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Journal of Virology, February 2001, p. 1220-1228, Vol. 75, No. 3
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1220-1228.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Antiapoptotic Function of Cdk9 (TAK/P-TEFb) in U937
Promonocytic Cells
Shannon M.
Foskett,1
Romi
Ghose,1
Derek Ng
Tang,2
Dorothy E.
Lewis,2 and
Andrew P.
Rice1,*
Department of Molecular Virology and
Microbiology1 and Department of
Immunology,2 Baylor College of Medicine,
Houston, Texas 77030
Received 15 May 2000/Accepted 3 November 2000
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ABSTRACT |
Cdk9 is the catalytic subunit of TAK (cyclinT1/P-TEFb), a cellular
protein kinase that mediates human immunodeficiency virus type 1 (HIV-1) Tat transcriptional activation function. To examine Cdk9
function in cells relevant to HIV-1 infection, we used a murine
leukemia virus retrovirus vector to transduce and overexpress the cDNA
of a dominant negative mutant Cdk9 protein (Cdk9-dn) in Jurkat T cells
and U937 promonocytic cells. In Jurkat cells, overexpression of Cdk9-dn
specifically inhibited Tat transactivation and HIV-1 replication but
had no inhibitory effect on induction of CD69, CD25, and interleukin-2
following T-cell activation. In U937 cells, overexpression of Cdk9-dn
sensitized cells to apoptosis, especially after phorbol myristate
acetate (PMA) treatment to induce differentiation to macrophage-like
cells. Because Cdk9 function is induced in PMA-treated U937 cells, Cdk9
may play an antiapoptotic role during monocyte differentiation.
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INTRODUCTION |
Human immunodeficiency virus type 1 (HIV-1) requires the viral transactivator protein Tat for efficient
elongation of the integrated proviral genome by RNA polymerase II. Tat
acts by recruitment of a cellular protein kinase, TAK (Tat-associated
kinase), to the TAR RNA element in nascent viral transcripts (reviewed
in references 8 and 37). TAK is composed of
Cdk9 as the catalytic subunit, cyclin T1 (cycT1) as a regulatory
subunit, and possibly other subunits that remain to be identified
(30, 39, 42, 44). TAK is closely related to the general
elongation factor P-TEFb (reviewed in reference 31).
Multiple P-TEFb complexes exist in human cells that contain Cdk9 and
differ according to their regulatory cyclin subunit: cyclins T1, T2a,
T2b, and possibly cyclin K (6, 30, 39). P-TEFb/TAK is
thought to activate elongation by hyperphosphorylating the
carboxyl-terminal domain of RNA polymerase II, thereby relieving
repression by negative factors that limit processivity (38,
41). It has been well established that TAK, in the cycT1/P-TEFb
complex, mediates Tat function (2, 7, 9, 12, 17, 24, 28, 32, 39, 43, 44).
P-TEFb was originally identified through in vitro transcription studies
in Drosophila melanogaster nuclear extracts
(25-27). From these in vitro studies, P-TEFb appears to
be required for efficient elongation of many cellular promoters. A
recent immunofluorescence analysis of Drosophila polytene
chromosomes with antibodies against cycT1 indicates that P-TEFb is
likely to be involved in regulation of many, but not all,
Drosophila genes (23). In human cells, Cdk9 in
the cycT1/P-TEFb complex is required for transcription of major
histocompatibility class II (MHC II) genes (19). Other than MHC II genes, there is currently little information as to which
human genes require P-TEFb function in vivo.
The major target cells for HIV-1 infection are CD4+
lymphocytes and macrophages. Regulation of TAK activity in these cell
types is potentially important to HIV-1 pathogenesis, since TAK may be
a limiting factor for HIV-1 replication under some conditions. TAK is
induced by activation of either peripheral blood lymphocytes (PBL) or
purified primary CD4+ T lymphocytes (11, 14,
42). TAK is also induced following differentiation of the
promonocytic cell lines U937 and HL-60 by phorbol myristate acetate
(PMA) treatment (14, 42). These observations suggest that
TAK may have distinct functions during T-cell activation and monocyte
differentiation. Activation of TAK in PBLs involves an increase in
levels of mRNA and protein for both Cdk9 and cycT1 (11,
14). In contrast, PMA-induced differentiation of promonocytic
cell lines involves a large increase in cycT1 protein through a
posttranscriptional mechanism, whereas Cdk9 protein levels are constant
before and after treatment with PMA (14). The difference
in expression of Cdk9 and cycT1 between PBLs and promonocytic cell
lines may highlight a difference in function for the proteins between
the two cell types.
In this study, we examined potential roles of Cdk9 in the activation of
T cells and the differentiation of monocytes. We report here that the
overexpression of a dominant negative Cdk9 protein in activated Jurkat
T cells has no apparent effect on induction of CD25, CD69, or
interleukin-2 (IL-2), three molecules known to be important for T-cell
function. By contrast, overexpression of a dominant negative Cdk9
protein in the promonocytic cell line U937 caused the cells to become
sensitive to apoptosis, especially following PMA treatment to induce
differentiation, suggesting that Cdk9 may have an antiapoptotic
function during monocyte differentiation.
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MATERIALS AND METHODS |
Cells.
Human promonocytic U937 and Jurkat T cell lines were
grown in RPMI-1640 (Gibco BRL) supplemented with 10% fetal bovine
serum and antibiotics. Densities of cultures were maintained between 2 × 105 and 8 × 105 cells/ml. 293T
cells were cultured in Dulbecco modified Eagle medium with high glucose
(Gibco BRL) supplemented with 10% fetal bovine serum and antibiotics.
Generation of retroviral vectors.
The cDNA of a dominant
negative mutant Cdk9, Cdk9-dn (12), was inserted into a
murine leukemia virus (MLV)-based retroviral vector, pBABEMN IRES GFP
(derived from pBABE-puro; Mike Rothenberg, Stanford University),
upstream to an internal ribosome entry site (IRES) followed by the cDNA
for green fluorescent protein (GFP). Pseudotyped MLV particles were
produced in 293T cells by cotransfection of the pBabe/Cdk9-dn plasmid,
pHit60, which expresses the gag and pol genes of
MLV, and a plasmid that expresses the vesicular stomatitis virus G
protein (35, 36). One day prior to transfections, a
confluent 10-cm culture dish of 293T cells was split 1:5, and on the
day of transfection, 25 µg of each of three plasmids (pBABE-Cdk9-dn or parental pBABE/GFP, pVSV-G, and pHit60) was cotransfected by a
standard calcium phosphate procedure. After 8 h, the calcium phosphate precipitate was washed from cells and the cells were treated
with 10% dimethyl sulfoxide in phosphate-buffered saline (PBS) for 2 min. Cells were cultured in 10 ml of complete media for 72 h, and
culture supernatants containing retroviruses were collected. Retrovirus
preparations were stored at 
70°C until use.
Generation of cell lines expressing Cdk9-dn.
Jurkat T cells
or U937 cells were infected at a multiplicity of infection of 0.4 with
retroviral vectors, and at 3 days postinfection, GFP+ cells
with the top 10% fluorescence intensity were sorted by flow cytometry
(EPICS-Altra; Beckman-Coulter). Postsort Jurkat T-cell pools and U937
pools were approximately 85 and 93% GFP+, respectively.
These GFP+ populations are referred to as sorted pools.
Limiting dilution of pool populations was used to generate clonal cell lines.
Immunoblots.
Cells were washed with PBS and lysed in EBC
buffer (50 mM Tris-HCl [pH 8.0], 120 mM NaCl, 0.5% Nonidet P-40, 5 mM dithiothreitol) containing protease inhibitors as described
previously (16). Protein concentrations were determined by
the Bio-Rad protein assay, and 15 µg of total protein was analyzed on
sodium dodecyl sulfate-9% polyacrylamide gels. Immunoblotting was
preformed by standard procedures by using enhanced chemiluminescence
for detection as described previously (15). Antibodies for
detection of Cdk9, cyclin T1, and TATA-binding protein (TBP) were
purchased from Santa Cruz Biotechnology.
Plasmid transfection assays and HIV-1 infections.
For HIV-1
Tat transactivation assays, 106 Jurkat Cdk9-dn clonal cells
were cotransfected with 1 µg of an HIV-1 long terminal repeat (LTR)
luciferase reporter plasmid, 3 µg of a simian virus 40 early promoter
-galactosidase (-Gal) reporter plasmid (pCH110; Pharmacia), and
either 1 µg of pCMV-Tat-1 or 1 µg of pCMV parental vector
(33). Transfections were performed with Superfect
Transfection Reagent (Qiagen) using the manufacturer's suggested
protocol. Lysates were prepared 48 h posttransfection. Luciferase
assays were performed with the Luciferase Assay System (Promega), and light units were measured using a TD20-e luminometer (Turner). For
-Gal assays, 100 µl of lysates was used in a standard enzyme assay. For human T-cell leukemia virus type 1 (HTLV-1) Tax
transactivation assays, 106 cells were cotransfected with 1 µg of an HTLV-1 LTR luciferase reporter (provided by Susan Marriott,
Baylor College of Medicine) and either 1 µg of GW1-Tax
(provided by Ron Javier, Baylor College of Medicine) or parental
vector. Cells were harvested 48 h posttransfection, and luciferase
and -Gal assays were performed as above.
For HIV-1 infections, 6 × 106 Jurkat Cdk9-dn and
control vector clonal cell lines were infected with pretitered HIV-1
NL4-3 at a final concentration of 200 ng/ml of p24 antigen. At 4 h
postinfection, cells were washed three times with PBS plus 2% fetal
bovine serum. Culture media were collected, and p24 concentrations were
determined by enzyme-linked immunosorbent assay (ELISA)
(Beckman-Coulter, Hialeah, Fla.). Cells remained more than 90% viable
throughout infections as determined by trypan blue exclusion.
Measurement of T-cell activation markers.
Jurkat cells were
activated with PMA (1 ng/ml) and ionomycin (1 µM) for 18 h.
Alternatively, 4.4 µg of anti-CD3 (Pharmingen) in 0.2 M sodium
bicarbonate was bound to 6-well plates overnight at 4°C. Binding
buffer was removed, and 106 cells plus 4.4 µg of soluble
anti-CD28 (Pharmingen) in 3 ml of media were added for 24 h. Cells
were collected and washed twice with PBS. Cells were stained with
either phycoerythrin-conjugated anti-CD69 or anti-CD25 antibodies
(Becton Dickinson) on ice for 30 min. Samples were analyzed by flow
cytometry using a Beckman-Coulter XL-MCL cytometer. A total of 10,000 events were collected.
For IL-2 induction, 5 × 10
6 Jurkat cells were
activated with PMA plus ionomycin as described above. Aliquots of
culture medium
were removed over a time course, and the IL-2
concentration was
measured by ELISA according to the manufacturer's
protocol (Endogen).
Samples were stored at
70°C prior to
assaying.
Apoptosis measurements in U937 cells.
U937 Cdk9-dn sorted
pool and clones, control vector pool and clones, and parental U937
cells were treated with PMA (1 ng/ml) for 24 or 48 h as described
for each experiment. Cells were collected from the plate and washed
twice with PBS. Cells were then ethanol fixed and stained with
propidium iodide (PI) (Sigma) (50 µg/ml); cells were treated with
RNase A (Sigma) (1 mg/ml) for 30 min at 37°C prior to analysis. Cells
were analyzed as described above for CD69 and CD25 markers. Annexin V
(Pharmingen) staining was performed according to the manufacturer's recommendations.
Caspase-3 activity in cell lysates was determined using a caspase-3
assay kit following the manufacturer's protocol (Pharmingen).
Cdk9-dn
pool, vector control pool, and parental U937 cells were
treated with
PMA as described above for 12, 24, or 48 h. Cells
were harvested,
and frozen pellets were stored at
70°C until
lysates were prepared.
Fluorescence was measured using a spectrofluorometer
(Perkin-Elmer
LS50B) with excitation at 380 nm and the emission
wavelength at 420 to
460
nm.
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RESULTS |
Generation of Jurkat T-cell clonal cell lines overexpressing
dominant negative Cdk9 protein.
To investigate potential roles of
Cdk9 in T-cell activation events, we used an MLV-based retroviral
vector to transduce the cDNA of a dominant negative Cdk9 mutant protein
(Cdk9-dn) into Jurkat T cells. The dominant negative Cdk9 protein
contains a substitution of asparagine for aspartic acid at residue 167, rendering the protein catalytically inactive (10). Upon
overexpression, the Cdk9-dn protein inhibits wild-type (wt) Cdk9
function as demonstrated by specific inhibition of Tat transactivation
of the HIV-1 LTR (12, 24). The mRNA expressing the Cdk9-dn
protein from the MLV LTR contains an IRES element at its 3' end
followed by the coding sequence of the GFP, allowing GFP to be used as
a marker for transduction of the Cdk9-dn cDNA. Cdk9-dn expression can
be distinguished from endogenous Cdk9 expression by incorporation of
the FLAG-epitope tag at the amino terminus of Cdk9-dn.
Jurkat T cells were infected with MLV vectors expressing both Cdk9-dn
and GFP or the parental MLV vector expressing only GFP.
Three days
postinfection, transduced cells that expressed GFP
were sorted by flow
cytometry. Clonal cell lines were generated
from the sorted pool by
limiting dilution. Forty clones transduced
with the Cdk9-dn cDNA were
evaluated in immunoblots for levels
of expression of the catalytic
mutant protein, and all clones
were found to express the Cdk9-dn
protein (data not shown). From
these clones, three cell lines
expressing the highest levels of
Cdk9-dn were chosen for subsequent
experiments. Five clones from
infection with the parental MLV vector
expressing GFP alone were
also generated, and two of these control cell
lines were used
for experiments. An immunoblot analysis of the three
Cdk9-dn and
two control cell lines is shown in Fig.
1. By quantitative Western
blot analysis
using twofold serial dilutions of cell extracts,
we estimate that
Cdk9-dn levels in clones 3, 30, and 43 were greater
than fourfold more
than those of endogenous Cdk9 (data not shown).
We observed no evidence
during the course of this study that expression
of the Cdk9-dn protein
had a negative effect on Jurkat T-cell
growth, since the percentages of
GFP-positive cells in the sorted
pool and clonal cell lines were stable
over a 3-month period.
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FIG. 1.
Jurkat T-cell clonal lines overexpress FLAG-Cdk9-dn.
Immunoblots to detect Cdk9 proteins were performed on lysates from the
indicated cell lines. FLAG-tagged Cdk9-dn and endogenous Cdk9 proteins
are indicated.
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In the immunoblots shown in Fig.
1 and
7, both the endogenous wt and
epitope-tagged Cdk9-dn proteins often resolved into a
pair of bands. We
have observed that resolution of Cdk9 proteins
into a doublet is
variable, and in some immunoblots only a single
form of Cdk9 was
observed. The explanation for Cdk9 proteins with
different
electrophoretic mobilities is not known at this time,
although it is
possible that these forms represent different phosphorylation
states of
Cdk9.
Cdk9-dn overexpression in Jurkat T cells inhibits HIV-1 LTR
transactivation by Tat.
To examine the effect of overexpression of
the catalytic mutant on Tat function, the Cdk9-dn clonal lines 3 and 30 and control clonal lines 7 and 10 were used in plasmid cotransfection
assays. The results shown in Fig. 2
indicate that Tat transactivation of the HIV-1 LTR was reduced more
than threefold in both Cdk9-dn lines relative to both control lines.
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FIG. 2.
Effect of Cdk9-dn overexpression on transactivation by
HIV-1 Tat and HTLV-1 Tax proteins. The indicated Jurkat Cdk9-dn or
vector control clonal lines were transfected with an HIV-1 LTR
luciferase reporter plasmid plus either a Tat expression or vector
control plasmid and a -Gal internal reference plasmid. Extracts were
prepared at 48 h posttransfection, luciferase expression was
normalized to -Gal expression, and Tat transactivation was
calculated. Jurkat Cdk9-dn or vector control clones were also
transfected with an HTLV-1 LTR luciferase reporter plasmid plus either
a Tax expression or vector control plasmid and a -Gal internal
reference plasmid. Tax transactivation was determined as described for
Tat transactivation.
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To examine the specificity of inhibition by Cdk9-dn overexpression, we
examined transactivation of the HTLV-1 LTR by its Tax
transactivator
protein. The Jurkat clonal cell lines were cotransfected
with an HTLV-1
luciferase reporter plasmid and a Tax expression
plasmid, and
luciferase expression was determined as described
above (Fig.
2).
Consistent with previous work (
12), overexpression
of the
Cdk9-dn protein did not inhibit Tax transactivation, since
activation
of the HTLVI LTR by Tax was similar between the Cdk9-dn
and control
cell
lines.
We also examined replication of HIV-1 NL4-3 over a 12-day time course
in Cdk9-dn cell lines 3 and 30 and vector control cell
lines 7 and 10. As determined by p24 content in culture supernatants,
HIV-1 replication
in the Cdk9-dn cell lines was significantly
below that in the control
cell lines (Fig.
3). Cdk9-dn clone 3
showed the lowest levels of HIV-1 replication, more than 185-fold
and
25-fold below those of control lines 7 and 10, respectively,
at days 8 and 12. Cdk9-dn clone 30 showed HIV-1 replication levels
that were at
least 14-fold less than those for control clone 7
at days 8 and 12 and
5-fold and approximately 3-fold less than
those for control clone 10 at
days 8 and 12, respectively. These
results confirm those of a previous
study in which the Cdk9-dn
protein was shown to inhibit HIV-1
replication in Jurkat T cells
(
5). We used flow cytometry
to examine CD4 expression levels
in the Cdk9-dn and control cell lines
used in the experiment presented
in Fig.
3. The percentages of cells in
each clonal line expressing
CD4 were similar, and the mean fluorescent
intensities of the
phycoerythrin-conjugated anti-CD4 antibody bound to
cells indicated
that all clonal lines expressed comparable amounts of
CD4 on the
cell surface (data not shown). It is therefore unlikely that
reduced
replication of HIV-1 in the Cdk9-dn cell lines is the
consequence
of reduced levels of CD4 on the cell surface. Taken
together,
the results shown in Fig.
2 and
3 indicate that the level of
the
Cdk9-dn protein in these clonal cell lines is sufficient to inhibit
Tat transactivation, thereby indicating that the function of the
wt
Cdk9 protein is inhibited.
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FIG. 3.
HIV-1 NL4-3 replication in Jurkat Cdk9-dn and vector
control clonal cell lines. The indicated cell lines were infected with
HIV-1 pNL4-3, and p24 levels in culture media were measured by ELISA at
the indicated time points.
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Cdk9-dn overexpression does not inhibit induction of CD69, CD25, or
IL-2 in activated Jurkat cell lines.
To determine whether Cdk9-dn
overexpression affects events associated with Jurkat T-cell activation,
Jurkat clonal cell lines were activated with PMA plus ionomycin or
anti-CD3 plus anti-CD28 antibodies, and induction of the activation
cell surface markers CD69 and CD25 was evaluated by flow cytometry
(Fig. 4). The results demonstrated no
significant effect of overexpression of the Cdk9-dn protein on
induction of CD25 or CD69 by either activation protocol. Although there
was some clonal variability, each Cdk9-dn clonal line demonstrated
induction of both markers to levels that are equivalent to those seen
for the vector control clonal line 7. In other experiments, control
clonal line 10 was found to express CD25 and CD69 after activation to
levels similar to those shown for the cell lines used for Fig. 4 (data
not shown).
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FIG. 4.
Induction of CD69 and CD25 in Jurkat T-cell lines. The
indicated Jurkat clones were activated either with anti-CD3 plus
anti-CD28 for 24 h or with PMA plus ionomycin for 18 h.
Expression of CD25 and CD69 were measured by flow cytometry. Black
areas represent marker expression before activation, and white areas
represent marker expression after activation. Fold induction values, as
determined by comparison of mean fluorescence intensities, of each
marker are the following: 6.4 (A), 3.7 (B), 37.6 (C), 204.3 (D), 2.4 (E), 2.1 (F), 21.3 (G), 192.0 (H), 4.8 (I), 10.5 (J), 10.4 (K), 459.9 (L), 10.6 (M), 12.2 (N), 43.2 (O), and 33.1 (P).
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Induction of IL-2, a hallmark of T-cell activation, was also measured
in the Cdk9-dn clonal cell lines. Cells were activated
with PMA plus
ionomycin, culture supernatants were removed over
a 24-h time course,
and IL-2 levels were determined by ELISA (Fig.
5). Each of the Cdk9-dn clonal lines and
the vector controls showed
strong induction of IL-2, indicating that
overexpression of the
Cdk9-dn protein had no significant inhibitory
consequence for
IL-2 induction. Taken together, these results suggest
that Cdk9
function is not required for induction of CD25, CD69, or IL-2
following activation of Jurkat T cells.
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FIG. 5.
Induction of IL-2 in Jurkat T cell lines. The indicated
Cdk9-dn and vector control Jurkat cell lines were activated with PMA
plus ionomycin. The concentration of IL-2 in the culture medium was
measured for duplicate samples by ELISA.
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Cdk9-dn expression renders U937 cells sensitive to apoptosis.
We were also interested in investigating whether Cdk9 may have a role
in monocyte differentiation. We therefore used the MLV retroviral
vector to transduce the Cdk9-dn cDNA into U937 promonocytic cells. U937
clonal lines expressing the Cdk9-dn protein were then generated by
limiting dilution. We observed under light microscopy that many of
these clonal cell lines spontaneously contained irregularly shaped
cells and the culture media contained high amounts of membranous debris, indicative of apoptosis. When treated with PMA to induce differentiation, many of the U937 clonal lines transduced with the
Cdk9-dn cDNA produced massive membrane blebs and cell debris in the
culture medium. To examine apoptosis in these clonal cell lines, PI
staining and flow cytometry were used to monitor cells with a
subdiploid DNA content (Fig. 6). Without
PMA treatment, for the parental U937 cells and the vector control clone
2, less than 1% of cells were in the apoptotic subdiploid DNA region, whereas for Cdk9-dn clones 6 and 11, approximately 7% of cells were in
the subdiploid DNA region. Upon PMA treatment for 24 h, this
percentage increased to 1.7 and 1.3 for the parental U937 cells and the
vector control clone 2, respectively, whereas the Cdk9-dn clonal lines
6 and 11 increased to 26.2 and 18.4%, respectively. By 48 h of
PMA treatment, the Cdk9-dn clonal lines contained massive cellular
debris and too few cells for analysis.
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FIG. 6.
PI staining of U937 clonal cell lines. Parental U937,
MLV vector control, and Cdk9-dn clonal lines 6 and 11 were treated with
PMA (1 ng/ml) for 24 h. DNA content was analyzed by PI staining
and flow cytometry. The subdiploid DNA regions are gated, and the
percentages of apoptotic cells are indicated.
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We chose not to further analyze these U937 clonal lines
expressing the Cdk9-dn protein because of concern over selection
pressures
that might result in second site mutations to allow survival
of
the cell lines. Therefore, we examined pools of U937 cells
expressing
the Cdk9-dn protein that were obtained by cell sorting. An
immunoblot
analysis of a sorted U937 pool demonstrates that Cdk9-dn
protein
is expressed at a relatively high level in these cells (Fig.
7).
We also evaluated expression levels
of cycT1 and TBP (
20) in
U937 pools and Jurkat clonal
lines that overexpress the Cdk9-dn
protein. Although there was some
variation in the levels of cycT1
(lane 3) and TBP (lane 6) in some
cells, there was no consistent
effect of Cdk9-dn expression on the
level of cycT1 or TBP.
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FIG. 7.
Expression of wt and Cdk9-dn, cyclin T1, and TBP
proteins in U937 sorted pools and Jurkat clonal lines. Immunoblots were
performed to measure Cdk9, cyclin T1, and TBP expression in lysates of
U937 parental cells or sorted U937 cell pools transduced with Cdk9-dn
cDNA or vector control and Jurkat Cdk9-dn cell lines 3 and 30 and
Jurkat control lines 7 and 10. FLAG-tagged Cdk9-dn and endogenous
proteins are indicated.
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Using PI staining and flow cytometry to measure subdiploid DNA content,
we observed very little difference among non-PMA-treated
parental U937,
the Cdk9-dn pool, and the MLV vector control pool
(Fig.
8). After 48 h of PMA treatment,
however, the Cdk9-dn pool
contained approximately threefold more cells
in the subdiploid
DNA region than either the vector control pool or the
parental
U937 cells. No significant difference was observed between the
PMA-treated vector control pool and the parental U937 cells.
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FIG. 8.
PI staining of U937 pool expressing Cdk9-dn protein.
Parental U937 or sorted pools of vector control U937 or Cdk9-dn U937
cells were treated with PMA for 48 h. DNA content was analyzed
with PI and flow cytometry. The subdiploid DNA regions are gated, and
the percentages of apoptotic cells are indicated.
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To confirm that the increase in subdiploid DNA content in the U937
Cdk9-dn pool was the result of apoptosis, PMA-treated cells
were
examined for PI and Annexin V binding. Annexin V staining
is indicative
of apoptosis, since it binds to phospholipid phosphatidylserine
that is
translocated to the cell surface at an early stage of
apoptosis. As
shown in Table
1, PMA treatment resulted
in an
increase in both PI and Annexin V staining for the Cdk9-dn pool
relative to the vector control pool. After 48 h of PMA treatment,
the Cdk9-dn pool contained 4.5-fold more cells with a subdiploid
DNA
content and 4-fold more Annexin V-positive cells than the
vector
control pool.
To further confirm that expression of the Cdk9-dn protein renders U937
cells sensitive to apoptosis upon treatment with PMA,
we measured
activation of caspase-3. Caspase-3 is a protease which
is an early
marker of apoptosis and cleaves other caspases and
additional protein
targets in the cytoplasm and nucleus. We observed
a sixfold increase in
caspase-3 activity in extracts from the
PMA-treated Cdk9-dn pool
relative to extracts from the vector
control pool or parental U937
cells (Fig.
9). No difference in
caspase-3 activity was seen between the vector control pool and
the
parental U937 cells. The results shown in Fig.
6,
8, and
9 and Table
1
indicate that expression of the Cdk9-dn protein renders
U937 cells
sensitive to apoptosis after PMA treatment.
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FIG. 9.
Caspase-3 activity in extracts of U937 cells. Parental
U937 cells, sorted pools of vector control U937 cells, or Cdk9-dn U937
cells were either untreated (top) or treated with PMA. Lysates were
prepared at 48 h posttreatment, and caspase-3 activity was
determined by the ability to cleave a synthetic tetrapeptide
fluorogenic substrate, Ac-DEVD-AMC, to release fluorescent
7-amino-4-methylcoumarin, which is measured by a spectrofluorometer.
Excitation wavelength was at 380 nm, and the emission was read between
420 and 460 nm.
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To examine whether expression of the Cdk9-dn protein might render
Jurkat T cells sensitive to apoptosis, we also analyzed
the Jurkat
Cdk9-dn and control clonal lines used in the experiments
presented in
Fig.
1 to
5 and described above. To induce apoptosis
in Jurkat cells,
cultures were treated with anti-Fas antibodies
or PMA plus ionomycin,
and apoptosis was evaluated by both PI
and Annexin V staining. We found
no significant difference in
the levels of spontaneous or induced
apoptosis between the Cdk9-dn
and control clonal cell lines (data not
shown).
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DISCUSSION |
Because small molecular inhibitors of Cdk9 can inhibit HIV-1
replication in vitro at concentrations that are not toxic to cells, it
has been suggested that the development of selective inhibitors of Cdk9
might lead to novel chemotherapeutic agents against HIV-1
(5). The elucidation of normal cellular functions of Cdk9
in CD4+ T cells and monocytes/macrophages is therefore
important for evaluating the feasibility of Cdk9 as a therapeutic
target. Additionally, elucidation of Cdk9 function in T cells and
monocytes/macrophages may be important for understanding HIV-1
pathogenesis, since infection may perturb these functions. In this
study, we utilized a dominant negative Cdk9 mutant protein to
investigate roles of Cdk9 in Jurkat T and U937 promonocytic cell lines.
We found that overexpression of the Cdk9-dn protein in U937 cells
renders cells sensitive to apoptosis, especially following PMA
treatment to induce differentiation. This finding suggests that Cdk9
may have an antiapoptotic function during monocyte differentiation.
Jurkat cell lines expressing the Cdk9-dn protein were found not to be
more sensitive to apoptosis than control Jurkat cell lines (data not
shown). However, before conclusions can be drawn about the cell-type
specificity of regulation of apoptosis by Cdk9, additional lymphoid
cell lines and other cell types will have to be examined.
The ability to readily undergo apoptosis is important to monocyte
homeostasis, since monocytes normally circulate in the blood for a
period of only a few days, during which time they either emigrate to
tissues and differentiate to macrophages or die through apoptosis
(13, 21). Previous studies of U937 and HL-60 cells showed
that Cdk9 catalytic activity is low in promonocytic cells due to
limiting amounts of the cycT1 regulatory subunit (14, 42).
The findings in this study suggest that a low level of cycT1 protein in
monocytes, and therefore a low level of Cdk9 function, may be important
for their requirement to readily undergo apoptosis in the absence of
differentiation. The mechanisms responsible for the increased rate of
apoptosis in U937 cells expressing the Cdk9-dn protein remain to be established.
The antiapoptotic function of Cdk9 could be due to a direct role in an
apoptosis pathway or to a block in the differentiation program of
monocytes by the Cdk9-dn protein. Previous work on monocyte
differentiation indicates that the cells have an intrinsic program to
differentiate when apoptosis is blocked by enforced expression of Bcl-2
(21). This might suggest that Cdk9 functions in the P-TEFb
complex to regulate transcription of genes, such as the gene for
p21WAF1, whose expression confers resistance to apoptosis
prior to monocyte differentiation (1, 40). An additional
possibility is that Cdk9 function is necessary for the differentiation
program of monocytes.
Our experiments with Jurkat T cells indicate that overexpression of a
Cdk9-dn protein to levels that inhibit Tat function does not inhibit
induction of CD69, CD25, and IL-2 following T-cell activation. Because
induction of each of these corresponding genes occurs at the
transcriptional level (3, 4, 18, 22, 34, 45), our rather
surprising results suggest that Cdk9 in the P-TEFb complex is not
required for their transcriptional induction. Since Tat has been shown
to sequester P-TEFb activity from MHC II genes (19), a
requirement for P-TEFb in the transcription of IL-2 would be expected
to have been detectable in our experiments. The lack of a requirement
for Cdk9 for IL-2 production in this study is consistent with the
observation that HIV-1 infection and Tat expression can actually
superinduce IL-2, suggesting that sequestration of Cdk9 and cyclin T1
in a complex with Tat does not inhibit some basic T-cell functions
(29).
Cdk9 in the P-TEFb complex is thought to be a general elongation factor
required for expression of many genes. We were therefore somewhat
surprised to observe that in Jurkat T cells transduced with the Cdk9-dn
cDNA, the percentages of GFP+ cells and therefore Cdk-9dn
expression in sorted pools and clonal lines were stable over a 3-month
period. It may be informative to utilize DNA microarray technology to
investigate how overexpression of Cdk9-dn affects global gene
expression in both activated and nonactivated Jurkat T cells.
The results of this study suggest that Cdk9 function is likely to
be crucial to the monocyte/macrophage life cycle. This raises the
possibility that HIV-1 infection might perturb normal monocyte homeostasis through sequestering Cdk9 for Tat function, leading to
pathogenic consequences. Because Cdk9 appears to play an important role
in monocyte apoptosis and differentiation, it may not be a feasible
therapeutic target in treatment of HIV-1 infection.
| |
ACKNOWLEDGMENTS |
We thank Richard Sutton for advice on retroviral vectors and Jeff
Scott for cell sorting and flow cytometry analysis.
The work was supported by grant AI35381 (A.P.R.) and the Center of AIDS
Research at BCM (AI 36211) from the National Institutes of Health.
S.M.F. was supported by Training Grant T32 AI07471 from the National
Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Mail Stop BCM-385, Houston, TX 77030. Phone: (713) 798-5774. Fax: (713) 798-3490. E-mail:
arice{at}bcm.tmc.edu.
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Journal of Virology, February 2001, p. 1220-1228, Vol. 75, No. 3
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1220-1228.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
