that this mechanism may account for the transcriptional effects of the drug. The present studies confirm that phosphorylation of RNA polymerase II is decreased in response to flavopiridol treatment (Figs. 3 and 5A). Addition experiments, however, suggest that inhibition of P-TEFb seems not to be the sole mechanism by which flavopiridol down-regulates target proteins. First, the effects of flavopiridol and the classic P-TEFb inhibitor DRB are distinct, with flavopiridol having much more limited effects at the mRNA and protein levels (Figs. 2 and 3). Second, flavopiridol down-regulates Mcl-1 under conditions that do not affect RNA polymerase II phosphorylation in some cancer cell lines (Fig. 5C). Third, flavopiridol was found to attenuate STAT3-directed transcription in a luciferase reporter assay relative to a control reporter plasmid (Fig. 1C), indicating at least some degree of selectivity of transcriptional effects of the agent. Thus, at least a portion of the effects of flavopiridol are apparently independent of P-TEFb inhibition. Nonetheless, the relative contributions of P-TEFb inhibition and disruption of transcription factor/DNA binding to flavopiridol-induced down-regulation of transcription in various circumstances remain to be more fully elucidated. Additionally, the relevance of the ability of flavopiridol to disrupt STAT3-directed transcription in vitro to observed effects in human clinical trials remains to be clarified through targeted translational studies.

In summary, the present studies indicate that flavopiridol can disrupt STAT3/DNA interactions, attenuate STAT3-directed transcription, and down-regulate levels of the STAT3 transcriptional target Mcl-1 at the transcriptional level. These observations provide a potential explanation for at least some of the transcriptional effects of flavopiridol independent of its effects on P-TEFb. Additionally, the observation that flavopiridol can be combined with another STAT3 pathway inhibitor (AG490) to achieve cytotoxic synergy has potential implication for future combination clinical trials involving flavopiridol. Perhaps most importantly, these collective observations not only provide a potential explanation for many of the previously unexplained effects of flavopiridol but also raise the possibility that flavopiridol might represent the first of a new class of target-directed small-molecule antineoplastic agents capable of disrupting survival-critical transcription factor/DNA interactions with some degree of selectivity.


   Acknowledgments
 
We thank Dr. Jacqueline Bromberg for kindly providing the used STAT3 luciferase reporter plasmid, John Reed for kindly providing anti-Mcl-1, Peter Mesner, Chris Hallgren, April Blajeski, Kriste Lewis, and Xue Wei Meng for helpful suggestions, and Deb Strauss for editorial assistance.


   Footnotes
 
Grant support: American Cancer Society grant RPG CCE-98842, NIH grant CA097129, and Jack Taylor Family Foundation.

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.

Note: Y.K. Lee and C.R. Isham contributed equally to this article.

3 T.J. Kottke and S.H. Kaufmann, unpublished observations. Back

Received 7/ 7/05; revised 9/12/05; accepted 10/25/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kisseleva T, Bhattacharya S, Braunstein J, Schindler CW. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene 2002;285:1–24.[CrossRef][Medline]
  2. Aaronson DS, Horvath CM. A road map for those who don't know JAK-STAT. Science 2002;296:1653–5.[Abstract/Free Full Text]
  3. Bromberg J, Darnell JR. The role of STATs in transcriptional control and their impact on cellular function. Oncogene 2000;96:2468–73.
  4. Bowman T, Garcia R, Turkson J, Jove R. STATs in oncogenesis. Oncogene 2000;19:2474–88.[CrossRef][Medline]
  5. Lin TS, Mahajan S, Frank DA. STAT signaling in the pathogenesis and treatment of leukemias. Oncogene 2000;19:2496–504.[CrossRef][Medline]
  6. Garcia R, Bowman T, Niu G, et al. Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells. Oncogene 2001;20:2499–513.[CrossRef][Medline]
  7. Song JI, Grandis JR. STAT signaling in head and neck cancer. Oncogene 2000;19:2489–95.[CrossRef][Medline]
  8. Nielsen M, Kaestel CG, Ericksen KW, et al. Inhibition of constitutively activated Stat3 correlates with altered Bcl-2/Bax expression and induction of apoptosis in mycosis fungoides tumor cells. Leukemia 1999;13:735–8.[CrossRef][Medline]
  9. Epling-Burnette PK, Liu JH, Catlett-Falcone R, et al. Inhibition of STAT3 signaling leads to apoptosis of leukemic large granular lymphocytes and decreased Mcl-1 expression. J Clin Invest 2001;107:351–62.[Medline]
  10. O'Shea JJ, Visconti R, Cheng TP, Gadina M. Jaks and Stats as therapeutic targets. Ann Rheum Dis 2000;59:i115–8.[Abstract/Free Full Text]
  11. Seidel HM, Lamb P, Rosen J. Pharmaceutical intervention in the JAK/STAT signaling pathway. Oncogene 2000;19:2645–56.[CrossRef][Medline]
  12. Buettner R, Mora LB, Jove R. Activated STAT signaling in human tumors provides novel molecular targets for therapeutic intervention. Clin Cancer Res 2002;8:945–54.[Abstract/Free Full Text]
  13. Shah MA, Kortmansky J, Motwani M, et al. A phase I clinical trial of the sequential combination of irinotecan followed by flavopiridol. Clin Cancer Res 2005;11:3836–45.[Abstract/Free Full Text]
  14. Johnson AJ, Lucas DM, Smith LL, et al. Clinical rebirth of flavopiridol in chronic lymphocytic leukemia (CLL): utilizing in vitro and in vivo pharmacodynamic measurements to improve efficacy and interrogate relevant mechanism(s) of action [abstract 1672]. Proc Am Assoc Cancer Res 2005;46:374.
  15. Van Veldhuizen PJ, Faulkner JR, Lara PN, Jr., et al. A phase II study of flavopiridol in patients with advanced renal cell carcinoma: results of Southwest Oncology Group Trial 0109. Cancer Chemother Pharmacol 2005;56:39–45.[Medline]
  16. Bible KC, Kaufmann SH. Flavopiridol (NSC 649890, L86-8275): a cytotoxic flavone that induces cell death in human lung carcinoma cells. Cancer Res 1996;56:4856–61.[Abstract/Free Full Text]
  17. Arguello F, Alexander M, Sterry JA, et al. Flavopiridol induces apoptosis of normal lymphoid cells, causes immunosuppression, and has potent antitumor activity in vivo against human leukemia and lymphoma xenografts. Blood 1998;91:2482–90.[Abstract/Free Full Text]
  18. Brüsselbach S, Nettelbeck DM, Sedlacek H-H, Müller R. Cell cycle-independent induction of apoptosis by the anti-tumor drug flavopiridol in endothelial cells. Int J Cancer 1998;77:146–52.[CrossRef][Medline]
  19. Parker BW, Senderowicz AM, Nieves-Neira W, Pommier Y, Sausville EA. DNA fragmentation and apoptosis of lymphoma and prostate cancer cell lines after flavopiridol treatment. Proc Am Assoc Cancer Res 1996;37:398.
  20. König A, Schwartz GK, Mohammad RM, Al-Katib A, Gabrilove JL. The novel cyclin-dependent kinase inhibitor flavopiridol downregulates Bcl-2 and induces growth arrest and apoptosis in chronic B-cell leukemia lines. Blood 1997;90:4307–12.[Abstract/Free Full Text]
  21. Schwartz GK, Farsi K, Maslak P, Kelsen DP, Spriggs D. Potentiation of apoptosis by flavopiridol in mitomycin-C-treated gastric and breast cancer cells. Clin Cancer Res 1997;3:1467–72.[Abstract]
  22. Parker BW, Kaur G, Nieves-Neira W, et al. Early induction of apoptosis in hematopoietic cell lines after exposure to flavopiridol. Blood 1998;91:458–65.[Abstract/Free Full Text]
  23. Byrd JC, Shinn C, Waselenko JK, et al. Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence upon functional p53. Blood 1998;92:3804–16.[Abstract/Free Full Text]
  24. Patel V, Senderowicz AM, Pinto D, Jr., et al. Flavopiridol, a novel cyclin dependent kinase inhibitor, suppresses the growth of squamous cell carcinomas of the head and neck by inducing apoptosis. J Clin Invest 1998;102:1674–81.[Medline]
  25. Schrump DS, Matthews W, Pinto D, Jr. Flavopiridol mediates cell cycle arrest and apoptosis in esophageal cancer cells. Clin Cancer Res 1998;4:2885–90.[Abstract]
  26. Drees M, Dengler WA, Roth T, et al. Flavopiridol (L86-8275): selective antitumor in vitro and activity in vivo for prostate carcinoma cells. Clin Cancer Res 1997;3:273–9.[Abstract]
  27. Sedlacek HH, Czech J, Naik R, et al. Flavopiridol (L86 8275; NSC 649890), a new kinase inhibitor for tumor therapy. Int J Oncol 1996;9:1143–68.
  28. Worland PJ, Kaur G, Stetler-Stevenson M, Sebers S, Sartor O, Sausville EA. Alteration of the phosphorylation state of p34cdc2 kinase by the flavone L86-8275 in breast carcinoma cells. Correlation with decreased H1 kinase activity. Biochem Pharmacol 1993;46:1831–40.[CrossRef][Medline]
  29. Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ. Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells. Cancer Res 1996;56:2973–8.[Abstract/Free Full Text]
  30. Kaur G, Stetler-Stevenson M, Sebers S, et al. Growth inhibition with reversible cell cycle arrest of carcinoma cells by flavone L86-8275. J Natl Cancer Inst 1992;84:1736–40.[Abstract/Free Full Text]
  31. Kaiser A, Nishi K, Gorin FA, Walsh DA, Bradbury EM, Schnier JB. The cyclin-dependent kinase (CDK) inhibitor flavopiridol inhibits glycogen phosphorylase. Arch Biochem Biophys 2001;386:179–87.[CrossRef][Medline]
  32. Oikonomakos NG, Schnier JB, Zographos SE, Skamnaki VT, Tsitsanou KE, Johnson LN. Flavopiridol inhibits glycogen phosphorylase by binding at the inhibitor site. J Biol Chem 2000;275:34566–73.[Abstract/Free Full Text]
  33. Chao S-H, Price DH. Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo. J Biol Chem 2001;276:31793–9.[Abstract/Free Full Text]
  34. Chao S-H, Fujinaga K, Marion JE, et al. Flavopiridol inhibits P-TEFb and blocks HIV-1 replication. J Biol Chem 2000;275:28345–8.[Abstract/Free Full Text]
  35. Bible KC, Bible RH, Jr., Kottke TJ, et al. Flavopiridol binds to duplex DNA. Cancer Res 2000;60:2419–28.[Abstract/Free Full Text]
  36. Carlson B, Lahusen T, Singh S, et al. Down-regulation of cyclin D1 by transcriptional repression in MCF-7 human breast carcinoma cells induced by flavopiridol. Cancer Res 1999;59:4634–42.[Abstract/Free Full Text]
  37. Kitada S, Andreeff M, Reed JC. Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia. Blood 2000;96:393–7.[Abstract/Free Full Text]
  38. Gray NS, Wodicka L, Thunnissen A-MWH, et al. Exploiting chemical libraries, structures, and genomics in the search for kinase inhibitors. Science 1998;281:533–8.[Abstract/Free Full Text]
  39. Zhou P, Levy NB, Xie H, et al. MCL1 transgenic mice exhibits a high incidence of B-cell lymphoma manifested as a spectrum of histologic subtypes. Blood 2001;97:3902–9.[Abstract/Free Full Text]
  40. Moulding DA, Giles RV, Spiller DG, White MR, Tidd DM, Edwards SW. Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells. Blood 2000;96:1756–63.[Abstract/Free Full Text]
  41. Kaufmann SH, Karp JE, Svingen PA, Krajewski S, Burke PJ, Gore SD. Elevated Expression of the apoptotic regulator Mcl-1 at the time of leukemic relapse. Blood 1998;91:991–1000.[Abstract/Free Full Text]
  42. Bible KC, Boerner SA, Kirkland K, et al. Characterization of an ovarian carcinoma cell line resistant to cisplatin and flavopiridol. Clin Cancer Res 2000;6:661–70.[Abstract/Free Full Text]
  43. Badley JE, Bishop GA, St. John T, Frelinger JA. A simple, rapid method for the purification of poly A+ RNA. Biotechniques 1988;6:114–5.[Medline]
  44. Alberts B, Herrick G. DNA-cellulose chromatography. Methods Enzymol 1971;21:198–217.
  45. Senderowicz AM, Messmann R, Arbuck S. A phase I trial of 1 hour infusion of flavopiridol (FLA), a novel cyclin-dependent kinase inhibitor, in patients with advanced neoplasms. Proc Am Soc Clin Oncol 2000;Abstract 796.
  46. Caruccio L, Banerjee R. An efficient method for simultaneous isolation of biologically active transcription factors and DNA. J Immunol Methods 1999;230:1–10.[CrossRef][Medline]
  47. Townsend KJ, Zhou P, Qian L, et al. Regulation of MCL1 through a serum response factor/Elk-1-mediated mechanism links expression of a viability-promoting member of the BCL2 family to the induction of hematopoietic cell differentiation. J Biol Chem 1999;274:1801–13.[Abstract/Free Full Text]
  48. Puthier D, Bataille R, Amiot M. IL-6 up-regulates Mcl-1 in human myeloma cells through JAK/STAT rather than Ras/MAP kinase pathway. Eur J Immunol 1999;29:3945–50.[CrossRef][Medline]
  49. Wang JM, Chao JR, Chen W, Kuo ML, Yen JJ, Yang-Yen HF. The antiapoptotic gene Mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway through a transcription factor complex containing CREB. Mol Cell Biol 1999;19:6195–206.[Abstract/Free Full Text]
  50. Schubert KM, Duronio V. Distinct roles for extracellular-signal-regulated protein kinase (ERK) mitogen-activated protein kinases and phosphatidylinositol 3-kinase in the regulation of Mcl-1 synthesis. Biochem J 2001;35:473–80.
  51. Huang HM, Huang CJ, Yen JJ. Mcl-1 is a common target of stem cell factor and interleukin-5 for apoptosis prevention activity via MEK/MAPK and PI-3K/Akt pathways. Blood 2000;96:1764–71.[Abstract/Free Full Text]
  52. Wei LH, Kuo ML, Chen CA, et al. The anti-apoptotic role of interleukin-6 in human cervical cancer is mediated by up-regulation of Mcl-1 through a PI 3-K/Akt pathway. Oncogene 2001;20:5799–809.[CrossRef][Medline]
  53. Kuo ML, Chuang SE, Lin MT, Yang SY. The involvement of PI 3-K/Akt-dependent up-regulation of Mcl-1 in the prevention of apoptosis of Hep3B cells by interleukin-6. Oncogene 2001;20:677–85.[CrossRef][Medline]
  54. Boucher MJ, Morisset J, Vachon PH, Reed JC, Laine J, Rivard N. MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-X(L), and Mcl-1 and promotes survival of human pancreatic cancer cells. J Cell Biochem 2000;79:355–69.[CrossRef][Medline]
  55. Croxton R, Ma Y, Song L, Haura EB, Cress WD. Direct repression of the Mcl-1 promoter by E2F1. Oncogene 2002;21:1359–69.[CrossRef][Medline]
  56. Akgul C, Moulding DA, White MR, Edwards SW. In vivo localization and stability of human Mcl-1 using green fluorescent protein (GFP) fusion proteins. FEBS Lett 2000;478:72–6.[CrossRef][Medline]
  57. Coffer PJ, Koenderman L, de Groot RP. The role of STATs in myeloid differentiation and leukemia. Oncogene 2000;19:2511–22.[CrossRef][Medline]
  58. Catlett-Falcone R, Landowski TH, Oshiro MM, et al. Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells. Immunity 1999;10:105–15.[CrossRef][Medline]
  59. Craig RW. MCL1 provides a window on the role of the BCL2 family in cell proliferation, differentiation and tumorigenesis. Leukemia 2002;16:444–54.[CrossRef][Medline]




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