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• W2081567150 abstract "Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor in clinical trials as a cancer therapy that has been recently shown to block human immunodeficiency virus Tat transactivation and viral replication through inhibition of positive transcription elongation factor b (P-TEFb). Flavopiridol is the most potent P-TEFb inhibitor reported and the first Cdk inhibitor that is not competitive with ATP. We examined the ability of flavopiridol to inhibit P-TEFb (Cdk9/cyclin T1) phosphorylation of both RNA polymerase II and the large subunit of the 5, 6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and found that the IC50determined was directly related to the concentration of the enzyme. We concluded that the flavonoid associates with P-TEFb with 1:1 stoichiometry even at concentrations of enzyme in the low nanomolar range. These results indicate that the apparent lack of competition with ATP could be caused by a very tight binding of the drug. We developed a novel immobilized P-TEFb assay and demonstrated that the drug remains bound for minutes even in the presence of high salt. Flavopiridol remained bound in the presence of a 1000-fold excess of the commonly used inhibitor DRB, suggesting that the immobilized P-TEFb could be used in a simple screening assay that would allow the discovery or characterization of compounds with binding properties similar to flavopiridol. Finally, we compared the ability of flavopiridol and DRB to inhibit transcription in vivo using nuclear run-on assays and concluded that P-TEFb is required for transcription of most RNA polymerase II molecules in vivo. Flavopiridol (L86-8275, HMR1275) is a cyclin-dependent kinase (Cdk) inhibitor in clinical trials as a cancer therapy that has been recently shown to block human immunodeficiency virus Tat transactivation and viral replication through inhibition of positive transcription elongation factor b (P-TEFb). Flavopiridol is the most potent P-TEFb inhibitor reported and the first Cdk inhibitor that is not competitive with ATP. We examined the ability of flavopiridol to inhibit P-TEFb (Cdk9/cyclin T1) phosphorylation of both RNA polymerase II and the large subunit of the 5, 6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and found that the IC50determined was directly related to the concentration of the enzyme. We concluded that the flavonoid associates with P-TEFb with 1:1 stoichiometry even at concentrations of enzyme in the low nanomolar range. These results indicate that the apparent lack of competition with ATP could be caused by a very tight binding of the drug. We developed a novel immobilized P-TEFb assay and demonstrated that the drug remains bound for minutes even in the presence of high salt. Flavopiridol remained bound in the presence of a 1000-fold excess of the commonly used inhibitor DRB, suggesting that the immobilized P-TEFb could be used in a simple screening assay that would allow the discovery or characterization of compounds with binding properties similar to flavopiridol. Finally, we compared the ability of flavopiridol and DRB to inhibit transcription in vivo using nuclear run-on assays and concluded that P-TEFb is required for transcription of most RNA polymerase II molecules in vivo. cyclin-dependent kinase positive transcription elongation factor b dichloro-1-β-d-ribofuranosylbenzimidazole DRB sensitivity-inducing factor human immunodeficiency virus SDS-polyacrylamide gel electrophoresis Flavopiridol is a potential anti-cancer therapeutic agent currently being tested in phase I and II clinical trials (1Senderowicz A.M. Sausville E.A. J. Natl. Cancer Inst. 2000; 92: 376-387Crossref PubMed Scopus (432) Google Scholar, 2Kelland L.R. Expert Opin. Investig. Drugs. 2000; 9: 2903-2911Crossref PubMed Scopus (126) Google Scholar). Previous evidence indicates that flavopiridol most effectively inhibits Cdk1,1 Cdk2, and Cdk4 withK is between 40 and 70 nm (1Senderowicz A.M. Sausville E.A. J. Natl. Cancer Inst. 2000; 92: 376-387Crossref PubMed Scopus (432) Google Scholar, 3Losiewicz M.D. Carlson B.A. Kaur G. Sausville E.A. Worland P.J. Biochem. Biophys. Res. Commun. 1994; 201: 589-595Crossref PubMed Scopus (337) Google Scholar, 4Carlson B.A. Dubay M.M. Sausville E.A. Brizuela L. Worland P.J. Cancer Res. 1996; 56: 2973-2978PubMed Google Scholar). The crystal structure of Cdk2-flavopiridol complexes reveals that the drug docks in the ATP-binding site, consistent with the fact that inhibition of Cdk2 is competitive with ATP (5De A.W.J. Mueller-Dieckmann H.J. Schulze-Gahmen U. Worland P.J. Sausville E. Kim S.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2735-2740Crossref PubMed Scopus (505) Google Scholar). Treatment with flavopiridol resulted in blocking cell cycle progression, promoting differentiation, and inducing apoptosis in various types of cancerous cells (1Senderowicz A.M. Sausville E.A. J. Natl. Cancer Inst. 2000; 92: 376-387Crossref PubMed Scopus (432) Google Scholar). In addition, an effect of flavopiridol on transcription of yeast and mammalian genes has been reported (6Gray N.S. Wodicka L. Thunnissen A.M. Norman T.C. Kwon S. Espinoza F.H. Morgan D.O. Barnes G. LeClerc S. Meijer L. Kim S.H. Lockhart D.J. Schultz P.G. Science. 1998; 281: 533-538Crossref PubMed Scopus (837) Google Scholar, 7Carlson B. Lahusen T. Singh S. Loaiza-Perez A. Worland P.J. Pestell R. Albanese C. Sausville E.A. Senderowicz A.M. Cancer Res. 1999; 59: 4634-4641PubMed Google Scholar). Based on these observations, we examined the effects of the inhibitor on transcription by RNA polymerase II using several in vitro assays. Our previous data demonstrates that flavopiridol specifically blocked the elongation phase of transcription through the inhibition of P-TEFb and was the most potent P-TEFb inhibitor with a K i of 3 nm (8Chao S.H. Fujinaga K. Marion J.E. Taube R. Sausville E.A. Senderowicz A.M. Peterlin B.M. Price D.H. J. Biol. Chem. 2000; 275: 28345-28348Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). Unlike its effects on the other Cdks, flavopiridol inhibition of P-TEFb was not competitive with ATP (8Chao S.H. Fujinaga K. Marion J.E. Taube R. Sausville E.A. Senderowicz A.M. Peterlin B.M. Price D.H. J. Biol. Chem. 2000; 275: 28345-28348Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). P-TEFb, comprised of Cdk9 and a cyclin subunit derived from one of three different genes, is required in the elongation control of RNA polymerase II (9Zhu Y. Pe'ery T. Peng J. Ramanathan Y. Marshall N. Marshall T. Amendt B. Mathews M.B. Price D.H. Genes Dev. 1997; 11: 2622-2632Crossref PubMed Scopus (605) Google Scholar, 10Wei P. Garber M.E. Fang S.M. Fischer W.H. Jones K.A. Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar, 11Gold M.O. Yang X. Herrmann C.H. Rice A.P. J. Virol. 1998; 72: 4448-4453Crossref PubMed Google Scholar, 12Peng J. Zhu Y. Milton J.T. Price D.H. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (441) Google Scholar, 13Fu T.J. Peng J. Lee G. Price D.H. Flores O. J. Biol. Chem. 1999; 274: 34527-34530Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 14Price D.H. Mol. Cell. Biol. 2000; 20: 2629-2634Crossref PubMed Scopus (562) Google Scholar). P-TEFb is a required cellular cofactor for activation of transcription of the HIV-1 genome by the viral transactivator Tat (9Zhu Y. Pe'ery T. Peng J. Ramanathan Y. Marshall N. Marshall T. Amendt B. Mathews M.B. Price D.H. Genes Dev. 1997; 11: 2622-2632Crossref PubMed Scopus (605) Google Scholar, 15Taube R. Fujinaga K. Wimmer J. Barboric M. Peterlin B.M. Virology. 1999; 264: 245-253Crossref PubMed Scopus (116) Google Scholar). Tat forms a complex with P-TEFb containing Cdk9 and cyclin T1 and the nascent transcript from the HIV-1 promoter (TAR), resulting in activating transcription by causing an increase in the number of RNA polymerase II molecules that synthesize full-length mRNAs (10Wei P. Garber M.E. Fang S.M. Fischer W.H. Jones K.A. Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar, 15Taube R. Fujinaga K. Wimmer J. Barboric M. Peterlin B.M. Virology. 1999; 264: 245-253Crossref PubMed Scopus (116) Google Scholar, 16Garber M.E. Wei P. KewalRamani V.N. Mayall T.P. Herrmann C.H. Rice A.P. Littman D.R. Jones K.A. Genes Dev. 1998; 12: 3512-3527Crossref PubMed Scopus (381) Google Scholar, 17Bieniasz P.D. Grdina T.A. Bogerd H.P. Cullen B.R. Mol. Cell. Biol. 1999; 19: 4592-4599Crossref PubMed Scopus (40) Google Scholar, 18Fujinaga K. Cujec T.P. Peng J. Garriga J. Price D.H. Grana X. Peterlin B.M. J. Virol. 1998; 72: 7154-7159Crossref PubMed Google Scholar, 19Marciniak R.A. Sharp P.A. EMBO J. 1991; 10: 4189-4196Crossref PubMed Scopus (263) Google Scholar). One study used a combination ofin vitro and in vivo assays to screen more than 100,000 compounds for the inhibitors of HIV Tat transcriptional activation. All the compounds identified, including DRB, were found to inhibit P-TEFb (IC50 values between 0.2 and 5 µm in vitro; IC50 values between 1 and 9.5 µm in cell culture), strongly suggesting that P-TEFb represents an attractive target for HIV therapeutics (20Mancebo H.S. Lee G. Flygare J. Tomassini J. Luu P. Zhu Y. Peng J. Blau C. Hazuda D. Price D.H. Flores O. Genes Dev. 1997; 11: 2633-2644Crossref PubMed Scopus (473) Google Scholar, 21Flores O. Lee G. Kessler J. Miller M. Schlief W. Tomassini J. Hazuda D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7208-7213Crossref PubMed Scopus (66) Google Scholar). Consistent with its ability to inhibit P-TEFb, flavopiridol blocked Tat transactivation in vitro and HIV-1 replication in both single-round and viral spread assays with an IC50 of less than 10 nm (8Chao S.H. Fujinaga K. Marion J.E. Taube R. Sausville E.A. Senderowicz A.M. Peterlin B.M. Price D.H. J. Biol. Chem. 2000; 275: 28345-28348Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). Unlike the current anti-HIV drugs that target the viral proteins and result in the selection and propagation of resistant strains, flavopiridol specifically inhibits a cellular factor, P-TEFb. Therefore, it is unlikely that resistant viral strains will arise under the treatment of flavopiridol. However, toxicity of flavopiridol is a concern because it affects normal cellular transcription. Clinical study indicates that the maximal tolerated dose of flavopiridol in cancer patients is between 200 and 400 nm, which results in diarrhea and a proinflammatory syndrome (22Senderowicz A.M. Headlee D. Stinson S.F. Lush R.M. Kalil N. Villalba L. Hill K. Steinberg S.M. Figg W.D. Tompkins A. Arbuck S.G. Sausville E.A. J. Clin. Oncol. 1998; 16: 2986-2999Crossref PubMed Scopus (381) Google Scholar). Our early results suggested that flavopiridol might be effective against HIV (8Chao S.H. Fujinaga K. Marion J.E. Taube R. Sausville E.A. Senderowicz A.M. Peterlin B.M. Price D.H. J. Biol. Chem. 2000; 275: 28345-28348Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). Because lower doses (10–20 nm) might alleviate serious side effects, flavopiridol should be evaluated as a potential HIV therapeutic agent. One of the important issues that needs to be resolved is how the drug specifically inhibits P-TEFb and why it is much more potent than any previously identified P-TEFb inhibitors. Here we show a 1:1 association between P-TEFb and flavopiridol. Utilizing a newly developed immobilized P-TEFb assay, we demonstrate much tighter binding of flavopiridol compared with DRB and the possible usage of the immobilized P-TEFb assay to characterize or screen for compounds with properties similar to flavopiridol. A second important issue is the effect of flavopiridol on normal cellular transcription. Nuclear run-on assays were performed to determine the extent of inhibition of RNA polymerase II in vivo at flavopiridol concentrations that ranged from those likely to be effective against HIV up to those used in chemotherapy. [γ-32P]ATP (>5000 Ci/mmol) and ribonucleoside triphosphates were from Amersham Pharmacia Biotech.Flavopiridol (Aventis Inc.) was dissolved in dimethyl sulfoxide (Me2SO) to 10 mm and stored at −80 °C. DRB (Sigma) was dissolved in ethanol to 10 mm and stored at −80 °C. All other chemicals were reagent grade. Recombinant human P-TEFb protein, comprised of cyclin T1 and His-tagged Cdk9, was expressed using a baculovirus expression system and purified as described previously (12Peng J. Zhu Y. Milton J.T. Price D.H. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (441) Google Scholar). To biotinylate P-TEFb, 10 µg of P-TEFb was incubated with 2 µg of biotin N-hydroxysuccinimide ester-water soluble (Vector Laboratories, Inc.) in 100 mm HEPES, pH 8.5 for 1–2 h at room temperature, followed by adding 15 µg of glycine to stop the biotinylation reaction. The protein mixture was loaded onto an Ni2+-nitrilotriacetic acid-agarose column (Qiagen) to separate the biotin-labeled P-TEFb and the free biotin reagent. After elution with 200 mm imidazole-containing buffer, the biotin-P-TEFb was mixed with M-280 streptavidin paramagnetic beads (Dynal Biotech) at 4 °C for 1 h. A magnetic concentrator (Dynal Biotech) was utilized to isolate and concentrate the immobilized P-TEFb. Kinase assays were as described in Chaoet al. (8Chao S.H. Fujinaga K. Marion J.E. Taube R. Sausville E.A. Senderowicz A.M. Peterlin B.M. Price D.H. J. Biol. Chem. 2000; 275: 28345-28348Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar) using Drosophila RNA polymerase II or DSIF as substrates. The production and purification of recombinant DSIF is described. 2D. Renner, Y. Yamaguchi, T. Wada, H. Handa, and D. Price, submitted for publication. Flavopiridol and DRB were serially diluted in 4% Me2SO and 16% ethanol, respectively, to give the indicated concentrations. The final concentration of Me2SO and ethanol in kinase assays was less than 1 and 4%, respectively. The drugs were incubated with soluble or immobilized P-TEFb at 30 °C for 10 min. In immobilized P-TEFb assays, the concentrations of the two drugs (90 µmDRB or 400 nm flavopiridol) were more than 10-fold greater than their IC50 values. After concentrating the beads and removing the unbound drugs, the immobilized P-TEFb was quickly washed twice with HGKE buffer (25 mm HEPES, pH 7.6, 15% glycerol, 0.1 mm EDTA, with the indicated concentrations of KCl). In both soluble and immobilized P-TEFb assays, subsequent phosphorylation reaction times were 10 min at 30 °C. Reactions were terminated by SDS loading buffer and analyzed by SDS-PAGE. The extent of phosphorylation was quantitated from dried gels using a Packard InstantImager. The IC50 values were calculated by fitting the data to a logistic dose-response curve using the program TableCurve (Jandel Scientific). Suspension HeLa cells were grown to the density of 4 × 105 cells/ml and then treated with the indicated amounts of flavopiridol or DRB for 1 h. All of the following operations were performed at 4 °C. 500 ml of cells were harvested by centrifugation at 500 × g for 10 min and then resuspended in 5 ml of Buffer A (0.32 m sucrose, 10 mm Tris, pH 7.5, 2 mm Mg(Ac)2, 1 mm dithiothreitol, 3 mm CaCl2, and 0.1% Triton X-100). The cells were broken using a Dounce homogenizer. Cell lysis was monitored by phase-contrast microscopy. The homogenate was diluted with 2 volumes of the sucrose cushion (1.9 msucrose, 10 mm Tris, pH 7.5, 5 mmMg(Ac)2, and 1 mm dithiothreitol), giving a final sucrose concentration of 1.3 m, and then layered over the sucrose cushion. The homogenate was centrifuged for 45 min at 22,000 × g. After carefully removing the sucrose solution, the white-colored nuclei were suspended in storage buffer (25% glycerol, 10 mm Tris, pH 7.5, 5 mmMg(Ac)2, and 5 mm dithiothreitol) at about 5 × 108 nuclei/ml at −80 °C. Transcription reactions were carried out as described previously (23Reeder R.H. Roeder R.G. J. Mol. Biol. 1972; 67: 433-441Crossref PubMed Scopus (168) Google Scholar). Each 200-µl reaction contained 1 × 107 nuclei from either treated or untreated cells. Reactions were carried out in 0.12 mKCl, 7 mm Mg(Ac)2, 25 µCi of [32P]GTP, 500 µm ATP, UTP, and CTP with or without 2 µg/ml α-amanitin. Temperature was maintained at 30 °C and duplicated timepoints (18 µl/point) were stopped at 0, 5, 10, 20, and 30 min by the addition of 57 µl of Sarkosyl solution (1% Sarkosyl, 0.1 m Tris, pH 8.0, 0.1 m NaCl, 10 mm EDTA, and 200 µg/ml tRNA). The stopped reactions were transferred onto Whatman DE81 paper, and the paper was washed four times with wash buffer (5% K2HPO4 and 0.3% Na4P2O7) for 10 min, followed by a 5-min water wash. Then the paper was briefly rinsed with 95% ethanol and allowed to dry completely. Radiation was quantitated using a liquid scintillation counter (Wallac). We have shown that flavopiridol is the most potent inhibitor of P-TEFb identified thus far and is able to block HIV transcription and replication efficiently (8Chao S.H. Fujinaga K. Marion J.E. Taube R. Sausville E.A. Senderowicz A.M. Peterlin B.M. Price D.H. J. Biol. Chem. 2000; 275: 28345-28348Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). Because our data suggested that flavopiridol should be evaluated as a potential HIV therapy, it was important to investigate the inhibitory mechanism of the drug on P-TEFb in detail. We calculated the amount of P-TEFb present in the previously published kinase reactions (8Chao S.H. Fujinaga K. Marion J.E. Taube R. Sausville E.A. Senderowicz A.M. Peterlin B.M. Price D.H. J. Biol. Chem. 2000; 275: 28345-28348Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar) and were surprised to find that the concentration of P-TEFb was very close to the IC50 values determined. Based on this observation, we decided to carry out flavopiridol inhibition studies in a series of reactions that contained different concentrations of the enzyme. If flavopiridol binding was very tight, lower concentrations of P-TEFb might result in lower IC50 values. We first performed kinase assays in the presence of increasing concentrations of flavopiridol and a constant amount ofDrosophila RNA polymerase II as substrate. P-TEFb is able to phosphorylate up to 100 different sites in the carboxyl-terminal domain of the large subunit of RNA polymerase II. Four different levels of P-TEFb (1×, 5×, 10×, and 25×) were used in these assays with 1× P-TEFb about 4 nm. The amount of 32P from [γ-32P]ATP incorporated into the large subunit of RNA polymerase II increased as the amount of P-TEFb increased (Fig.1A). Note that the autoradiographs chosen for Fig. 1 A do not necessarily represent equal exposure times. Flavopiridol inhibited phosphorylation at all concentrations of P-TEFb tested (Fig. 1). However, inhibition of kinase activity occurred at lower concentrations of flavopiridol when low levels of P-TEFb were used (Fig. 1 B). To determine whether the unusual dependence of the IC50 on the concentration of P-TEFb was attributed to the large number of phosphorylation sites present in the carboxyl-terminal domain, an identical experiment was carried out using DSIF as substrate. Only a few serine/threonine residues in the carboxyl-terminal region of the large subunit of DSIF are phosphorylated by P-TEFb (24Kim J.B. Sharp P.A. J. Biol. Chem. 2001; 276: 12317-12323Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 25Ping Y.H. Rana T.M. J. Biol. Chem. 2000; 276: 12951-12958Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Fig.2shows that the phosphorylation of DSIF was similar to that with RNA polymerase II. Phosphorylation increased with increasing P-TEFb, indicating that the substrate was not limiting (data not shown). As found with RNA polymerase II, flavopiridol was able to inhibit phosphorylation of DSIF more easily when less P-TEFb was used (Fig. 2). It is clear from the plot of the percent of activity remaining at each flavopiridol concentration that the 50% inhibition points increased as the amount of P-TEFb increased (Fig.2 B). These data indicate that the unusual inhibitory properties of flavopiridol on P-TEFb are substrate-independent. The fact that the amount of flavopiridol needed to inhibit P-TEFb changed with the amount of P-TEFb used suggested that flavopiridol might be acting stoichiometrically with the kinase. The concentration of the purified recombinant P-TEFb was estimated using absorbance at 280 nm and an extinction coefficient calculated from the composition of the protein. IC50 values were determined at each concentration of P-TEFb used in the experiments in Figs. 1 and 2. For each flavopiridol titration the IC50 value calculated was roughly half of the concentration of P-TEFb. This striking result was visualized by plotting IC50 versus the amount of P-TEFb (Fig. 3). An average of the two sets of data described a straight line with an intercept at 0.9 nm and a slope of 0.4. A comparison of IC50 to half of the concentration of P-TEFb, giving the IC50, indicates that at the concentrations tested, one molecule of flavopiridol inhibited [1/(2 × 0.4)] or 1.25 molecules of P-TEFb. It is possible that our calculation of the concentration of P-TEFb was off by 25%, because part of the calculation was an estimation of the percent purity of the preparation. Because of this possible error and because it is unlikely that flavopiridol could inhibit P-TEFb at less than a 1:1 molar ratio, we tentatively conclude that there is stoichiometric binding of the drug to P-TEFb. Because binding is 1:1 even at 4 nm P-TEFb, it is likely that flavopiridol has a subnanomolar dissociation constant. As a control similar assays to those in Figs. 1 and 2 were carried out using DRB as the kinase inhibitor, and the tight correlation between the drug and the enzyme concentration was not observed (data not shown). To study the unique binding of flavopiridol and to examine the inhibitory properties of flavopiridol and DRB in greater detail, we developed an immobilized P-TEFb assay. This assay allowed us to investigate how tightly the drug binds to P-TEFb by rapidly washing the drug-P-TEFb complexes multiple times without losing significant amounts of P-TEFb. The protocol used to generate immobilized P-TEFb is summarized in Fig. 4A. First, the His-tagged recombinant P-TEFb was biotinylated through a covalent interaction between a water-soluble biotin-N-hydroxysuccinimide ester and at least one of the lysine residues in the protein. There are 29 and 52 lysine residues present in Cdk9 and cyclin T1, respectively. The biotinylation process resulted in a loss of about 70% of the kinase activity of P-TEFb (Fig.4 B). However, it is not clear whether the loss was due to inactivation specifically by biotinylation of critical residues or nonspecifically because of the conditions of the reaction. The biotinylated P-TEFb was then repurified using an Ni2+column to remove the free biotin reagent. This step resulted in a loss of 90% of the kinase activity (Fig. 4 B). This was probably due to nonspecific association of the small amount of protein to the resin or tubes, because no kinase activity was detected in the flow-through of the Ni2+ column (Fig. 4 B). The purified biotin-P-TEFb was then bound to streptavidin-conjugated paramagnetic beads. Most of the P-TEFb bound to the beads (Fig.4 B). Taking into account losses at all steps, about 5% of the original enzymatic activity was recovered immobilized to the beads. Such a loss of activity was expected because of the numerous steps involved, the chemical modification of the enzyme, and the immobilization that would limit diffusion of the enzyme in the kinase assay. Although the loss of kinase activity was significant, the final preparation was able to easily generate a strong signal in the kinase assay. To investigate the difference between DRB and flavopiridol association with P-TEFb, immobilized P-TEFb was incubated with DRB or flavopiridol individually for 10 min. The concentrations of DRB and flavopiridol utilized were more than 10-fold greater than their IC50values on P-TEFb (Fig. 5A). After isolating the kinase and removing the unbound drugs using a magnetic concentrator, immobilized P-TEFb was washed twice with a low salt buffer (30 mm KCl). If the drugs were not bound to P-TEFb tightly, the washing steps would cause a 1:40,000 dilution, and the resulting concentrations of the drugs would not be able to inhibit the activity of P-TEFb. As shown in Fig. 5 A, washing allowed the recovery of activity from DRB-treated P-TEFb. However, only 20% of the activity was recovered from flavopiridol-treated P-TEFb (Fig.5 A). Similar results (data not shown) were obtained even when the beads were washed up to four times (200-fold dilution each time). Clearly, flavopiridol is very tightly bound to P-TEFb. We then investigated the effects of ionic strength on drug binding. The drug-treated immobilized kinases were washed twice with buffer containing different concentrations of KCl (0, 30, 100, or 300 mm). All of the kinase activity was recovered from DRB-treated beads regardless of the salt concentration (Fig.5 B), indicating that the weak interaction between DRB and P-TEFb is not stabilized by salt. Activity was not recovered from flavopiridol-treated beads at any of the salt concentrations tested (Fig. 5 B). This suggests that the strong interaction between flavopiridol and P-TEFb may be mediated by hydrophobic contacts. It occurred to us that it might be possible to select compounds that bound tightly to P-TEFb from a mixture of compounds. This would be useful in screening for strong P-TEFb inhibitors by looking for mixtures that resulted in inhibition of the kinase activity of immobilized P-TEFb after washing and then identifying the strong binding component. One potential problem is that a given mixture might contain a number of weak P-TEFb inhibitors that would compete with the strong inhibitor binding. To examine the practicality of detecting a tightly binding compound such as flavopiridol in the presence of other potentially competing compounds, we investigated whether DRB would inhibit flavopiridol binding to immobilized P-TEFb. We have already shown that DRB could be removed from immobilized P-TEFb with several washing steps (Fig. 5), but it was possible that it might compete with flavopiridol during the binding step, especially if it were present in a large excess. As expected, incubation of 400 nm flavopiridol with immobilized P-TEFb inhibited the kinase activity after two wash steps (Fig. 6A). Incubation with up to 400 µm DRB alone did not significantly inhibit P-TEFb after two wash steps. When 400 nm flavopiridol was included in the incubation with the same increasing amounts of DRB, P-TEFb activity after the two wash steps was inhibited to the same extent as when flavopiridol was in the incubation alone. This indicates that DRB does not significantly compete with flavopiridol for binding to P-TEFb even when present in a 1000-fold excess (Fig. 6 B). One important aspect of the evaluation of flavopiridol as an HIV therapy is the determination of its effect on normal cellular transcription. To address this issue and to demonstrate that the effects seen are caused by inhibition of P-TEFb, we performed nuclear run-on assays using nuclei isolated from HeLa cells treated with different amounts of either flavopiridol or DRB. The effect of flavopiridol could be determined directly, and the idea that P-TEFb was the target was strengthened by the use of two different P-TEFb inhibitors that had IC50 values against P-TEFb that differed by about three orders of magnitude. Both compounds have been shown to inhibit P-TEFb more effectively than any other cyclin-dependent kinases. Nuclear run-on assays were chosen over techniques using DNA microarrays because the readout from nuclear run-on assays is direct and minimizes secondary effects such as mRNA stability and indirect effects caused by inhibition of genes whose products regulate transcription of other genes. The cells were treated with the P-TEFb inhibitors for only 1 h before the nuclei were isolated. P-TEFb controls the transition into productive elongation of complexes that are blocked shortly after initiation by the action of negative elongation factors. If P-TEFb is inhibited shortly after the addition of the compound, RNA polymerase II molecules that initiate after the addition of the compound would not be able to make the transition and should exhibit decreased incorporation of radioisotope into RNA. However, polymerases that made the transition into productive elongation before the drug was added would be able to continue to carry out elongation even in the presence of the inhibitor (26Egyhazi E. Ossoinak A. Pigon A. Holmgren C. Lee J.M. Greenleaf A.L. Chromosoma. 1996; 104: 422-433Crossref PubMed Google Scholar). The 1-h treatment used would be effective for inhibiting all transcription from genes whose lengths are less than about 90,000 base pairs because the transcription ratein vivo is about 1500 nucleotides/min. Longer genes would still have elongating polymerases even after 1 h. Longer incubation times could have been chosen, but we wanted to minimize secondary effects. After 1 h of inhibition, mRNA levels should not have changed, thereby reducing the possibility of altering protein levels that might affect transcription. Nuclei were isolated from HeLa cells treated with mock drug or different levels of either flavopiridol or DRB for 1 h and then subjected to the nuclear run-on procedure. Reactions were performed either in the absence or presence of 2 µg/ml α-amanitin to inhibit RNA polymerase II directly. Each experiment compared nuclei obtained from control cells to identical cells treated with the P-TEFb inhibitor. In all control experiments, 70–80% of the total counts incorporated were sensitive to α-amanitin, indicating t" @default.
• W2081567150 created "2016-06-24" @default.
• W2081567150 creator A5067390762 @default.
• W2081567150 creator A5074762963 @default.
• W2081567150 date "2001-08-01" @default.
• W2081567150 modified "2023-10-10" @default.
• W2081567150 title "Flavopiridol Inactivates P-TEFb and Blocks Most RNA Polymerase II Transcription in Vivo" @default.
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