|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2896-2904
Effects of Guanine Nucleotide Depletion on Cell Cycle
Progression in Human T Lymphocytes
By
Josée Laliberté,
Ann Yee,
Yue Xiong, and
Beverly S. Mitchell
From the Departments of Pharmacology, Biochemistry, and Biophysics,
University of North Carolina at Chapel Hill, Chapel Hill, NC; the
Department of Medicine, University of North Carolina at Chapel Hill,
Chapel Hill, NC; and the Lineberger Comprehensive Cancer Center,
University of North Carolina at Chapel Hill, Chapel Hill, NC.
 |
ABSTRACT |
Depletion of guanine nucleotide pools after inhibition of inosine
monophosphate dehydrogenase (IMPDH) potently inhibits DNA synthesis by
arresting cells in G1 and has been shown to induce the differentiation
of cultured myeloid and erythroid cell lines, as well as chronic
granulocytic leukemic cells after blast transformation. Inhibitors of
IMPDH are also highly effective as immunosuppressive agents. The
mechanism underlying these pleiotropic effects of depletion of guanine
nucleotides is unknown. We have examined the effects of mycophenolic
acid (MPA), a potent IMPDH inhibitor, on the cell cycle progression of
activated normal human T lymphocytes. MPA treatment resulted in the
inhibition of pRb phosphorylation and cell entry into S phase. The
expression of cyclin D3, a major component of the cyclin-dependent
kinase (CDK) activity required for pRb phosphorylation, was completely
abrogated by MPA treatment of T cells activated by interleukin-2 (IL-2)
and leucoagglutinin (PHA-L), whereas the expression of cyclin D2, CDK6,
and CDK4 was more mildly attenuated. The direct kinase activity of a
complex immunoprecipitated with anti-CDK6 antibody was also inhibited. In addition, MPA prevented the IL-2-induced elimination of
p27Kip1, a CDK inhibitor, and resulted in the retention of
high levels of p27Kip1 in IL-2/PHA-L-treated T cells bound
to CDK2. These results indicate that inhibition of the de novo
synthesis of guanine nucleotides blocks the transition of normal
peripheral blood T lymphocytes from G0 to S phase in early- to mid-G1
and that this cell cycle arrest results from inhibition of the
induction of cyclin D/CDK6 kinase and the elimination of
p27Kip1 inhibitory activity.
| |
INTRODUCTION |
INOSINE-5'-MONOPHOSPHATE
dehydrogenase (IMPDH; EC 1.1.1.205) is a rate-limiting
enzyme required for the de novo synthesis of guanine ribo- and
2'-deoxyribonucleotides. This enzyme has been shown to have higher
activity in proliferating than in quiescent cells1,2 and
inhibitors of IMPDH are used as chemotherapeutic and, more recently, as
highly effective immunosuppressive agents.3-6 Inhibition of
IMPDH activity has also been shown to induce the differentiation of
cultured myeloid and erythroid cell lines,7-9 and
preliminary studies have indicated that inhibition of IMPDH activity
can lead to the differentiation of blast cells in patients with chronic
myelocytic leukemia (CML) in blast
crisis.10,11 However, the mechanism whereby depletion of
guanine nucleotides by IMPDH inhibitors limits cell
proliferation and/or induces differentiation is unknown.
Studies with mizoribine, an IMPDH inhibitor and clinically effective
immunosuppressive agent,12 have shown that depletion of
guanine nucleotides in T lymphocytes inhibits the entry of these cells
on activation into the S phase of the cell cycle without inhibiting
early G1 events such as expression of c-Myc, c-Myb, interleukin-2
(IL-2), or the IL-2 receptor.13 These results imply that
guanine nucleotide depletion may have direct consequences on cell cycle
progression in G1 that affect the ability of cells to commit to cell
division.
The objective of the present study is to elucidate the mechanism(s) by
which guanine nucleotide pool depletion triggers cell-cycle arrest at
or before the point of initiation of DNA synthesis. The control of
cell-cycle progression is finely ordered and regulated through a series
of events that function in part to prevent cells with inadequate
metabolites for replication from entering S phase.14-16 A
major component of this control is the phosphorylation status of the Rb
protein, which in turn is dependent on the activities of a series of
cyclin-dependent kinases (CDKs). Phosphorylation of pRb by CDK4/6 in
association with the D cyclins and by CDK2 in association with cyclin E
results in release of the E2F-family of transcription factors and the
increased expression of genes encoding enzymes required for DNA
synthesis.15,17-21 The cell cycle is also negatively
regulated by a series of cyclin-dependent kinase inhibitors (CKIs),
which fall into two classes: the INK4 family (p15, p16, p18, and p19)
and the p21 family (p21Waf1, p27Kip1,
p57Kip2).22-24 One of the roles of these
inhibitors is the tight regulation of the cyclin D/CDK complex in G1
and it is only when the level of cyclin D/CDK exceeds the inhibitory
threshold that the kinase activity is measurable and cells progress
through the restriction point.20,23
To elucidate the mechanisms underlying cell-cycle arrest in response to
depletion of guanine nucleotides in nontransformed cells, we have
examined the effect of the IMPDH inhibitor mycophenolic acid (MPA) on
cell-cycle progression in activated normal human T lymphocytes. It was
our goal to determine whether depletion of this important metabolic
pool leads to a specific effect on pRb phosphorylation and whether such
an effect might be mediated by alterations in CDK activities
and/or CKI expression.
| |
MATERIALS AND METHODS |
Reagents.
RPMI 1640 tissue culture medium, penicillin, streptomycin, fetal bovine
serum (FBS), and interleukin-2 (IL-2) were purchased from Gibco BRL
(Grand Island, NY). Leucoagglutinin (PHA-L), MPA, 1- -D-arabinofuranosylcytidine (ara-C), hydroxyurea, aphidicolin, 5-fluorouracil (5-FU), and propidium iodide were purchased from Sigma
(St Louis, MO) and 8-aminoguanosine from Calbiochem (La Jolla,
CA).
Isolation of peripheral blood T lymphocytes.
Buffy coats from normal donors were obtained from the American Red
Cross (Charlotte, NC) and the mononuclear cells were isolated by
density-gradient centrifugation using Histopaque 1077 (Sigma). Cells at
the interface were removed, washed with phosphate-buffered saline
(PBS), and resuspended in RPMI 1640 medium containing 10% heat-inactivated FBS, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. Monocytes were depleted by culture dish adherence. For
metabolic labeling with [35 S]methionine, cells were
washed once with prewarmed PBS, and resuspended in labeling medium
(methionine-cysteine-free Dulbecco's modified Eagles medium [ICN,
Costa Mesa, CA]) supplemented with 10% dialyzed FBS (Gibco). After 30 minutes of incubation with the labeling medium,
[35S]methionine (ICN) was added to the medium (100 to 125 µCi/mL) and the incubation continued for 4 hours before lysis.
Cell culture.
Cells were cultured at a density of 2.5 × 106/mL and
were stimulated by adding 10 U/mL of IL-2 and 5 µg/mL of PHA-L.
Incorporation of [3H]thymidine (Amersham, Arlington
Heights, IL) into DNA in resting and activated cells was determined as
a measure of proliferation. Cells were collected by centrifugation at
various times after treatment with 1 µmol/L MPA, 50 µmol/L
guanosine and 100 µmol/L 8-aminoguanosine, 250 µmol/L hydroxyurea,
10 µmol/L ara-C or 1 µg/mL aphidicolin.
Cell-cycle analysis.
For flow-cytometric analysis (FACS) of DNA content, 5 × 106 cells were suspended in 5 mL of cold 70% ethanol and
stored at  20°C until FACS analysis. Cells were spun down,
rinsed once with 2% bovine serum albumin (BSA) in PBS, and resuspended
in 0.5 mL of PBS. The day before analysis, 50 µg of propidium iodide
and 100 µg of RNase were added to each sample. DNA fluorescence was measured by FACS using a FACScan flow (Becton-Dickinson
Immunocytometry, Mountain View, CA) and percentages of cells within the
G1, S, and G2/M phases of the cell cycle were determined by the ModFit Cell-Cycle Analysis program (Verity Software, Topsham,
ME).
Immunoblots.
T lymphocytes were washed with ice-cold PBS and collected in
microcentrifuge tubes for lysis. The lysis buffer contained 50 mmol/L
Tris-HCl pH 7.5, 150 mmol/L NaCl, 0.5% NP-40, 1 mmol/L Na3VO3, 1 mmol/L dithiolthreitol (DTT), 50 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 25 µg/mL
leupeptin, 1 mmol/L benzamidine, 10 µg/mL trypsin inhibitor, and, for
the pRb immunoblot, 10 nmol/L okadaic acid. After centrifugation at
15,000g for 10 minutes, the protein content in the supernatant
was assayed by the Bradford method using Coomassie dye (Biorad) and BSA
as standard. Equal amounts of protein were separated on a sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [7.5%
for pRb, 15% for p27Kip1, and 12.5% for all other
separations] and transferred to polyvinylidene difluoride (PVDF)
membranes (Immobilon, Bedford, MA). The membrane was
blocked with 5% nonfat milk in 0.1% Tween 20 in TBS (TBST) and
incubated overnight with primary antibody. Primary mouse monoclonal antibodies against human pRb and p53 (Pharmagen, San Diego, CA) were
used at 1 µg/mL in blocking solution. All other antibodies were
rabbit polyclonal antisera as previously described.25,26 Membranes were then washed five times with TBST (0.05% to 0.2% Tween)
and the appropriate secondary antibody (1:2,000 to 1:10,000 dilution of
either horseradish peroxidase-linked sheep antimouse immunoglobulin or
horseradish peroxidase-linked donkey antirabbit immunoglobulin
[Amersham]). Detections were performed using the ECL
chemiluminescence system (Amersham).
Immnunoprecipitation.
Cells were collected, washed, and lysed as in the immunoblot method.
After protein determination, identical amounts of protein from the
lysates were immunoprecipitated with the corresponding antibody. For
peptide competition, 1 µg of synthetic peptide was preincubated with
1 µL of specific antibody (2 µg of peptide were used with affinity
purified antibody) before immunoprecipitation. Protein A agarose
(Pierce, Rockford, IL or Gibco) was then added and immunoprecipitates
were washed three to four times with lysis buffer, resuspended in
sodium dodecyl sulfate (SDS) sample buffer, and separated on
SDS-polyacrylamide gels. For [35S]methionine-labeled
extracts, total cell lysates from identical numbers of cells were
immunoprecipitated and polyacrylamide gels were enhanced with Enhancer
(Dupont, Newtown, CT) before exposure to radiograph films at
70°C.
Cyclin-dependent kinase assay.
The procedure for the CDK kinase assay was performed as
described.26 Samples were immunoprecipitated for 2 hours as
described above. For CDK6 kinase activity, affinity-purified antibodies were used. After addition of protein A agarose, beads were washed twice
with cold (4°C) lysis buffer and twice with cold kinase buffer (50 mmol/L HEPES, pH 7, 10 mmol/L MgCl2 , 5 mmol/L
MnCl2, and 1 mmol/L DTT). For the last wash, 5 µmol/L
adenosine triphosphate (ATP) was added. Reactions were initiated by
adding substrate protein (histone H1 or GST-Rb) and 5 µCi of [ -32P]ATP (Amersham, 3000 Ci/mmol) in a final
volume of 30 µL. Samples were incubated for 30 minutes at 30°C.
The phosphorylated proteins were then electrophoresed on
SDS-polyacrylamide gels and the gels stained with Coomassie blue,
dried, and exposed to radiograph films at 70°C.
| |
RESULTS |
Mycophenolic acid (MPA) induces a G1 arrest in activated T lymphocytes.
To determine the effect of the highly specific IMPDH inhibitor MPA on
cell-cycle progression, flow-cytometry analysis of stimulated T
lymphocytes was performed in the absence or presence of 1 µmol/L drug. Stimulation of human T lymphocytes with IL-2 and PHA-L for 48 hours results in the cell-cycle distribution shown in
Fig 1B with 64% of the cells in G1 and
29% in S phase. [H3]Thymidine incorporation data are
consistent with these results, showing DNA synthesis after 30 hours of
stimulation with IL-2 and PHA-L (data not shown). Treatment with 1 µmol/L MPA arrested cell-cycle progression and resulted in the
accumulation of 99% of the cells in G1 (Fig 1C). The addition of
guanosine and 8-aminoguanosine to the cells treated with MPA, at
concentrations previously shown to replete guanine nucleotide pools via
the salvage pathway,27 prevented the G0/G1 block and
allowed the induction of DNA synthesis as judged both by FACS analysis
(Fig 1D) and by [H3]thymidine incorporation studies (data
not shown). Of interest, however, is the observation that these cells
remained in S phase without entry into G2/M for up to 72 hours.
View larger version (33K):
[in this window]
[in a new window]
| Fig 1.
FACS analysis of the effects of MPA on cell cycle
progression in stimulated human T lymphocytes: (A) Resting T
lymphocytes; (B) T lymphocytes stimulated with IL-2/PHA-L for 48 hours;
(C) T lymphocytes stimulated with IL-2/PHA-L and cocultured with 1 µmol/L of MPA; and (D) T lymphocytes stimulated with IL-2/PHA-L and
cocultured with 1 µmol/L of MPA, 50 µmol/L of guanosine and 100 µmol/L of 8-aminoguanosine under conditions previously shown to
increase guanine nucleotide pools.13 Cells were analyzed by
FACS for DNA content and cell-cycle distribution, as outlined in the
Materials and Methods section.
|
|
Inhibition of pRb phosphorylation by MPA.
Figure 2 shows the effects of stimulation
of peripheral blood T lymphocytes with IL-2 and PHA-L on the
phosphorylation status of pRb. Stimulation for 24 hours led to several
hyperphosphorylated forms of pRb, as evidenced by multiple bands on the
immunoblot (Fig 2, lane 2). The presence of 1 µmol/L MPA (Fig 2, lane
3) prevented the phosphorylation of pRb, showing that MPA arrests the
cell cycle by inhibiting the activity of the kinases responsible for
the pRb phosphorylation before the onset of DNA synthesis. The addition
of guanosine and 8-aminoguanosine prevented the effect of MPA on pRb
phosphorylation (Fig 2, lane 4). Ara-C slightly inhibited the
phosphorylation of pRb, whereas hydroxyurea and aphidicolin, known
inhibitors of DNA synthesis, did not inhibit Rb phosphorylation ( Fig
2, lanes 5 to 7).
View larger version (21K):
[in this window]
[in a new window]
| Fig 2.
Effect of MPA on pRb phosphorylation. Immunoblot analysis
of pRb in resting peripheral blood T lymphocytes (lane 1), T
lymphocytes stimulated with IL-2/PHA-L alone (lane 2) or in combination
with 1 µmol/L MPA (lane 3), 1 µmol/L MPA plus 50 µmol/L guanosine
and 100 µmol/L 8-aminoguanosine (lane 4), 250 µmol/L hydroxyurea
(lane 5), 10 µmol/L ara-C (lane 6), and 1 µg/mL aphidicolin (lane
7). Cells were harvested after 24 hours of treatment and drugs were added at time 0.
|
|
Inhibition of CDK2- and CDC2-associated kinase
activities by MPA.
Because the activation of CDK2 by cyclin E, and eventually by cyclin A,
is essential for the G1/S transition,28 we performed in
vitro enzymatic assays of immunoprecipitated CDK2 and CDC2 using
histone H1 as a substrate. As shown in Fig
3, CDK2 activity was detected at a low level in resting cells, most
likely due to some degree of intrinsic activation. CDK2 and CDC2
kinases were both highly active after 48 hours of stimulation with IL-2 and PHA-L in control cells (lanes 3 and 10). Treatment with MPA resulted in complete inhibition of CDK2 and CDC2 activities at these
time points (lanes 6 and 13).
View larger version (18K):
[in this window]
[in a new window]
| Fig 3.
Inhibition of CDK2- and CDC2-associated kinase activities
by MPA. Immunoprecipitations were performed using lysates from human T
lymphocytes before and after IL-2/PHA-L stimulation and anti-CDK2 (lanes 1 to 7) or anti-CDC2 (lanes 8 to 14) antibodies. Competitive peptides were added to the 48-hour samples to control for the specificity of the assay. The immune complexes were incubated in kinase
buffer containing [-32P]ATP and histone H1 for 30 minutes, separated on a SDS-PAGE and exposed to radiograph film, as
outlined in the Materials and Methods section.
|
|
Effects of MPA on p21Waf1, p27Kip1, and p53
expression.
p21Waf1 is a CKI that can bind to and inhibit multiple
cyclin/CDK complexes, including CDK2.29,30 To determine
whether p21Waf1 mediated the loss of CDK2 activity, protein
levels were determined by immunoprecipitation of
35S-labeled cell lysates using an antibody against
p21Waf1 and analysis by SDS-PAGE. As shown in
Fig 4A, 24 hours of treatment with 1 µmol/L MPA does not induce p21Waf1 (lanes 3 to 4), in
contrast to the strong induction with 5-FU (lane 7) and ara-C (lane 9).
Both 5-FU and ara-C also result in coimmunoprecipitation of CDK6, as
determined by its migration on direct Western blot analysis, with
p21Waf1 that is not seen in the control lanes (lanes 8 and
10). After 42 hours of stimulation (Fig 4B), the presence of MPA did
result in p21Waf1 expression (Fig 4B, lane 3) and low
levels of CDK6. Because pRb phosphorylation was completely inhibited at
24 hours, however, we interpret these results to be a consequence
rather than a cause of inhibition of cell cycle progression.
View larger version (68K):
[in this window]
[in a new window]
View larger version (70K):
[in this window]
[in a new window]
| Fig 4.
Immunoprecipitation analysis of
[35S]methionine-labeled lysates using
anti-p21Waf1 antibody.
[35S]methionine-labeled lysates of T lymphocytes
stimulated for 24 hours (A) and 42 hours (B) were immunoprecipitated
with anti-p21Waf1 antibody (lanes 1, 3, 5, 7, and 9) or
with preimmune serum (lanes 2, 4, 6, 8, and 10). Cells were treated
with the drugs indicated from time 0 (1 µmol/L MPA, 50 µmol/L
guanosine, 100 µmol/L 8-aminoguanosine, 25 µg/mL 5-FU, 10 µmol/L
ara-C). The mobility of protein molecular weight standards
p21Waf1 and CDK6 are indicated.
|
|
Because the induction of p53 has been shown to induce
p21Waf1 expression,30-32 Western blot analysis
of p53 was performed under similar conditions
(Fig 5). MPA treatment resulted in a very
low level of p53 expression at 24 hours (lane 3) whereas ara-C
treatment (lane 6) resulted in strongly increased p53 expression. Ara-C treatment has previously been shown to increase p53
levels33,34 which increased p21Waf1
transcription.31 In contrast, direct inhibitors of DNA
synthesis such as hydroxyurea (lane 5) and aphidicolin (lane 7) did not alter p53 expression.
View larger version (17K):
[in this window]
[in a new window]
| Fig 5.
Effect of MPA on p53 expression. p53 immunoblot analysis
of isolated peripheral blood T lymphocytes in the resting state (lane 1), or after stimulation with IL-2/PHA-L alone (lane 2) or in combination with 1 µmol/L MPA (lane 3), 1 µmol/L MPA, 50 µmol/L guanosine and 100 µmol/L 8-aminoguanosine (lane 4), 250 µmol/L hydroxyurea (lane 5), 10 µmol/L ara-C (lane 6), and 1 µg/mL
aphidicolin (lane 7). Cells were harvested after 24 hours of
treatment.
|
|
The CKI p27Kip1 has also been implicated in the control of
the cell-cycle progression in T lymphocytes and appears to be important in the pharmacological effects of rapamycin, a potent immunosuppressive drug.35 Activation of peripheral blood T lymphocytes with
IL-2 and PHA-L resulted in a gradual decrease in the expression of p27Kip1 (Fig 6A, lanes 1 to 6);
in the presence of MPA, however, p27Kip1 expression was
maintained at substantially higher levels over the entire 24-hour
period (Fig 6A, lanes 7 to 11). These results suggest that MPA
treatment prevents or retards the degradation of p27Kip1
that is normally seen with the cell-cycle progression from G1 to S. In
addition, lysates immunoprecipitated with anti-p27Kip1
showed a significant amount of CDK2 associated with p27Kip1
in MPA-treated as compared with control cells at 48 hours (Fig 6B,
lanes 5 and 9). These data strongly suggest that the inhibition of CDK2
activity is due to a p27Kip1 inhibitory effect (Fig 3,
lanes 6 to 7), as well as to the lack of CDK2 induction, because the
level of CDK2 by Western blot analysis is less at both 24 and 48 hours
in treated as compared with control cells (Fig 6B, lanes 6 and 10).
These results indicate an inhibitory effect of MPA on CDK2 induction as
well as on kinase activity.
View larger version (13K):
[in this window]
[in a new window]
View larger version (15K):
[in this window]
[in a new window]
| Fig 6.
Effect of MPA on p27Kip1 expression. (A)
Western blot analysis of lysates immunoprecipitated with
p27Kip1 antibody and detected with the same
p27Kip1 antibody. Resting T lymphocytes (lane 1) were
stimulated by the addition of IL-2/PHA-L in absence (lanes 2 to 6) or
presence (lanes 7 to 11) of 1 µmol/L MPA for 4, 8, 12, 20, and 24 hours. (B) Western blot analysis of lysates immunoprecipitated with
p27Kip1 antibody and probed with CDK2 antibody. T
lymphocytes were stimulated with IL-2/PHA for 24 and 48 hours in
absence (lanes 1 to 6) or presence of 1 µmol/L MPA (lanes 7 to 10).
|
|
Effect of MPA on the formation of the cyclin D/CDK6 complex.
The D-type cyclins are synthesized as part of an early response to
growth factor stimulation and act as regulatory subunits for CDK4 and
CDK6.20,36,37 In Fig 7, we
analyzed the expression of cyclin D2, cyclin D3, and CDK6, the major-D
cyclins and associated kinase in human peripheral blood T
lymphocytes,37,38 after T-cell activation. These
immunoblots show that cyclin D2 is induced by 12 hours after
T-lymphocyte stimulation (Fig 7A, lanes 1 to 4) followed by cyclin D3
(Fig 7B, lanes 1 to 4), as has been shown in previous
studies.36,37 The level of CDK6 also increased markedly by
20 hours after stimulation (Fig 7C, lanes 1 to 4), consistent with
published data.37,38 MPA treatment somewhat diminished the
induction of cyclin D2 and CDK6 (Fig 7AC, lanes 5 to 7), as well as
that of the much less abundant CDK4 (data not shown), but totally
abrogated the induction of cyclin D3 in T cells (Fig 7B, lanes 5 to 7).
View larger version (36K):
[in this window]
[in a new window]
| Fig 7.
Effect of MPA on cyclin D2, cyclin D3, and CDK6 levels.
Western blot analysis of cyclin D2 (A), cyclin D3 (B), and CDK6 (C) proteins were performed on extracts of isolated peripheral blood T
lymphocytes in the resting state (lane 1), or after stimulation with
IL-2/PHA-L for 12, 20, 24, and 36 hours in absence (lanes 2 to 4) or
presence (lanes 5 to 7) of 1 µmol/L MPA.
|
|
We then asked whether MPA affected the association of CDK6 with the
D-cyclins and/or other potentially complexed proteins. Immunoprecipitations of 35S-labeled cell lysates performed
using antibodies against CDK6 showed that CDK6 is highly associated
with the D cyclins after 42 hours of stimulation with IL-2 and PHA-L
(Fig 8A, lane 1). The addition of MPA before incubation markedly
reduced the amount of CDK6 and the associated amount of D cyclin (lane
3), but did not alter the pattern of their association; whereas, the
addition of guanosine and 8-aminoguanosine resulted in levels
equivalent to those in control cells (lane 5). No additional proteins
were found associated with the cyclin D/CDK6 complex in cells treated with MPA. These results were extended to steady-state levels of cyclin
D3 in association with CDK6
(Fig 8B).
Immunoprecipitation with anti-CDK6 antibody showed a virtual absence of
cyclin D3 at 24 hours (lane 4) and complete restoration with guanine
and 8-aminoguanosine (lane 6).
View larger version (42K):
[in this window]
[in a new window]
View larger version (13K):
[in this window]
[in a new window]
| Fig 8.
Effect of MPA on CDK6 complex formation. (A)
[35S]methionine-labeled lysates of stimulated T
lymphocytes were immunoprecipitated with anti-CDK6 antibody
(stimulation for 42 hours) with or without preincubation with the
competing peptide. Cells were stimulated with IL-2/PHA-L in absence
(lanes 1 to 2) or presence of 1 µmol/L MPA (lanes 3 to 4) and 50 µmol/L guanosine and 100 µmol/L 8-aminoguanosine (lanes 5 to 6).
(B) Western blot analysis of lysates immunoprecipitated with CDK6
antibody and detected with cyclin D3 antibody. Resting T lymphocytes
(lane 1) were stimulated with IL-2/PHA-L for 24 and 48 hours in absence
(lanes 2 to 3) or presence of 1 µmol/L MPA (lanes 4 to 5) and 50 µmol/L guanosine and 100 µmol/L 8-aminoguanosine (lanes 6 to 7).
The mobility of protein molecular weight standards and relevant
proteins are indicated.
|
|
In order to ascertain whether inhibition of the induction of cyclin D3
and CDK6 affected the kinase activity for pRb phosphorylation, we
directly measured the phosphorylation of a GST-Rb fusion protein by
immunoprecipitates obtained with anti-CDK6 antibody. The pRb kinase
activity of the immunoprecipitates increased in T cells after 24 hours
and to a greater extent after 48 hours of activation in control
cultures (Fig 9, lanes 1 to 6). Treatment
of cells with MPA greatly decreased the activity of the complex at 24 hours, wherease low-level activity was observed at 48 hours (Fig 9,
lanes 7 and 9). These data confirm that MPA treatment results in marked inhibition of pRb phosphorylating activity by the cyclin D/CDK6 complex
in vitro. The small amount of residual activity at 48 hours may be due
to complex formation with cyclin D2. We were unable to identify
p27Kip1 in association with the anti-CDK6 immunoprecipitate
by Western blot analysis, leading us to believe that the reduction in
cyclin D3 levels is the primary event leading to loss of CDK activity.
View larger version (19K):
[in this window]
[in a new window]
| Fig 9.
Effect of MPA on CDK6 kinase activity. CDK6-associated
kinase activity using affinity-purified antisera against CDK6 and a GST-Rb fusion protein as substrate. Cells were stimulated with IL-2/PHA-L for 24 and 48 hours in absence (lanes 1 to 6) or in presence
of 1µmol/L MPA (lanes 7 to 10). Immunoprecipitations were performed
with or without incubation with the competing peptide for CDK6. The
immune complexes were incubated in kinase buffer containing
[-32P]ATP and separated by SDS-PAGE and exposed to
radiograph film, as outlined in the Materials and Methods section.
|
|
| |
DISCUSSION |
The provision of an adequate supply of guanine nucleotides is clearly
of generalized importance to many aspects of cellular metabolism and
depletion of this nucleotide pool by inhibition of the de novo purine
biosynthetic pathway has been shown to result in cell-cycle arrest in
G1 in T lymphocytes.13 In addition, pharmacologic depletion
of GTP induced by inhibitors of IMPDH has been shown to inhibit the
initiation of DNA replication by preventing RNA-primed DNA synthesis
both in vitro and in T-lymphoblast cell lines.39 Although
each of these observations is of potential relevance to the
well-documented immunosuppressive effects of IMPDH inhibitors, the
underlying mechanisms by which depletion of this essential nucleotide
pool results in these changes are unclear. To understand the
relationship between the depletion of guanine nucleotides and
cell-cycle arrest leading to the inhibition of DNA synthesis, we have
investigated the effects of MPA, a highly selective IMPDH inhibitor, on
the expression and activity of cell-cycle-regulating proteins during
T-lymphocyte activation.
We have shown that the depletion of guanine nucleotides directly
induces a block in cell-cycle progression in G1 and that the addition
of guanosine in conjunction with 8-aminoguanosine to replete GTP levels
completely abrogates this arrest. Of further interest is the
observation that the resulting increase in guanine nucleotide levels
resulting from the addition of these nucleosides leads to a marked
increase in the percentage of activated T cells remaining in S phase at
time points up to 48 hours. These results are similar to data obtained
in cultured Epstein-Barr virus transformed B lymphoblasts, mature
T-cell leukemic cell lines40 and murine melanoma
cells,41 and correlate with the induction by guanosine of
increased incorporation of 3H-thymidine into
DNA.13 The corresponding decrease in the number of cells
remaining in G0/G1 and in G2/M validates the reversal of the G1 block
induced by MPA and suggests that the elevation of guanine nucleotide
pools caused by the salvage of guanine to GMP results in a block in the
exodus of cells from S phase. This observation has yet to be explained.
In contrast, we are able to correlate the G1 arrest induced by MPA
during T-cell activation with the inhibition of pRb phosphorylation resulting in inhibition of the release of the E2F family of
transcription factors that are required for entry into S phase.
Numerous studies have examined the pharmacologic effects of agents that
both arrest proliferating cells at or before the G1/S transition and
inhibit pRb phosphorylation.42-46 These agents include
drugs such as prostaglandin A2, staurosporine,
1,25-dihydroxyvitamin D3, and rapamycin and the experiments
have generally been performed on rapidly proliferating cell lines in
culture. Only one study has investigated the effects of depletion of
normal nucleotide pools on the transition of noncycling, nontransformed
cells from G0 to S phase. Linke et al42 used serum-deprived
normal fibroblasts to show that an inhibitor of pyrimidine nucleotide
biosynthesis, n-phosphonacetyl-L-aspartate or PALA, inhibited the
phosphorylation of pRb that normally occurs after serum repletion. This
effect was shown to be p53-dependent in that p53-deficient fibroblasts
were resistant to cell-cycle arrest; in addition, the inhibition of pRb
phosphorylation correlated directly with the expression of both p53 and
its downstream CDK inhibitor, p21Waf1. The authors
concluded that the nucleotide deprivation induced by PALA and similar
agents caused a reversible quiescent state that is induced by a lack of
pRb phosphorylation and is mediated through p53.
Our data strongly suggest that there are other mechanisms resulting in
cell-cycle arrest that are caused by nucleotide depletion during the
activation of peripheral blood T lymphocytes. Although we observed an
increase in p53 protein levels in MPA-treated T cells, it was at very
low levels and no measurable increase in p21Waf1/Cip1
occurred at 24 hours, despite strong induction by both 5-FU and ara-C.
In contrast, there was a striking and complete inhibition of CDK2
histone phosphorylating activity induced by MPA, indicating an
inhibitory effect of guanine nucleotide depletion at this level. However, the extent to which the inhibition of CDK2 was a primary, as
opposed to a secondary, effect of MPA treatment was unclear and we
elected to examine CDK activity expressed earlier in the cell cycle. It
is known that the expression of specific cyclins and CDKs vary
according to cell type. In primary human peripheral blood T
lymphocytes, cyclin D2 and cyclin D3 complex with CDK4 and CDK6 with
initiation of kinase activity in mid-G1 after 12 to 16 hours of mitogen
stimulation.38 In our study, the addition of IL-2 plus
mitogen rapidly induced the predominant expression of cyclins D2, D3,
and CDK6 corresponding with the induction of CDK6 kinase activity,
observed in control cultures at 24 and 48 hours. MPA decreased the
induction of both cyclin D2 and CDK6, but completely abrogated the
induction of cyclin D3, markedly reducing the ability of the complex to
phosphorylate an Rb substrate in an in vitro assay. In mammalian cells,
the phosphorylation of Rb is initiated by the cyclin D-dependent
kinases and is accelerated by the cyclin E-CDK2 complex.21
The inhibitory effect of MPA on both of these complexes can thus
explain the lack of Rb phosphorylation in T cells. Although one
possible explanation for these effects is an overall reduction in
protein synthesis, the effect of MPA appears to be selective in that
the level of incorporation of 35S-methionine into newly
synthesized protein in T-cell lysates was not diminished under
identical conditions.
To investigate other potentially selective mechanisms for the induction
of cell cycle arrest in T cells, we analyzed the effects of MPA on the
expression of p27Kip1. Stimulation of T lymphocytes with
IL-2 alone leads to the rapid elimination of this inhibitor, which
binds to both CDK6 and CDK235,47 The level of
p27Kip1 protein in human cells is tightly regulated by two
ubiquitin-conjugating enzymes and by cyclin E/CDK2-dependent
phosphorylation.48, 49 Rapamycin, a potent
immunosuppressive agent, has been shown both to prevent the activation
of cyclin E/CDK2 kinase activity and to prevent the degradation of
p27Kip1 found with normal T-cell activation.35
These observations raise the distinct possibility that inhibition of
p27Kip1 degradation might underlie the activity of other
immunosuppressive agents. Our immunoblot data indeed show that guanine
nucleotide depletion significantly retards the degradation of
p27Kip1 after T-cell activation. In addition, in
experiments not shown, the addition of MPA for 24 hours after 24 hours
of IL-2/PHA-L treatment significantly increased the level of
p27Kip1 over that found in control cells, indicating that
guanine nucleotide depletion can also increase p27Kip1
levels in preactivated T cells. A plausible explanation for these results is that the diminished degradation of p27Kip1
induced by MPA during T-cell activation, in conjunction with the lack
of induction of cyclins D2, D3, and CDK6, leads to an increase in the
amount of p27Kip1 available for inhibiting cyclin E/CDK2
activity. Thus, the relative ratio of p27Kip1 to other cell
cycle intermediates may be a major determinant of cell-cycle
progression in these cells, particularly when the amount of CDK2 is
also reduced. Whether guanine nucleotide depletion plays any direct
role in the ubiquitin-dependent proteolysis of p27Kip1 in
the inhibition of cyclin E/CDK2 kinase activity that relates to the
proteolysis of this protein, or in another as yet undefined GTP-dependent pathway regulating p27Kip1 levels, is not
known at present.50-52
In summary, guanine nucleotide depletion induced by inhibitors of the
enzyme IMPDH has a number of biologic effects including the induction
of cell differentiation in many cultured cell models and the induction
of cell cycle arrest in others. The striking selectivity of IMPDH
inhibitors as immunosuppressive agents in vivo supports the concept
that T lymphocytes may have specific mechanisms for inducing cell-cycle
arrest in response to depletion of these metabolites that differentiate
them from other cell types. The effector mechanisms for inhibiting
T-cell activation include inhibition of the induction of cyclin D3, and
to a lesser extent cyclin D2 and CDK6 levels, and decreased degradation
of p27Kip1, with concomitant inhibition of CDK2 activity
and inhibition of pRb phosphorylation. A point for speculation is
whether a single sensing mechanism might exist in early- to mid-G1 that
monitors guanine nucleotide levels and results in a downstream dual
effect on the levels of cyclin D3/CDK6 as well as p27Kip1.
Whether such a mechanism might involve highly specific phosphorylation reactions using GTP as a phosphate donor or an as yet undefined signal
transduction pathway remains to be investigated.
| |
FOOTNOTES |
Submitted June 6, 1997;
accepted November 25, 1997.
A.Y. is supported by a National Institutes of Health Postdoctoral Grant
to the Lineberger Comprehensive Cancer Center. Y.X. is a recipient of
American Cancer Society Junior Faculty Award and is a Pew Scholar in
Biomedical Science. This study was supported by National Institutes of
Health Grants No. 1-R01-CA64192 (B.S.M.) and No. CA-65572 (Y.X).
Address reprint requests to Beverly S. Mitchell, MD, Departments of
Pharmacology and Medicine, Lineberger Comprehensive Cancer Center,
University of North Carolina, Chapel Hill, NC 27599-7295.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
REFERENCES |
1.
Jackson RC,
Weber G:
IMP dehydrogenase, an enzyme linked with proliferation and malignancy.
Nature
256:331,
1975[Medline]
[Order article via Infotrieve]
2.
Natsumeda Y,
Ikegami T,
Murayama K,
Weber G:
De novo guanylate synthesis in the commitment to replication in hepatoma 3924A cells.
Cancer Res
48:507,
1988[Abstract/Free Full Text]
3.
Kokado Y,
Ishibashi M,
Jiang H,
Takahara S,
Sonoda T:
A new triple-drug induction therapy with low dose cyclosporine, mizoribine and prednisolone in renal transplantation.
Transplant Proc
21:1575,
1989[Medline]
[Order article via Infotrieve]
4.
Mita K,
Akiyama N,
Nagao T,
Sugimoto H,
Inoue S,
Osakabe T,
Nakayama Y,
Yokota K,
Sato K,
Uchida H:
Advantages of mizoribine over azathioprine in combination therapy with cyclosporine for renal transplantation.
Transplant Proc
22:1679,
1990[Medline]
[Order article via Infotrieve]
5.
Allison AC,
Almquist SJ,
Muller CD,
Eugui EM:
In vitro immunosuppressive effects of mycophenolic acid and an ester pro-drug, RS-61443.
Transplant Proc
23:10,
1991[Medline]
[Order article via Infotrieve]
6.
Dayton JS,
Turka LA,
Thompson CB,
Mitchell BS:
Comparison of the effects of mizoribine with those of azathioprine, 6-mercaptopurine, and mycophenolic acid on T lymphocyte proliferation and purine ribonucleotide pools.
Mol Pharmacol
41:671,
1992[Abstract]
7.
Knight RD,
Mangum J,
Lucas DL,
Cooney DA,
Khan EC,
Wright DG:
Inosine monophosphate dehydrogenase and myeloid cell maturation.
Blood
69:634,
1987[Abstract/Free Full Text]
8.
Kharbanda SM,
Sherman ML,
Spriggs DR,
Kufe DW:
Effects of tiazofurin on protooncogene expression during HL60 cell differentiation.
Cancer Res
48:5965,
1988[Abstract/Free Full Text]
9.
Lucas DL,
Robins RK,
Knight RD,
Wright DG:
Induced maturation of the human promyelocytic leukemia cell line, HL60, by 2--D-ribofuranosylselenazole-4-carboxamide.
Biochem Biophys Res Commun
115:971,
1983[Medline]
[Order article via Infotrieve]
10.
Tricot GJ,
Jayaram HN,
Nichols CR,
Pennington K,
Lapis E,
Weber G,
Hoffman R:
Hematological and biochemical action of tiazofurin in a case of refractory acute myeloid leukemia.
Cancer Res
47:4988,
1987[Abstract/Free Full Text]
11.
Tricot GJ,
Jayaram HN,
Lapis E,
Natsumeda Y,
Nichols CR,
Kneebone P,
Heerema N,
Weber G,
Hoffman R:
Biochemically directed therapy of leukemia with tiazofurin, a selective blocker of inosine 5'-phosphate dehydrogenase activity.
Cancer Res
49:3696,
1989[Abstract/Free Full Text]
12.
Kusaba R,
Otubo O,
Sugimoto H,
Takahashi I,
Yamada Y,
Yamauchi J,
Akiyama N,
Inou T:
Immunosuppressive effect of bredinin in the management of patients with renal transplantation.
Proc Eur Dialysis Transplant Assoc
18:420,
1981
13.
Turka LA,
Dayton J,
Sinclair G,
Thompson CB,
Mitchell BS:
Guanine ribonucleotide depletion inhibits T cell activation: Mechanism of action of the immunosuppressive drug mizoribine.
J Clin Invest
87:940,
1991
14.
Pardee AB:
G1 events and regulation of cell proliferation.
Science
246:603,
1989[Abstract/Free Full Text]
15.
Weinberg RA:
The retinoblastoma protein and cell cycle control.
Cell
81:323,
1995[Medline]
[Order article via Infotrieve]
16.
Elledge SJ:
Cell cycle checkpoints: preventing an identity crisis.
Science
274:1664,
1996[Abstract/Free Full Text]
17.
Ewen ME,
Sluss HK,
Sherr CJ,
Matsushime H,
Kato J,
Linvingston DM:
Functional interactions of the retinoblastoma protein with mammalian D-type cyclins.
Cell
73:487,
1993[Medline]
[Order article via Infotrieve]
18.
Matsushime H,
Quelle DE,
Shurtleff SA,
Shibuya M,
Sherr CJ,
Kato J-Y:
D-type cyclin-dependent kinase activity in mammalian cells.
Mol Cell Biol
14:2066,
1994[Abstract/Free Full Text]
19.
Hinds PW,
Weinberg RA:
Tumor suppressor genes.
Curr Opin Genet Dev
4:135,
1994[Medline]
[Order article via Infotrieve]
20.
Sherr CJ:
D-type cyclins.
Trends Biochem Sci
20:187,
1995[Medline]
[Order article via Infotrieve]
21.
Sherr CJ:
Cancer cell cycles.
Science
274:1672,
1996[Abstract/Free Full Text]
22.
Morgan DO:
Principles of CDK regulation.
Nature
374:131,
1995[Medline]
[Order article via Infotrieve]
23.
Sherr CJ,
Roberts JM:
Inhibitors of mammalian G1 cyclin-dependent kinases.
Genes Dev
9:1149,
1995[Free Full Text]
24.
Xiong Y:
Why are there so many CDK inhibitors?
Biochim Biophys Acta
1288:01,
1996[Medline]
[Order article via Infotrieve]
25.
Franklin DS,
Xiong Y:
Induction of p18INK4c and its predominant association with CDK4 and CDK6 during myogenic differentiation.
Mol Biol Cell
7:1587,
1996[Abstract]
26.
Phelps DE,
Xiong Y:
Assay for activity of mammalian cyclin D-dependent kinases CDK4 and CDK6.
Methods Enzymol
283:194,
1997[Medline]
[Order article via Infotrieve]
27.
Sidi Y,
Mitchell BS:
Z-nucleotide accumulation in erythrocytes from Lesch-Nyhan patients.
J Clin Invest
76:2416,
1985
28.
Ohtsubo M,
Theodoras AM,
Schumacher J,
Roberts JM,
Pagano M:
Human cyclin E, a nuclear protein essential for the G1-to-S phase transition.
Mol Cell Biol
15:2612,
1995[Abstract]
29.
Harper JW,
Adami GR,
Wei N,
Keyomarsi K,
Elledge SJ:
The p21Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.
Cell
75:805,
1993[Medline]
[Order article via Infotrieve]
30.
Xiong Y,
Hannon GJ,
Zhang H,
Casso D,
Kobayashi R,
Beach D:
p21 is a universal inhibitor of cyclin kinases.
Nature
366:701,
1993[Medline]
[Order article via Infotrieve]
31.
El-Deiry W,
Tokino T,
Velculescu VE,
Levy DB,
Parsons R,
Trent JM,
Lin D,
Mercer WE,
Kinzler KW,
Vogelstein B:
WAF1, a potential mediator of p53 tumor suppression.
Cell
75:817,
1993[Medline]
[Order article via Infotrieve]
32.
Duclic V,
Kaufmann WK,
Wilson SJ,
Tlsty TD,
Lees E,
Harper JW,
Elledge SJ,
Reed SI:
p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest.
Cell
76:1013,
1994[Medline]
[Order article via Infotrieve]
33.
Yuan Z-M,
Huang Y,
Fan M-M,
Sawyers C,
Kharbanda S,
Kufe D:
Genotoxic drugs induce interaction of the c-Abl tyrosine kinase and the tumor suppressor protein p53.
J Biol Chem
271:26457,
1996[Abstract/Free Full Text]
34.
Enokido Y,
Araki T,
Aizawa S,
Hatanaka H:
p53 involves cytosine arabinoside-induced apoptosis in cultured cerebellar granule neurons.
Neurosci Lett
203:1,
1996[Medline]
[Order article via Infotrieve]
35.
Nourse J,
Firpo E,
Flanagan WM,
Coats S,
Polyak K,
Lee M-H,
Massague J,
Crabtree GR,
Roberts JM:
Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin.
Nature
372:570,
1994[Medline]
[Order article via Infotrieve]
36.
Ajchenbaum F,
Ando K,
DeCaprio JA,
Griffin JD:
Independent regulation of human D-type cyclin gene expression during G1 phase in primary human T lymphocytes.
J Biol Chem
268:4113,
1993[Abstract/Free Full Text]
37.
Lucas JJ,
Szepesi A,
Modiano JF,
Domenico J,
Gelfand EW:
Regulation of synthesis and activity of the PLSTIRE protein (cyclin-dependent kinase 6 (cdk6)), a major cyclin D-associated cdk4 homologue in normal human T lymphocytes.
J Immunol
154:6275,
1995[Abstract]
38.
Meyerson M,
Harlow E:
Identification of G1 kinase activity for cdk6, a novel cyclin D partner.
Mol Cell Biol
14:2077,
1994[Abstract/Free Full Text]
39.
Catapano CV,
Dayton JS,
Mitchell BS,
Fernandes DJ:
GTP Depletion induced by IMP dehydrogenase inhibitors blocks RNA-primed DNA synthesis.
Mol Pharmacol
47:948,
1995[Abstract]
40. Sidi Y, Hudson JL, Mitchell BS: Effects of guanine
ribonucleotide accumulation on the metabolism and cell cycle of human
lymphoid cells. Cancer Res 45:1985
41.
Sidi Y,
Panet C,
Cyjon A,
Fenig E,
Beery E,
Nordenberg J:
Guanosine potentiates the antiproliferative effect of cytosine--D-arabinofuranoside in melanoma cell lines.
Cancer Invest
11:523,
1993[Medline]
[Order article via Infotrieve]
42.
Linke SP,
Clarkin KC,
Di Leonardo A,
Tsou A,
Wahl GM:
A reversible, p53-dependent G0/G1 cell cycle arrest induced by ribonucleotide depletion in the absence of detectable DNA damage.
Genes Dev
10:934,
1996[Abstract/Free Full Text]
43.
Gorospe M,
Liu Y,
Xu Q,
Chrest FJ,
Holbrook NJ:
Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human breast carcinoma cells by prostaglandin A2.
Mol Cell Biol
16:762,
1996[Abstract]
44.
Schnier JB,
Gadbois DM,
Nishi K,
Bradbury EM:
The kinase inhibitor staurosporine induces G1 arrest at two points: Effect on retinoblastoma protein phosphorylation and cyclin-dependent kinase 2 in normal and transformed cells.
Cancer Res
54:5959,
1994[Abstract/Free Full Text]
45.
Wang QM,
Jones JB,
Studzinski GP:
Cyclin-dependent kinase inhibitor p27 as a mediator of the G1-S phase block induced by 1,25-dihydroxyvitamin D3 in HL60 cells.
Cancer Res
56:264,
1996[Abstract/Free Full Text]
46.
Marx SO,
Jayaraman T,
Go LO,
Marks AR:
Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells.
Circ Res
76:412,
1995[Abstract/Free Full Text]
47.
Firpo EJ,
Koff A,
Solomon MJ,
Roberts JM:
Innactivation of a cdk2 inhibitor during interleukin2-induced proliferation of human T lymphocytes.
Mol Cell Biol
14:4889,
1994[Abstract/Free Full Text]
48.
Pagano M,
Tam SW,
Theodoras AM,
Beer-Romero P,
Del Sal G,
Chau V,
Yew PR,
Draetta GF,
Rolfe M:
Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27.
Science
269:682,
1995[Abstract/Free Full Text]
49.
Sheaff RJ,
Groudine M,
Gordon M,
Roberts JM,
Clurman BE:
Cyclin E-CDK2 is a regulator of p27 Kip1.
Genes Dev
11:1464,
1997[Abstract/Free Full Text]
50.
Leone G,
DeGregori J,
Sears R,
Jakoi L,
Nevins JR:
Myc and Ras collaborate in inducing accumulation of active cyclin E/CDK2 and E2F.
Nature
387:422,
1997[Medline]
[Order article via Infotrieve]
51.
Fan J,
Bertino JR:
K-ras modulates the cell cycle via both positive and negative regulatory pathways.
Oncogene
14:2595,
1997[Medline]
[Order article via Infotrieve]
52.
Aktas H,
Cai H,
Cooper GM:
Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the CDK inhibitor p27Kip1.
Mol Cell Biol
17:3850,
1997[Abstract]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
X.-X. Sun, M.-S. Dai, and H. Lu
Mycophenolic Acid Activation of p53 Requires Ribosomal Proteins L5 and L11
J. Biol. Chem.,
May 2, 2008;
283(18):
12387 - 12392.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. I. van Leuven, R. Franssen, J. J. Kastelein, M. Levi, E. S. G. Stroes, and P. P. Tak
Systemic inflammation as a risk factor for atherothrombosis
Rheumatology,
January 1, 2008;
47(1):
3 - 7.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S I van Leuven, J J P Kastelein, M R Hayden, and E S Stroes
Mycophenolate mofetil as an immunomodulatory silver bullet in atherogenesis?
Lupus,
November 1, 2006;
15(11_suppl):
11 - 17.
[Abstract]
[PDF]
|
|
|
|
|
|
|
S. I. van Leuven, J. J.P. Kastelein, A. C. Allison, M. R. Hayden, and E. S.G. Stroes
Mycophenolate mofetil (MMF): Firing at the atherosclerotic plaque from different angles?
Cardiovasc Res,
February 1, 2006;
69(2):
341 - 347.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Goulvestre, C. Chereau, C. Nicco, L. Mouthon, B. Weill, and F. Batteux
A Mimic of p21WAF1/CIP1 Ameliorates Murine Lupus
J. Immunol.,
November 15, 2005;
175(10):
6959 - 6967.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. J. Gu, L. Santiago, and B. S. Mitchell
Synergy between imatinib and mycophenolic acid in inducing apoptosis in cell lines expressing Bcr-Abl
Blood,
April 15, 2005;
105(8):
3270 - 3277.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Quemeneur, L. Beloeil, M.-C. Michallet, G. Angelov, M. Tomkowiak, J.-P. Revillard, and J. Marvel
Restriction of De Novo Nucleotide Biosynthesis Interferes with Clonal Expansion and Differentiation into Effector and Memory CD8 T Cells
J. Immunol.,
October 15, 2004;
173(8):
4945 - 4952.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Diez-Juan, P. Perez, M. Aracil, D. Sancho, A. Bernad, F. Sanchez-Madrid, and V. Andres
Selective inactivation of p27Kip1 in hematopoietic progenitor cells increases neointimal macrophage proliferation and accelerates atherosclerosis
Blood,
January 1, 2004;
103(1):
158 - 161.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. J. Gu, A. K. Tolin, J. Jain, H. Huang, L. Santiago, and B. S. Mitchell
Targeted Disruption of the Inosine 5'-Monophosphate Dehydrogenase Type I Gene in Mice
Mol. Cell. Biol.,
September 15, 2003;
23(18):
6702 - 6712.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. J. Gu, K. Gathy, L. Santiago, E. Chen, M. Huang, L. M. Graves, and B. S. Mitchell
Induction of apoptosis in IL-3-dependent hematopoietic cell lines by guanine nucleotide depletion
Blood,
June 15, 2003;
101(12):
4958 - 4965.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Quemeneur, L.-M. Gerland, M. Flacher, M. Ffrench, J.-P. Revillard, and L. Genestier
Differential Control of Cell Cycle, Proliferation, and Survival of Primary T Lymphocytes by Purine and Pyrimidine Nucleotides
J. Immunol.,
May 15, 2003;
170(10):
4986 - 4995.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Quemeneur, M. Flacher, L.-M. Gerland, M. Ffrench, J.-P. Revillard, and N. Bonnefoy-Berard
Mycophenolic Acid Inhibits IL-2-Dependent T Cell Proliferation, But Not IL-2-Dependent Survival and Sensitization to Apoptosis
J. Immunol.,
September 1, 2002;
169(5):
2747 - 2755.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. I. Kovalev, D. S. Franklin, V. M. Coffield, Y. Xiong, and L. Su
An Important Role of CDK Inhibitor p18INK4c in Modulating Antigen Receptor-Mediated T Cell Proliferation
J. Immunol.,
September 15, 2001;
167(6):
3285 - 3292.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Metz, S. Holland, L. Johnson, E. Espling, M. Rabaglia, V. Segu, J. S. Brockenbrough, and P. O. Tran
Inosine-5'-Monophosphate Dehydrogenase Is Required for Mitogenic Competence of Transformed Pancreatic {beta} Cells
Endocrinology,
January 1, 2001;
142(1):
193 - 204.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Millan, F. Oppenheimer, M. Brunet, J. Vilardell, I. Rojo, J. Vives, and J. Martorell
Assessment of Mycophenolic Acid-induced Immunosuppression: A New Approach
Clin. Chem.,
September 1, 2000;
46(9):
1376 - 1383.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O Engelking, L. Fedorov, R Lilischkis, V ter Meulen, and S Schneider-Schaulies
Measles virus-induced immunosuppression in vitro is associated with deregulation of G(1) cell cycle control proteins
J. Gen. Virol.,
July 1, 1999;
80(7):
1599 - 1608.
[Abstract]
|
|
|
|
|
|
|
D. H. Munn, E. Shafizadeh, J. T. Attwood, I. Bondarev, A. Pashine, and A. L. Mellor
Inhibition of T Cell Proliferation by Macrophage Tryptophan Catabolism
J. Exp. Med.,
May 3, 1999;
189(9):
1363 - 1372.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract