Inhibition of HIV-1 replication by P-TEFb inhibitors DRB, seliciclib and flavopiridol correlates with release of free P-TEFb from the large, inactive form of the complex
- Sebastian Biglione†1, 5,
- Sarah A Byers†1, 6,
- Jason P Price2,
- Van Trung Nguyen4,
- Olivier Bensaude4,
- David H Price1, 3 and
- Wendy Maury1, 2Email author
© Biglione et al; licensee BioMed Central Ltd. 2007
Received: 23 April 2007
Accepted: 11 July 2007
Published: 11 July 2007
The positive transcription elongation factor, P-TEFb, comprised of cyclin dependent kinase 9 (Cdk9) and cyclin T1, T2 or K regulates the productive elongation phase of RNA polymerase II (Pol II) dependent transcription of cellular and integrated viral genes. P-TEFb containing cyclin T1 is recruited to the HIV long terminal repeat (LTR) by binding to HIV Tat which in turn binds to the nascent HIV transcript. Within the cell, P-TEFb exists as a kinase-active, free form and a larger, kinase-inactive form that is believed to serve as a reservoir for the smaller form.
We developed a method to rapidly quantitate the relative amounts of the two forms based on differential nuclear extraction. Using this technique, we found that titration of the P-TEFb inhibitors flavopiridol, DRB and seliciclib onto HeLa cells that support HIV replication led to a dose dependent loss of the large form of P-TEFb. Importantly, the reduction in the large form correlated with a reduction in HIV-1 replication such that when 50% of the large form was gone, HIV-1 replication was reduced by 50%. Some of the compounds were able to effectively block HIV replication without having a significant impact on cell viability. The most effective P-TEFb inhibitor flavopiridol was evaluated against HIV-1 in the physiologically relevant cell types, peripheral blood lymphocytes (PBLs) and monocyte derived macrophages (MDMs). Flavopiridol was found to have a smaller therapeutic index (LD50/IC50) in long term HIV-1 infectivity studies in primary cells due to greater cytotoxicity and reduced efficacy at blocking HIV-1 replication.
Initial short term studies with P-TEFb inhibitors demonstrated a dose dependent loss of the large form of P-TEFb within the cell and a concomitant reduction in HIV-1 infectivity without significant cytotoxicity. These findings suggested that inhibitors of P-TEFb may serve as effective anti-HIV-1 therapies. However, longer term HIV-1 replication studies indicated that these inhibitors were more cytotoxic and less efficacious against HIV-1 in the primary cell cultures.
During HIV-1 replication, the host polymerase (Pol II) is recruited to the viral promoter within the long terminal repeat (LTR) and initiates transcription . Pol II initiates transcription, but elongation of most of the transcripts is blocked by negative elongation factors [2, 3]. The HIV-1 transcription transactivator Tat binds to the bulge of the HIV-1 RNA stem loop termed TAR that is found in all nascent HIV-1 messages and recruits positive transcription elongation factor b (P-TEFb) to the LTR [reviewed in [4, 5]]. P-TEFb phosphorylates both the carboxyl-terminal domain (CTD) of Pol II  and the negative elongation factors [2, 7] allowing Pol II to transition from abortive to productive elongation .
P-TEFb is found within a cell in two forms referred to as large and free forms [9, 10]. The kinase active, free form contains Cdk9 and one of several cyclin regulatory subunits, cyclin T1, cyclin T2a, cyclin T2b or cyclin K, with cyclin T1 being the predominantly associated cyclin in many cell types [11, 12]. The kinase inactive, large form of P-TEFb additionally contains 7SK RNA [9, 10] and hexamethylene bisacetamide-induced protein 1 (HEXIM1) [13, 14] or HEXIM2 . In HeLa cells, between 50% and 90% of P-TEFb is present in the large form of the complex while the remainder of P-TEFb is in the kinase active, free form [9, 10, 14, 15]. It is hypothesized that the large form of P-TEFb serves a reservoir for the free form.
All currently approved anti-HIV therapies target viral proteins that have been shown to rapidly evolve under the selective pressure of highly active anti-retroviral therapy (HAART) [16–18]. Mutations in the viral genome that decrease the effectiveness of HAART arise as a result of the selection of random mutations generated by the lack of proofreading activity in HIV reverse transcriptase [17, 19] and by G to A hypermutation that is believed to result from APOBEC3G restriction . Thus, identification and characterization of additional anti-virals is a necessity. Anti-virals against cellular targets that are required for virus replication may prove to be highly effective. Furthermore, evolution of HIV resistance to this group of compounds might be less likely. Consistent with this possibility, an extensive 6 month study aimed at generating a HIV-1 strain resistant to the cyclin-dependent kinase inhibitor, roscovitine, proved unsuccessful .
Targeting P-TEFb kinase activity as an anti-HIV therapy is potentially attractive, but has not been extensively evaluated. The P-TEFb inhibitors DRB and flavopiridol have been demonstrated to effectively inhibit HIV Tat-dependent transcription in cell lines [22, 23]. Limited studies of the effect of these inhibitors on HIV replication demonstrate a significant reduction of replication at concentrations with limited cytotoxicity [22, 23]. The anti-retroviral activity of roscovitine or the R-enantiomer of roscovitine (seliciclib or Cyc202) has also been explored. This inhibitor has a spectrum of inhibitory activities against a number of cyclin dependent kinases including Cdk 1, 2, 7 and 9 . A previous examination of the effect of seliciclib on HIV replication had focused on its inhibition of Cdk2 activity .
The use of P-TEFb inhibitors as chemotherapeutic agents against cancers has also been proposed . Flavopiridol and seliciclib showed modest cytotoxicity when tested in clinical trials against different kinds of cancers [reviewed on ]. In phase II cancer clinical trials, fatigue, venous thromboses and diarrhea were the primarily side effects of flavopiridol infusions that achieved plasma flavopiridol levels of approximately 400 nM during a 72 hour treatment period [28–31]. Phase II monotherapy trials with flavopiridol have proved disappointing  and newer studies have combined flavopiridol with other chemotherapeutic agents [32, 33]. Seliciclib has recently been tested as a chemotherapeutic agent in Phase I trials and was shown to cause fatigue and elevated creatinine at the highest tested doses that achieved maximal plasma levels of 2 to 4 μg/ml [24, 34].
In this study, we sought to characterize the anti-HIV activity of the cyclin-dependent kinase inhibitors DRB, flavopiridol and seliciclib. In HeLa cells, we found that the anti-HIV activity of these compounds correlated with concentrations that released free P-TEFb from the large form of the complex. These concentrations were not cytotoxic to cells despite the known requirement of P-TEFb activity for Pol II-dependent transcript elongation. However, the concentration of these compounds that was needed to inhibit HIV replication in PBLs and MDMs was higher. Compound cytotoxicity was also greater in these primary cells decreasing the likely utility of these compounds in controlling HIV replication in infected individuals.
Inhibition of Cdk9 kinase activity by P-TEFb inhibitors
Treatment of cells with DRB leads to release of P-TEFb from the large form
To examine the effect of DRB treatment of cells, we treated HeLa cells with increasing concentrations of DRB and analyzed the quantity of large and free forms of P-TEFb within the cell 1 hour later. Glycerol gradient fractionation of lysates followed by immunoblotting of the fractions has been shown to reproducibly separate the forms of P-TEFb with the larger molecular weight form sedimenting with higher concentrations of glycerol than the free form [10, 15, 35]. Quantitative analysis of the immunoblots provides an accurate representation of the ratio of large to free form of P-TEFb in cells. Increasing concentrations of DRB resulted in a shift in the ratio of P-TEFb forms (Fig. 1C). In the absence of DRB, approximately 60% of Cdk9 and 70% of cyclin T1 were located in the denser fractions (fractions 8–11) containing the large form of P-TEFb. In the presence of the highest concentration of DRB tested, 10 μM, only about 20% of P-TEFb subunits were left in the large form of the complex. By plotting the quantity of Cdk9 and cyclin T1 present in the large form of P-TEFb in the presence of DRB, it was observed that approximately 3 μM DRB caused a 50% reduction in large form within the cell (Fig. 1D). This gradual release of P-TEFb from the large form as DRB was increased suggests that the cells are trying to compensate for the loss of P-TEFb activity by releasing more active P-TEFb from the large form.
The free and large forms of P-TEFb are extracted from cell nuclei at different ionic strengths
We were interested in determining if a similar correlation between kinase inhibition and loss of large P-TEFb was found in cells treated with other P-TEFb inhibitors. However, glycerol gradient sedimentation studies require large numbers of cells and are reagent and time intensive. The development of an efficient and rapid method to examine the ratio of large to free form of P-TEFb would allow for the examination of small populations of cells or the simultaneous characterization of many treatments.
P-TEFb inhibitors shift the ratio of free to large P-TEFb forms in cells
Summary of results
IC50 of kinase inhibition:
IC50 of loss of large form from HeLa cells:
5 +/- 3
2.7 +/- 1
12 +/- 4
4.2 +/- 1.6
3.2 +/- 1.4
26 +/- 6
IC50 of inhibition of HIV in HeLa 37 cells:
Hela37 cell cytotoxicity (LD50):
Hela37 therapeutic index:
PBL therapeutic index:
MDM therapeutic index:
Inhibition of HIV-1 infection by non-cytotoxic concentrations of P-TEFb inhibitors
Flavopiridol inhibits long-term HIV-1 replication in PBLs and MDMs
Here, we developed a new approach for determining the ratio of large to free form of P-TEFb based on the differential salt extractability of the two forms of the complex. The differential salt extractability of the free and large P-TEFb forms provides a simple and rapid method for the separation of the two forms. We used differential salt extraction to demonstrate that P-TEFb inhibitors caused a dose dependent release of P-TEFb from its inactive, large form. Importantly, ability of HIV-1 to replicate in short term assays correlated with the amount of the large form of P-TEFb remaining. Findings from short term infectivity studies suggested that these P-TEFb inhibitors might be effective anti-HIV therapies. In these assays, flavopiridol had the most promising therapeutic index against HIV-1. However, in longer term HIV-1 replication assays in primary cells the IC50 values for flavopiridol inhibition were higher than those found in the short term assays and an increase in cytotoxicity reduced the T.I. in MDMs to less than two.
HAART regimens currently target viral proteins including the HIV-1 protease and reverse transcriptase enzymes . One of the problems that HAART therapy faces is the development of resistant strains of HIV-1 that arise due to a high rate of viral mutation. A possible advantage of targeting a cellular protein such as P-TEFb is to avoid the generation of drug-resistant strains of virus. The limited studies performed to date with roscovitine suggest that resistant viruses against this kinase inhibitor may arise slowly if at all in tissue culture . Including a P-TEFb inhibitor in HAART would decrease Tat-dependent transcription while potentially leading to a lower incidence of drug-resistant strains of HIV due to the stringent requirement of cellular P-TEFb for productive HIV-1 transcription [41–43]. Thus, we investigated the efficacy of three P-TEFb inhibitors against HIV-1.
Our studies demonstrate that the P-TEFb inhibitors DRB, flavopiridol and seliciclib inhibit HIV-1 infectivity in HeLa37 cells and to a lesser extent in longer replication studies performed in PBLs and MDMs. The IC50 of 9.5 nM for flavopiridol inhibition obtained during our single-round infectivity studies was consistent with the previously reported inhibition of HIV-1HXB2 infection in Sx22-1 indicator cells . Likewise, the IC50 and LD50 values we obtained for DRB were similar to previously reported values on inhibition of Tat-dependent transcription  and for the inhibition of virus replication by seliciclib [23, 25].
The P-TEFb inhibitor flavopiridol blocked HIV replication in MDMs and PBLs with a lower therapeutic index than that found in HeLa37 cell studies due to both a higher IC50 and lower LD50 values. Flavopiridol is the most effective and specific P-TEFb inhibitor currently identified [22, 39, 44] and yet efficacy of flavopiridol against HIV at non-cytotoxic concentrations is not promising. While clinical chemotherapeutic trials achieved transient plasma concentrations of flavopiridol 8 to 10 fold higher than the anti-viral IC50 values we obtained in primary cells, our findings suggest that consistent plasma levels of flavopiridol of 100 nM or higher would be needed to effectively impact HIV-1 replication and maintaining such levels would be associated with unacceptable levels of toxicity.
Treatment of cells with P-TEFb inhibitors shifted the ratio of free to large form of P-TEFb within cells as the inhibitors blocked kinase activity. P-TEFb may be released from the large form of the complex to compensate for the loss of P-TEFb activity. The P-TEFb inhibitor-induced reduction in the amount of large P-TEFb correlated with inhibition of viral replication suggesting the possibility that large P-TEFb is necessary for HIV-1 replication. Consistent with this possibility, a recent study indicates that HIV-1 Tat is able to recruit P-TEFb out of the large form thereby reducing the quantity of large form within HIV-1 infected cells . Alternatively, the large form of P-TEFb may not be required for viral replication. Instead, specific levels of P-TEFb activity within the cell maybe critical for HIV transcription. Thus, eliminating the kinase activity reduces HIV-1 transcription in parallel. In this model, the total amount of P-TEFb kinase activity required for HIV-1 replication is greater than that needed for cellular transcription and release of P-TEFb from the large form is not sufficient to compensate for the inhibitor-induced loss in Cdk9 activity. To address the role of the large form of P-TEFb in HIV replication, a previous study reduced 7SK levels within the cell by siRNA causing a reduction in large P-TEFb . The reduction in the quantity of large form of P-TEFb did not affect HIV-1 transcription and replication . This finding suggests that the large form of P-TEFb does not play a critical role in HIV-1 transcription and that alterations in the fine balance of kinase active P-TEFb within the cell is responsible for the loss of HIV replication.
Finally, the development of a new salt extraction method that allows separation of large and free P-TEFb may prove useful for future studies. This assay is both less laborious than glycerol gradient fractionation and requires fewer cells. The differential salt extractability of P-TEFb is presumably based on the tight association that free P-TEFb has with chromatin [47–51]. The large form of P-TEFb may be untethered and free to move about the nucleus, perhaps to deliver P-TEFb to where it is needed while maintaining Cdk9 in its inactive state would minimize off target phosphorylations. Alternatively, since P-TEFb can localize to nuclear speckles , differential salt extractability might be due to differential localization of the large and free forms of the complex. This latter alternative is unlikely since P-TEFb localization is not altered by DRB treatments that completely inhibit transcription and disrupt large form of P-TEFb . Therefore, we propose that the free form of P-TEFb is retained in the nucleus under low salt conditions due to its involvement in transcription and association with chromatin through numerous interactions with transcription factors [47–51]. All inhibitors of Pol II elongation that have been tested (flavopiridol, DRB, actinomycin D, ultraviolet irradiation) cause the release of P-TEFb from the large form, but the mechanism of release of is not currently understood . Future studies aimed at uncovering mechanistic details of this process would be facilitated by the new method described here.
Here, we developed a rapid assay that allowed us to quantitatively determine the amount of large and free forms of P-TEFb present in cells. Using this assay, we found that three P-TEFb inhibitors reduced the amount of the large form of P-TEFb in a dose dependent manner. Furthermore, initial short term studies with P-TEFb inhibitors demonstrated that loss of the large form of P-TEFb correlated with a reduction in HIV-1 infectivity without significant cytotoxicity. HIV-1 replication studies in primary cell cultures indicated that these inhibitors were more cytotoxic and less efficacious against HIV-1. How effective P-TEFb inhibitors would be at blocking HIV-1 replication in vivo is not clear.
HeLa S3 and HeLa37 cells (which exogenously express CD4 and CCR5)  were grown in DMEM with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin. HeLa37 cells were a gift from Dr. David Kabat (Oregon Health & Science University, Portland, OR). Jurkat cells (ATCC #TIB 152) were grown in RPMI with 10% fetal calf serum and 1% penicillin/streptomycin. All cells were grown at 37°C and 5% CO2.
Compounds and antibodies
DRB was obtained from Sigma and resuspended in ethanol to generate a 10 mM stock solution. Seliciclib (R-roscovitine) was obtained from Cyclacel (Dundee, Scotland, UK) and resuspended in DMSO to generate 10 mM stock solutions. Flavopiridol was obtained from NIH AIDS Research and Reference Reagent Program (Cat. #9925) and diluted in DMSO to generate a 10 mM stock solution. All compounds were aliquoted and stored at -80°C. Anti-Cdk9 (T-20), anti-cyclin T1 (T18) and anti-cyclin T2 rabbit polyclonal antibodies were obtained from Santa Cruz. Anti-Cdk7, anti-cyclin H and anti-p62 mouse monoclonal antibodies were a kind gift from J.M. Egly (Strasbourg, France).
Generation of HIV
Virus generated from the dual-tropic molecular clone of HIV-1p256  was used through out this study. p256 contains the V3 region from a patient isolate inserted into HIV-1pNL4-3 backbone . 293T cells were seeded at 5 × 105 cells per well in a six-well tray a day before transfection. Cells were transfected with 7 μg of p256 proviral DNA expressing plasmid using the calcium phosphate procedure to generate HIV-1p256 viral stocks . Virus-containing supernatants were collected at 24, 48, 72 and 96 hours post-transfection. Virus production was measured by titering the virus-containing, cell-free supernatants on HeLa37 cells using single-hit infectivity assays described below.
HIV single-hit infectivity assay
Short-term, single hit infectivity studies were performed as previously described . HeLa37 cells were plated in a 48-well tray and triplicate wells were infected with a dual-tropic HIV-1p256 and serial dilutions of P-TEFb inhibitor for 40 hours. The cells were fixed with 75% acetone/25% H2O and immunostained for HIV-1 antigens using human anti-HIV serum (a gift from Dr. Jack Stapleton, Univ. of Iowa) and HRP-conjugated goat anti-human IgG followed by staining with 3-amino-9-ethylcarbazole (AEC). The HIV-1 antigen-positive cells were counted. Experiments were repeated at least three times with each drug concentration in triplicate. Results are represented as the means and standard errors of the mean of the percent of control values (the number of HIV-1 positive cells in the presence of P-TEFb inhibitors/the number of HIV-1 positive cells in untreated wells).
Primary cell isolation, maintenance and infection with HIV
Human monocyte derived macrophages (MDMs) and peripheral blood lymphocytes (PBLs) cells were isolated from 350 ml of peripheral blood from healthy, HIV negative donors. Peripheral blood mononuclear cells (PBMCs) were isolated as previously described . Briefly, PBMCs were separated by centrifugation in lymphocyte separation medium (ICN Biomedicals, Solon, Ohio). The separated PBMCs were placed on gelatin and fibronectin-coated flasks in order to separate monocytes from mononuclear cells. Adherent monocytes were lifted with EDTA, washed and plated at a density of 1 × 106 per well in 48-well trays for infectivity and cytotoxicity studies. Monocytes were differentiated for 5 days in DMEM with 10% FCS, 10% human serum and 1% penicillin/streptomycin in order to generate monocyte-derived macrophages prior to HIV infections and drug treatment. PBLs were treated with 5 μg/ml of phytohaemagglutinin (PHA) for 72 hours prior to HIV infection and drug treatment. PHA-treated PBLs were plated at a density of 1 × 106 per well in 48-well trays and maintained in RPMI 1640 with 10% FCS, 1% Penicillin/Streptomycin and 10 units/ml of recombinant IL-2. Viral infection was performed in MDMs and PBLs by adding 10,000 RT units of HIV-1p256 stock per 1 × 106 cells. During long term studies in primary cells, supernatants were collected at 4, 8, 12 and 16 days post-infection, frozen at -80°C until analyzed and media was refreshed. Inhibition of HIV replication by flavopiridol in PBLs was determined in 3 independent donors and each flavopirdol concentration was tested in triplicate. Inhibition of HIV replication by flavopiridol in MDMs was determined by pooling data from 3 independent donors. A minimum of 3 data points for each flavopiridol concentration was taken into account when generating the IC50 curve for MDMs.
Cell viability assays
The impact of the P-TEFb inhibitors on cell viability was measured by ATPlite (Perkin Elmer). These cytotoxicity studies were performed as recommended by manufacturer utilizing a substrate solution that emits light in a manner proportional to the ATP present in each sample. Cells were plated in a 48-well format. Cells were treated with serial dilutions of the P-TEFb inhibitors and maintained for the indicated period of time. Mammalian cell lysis buffer was added to lyse the cells, followed by addition of the substrate solution. The amount of light produced in each well was measured in a TopCountR Microplate Scintillation and Luminescence Counter (Packard Instruments). Cytotoxicity experiments in HeLa37 cells were repeated at least three times with triplicates of each drug concentration. The results are represented as the means and standard errors of the mean of the percent of control values (the ATPLite values in the presence of P-TEFb inhibitors/the ATPLite values of untreated wells). Cytotoxicity studies in PBLs were performed in three independent donors and each flavopiridol concentration was tested in triplicate. The LD50 of flavopiridol in MDMs was determined by pooling data from 2 independent donors.
P-TEFb kinase assays
Kinase reactions were carried out with recombinant, purified P-TEFb (Cdk9/cyclin T1)  and either DSIF subunit Spt5 or Pol II CTD as the substrate as previously described . Kinase reactions contained 34 mM KCl, 20 mM HEPES pH 7.6, 7 mM MgCl2, 15 μM ATP, 1.3 μCi of [γ-32P]-ATP (Amersham) and 1 μg BSA. The reactions were incubated for 20 minutes at 30°C and stopped by addition of SDS-PAGE loading buffer. Reactions were resolved on a 7.5% SDS-PAGE gel. The dried gel was subjected to autoradiography. Quantitation was performed using an InstantImager (Packard) and data was normalized to the DMSO control. The data was fitted to a dose-response curve using TableCurve (Jandel Scientific) in order to determine the IC50.
Glycerol gradient fractionation of cell lysates
HeLa cells were grown in 100 ml of DMEM with 10% FCS to a density of 4 × 105 cells/ml in spinner flasks. The cells were treated for 1 hour with no P-TEFb inhibitor or serial dilutions of DRB ranging from 0.1 to 10 μM. Cell lysates were prepared in Buffer A (10 mM KCl, 10 mM MgCl2, 10 mM HEPES, 1 mM EDTA, 1 mM DTT, 0.1% PMSF and EDTA-free complete protease inhibitor cocktail (Roche)) containing 150 mM NaCl and 0.5% NP-40. The lysates were clarified by centrifugation at 20,000 g for 10 minutes at 4°C. The supernatant was layered on top of a 5–45% glycerol gradient containing 150 mM NaCl. Gradients were spun at 190,000 g for 16 hours using a SW-55Ti rotor. The fractions were analyzed for the presence of P-TEFb complexes by immunoblotting with anti-cyclin T1 and anti-Cdk9 antibodies (Santa Cruz). Following incubation with the appropriate HRP-conjugated secondary antibodies, the blots were developed using SuperSignal DuraWest (Pierce). The western blots were imaged using a cooled CCD camera (UVP) and the amount of P-TEFb in the large and free form was quantitated using LabWorks 4.0 software.
Separation of large and free forms of P-TEFb by differential salt extraction
HeLa37 and Jurkat cells were treated with serial dilutions of DRB, flavopiridol or seliciclib concentrations for 1 hour. The cytosolic extracts were prepared by resuspending the cells in 80 μl of Buffer A (10 mM KCl, 10 mM MgCl2, 10 mM HEPES, 1 mM EDTA, 1 mM DTT, 0.1% PMSF and EDTA-free complete protease inhibitor cocktail (Roche)) with 0.5% NP-40 for 10 minutes on ice. The nuclei were spun down at 5,000 g for 5 minutes and the supernatant was saved as the cytosolic extract (CE). The nuclei were washed once with 200 μl of Buffer A with 0.5% NP-40 and re-pelleted. The nuclei were resuspended in 80 μl of Buffer B (450 mM NaCl, 1.5 mM MgCl2, 20 mM HEPES, 0.5 mM EDTA, 1 mM DTT, 0.1% PMSF and EDTA-free complete protease inhibitor cocktail (Roche)) and incubated on ice for 10 minutes. The lysates were clarified by centrifugation at 20,000 g for 10 minutes. The supernatant was saved as the nuclear extract (NE). Western blotting was performed with one fifth of the samples and the fraction of Cdk9 and cyclin T1 in the cytosolic and nuclear extracts was determined by imaging the chemiluminescent signal using a cooled CCD camera (UVP). The signal was quantitated using LabWorks 4.0 software and the data fit to a logistic dose response curve using TableCurve (Jandel Scientific) to determine the IC50 for loss of the large, low salt extractable form of P-TEFb.
Reverse transcriptase assays
Reverse transcriptase (RT) assays were performed on supernatants from HIV-1p256 infected cells as previously described . Briefly, cell-free supernatant from infected cells were added to a mix containing 50 mM Tris (pH 7.8), 75 mM KCl, 2 mM DTT, 5 mM MgCl2, 0.05% NP-40, 5 μg poly(A), 4 μg poly(d) (T12-18) and 10 μCi/ml 32P-TTP. The mixture was incubated at 37°C for 3.5 hours and then blotted onto DE81 paper. The DE81 paper was washed 4 times with 3× SSPE and the amount of radioactivity that was incorporated into negative strand DNA was quantified with an InstantImager (Packard Instruments).
All HIV infectivity and cytotoxicity data are represented as the percent of control values to allow comparisons of separate experiments. The mean of the values obtained in the infectivity studies were determined by averaging the individual experimental data points for each drug concentration. Error bars on graphs represent the calculated standard error for each drug dilution. Determination of IC50 and LD50 values was performed using TableCurve (Jandel Scientific).
human immunodeficiency virus-1
positive transcription elongation factor b
cyclin dependent kinase 9
carboxyl terminal domain
DRB-sensitive inhibitory factor
hexamethylene bisacetamide-induced protein
- Pol II:
long terminal repeat
peripheral blood lymphocytes
monocyte derived macrophages
highly active anti-retroviral therapy.
This work was supported by NIH grants AI54340 and NIH AI54340-03S1 to D.H.P and W.M. and a grant from Association pour la Recherche sur le Cancer, Agence Nationale de Recherche sur le SIDA to O.B. Flavopiridol and recombinant IL-2 were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.
- Cujec TP, Cho H, Maldonado E, Meyer J, Reinberg D, Peterlin BM: The human immunodeficiency virus transactivator Tat interacts with the RNA polymerase II holoenzyme. Mol Cell Biol. 1997, 17 (4): 1817-1823.PubMed CentralView ArticlePubMedGoogle Scholar
- Fujinaga K, Irwin D, Huang Y, Taube R, Kurosu T, Peterlin BM: Dynamics of human immunodeficiency virus transcription: P-TEFb phosphorylates RD and dissociates negative effectors from the transactivation response element. Mol Cell Biol. 2004, 24 (2): 787-795. 10.1128/MCB.24.2.787-795.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Ivanov D, Kwak YT, Guo J, Gaynor RB: Domains in the SPT5 protein that modulate its transcriptional regulatory properties. Mol Cell Biol. 2000, 20 (9): 2970-2983. 10.1128/MCB.20.9.2970-2983.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Price DH: P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol Cell Biol. 2000, 20 (8): 2629-2634. 10.1128/MCB.20.8.2629-2634.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhou Q, Yik JH: The Yin and Yang of P-TEFb regulation: implications for human immunodeficiency virus gene expression and global control of cell growth and differentiation. Microbiol Mol Biol Rev. 2006, 70 (3): 646-659. 10.1128/MMBR.00011-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Marshall NF, Peng J, Xie Z, Price DH: Control of RNA polymerase II elongation potential by a novel carboxyl-terminal domain kinase. J Biol Chem. 1996, 271 (43): 27176-27183. 10.1074/jbc.271.43.27176.View ArticlePubMedGoogle Scholar
- Yamada T, Yamaguchi Y, Inukai N, Okamoto S, Mura T, Handa H: P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation. Mol Cell. 2006, 21 (2): 227-237. 10.1016/j.molcel.2005.11.024.View ArticlePubMedGoogle Scholar
- Peterlin BM, Price DH: Controlling the elongation phase of transcription with P-TEFb. Mol Cell. 2006, 23 (3): 297-305. 10.1016/j.molcel.2006.06.014.View ArticlePubMedGoogle Scholar
- Yang Z, Zhu Q, Luo K, Zhou Q: The 7SK small nuclear RNA inhibits the CDK9/cyclin T1 kinase to control transcription. Nature. 2001, 414 (6861): 317-322. 10.1038/35104575.View ArticlePubMedGoogle Scholar
- Nguyen VT, Kiss T, Michels AA, Bensaude O: 7SK small nuclear RNA binds to and inhibits the activity of CDK9/cyclin T complexes. Nature. 2001, 414 (6861): 322-325. 10.1038/35104581.View ArticlePubMedGoogle Scholar
- Peng J, Zhu Y, Milton JT, Price DH: Identification of multiple cyclin subunits of human P-TEFb. Genes Dev. 1998, 12 (5): 755-762.PubMed CentralView ArticlePubMedGoogle Scholar
- Fu TJ, Peng J, Lee G, Price DH, Flores O: Cyclin K functions as a CDK9 regulatory subunit and participates in RNA polymerase II transcription. J Biol Chem. 1999, 274 (49): 34527-34530. 10.1074/jbc.274.49.34527.View ArticlePubMedGoogle Scholar
- Yik JH, Chen R, Nishimura R, Jennings JL, Link AJ, Zhou Q: Inhibition of P-TEFb (CDK9/Cyclin T) kinase and RNA polymerase II transcription by the coordinated actions of HEXIM1 and 7SK snRNA. Mol Cell. 2003, 12 (4): 971-982. 10.1016/S1097-2765(03)00388-5.View ArticlePubMedGoogle Scholar
- Michels AA, Nguyen VT, Fraldi A, Labas V, Edwards M, Bonnet F, Lania L, Bensaude O: MAQ1 and 7SK RNA interact with CDK9/cyclin T complexes in a transcription-dependent manner. Mol Cell Biol. 2003, 23 (14): 4859-4869. 10.1128/MCB.23.14.4859-4869.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Byers SA, Price JP, Cooper JJ, Li Q, Price DH: HEXIM2, a HEXIM1-related protein, regulates positive transcription elongation factor b through association with 7SK. J Biol Chem. 2005, 280 (16): 16360-16367. 10.1074/jbc.M500424200.View ArticlePubMedGoogle Scholar
- Temesgen Z, Warnke D, Kasten MJ: Current status of antiretroviral therapy. Expert Opin Pharmacother. 2006, 7 (12): 1541-1554. 10.1517/146565184.108.40.2061.View ArticlePubMedGoogle Scholar
- D'Aquila RT, Schapiro JM, Brun-Vezinet F, Clotet B, Conway B, Demeter LM, Grant RM, Johnson VA, Kuritzkes DR, Loveday C, Shafer RW, Richman DD: Drug resistance mutations in HIV-1. Top HIV Med. 2003, 11 (3): 92-96.PubMedGoogle Scholar
- Tang JW, Pillay D: Transmission of HIV-1 drug resistance. J Clin Virol. 2004, 30 (1): 1-10. 10.1016/j.jcv.2003.12.002.View ArticlePubMedGoogle Scholar
- Menendez-Arias L: Molecular basis of fidelity of DNA synthesis and nucleotide specificity of retroviral reverse transcriptases. Prog Nucleic Acid Res Mol Biol. 2002, 71: 91-147.View ArticlePubMedGoogle Scholar
- Malim MH: Natural resistance to HIV infection: The Vif-APOBEC interaction. Comptes rendus biologies. 2006, 329 (11): 871-875. 10.1016/j.crvi.2006.01.012.View ArticlePubMedGoogle Scholar
- Wang D, de la Fuente C, Deng L, Wang L, Zilberman I, Eadie C, Healey M, Stein D, Denny T, Harrison LE, Meijer L, Kashanchi F: Inhibition of human immunodeficiency virus type 1 transcription by chemical cyclin-dependent kinase inhibitors. J Virol. 2001, 75 (16): 7266-7279. 10.1128/JVI.75.16.7266-7279.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Chao SH, Fujinaga K, Marion JE, Taube R, Sausville EA, Senderowicz AM, Peterlin BM, Price DH: Flavopiridol inhibits P-TEFb and blocks HIV-1 replication. J Biol Chem. 2000, 275 (37): 28345-28348. 10.1074/jbc.C000446200.View ArticlePubMedGoogle Scholar
- Mancebo HS, Lee G, Flygare J, Tomassini J, Luu P, Zhu Y, Peng J, Blau C, Hazuda D, Price D, Flores O: P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. Genes Dev. 1997, 11 (20): 2633-2644.PubMed CentralView ArticlePubMedGoogle Scholar
- Benson C, White J, De Bono J, O'Donnell A, Raynaud F, Cruickshank C, McGrath H, Walton M, Workman P, Kaye S, Cassidy J, Gianella-Borradori A, Judson I, Twelves C: A phase I trial of the selective oral cyclin-dependent kinase inhibitor seliciclib (CYC202; R-Roscovitine), administered twice daily for 7 days every 21 days. Br J Cancer. 2007, 96 (1): 29-37. 10.1038/sj.bjc.6603509.PubMed CentralView ArticlePubMedGoogle Scholar
- Agbottah E, de La Fuente C, Nekhai S, Barnett A, Gianella-Borradori A, Pumfery A, Kashanchi F: Antiviral activity of CYC202 in HIV-1-infected cells. J Biol Chem. 2005, 280 (4): 3029-3042. 10.1074/jbc.M406435200.View ArticlePubMedGoogle Scholar
- Senderowicz AM, Headlee D, Stinson SF, Lush RM, Kalil N, Villalba L, Hill K, Steinberg SM, Figg WD, Tompkins A, Arbuck SG, Sausville EA: Phase I trial of continuous infusion flavopiridol, a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasms. J Clin Oncol. 1998, 16 (9): 2986-2999.PubMedGoogle Scholar
- Schang LM: Effects of pharmacological cyclin-dependent kinase inhibitors on viral transcription and replication. Biochim Biophys Acta. 2004, 1697 (1-2): 197-209.View ArticlePubMedGoogle Scholar
- Innocenti F, Stadler WM, Iyer L, Ramirez J, Vokes EE, Ratain MJ: Flavopiridol metabolism in cancer patients is associated with the occurrence of diarrhea. Clin Cancer Res. 2000, 6 (9): 3400-3405.PubMedGoogle Scholar
- Stadler WM, Vogelzang NJ, Amato R, Sosman J, Taber D, Liebowitz D, Vokes EE: Flavopiridol, a novel cyclin-dependent kinase inhibitor, in metastatic renal cancer: a University of Chicago Phase II Consortium study. J Clin Oncol. 2000, 18 (2): 371-375.PubMedGoogle Scholar
- Schwartz GK, Ilson D, Saltz L, O'Reilly E, Tong W, Maslak P, Werner J, Perkins P, Stoltz M, Kelsen D: Phase II study of the cyclin-dependent kinase inhibitor flavopiridol administered to patients with advanced gastric carcinoma. J Clin Oncol. 2001, 19 (7): 1985-1992.PubMedGoogle Scholar
- Kouroukis CT, Belch A, Crump M, Eisenhauer E, Gascoyne RD, Meyer R, Lohmann R, Lopez P, Powers J, Turner R, Connors JM: Flavopiridol in untreated or relapsed mantle-cell lymphoma: results of a phase II study of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol. 2003, 21 (9): 1740-1745. 10.1200/jco.2003.09.057.View ArticlePubMedGoogle Scholar
- Kim JC, Saha D, Cao Q, Choy H: Enhancement of radiation effects by combined docetaxel and flavopiridol treatment in lung cancer cells. Radiother Oncol. 2004, 71 (2): 213-221. 10.1016/j.radonc.2004.03.006.View ArticlePubMedGoogle Scholar
- Bible KC, Lensing JL, Nelson SA, Lee YK, Reid JM, Ames MM, Isham CR, Piens J, Rubin SL, Rubin J, Kaufmann SH, Atherton PJ, Sloan JA, Daiss MK, Adjei AA, Erlichman C: Phase 1 trial of flavopiridol combined with cisplatin or carboplatin in patients with advanced malignancies with the assessment of pharmacokinetic and pharmacodynamic end points. Clin Cancer Res. 2005, 11 (16): 5935-5941. 10.1158/1078-0432.CCR-04-2566.View ArticlePubMedGoogle Scholar
- Laurence V: Preliminary results of an ongoing phase I and pharmacokinetics study of CYC202, a novel oral cyclin-dependent kinases inhibitor, in patients with advanced malignancies. European Journal of Cancer. 2002, 38 (supplement 7): S49-Google Scholar
- Li Q, Price JP, Byers SA, Cheng D, Peng J, Price DH: Analysis of the large inactive P-TEFb complex indicates that it contains one 7SK molecule, a dimer of HEXIM1 or HEXIM2, and two P-TEFb molecules containing Cdk9 phosphorylated at threonine 186. J Biol Chem. 2005, 280 (31): 28819-28826. 10.1074/jbc.M502712200.View ArticlePubMedGoogle Scholar
- Shore SM, Byers SA, Maury W, Price DH: Identification of a novel isoform of Cdk9. Gene. 2003, 307: 175-182. 10.1016/S0378-1119(03)00466-9.View ArticlePubMedGoogle Scholar
- Yang Z, Yik JH, Chen R, He N, Jang MK, Ozato K, Zhou Q: Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol Cell. 2005, 19 (4): 535-545. 10.1016/j.molcel.2005.06.029.View ArticlePubMedGoogle Scholar
- Jang MK, Mochizuki K, Zhou M, Jeong HS, Brady JN, Ozato K: The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol Cell. 2005, 19 (4): 523-534. 10.1016/j.molcel.2005.06.027.View ArticlePubMedGoogle Scholar
- Chao SH, Price DH: Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo. J Biol Chem. 2001, 276 (34): 31793-31799. 10.1074/jbc.M102306200.View ArticlePubMedGoogle Scholar
- Meadows DC, Gervay-Hague J: Current developments in HIV chemotherapy. ChemMedChem. 2006, 1 (1): 16-29. 10.1002/cmdc.200500026.View ArticlePubMedGoogle Scholar
- Schang LM, Bantly A, Knockaert M, Shaheen F, Meijer L, Malim MH, Gray NS, Schaffer PA: Pharmacological cyclin-dependent kinase inhibitors inhibit replication of wild-type and drug-resistant strains of herpes simplex virus and human immunodeficiency virus type 1 by targeting cellular, not viral, proteins. J Virol. 2002, 76 (15): 7874-7882. 10.1128/JVI.76.15.7874-7882.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- de la Fuente C, Maddukuri A, Kehn K, Baylor SY, Deng L, Pumfery A, Kashanchi F: Pharmacological cyclin-dependent kinase inhibitors as HIV-1 antiviral therapeutics. Curr HIV Res. 2003, 1 (2): 131-152. 10.2174/1570162033485339.View ArticlePubMedGoogle Scholar
- Provencher VM, Coccaro E, Lacasse JJ, Schang LM: Antiviral drugs that target cellular proteins may play major roles in combating HIV resistance. Curr Pharm Des. 2004, 10 (32): 4081-4101. 10.2174/1381612043382422.View ArticlePubMedGoogle Scholar
- Chen R, Keating MJ, Gandhi V, Plunkett W: Transcription inhibition by flavopiridol: mechanism of chronic lymphocytic leukemia cell death. Blood. 2005, 106 (7): 2513-2519. 10.1182/blood-2005-04-1678.PubMed CentralView ArticlePubMedGoogle Scholar
- Barboric M, Yik JH, Czudnochowski N, Yang Z, Chen R, Contreras X, Geyer M, Matija Peterlin B, Zhou Q: Tat competes with HEXIM1 to increase the active pool of P-TEFb for HIV-1 transcription. Nucleic Acids Res. 2007Google Scholar
- Haaland RE, Herrmann CH, Rice AP: siRNA depletion of 7SK snRNA induces apoptosis but does not affect expression of the HIV-1 LTR or P-TEFb-dependent cellular genes. J Cell Physiol. 2005, 205 (3): 463-470. 10.1002/jcp.20528.View ArticlePubMedGoogle Scholar
- Wittmann BM, Fujinaga K, Deng H, Ogba N, Montano MM: The breast cell growth inhibitor, estrogen down regulated gene 1, modulates a novel functional interaction between estrogen receptor alpha and transcriptional elongation factor cyclin T1. Oncogene. 2005, 24 (36): 5576-5588. 10.1038/sj.onc.1208728.View ArticlePubMedGoogle Scholar
- Eberhardy SR, Farnham PJ: Myc recruits P-TEFb to mediate the final step in the transcriptional activation of the cad promoter. J Biol Chem. 2002, 277 (42): 40156-40162. 10.1074/jbc.M207441200.View ArticlePubMedGoogle Scholar
- Kanazawa S, Okamoto T, Peterlin BM: Tat competes with CIITA for the binding to P-TEFb and blocks the expression of MHC class II genes in HIV infection. Immunity. 2000, 12 (1): 61-70. 10.1016/S1074-7613(00)80159-4.View ArticlePubMedGoogle Scholar
- Kanazawa S, Soucek L, Evan G, Okamoto T, Peterlin BM: c-Myc recruits P-TEFb for transcription, cellular proliferation and apoptosis. Oncogene. 2003, 22 (36): 5707-5711. 10.1038/sj.onc.1206800.View ArticlePubMedGoogle Scholar
- Luecke HF, Yamamoto KR: The glucocorticoid receptor blocks P-TEFb recruitment by NFkappaB to effect promoter-specific transcriptional repression. Genes Dev. 2005, 19 (9): 1116-1127. 10.1101/gad.1297105.PubMed CentralView ArticlePubMedGoogle Scholar
- Herrmann CH, Mancini MA: The Cdk9 and cyclin T subunits of TAK/P-TEFb localize to splicing factor-rich nuclear speckle regions. J Cell Sci. 2001, 114 (Pt 8): 1491-1503.PubMedGoogle Scholar
- Reed-Inderbitzin E, Maury W: Cellular specificity of HIV-1 replication can be controlled by LTR sequences. Virology. 2003, 314 (2): 680-695. 10.1016/S0042-6822(03)00508-7.View ArticlePubMedGoogle Scholar
- Chesebro B, Wehrly K, Nishio J, Perryman S: Mapping of independent V3 envelope determinants of human immunodeficiency virus type 1 macrophage tropism and syncytium formation in lymphocytes. J Virol. 1996, 70 (12): 9055-9059.PubMed CentralPubMedGoogle Scholar
- Byers SA, Schafer B, Sappal DS, Brown J, Price DH: The antiproliferative agent MLN944 preferentially inhibits transcription. Mol Cancer Ther. 2005, 4 (8): 1260-1267. 10.1158/1535-7163.MCT-05-0109.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.