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J. Biol. Chem., Vol. 276, Issue 34, 31793-31799, August 24, 2001
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and§¶
From the Molecular Biology Program and the
§ Department of Biochemistry, University of Iowa, Iowa
City, Iowa 52242
Received for publication, March 14, 2001, and in revised form, June 15, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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- Flavopiridol is a potential anti-cancer therapeutic agent
currently being tested in phase I and II clinical trials (1, 2).
Previous evidence indicates that flavopiridol most effectively inhibits
Cdk1,1 Cdk2, and Cdk4 with
Kis between 40 and 70 nM (1, 3, 4). 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 (5). Treatment with flavopiridol
resulted in blocking cell cycle progression, promoting differentiation,
and inducing apoptosis in various types of cancerous cells (1). In
addition, an effect of flavopiridol on transcription of yeast and
mammalian genes has been reported (6, 7). 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 Ki of 3 nM (8). Unlike its effects on the other Cdks, flavopiridol
inhibition of P-TEFb was not competitive with ATP (8).
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 (9-14). P-TEFb is a required cellular cofactor for
activation of transcription of the HIV-1 genome by the viral transactivator Tat (9, 15). 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 (10, 15-19). One study used a combination of
in 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 (20, 21).
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 (8).
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 (22). Our early results
suggested that flavopiridol might be effective against HIV (8). 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.
Materials--
[ Generation of Immobilized P-TEFb--
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 (12). 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 Assay--
Kinase assays were as described in Chao
et al. (8) using Drosophila RNA polymerase II or
DSIF as substrates. The production and purification of recombinant DSIF
is described.2 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 µM
DRB 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).
Nuclear Run-on Assays--
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 M
sucrose, 10 mM Tris, pH 7.5, 5 mM
Mg(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 mM
Mg(Ac)2, and 5 mM dithiothreitol) at about
5 × 108 nuclei/ml at One to One Binding of Flavopiridol with P-TEFb--
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 (8). 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 (8) 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 of
Drosophila 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
[
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 (24, 25). Fig.
2 shows 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.
2B). 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).
Flavopiridol Tightly Associates with P-TEFb through a Hydrophobic
Interaction--
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.
4B). 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. 4B). 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. 4B). The
purified biotin-P-TEFb was then bound to streptavidin-conjugated
paramagnetic beads. Most of the P-TEFb bound to the beads (Fig.
4B). 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 IC50
values 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. 5A, 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.
5A). 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.
5B), 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. 5B). This suggests that the strong interaction between
flavopiridol and P-TEFb may be mediated by hydrophobic contacts.
Potential Use of Immobilized P-TEFb in Drug Screening--
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. 6B).
P-TEFb Requirement for Cellular Transcription--
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 (26). 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 rate
in 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 The results presented here help to explain the unusual properties
of flavopiridol action on P-TEFb. We previously showed that flavopiridol inhibition of P-TEFb (Cdk9/cyclinT1) was not competitive with ATP (8). This result was surprising because the inhibition of Cdk1
and Cdk4 by flavopiridol was shown to be competitive with ATP (3, 4).
Furthermore, a crystal structure with flavopiridol and Cdk2 indicated
that the drug binds to the ATP binding site (5). Kinetic analysis of
flavopiridol inhibition of P-TEFb best fit an uncompetitive model (8).
Uncompetitive inhibition is characterized by binding of the inhibitor
to the enzyme/substrate complex. However, the characteristics of
uncompetitive inhibition are inconsistent with the competitive
inhibition of other Cdks by flavopiridol and the crystal structure
with the drug. Because it is unlikely that the mechanism of
flavopiridol inhibition of Cdk9 is different from Cdk1 and Cdk4, we
initially suggested that flavopiridol binds to the ATP site of Cdk9
very tightly and consequently inactivates the enzyme (8). The results
presented here support this idea. Flavopiridol binds to P-TEFb with 1:1
stoichiometry, and this tight binding inactivates the enzyme. It is
possible that flavopiridol inhibition of Cdk9 is competitive with ATP, but we have been unable to find conditions in which the competition can
be demonstrated. The Ki for flavopiridol of 3 nM calculated earlier (8) is four orders of magnitude below
the Km for ATP, and our results here suggest that
the actual Ki is even lower. The
Ki determined earlier was obtained with reactions
containing about 20 nM P-TEFb, and we showed here that
flavopiridol inhibition is directly affected by the concentration of
P-TEFb. Had a lower concentration of P-TEFb been used, a lower
Ki would have been determined.
The immobilized enzyme assay described here provided further evidence
of a tight association of flavopiridol with P-TEFb. In addition to the
experiments presented here an effort was made to determine the off-rate
of flavopiridol, but the binding properties of the drug made this
difficult (data not shown). Even when immobilized P-TEFb/flavopiridol
complexes were washed to remove free drug and then resuspended at a
P-TEFb concentration below 1 nM, the drug remained bound
and the enzyme remained inhibited. Shorter and longer kinase reactions
were tried, but even though the immobilized P-TEFb was diluted into the
reactions it gave the same results (data not shown). Long incubations
or long reactions did not allow much reactivation of the enzyme,
because even if the drug dissociated it then rebound. It was possible
to observe partial re-activation of immobilized P-TEFb inhibited by
flavopiridol, however this required continually washing the beads. All
of our results with immobilized P-TEFb/flavopiridol complexes suggest
that the dissociation constant for the drug is significantly below 1 nM, but other methods are needed to accurately measure this parameter.
The properties we have determined for flavopiridol make it attractive
as a P-TEFb inhibitor in vitro and in vivo.
Because of the conservation of the structure and function of kinases in general, it was thought that kinase inhibitors would lack specificity (27, 28). Flavopiridol has been shown to inhibit a number of
cyclin-dependent kinases (1). However, the biochemical
properties of the drug suggest that under appropriate conditions it can
be used to selectively inhibit P-TEFb. This stems primarily from the
extreme sensitivity of P-TEFb to flavopiridol and its ability to bind
tightly to the flavonoid. In a mixture of kinases flavopiridol would
first associate with and inhibit P-TEFb. Only after P-TEFb had been
titrated would free flavopiridol be available to inhibit other kinases.
In this way at low concentrations of flavopiridol, the drug would be
sequestered by P-TEFb from interactions with other kinases. The lack of
competition of ATP with flavopiridol action on P-TEFb further
accentuates the difference between P-TEFb and other kinases in
vivo. ATP does not significantly interfere with the interaction of
flavopiridol and P-TEFb (8), but normal cellular levels of ATP
significantly reduce the ability of the drug to inhibit other
cyclin-dependent kinases.
It is important to determine whether the antiproliferative effects of
flavopiridol are due to inhibition of P-TEFb or other cyclin-dependent kinases as originally proposed. If P-TEFb
is the effective target it is likely that flavopiridol is a very good
drug because of its selectivity at low concentrations. However, if the
effective targets of flavopiridol are Cdks controlling the cell cycle,
it is likely that better Cdk inhibitors can be designed, which inhibit
those kinases and do not affect P-TEFb. We favor the idea that the
antiproliferative effect of flavopiridol is mediated through P-TEFb
inhibition. The best evidence for this comes from a screen for
compounds that block HIV Tat transactivation, which uncovered a number
of P-TEFb inhibitors (20). P-TEFb is required for HIV Tat
transactivation (9) and all of the inhibitors found so far (including
flavopiridol) exhibit cytotoxic effects at about 15-fold higher
concentrations than those required to block Tat transactivation (8,
20). The mechanism of enhanced sensitivity of HIV to P-TEFb inhibitors
is not understood but is well documented in studies using P-TEFb
inhibitors or incorporating the expression of an inactive Cdk9 subunit
of P-TEFb (14, 20, 21). P-TEFb inhibitors such as DRB and flavopiridol
inhibit transcription by RNA polymerase II and cause apoptosis in a
variety of cell lines and tumors (2, 14, 29, 30). Several flavopiridol analogues have been generated and tested on Cdk1, 2, and 4 (31, 32).
The effects of these flavopiridol analogues on P-TEFb should be
examined. Comparison of the pattern of inhibition of a number of
compounds on P-TEFb and other Cdks with the pattern of inhibition of
cellular growth may help determine whether P-TEFb is an effective antiproliferative drug target.
The immobilized P-TEFb assays described here should prove useful in
uncovering basic parameters of P-TEFb function and inhibitor action. We
have shown that flavopiridol can be selected from a mixture including a
1000-fold excess of DRB, a known inhibitor of P-TEFb (Fig.
6B). This proves that the assay could be useful in screening
for other compounds of similar potency to flavopiridol. In one
conventional screen over 100,000 compounds were examined, and none were
as potent as flavopiridol (20). It is likely that a significantly
greater number of compounds would need to be screened to find another
drug like flavopiridol. Mixtures of 1000 compounds could be screened in
a single assay for the ability to maintain inhibition of P-TEFb after
washing (Fig. 6B). If the drug mixtures were available one
individual could manually assay 50,000-100,000 compounds a day. If a
mixture was found to contain an inhibitor like flavopiridol it could be
deconvoluted to identify the compound. The immobilized P-TEFb assay
could also prove useful in quickly examining the association and
inhibitory properties of compounds generated from flavopiridol in
attempts to create drugs with better pharmacokinetics.
Two important conclusions can be drawn from the nuclear run-on
experiments presented here. First, at concentrations of flavopiridol that block HIV replication, there was no detectable effect on the
transcription of cellular genes. It is possible that a small number of
genes are dramatically inhibited by low levels of the drug, but this is
not a significant fraction of the total RNA polymerase II transcription
and, therefore, was not detected. Such hypersensitive cellular genes
may be identified using microarray techniques, but if there are such
genes it is unlikely that they have essential functions because there
is no noticeable change in cell growth in the presence of low levels of
flavopiridol. Even though the mechanism is not known it is possible
that the hypersensitivity of HIV transcription to P-TEFb inhibitors can be exploited by using flavopiridol as an HIV therapy. A second important observation made is that two P-TEFb inhibitors blocked most
transcription by RNA polymerase II. This indicates that P-TEFb is
globally required. The nuclear run-on assay avoids many potential secondary effects because it is direct and can be performed quickly after drug treatment. In the future we plan to examine the
transcription of many specific genes to determine whether there are
genes that do not utilize the elongation control machinery and if
different genes exhibit differential sensitivity to P-TEFb inhibitors.
-D-ribofuranosylbenzimidazole (DRB)
sensitivity-inducing factor and found that the IC50
determined 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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.
80 °C. Transcription
reactions were carried out as described previously (23). Each 200-µl
reaction contained 1 × 107 nuclei from either treated
or untreated cells. Reactions were carried out in 0.12 M
KCl, 7 mM Mg(Ac)2, 25 µCi of
[32P]GTP, 500 µM ATP, UTP, and CTP with or
without 2 µg/ml 
-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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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. 1A 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. 1B).
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Fig. 1.
Effect of P-TEFb concentration on
flavopiridol inhibition of RNA polymerase II phosphorylation.
Kinase assays were performed as described under "Experimental
Procedures" using RNA polymerase II as the substrate and four levels
of P-TEFb (Cdk9/cyclin T1). 1× P-TEFb is 4 nM.
A, autoradiographs from SDS-PAGE analysis of kinase
reactions. Only the large subunit of RNA polymerase II is shown.
Exposure times for individual autoradiographs are not equal.
B, plot showing quantitation of the phosphorylated
polymerase.
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Fig. 2.
Effect of P-TEFb concentration on
flavopiridol inhibition of DSIF phosphorylation. Kinase assays
were performed as described under "Experimental Procedures" using
DSIF as the substrate and four levels of P-TEFb (Cdk9/cyclin T1). 1×
P-TEFb is 4 nM. A, autoradiographs from SDS-PAGE
analysis of kinase reactions. Only the large subunit of DSIF is shown.
Exposure times for individual autoradiographs are not equal.
B, plot showing quantitation of the phosphorylated
DSIF.
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Fig. 3.
Stoichiometry between flavopiridol and
P-TEFb. IC50 values were calculated as described in
"Experimental Procedures" for each flavopiridol titration shown in
Figs. 1 and 2. The plot shows the correlation between concentrations of
P-TEFb and IC50 values.
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Fig. 4.
Generation and characterization of
immobilized P-TEFb. A, diagram summarizing the
generation of immobilized P-TEFb. Briefly, His-tagged recombinant
P-TEFb was biotinylated, repurified using a nickel column, and then
bound to streptavidin-coated paramagnetic beads. See "Experimental
Procedures" for details. The percentage of kinase activity remaining
is indicated in parentheses. B, quantitation of activity
remaining at the indicated steps in the process. An autoradiograph from
the kinase assay carried out using RNA polymerase II as substrate is
shown here. Indicated relative amounts of material at each step were
assayed. P-TEFb, unmodified P-TEFb used as starting
material; Biotin, mixture of biotinylated P-TEFb and free
biotin agent (an aliquot of the biotinylation reaction); Ni
col., nickel column flow-through (Free) or bound
(B) material; Strep. bead, paramagnetic
streptavidin beads unbound (Free) or bound (B)
material.
View larger version (44K):
[in a new window]
Fig. 5.
Analysis of the interaction of DRB and
flavopiridol with immobilized P-TEFb. A, kinase assays
using immobilized P-TEFb after preincubation with DRB or flavopiridol
as indicated. The concentrations of the drugs utilized in the assays
were 10-fold greater than the IC50 values of the unmodified
P-TEFb (90 µM DRB and 400 nM flavopiridol).
As described in detail in "Experimental Procedures," the beads were
washed quickly with 30 mM HGKE and then subjected to a
10-min kinase assay using RNA polymerase II as substrate. Drugs were
included and washes performed as indicated. B, effect of
raising salt concentration during the wash step. Immobilized P-TEFb was
incubated with DRB or flavopiridol as indicated and then subjected to
two quick washes with HGKE buffer containing 0, 30, 100, or 300 mM KCl. Kinase assays were for 10 min using RNA polymerase
II as substrate.
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Fig. 6.
Effect of DRB on flavopiridol binding to
immobilized P-TEFb. A, immobilized P-TEFb was
preincubated with the indicated amounts of flavopiridol or DRB, alone
or in combination. After two washes the beads were assayed in a
standard kinase assay with RNA polymerase II as substrate.
B, diagram of results showing that DRB does not inhibit the
binding of flavopiridol to immobilized P-TEFb.
-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 -amanitin, indicating that RNA
polymerase II transcription accounted for most of the transcription
seen (Fig. 7). Infection of cultured cells by HIV has been shown to be inhibited by flavopiridol
(IC50 8 nM), but at 10, 30 (Fig. 7,
A and B), and even 100 nM (Fig. 7C) flavopiridol, very little effect on cellular
transcription was seen. A different result was obtained when cells were
treated with 300 nM flavopiridol. Total transcription was
dramatically inhibited. This was attributed to inhibition of RNA
polymerase II, because transcription in the presence of -amanitin by
RNA polymerase I and III was unaffected (Fig. 7D). Global
inhibition of RNA polymerase II transcription was also seen when the
cells were treated with 50 µM DRB (Fig. 7E).
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Fig. 7.
Effect of flavopiridol and DRB on cellular
transcription. Nuclear run-on assays were performed on nuclei
isolated from control cells (circles) or cells treated for
1 h with the indicated amounts of flavopiridol or DRB
(squares). Nuclei from treated or untreated cells were
assayed in the absence (filled) or presence
(open) of -amanitin. Details are described under
"Experimental Procedures." Each timepoint was done in duplicate,
and error bars indicate the range of the two
determinations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENT |
|---|
We thank Grace Roberts (GlaxoSmithKline) for suggesting that the IC50 values we first reported might be reflecting the concentration of the P-TEFb in the reactions.
| |
FOOTNOTES |
|---|
* This work was supported by National Institute of Heath Grants AI43691 and GM35500.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence could be addressed. Tel.: 319-335-7910; E-mail: david-price@uiowa.edu.
Published, JBC Papers in Press, June 28, 2001, DOI 10.1074/jbc.M102306200
2 D. Renner, Y. Yamaguchi, T. Wada, H. Handa, and D. Price, submitted for publication.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
Cdk, cyclin-dependent kinase;
P-TEFb, positive transcription
elongation factor b;
DRB, dichloro-1--D-ribofuranosylbenzimidazole;
DSIF, DRB
sensitivity-inducing factor;
HIV, human immunodeficiency virus;
SDS-PAGE, SDS-polyacrylamide gel electrophoresis.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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