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Department of Pathology and Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-mediated
antiproliferation. First, similar phenotypes were also observed in
fibroblasts and pre-B cells derived from STAT1-/- mice,
which do not produce IFN-. Second, comparisons with cells lacking
the gene for IFN- or with cells treated with neutralizing Abs to
IFN- only partially mimicked the STAT1-/- phenotype.
Interestingly, the kinetics of degradation of p27kip1, a
CDK inhibitor, following TCR ligation were faster, and, concomitantly,
the up-regulation of CDK2 kinase activity and protein levels were
increased in stimulated T cells of STAT1-/- mice relative
to those of wild-type mice. Furthermore, STAT1-/- animals
were more susceptible to carcinogen-induced thymic tumors, a possible
consequence of altered T cell growth and/or survival. These results
demonstrate an essential role for STAT1 for lymphocyte survival and
proliferation that is only partially dependent on IFN-
signaling. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(1). Although the mechanisms
for triggering apoptosis by these reagents are distinct, they are all
mediated by a group of functionally related proteases, termed caspases
(2, 3). Caspases are present in cells as inactive
proenzymes and are activated by proteolytic processing into two
subunits in response to apoptotic stimulation. Fourteen mammalian
caspases have been identified thus far, and these enzymes are involved
both in apoptosis and in cytokine protein processing
(4, 5, 6, 7, 8, 9). Modulation of both the levels and activities of
caspases can affect cell survival. Bcl-2 family, consisting of both proapoptotic and antiapoptotic members, acts as a checkpoint upstream of caspases and mitochondrial dysfunction (10). The proapoptotic members, such as bcl-2-associated x protein (bax), bcl-2 antagonist/killer (bak)3, bcl-xL/bcl-2-associated death promoter (bad), and bcl-xS can form homodimers or heterodimers with the antiapoptotic members, including bcl-2, bcl-xL, myeloid cell leukemia sequence 1 (mcl-1), and bfl-1, through conserved domains bcl-2 homology (BH)1, BH2, and BH3 (11). The overall ratio of the proapoptotic molecules to antiapoptotic molecules determines the susceptibility to a death stimulus. Within the thymus, bcl-2 is expressed in nearly all CD4+ or CD8+ single positive thymocytes but not CD4+CD8+ double positive thymocytes (12), possibly leaving them more susceptible to apoptosis. In addition, bcl-2 is highly expressed in mature peripheral T cells in the spleen and lymph nodes. In contrast, bcl-xL is highly expressed in the immature double positive thymocytes but absent from mature single positive thymocytes (13). Similarly, bcl-2 and bcl-xL have a reciprocal pattern of expression in B cell compartment where bcl-2 is high in pro-B cells in the bone marrow and mature B cells in the periphery (14). Conversely, bcl-xL is high in pre-B cells but down-regulated in mature B cells (15). The reciprocal expression patterns of bcl-2 and bcl-xL indicates that their tight regulation is likely critical for providing surviving signals for lymphocytes during development.
Proliferation of T lymphocytes occurs in central lymphoid organs during positive selection as well as in the periphery during clonal expansion in response to foreign Ags. Peripheral T lymphocytes are normally resting and require two sequential mitogenic signals to reenter the cell cycle and proliferate from this quiescent state. The first signal is provided by stimulation of TCR, which induces the synthesis of proteins necessary for progression from G0 to G1. TCR stimulation alone, however, is not sufficient to promote the activation of G1 cyclin-CDK2 complexes, at least in part due to inhibition by p27Kip1, a CDK inhibitor (16). The second mitogenic signal, which can be provided by IL-2 or other mitogenic cytokines, leads to inactivation and degradation of the CDK inhibitor, allowing progression into S phase (17, 18).
Proliferation of T cells in response to Ag is mediated primarily by an
autocrine/paracrine growth pathway. The principle autocrine cytokines
for T cells are IL-2 and IL-4 (19). Activated T cells
transiently increase the transcription of several genes, including IL-2
and IL-2 receptors, that are essential for T cell proliferation and
function. The up-regulated IL-2R -chain (CD25) associates with
constitutively produced IL-2R ß- and -chains to form high affinity
receptors that are capable of stimulating T cell proliferation in
response to physiologically relevant concentrations of IL-2
(20). IL-2 signaling subsequently leads to activation of
several STAT proteins. STAT5, activated by IL-2, plays a critical role
in IL-2-mediated CD25 induction such that
STAT5-/- lymphocytes show impaired induction of
high affinity receptors and markedly reduced proliferation in response
to IL-2 (21, 22). Although it has been suggested that
STAT5 might be activated in response to Ag, such activation is probably
completely dependent on cytokine stimulation (23).
Similarly, STAT6 is required for IL-4-dependent lymphocyte
proliferation. STAT6-/- lymphocytes show
reduced proliferation in response to IL-4, due in part to increased
protein levels of the CDK inhibitor p27Kip1
following IL-4 stimulation (24). In contrast, STAT1, a
major mediator of IFN-, was reported to induce the CDK inhibitor
p21WAF1 in fibroblast cells, in response to
IFN-, and was suggested to be involved in cell growth arrest
(25). However, the role of p21 in the cell cycle
regulation of lymphocytes is controversial, since p21 is also induced
during the proliferative response to TCR stimulation
(18).
In this report, we studied the role of STAT1 in primary T cell
physiology, using mice lacking the STAT1 gene. These experiments showed
that STAT1 modulates lymphocyte survival/apoptosis and proliferation.
Moreover, although some of these effects can be ascribed to the action
of IFN-, an IFN--independent action of STAT1, possibly mediated
by IFN-, was also revealed by these studies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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STAT1-/- mice lacking STAT1
(26), IFNAR-/- mice lacking IFN
type I receptors (27), IFNGR-/-
mice lacking IFN type II receptors (28), and GKO mice
lacking IFN- (29) were compared with wild-type mice of
the same strain background and were housed under specific pathogen-free
conditions. All work with animals was approved by the Institutional
Animal Care and Use Committee of New York University.
In vitro survival assay
Freshly isolated splenocytes (2 x 106) were seeded in 12-well plates for different periods of time. Viable cells were scored and enumerated by trypan blue exclusion. The in vitro cultured splenocytes were fixed with cold 70% ethanol overnight, stained with propidium iodide at 40 µg/ml for 30 min at room temperature, and analyzed by flow cytometry.
RNase protection assay
RNA probes were prepared using mouse apoptosis (mAPO)-1 and mAPO-2 template sets according to the manufacturer’s instructions (PharMingen, San Diego, CA). For mAPO-1, 3 x 105 cpm of mixed probes specific for mouse caspase 1 (ICE), caspase 2 (ICHL/S), caspase 3 (YAMA, CPP32), caspase 6 (Mch2), caspase 7 (Mch3), caspase 8 (FLICE, Mch-5), caspase 11, caspase 12, caspase 14, and two house-keeping genes, L32 and GAPDH, were hybridized at 56°C overnight with 20 µg of total RNA prepared from splenocytes. For mAPO-2, 2.5 x 105 cpm of mixed probe specific for mouse bcl-W, bfl-1, bcl-x, bak, bax, bcl-2, bad, and two house-keeping genes, L32 and GAPDH, were hybridized at 56°C overnight with 15 µg of total RNA prepared from splenocytes. Unhybridized ssRNA was then digested with a mixture of RNases A and T1, and protected RNA was resolved in 6% sequencing gels followed by autoradiography and quantitation by phosphor imager.
Enrichment and stimulation of T lymphocytes and Western blot
Freshly prepared splenocytes of wild-type or STAT1-/- mice were stained with rat anti-B220 Ab (Caltag, South San Francisco, CA) followed by two washes with washing buffer (0.2% BSA containing 1x PBS) then incubated with anti-rat Ig-coated Dynalbeads (Dynal, Oslo, Norway) for 30 min at 4°C. Purified T cell were left untreated or stimulated with plate-bound 2C11 anti-CD3 Ab (10 µg/ml) plus IL-2 (50 U/ml) for 24 h or 48 h. Nuclear extracts were prepared as described (30), and equal amount of proteins were loaded and resolved in 12% SDS-PAGE, transferred to nitrocellulose membrane, and blotted with Abs to p27 (Transduction Laboratories, Lexington, KY), CDK2, and Cyclin A (gifts from Dr. Michele Pagano, New York University, New York).
Histone H1 kinase assay
In vitro kinase assay for CDK2 was performed as described
(17). In brief, equal amounts of whole cell extracts
prepared from purified T cells left untreated or stimulated with
plate-bound 2C11 anti-CD3 Ab (10 µg/ml) plus IL-2 (50 U/ml) for
5 h or 24 h were subject to immunoprecipitation with
anti-CDK2 Ab-conjugated protein A-Sepharose. Following three washes
with lysis buffer and 1 wash with kinase buffer (50 mM Tris (pH 7.5),
10 mM MgCl2, 1 mM DTT), the beads were
resuspended in 20 µl of kinase buffer and incubated with histone H1
(5 µg/reaction) and [-32P]ATP (20 µM) at
30°C for 20 min. The reactions were boiled in sample buffer, resolved
by 12% SDS-PAGE, transferred to nitrocellulose membranes, and detected
by autoradiography.
In vitro proliferation assays
Lymphocytes (1 x 105) were seeded on
anti-CD3 (2C11)-coated 96-well plates (2 µg/ml) in the presence
of human IL-2 (50 U/ml). [3H]Thymidine (1
µCi) was added 24 h before harvest. Where indicated, IFN-
(500 unit/ml) (Boehringer Mannheim, Indianapolis, IN) or
affinity-purified XMG1.2 anti-IFN- (50 µg/ml) Ab was added
into culture medium at the beginning of the treatment with
anti-CD3 Ab.
Fibroblasts were cultured in DMEM supplemented with 10% calf serum. Viable cells from parallel cultures were counted at the indicated times by trypan blue exclusion. Cell cycle parameters were determined by flow cytometry of propidium iodide-stained cells harvested from asynchronously growing cultures (31, 32), and the mean percentage of cells in S phase was averaged for 2 STAT1-/- and three wild-type cell lines, each measured in triplicate.
Thymic tumor induction
Groups of 30 wild-type or STAT1-/- mice were injected i.p. with N-nitroso-N-methylurea (75 mg/kg), as described (33). Mice that died from disease were found at necropsy to have multifocal thymic tumors, often with visible liver metastases (data not shown).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The majority of circulating peripheral lymphocytes are quiescent
and consist of both naive and memory cells. Although the life span of
these two populations differs, both can be measured in weeks or months.
However, the persistence of resting cells is dependent on survival
signals received from the environment through both cytokine and Ag
receptors (34). Removal of quiescent lymphocytes from the
microenvironment of lymph nodes or spleen results in rapid induction of
apoptosis. We investigated the involvement of STAT1 in this process.
Lymphocytes from wild-type and STAT1-/- mice
were cultured in the absence of added cytokines or growth factors.
Following 4 days of culture, only about 25% of wild-type
lymphocytes survived, indicating significant induction of apoptosis. In
contrast to wild-type cells, lymphocytes from
STAT1-/- mice showed enhanced survival (Fig. 1
A), with about 60% of these
cells still viable after 4 days, indicating an approximately
2-fold increase in cell survival in the absence of STAT1.
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B). In contrast, an
approximate 30% decrease in the apoptotic rate was observed for
STAT1-/- lymphocytes, resulting in survival of
greater than 75% of the cells after 24 h, and this difference was
maintained after 48 h with greater than 65% of
STAT1-/- cells still alive. Interestingly, a
small but reproducible increase in cell proliferation was also observed
in the STAT1-/- population, as judged by the
fraction of cells in mitosis with a 4n DNA content. The decreased
apoptotic rate measured in STAT1-/- cells may
explain the observed enhanced survival; however, it did not appear to
result from a defect in Fas-dependent killing since cells from both
strains of mice were equally susceptible to anti-CD3-induced cell
death (data not shown). Decreased caspase 1 and caspase 11 mRNA but not bcl-2 gene family in STAT1-/- lymphocytes
Members of the caspase and bcl-2 families play pivotal roles in
cell survival and/or apoptosis. We first examined the levels of caspase
family mRNA in resting lymphocytes from wild-type and
STAT1-/- mice to see whether changes in
their levels might explain the increased survival or decreased
apoptosis observed in mutant cells. Total RNA prepared from spleen
cells of wild-type or STAT1-/- mice was
hybridized with RNase protection probes specific for nine different
caspases. As shown in Fig. 2A,
the mRNA levels of both caspase 1 and caspase 11 were significantly
reduced in STAT1-/- cells whereas other
caspases were expressed at comparable levels in both
STAT1-/- and wild-type cells. STAT1 is
activated by both type I and type II IFN; therefore, caspase mRNA
levels were also examined in animals specifically deficient in
signaling by each IFN family. The levels of caspase 11 were
decreased in lymphocytes from animals deficient in either receptor.
Caspase 1 mRNA was only significantly reduced in
IFNGR-/- cells. Similar to the effect of STAT1
loss, expression of other caspases was largely unaffected by impaired
IFN signaling. Therefore, the basal levels of caspase 1 and 11 appear
to rely on IFN signaling through STAT1 in vivo, with caspase 11
requiring both IFN- and IFN- for full expression and caspase 1
requiring only IFN-.
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B). In contrast, comparisons of mRNA levels in spleen and
thymus showed that bcl-xL was expressed at
significantly higher levels in the thymus (data not shown, and Ref.
36), suggesting differential roles of bcl-2 genes in
resting T and B lymphocytes. Nonetheless, mRNA levels of the bcl-2 gene
family were comparable in wild-type and mutant mice (Fig. 2B), suggesting that expression of these genes did not
account for increased survival of STAT1-/-
lymphocytes. Enhanced proliferation of STAT1-/- lymphocytes
IFN- is secreted by activated T lymphocytes and is known to
have a potent antiproliferative effect. We asked whether disruption
of STAT1 might relieve the inhibitory effect of IFN- and lead
to enhanced proliferation. Splenocytes from wild-type and
STAT1-/- mice were treated with anti-CD3
Ab, and their proliferation in vitro was monitored by
[3H]thymidine incorporation. Compared with the
proliferation profile of wild-type lymphocytes,
STAT1-/- lymphocytes showed enhanced growth
(Fig. 3A). To test the
potential involvement of IFN- in the growth regulation of wild-type
cells, proliferation of wild-type and STAT1-/-
cells was compared with those from mice missing the IFN- gene (GKO).
As would be expected given the antiproliferative capacity of IFN-,
GKO lymphocytes also exhibited higher growth than wild-type,
particularly after prolonged incubation (Fig. 3A).
Interestingly, however, growth of GKO cells, while greater than
wild-type, did not equal the growth of STAT1-/-
cells, suggesting that their growth was still limited by a
STAT1-dependent process not mediated by IFN-.
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-dependent and -independent regulation
of lymphocyte proliferation was obtained by culturing cells with added
cytokine or with neutralizing Ab to IFN-. Addition of IFN- to
cultures of GKO cells reduced their proliferation to levels equivalent
to wild-type cells (Fig. 3B), demonstrating that their
enhanced growth was due to absence of IFN--mediated inhibition. As
expected, addition of IFN- to cultures of
STAT1-/- cells had no effect on their
proliferation. Likewise, neutralization of secreted IFN- with Ab
increased the proliferation of wild-type cells equal to the growth of
GKO cells (Fig. 3C) but had no effect on the growth of
either GKO or STAT1-/- cells. These results
demonstrate that IFN- secreted by proliferating wild-type
lymphocytes inhibits their growth, especially after extended culture
when it accumulates above some minimal threshold level. GKO cells,
unable to secrete IFN-, or STAT1-/- cells,
unable to respond to it, are unaffected by this autocrine inhibition.
However, the growth of STAT1-/- cells is
further deregulated in a manner independent of the action of IFN-
since these cells proliferated more than cells deprived of IFN-. STAT1 affects molecules that regulate the cell cycle
Since TCR ligation induces resting T cells to undergo cell cycle
progression, we investigated whether molecules involved in the
regulation of the cell cycle were affected in the absence of STAT1. To
avoid the interference of nonproliferating lymphocytes, mainly B cells,
freshly isolated splenocytes were enriched for T cells by removing
B220-positive cells using anti-B220 Ab and magnetic beads. The
levels of CDKs, CDK inhibitors, and cyclins were monitored after
stimulation with anti-CD3 Ab plus IL-2 for 24 h or 48 h.
The kinetics of reduction of nuclear p27 were significantly enhanced in
STAT1-/- cells relative to wild-type cells
(Fig. 4A , first panel). After
stimulation for 24 h, nuclear p27 in
STAT1-/- cell decreased to a minimal level,
whereas around 50% of the protein remained in wild-type cells,
suggesting that STAT1-/- cells were released
from p27 inhibition into the cell cycle earlier or more efficiently
than wild-type cells. The mRNA and protein levels of another CDK
inhibitor, p21WAF1, however, were similar in both
wild-type and STAT1-/- cells after stimulation
(data not shown). The protein levels of nuclear CDK2 were significantly
increased in both wild-type and STAT1-/- cells
after stimulation for 48 h (Fig. 4A, second
panel). Interestingly, however, there was about 4-fold more
nuclear CDK2 protein in STAT1-/- cells than in
wild-type cells (Fig. 4A, second panel). The
induced levels cyclin A, E, and D3 were comparable between wild-type
and STAT1-/- cells (Fig. 4A,
third panel, and data not shown).
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B, increased histone kinase
activity was observed 24 h after stimulation of wild-type cells.
In contrast, STAT1-/- cells showed increased
activity at 5 h of stimulation. At 24 h, the activity was
increased significantly by almost 2-fold that of wild-type cells. An
equivalent amount of CDK2 protein was observed in both wild-type and
STAT1-/- cells at 24 h (Fig. 4A, second panel), suggesting that the increased
CDK2 kinase activity at this early time point in
STAT1-/- cells was due to a diminished
inhibitory effect of p27 (Fig. 4A, first
panel). These results suggest that a more rapid decrease of a CDK
inhibitor followed by a higher increase of a CDK protein contribute to
enhanced proliferation of T cells devoid of STAT1.
Enhanced proliferation and survival of T lymphocytes in vitro
prompted us to examine possible in vivo consequences of the lack of
STAT1-mediated growth control. Although no major alterations in the
steady-state abundance of lymphocytes were observed,
STAT1-/- mice showed increased susceptibility
to carcinogen-induced thymic tumor induction. Groups of wild-type
and STAT1-/- mice were challenged with
N-nitroso-N-methylurea and monitored for tumor
induction. Tumor induction occurred earlier and more frequently in
STAT1-/- mice than in control mice treated with
carcinogen. The mean latency for tumor induction in wild-type mice was
139 ± 4 days whereas mean latency occurred in
STAT1-/- mice after 118 ± 4 days
(Fig. 5A). The tumor cells
were mainly immature T lymphocytes in both wild-type and
STAT1-/- mice (data not shown, and Ref.
33). All animals succumbing to disease showed similar
thymic tumors at necropsy, with no gross differences in tumor histology
between mutant and wild-type mice (data not shown).
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B). In
addition, fibroblast lines established from
STAT1-/- embryos proliferated more rapidly that
wild-type cells, exhibiting a doubling time of 
14 h vs 22 h for
wild-type cells. A significantly higher fraction of cells in S phase
was observed for asynchronously growing cultures of
STAT1-/- cells compared with that of wild-type
cells (Fig. 5C). Because neither of these cell types is
capable of producing IFN- (37), these results again
point to STAT1-dependent growth regulation that is independent of the
action of IFN-. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or
IFN- that signal through STAT1 and exert broad activities on
lymphocyte functions. Overlap between loss of STAT1 and loss of IFN
receptors suggests a role for constitutive IFN signaling in the
maintenance of lymphocyte function even in the absence of inflammation.
Interestingly, however, evidence of IFN--independent functions in
lymphocytes that are nonetheless still dependent on STAT1 was also
obtained. It is likely that some of these effects reflect an important
role for IFN-/IFN-ß in lymphocyte functions normally ascribed to
IFN- (38) but the possibility also exists that some
STAT1-dependent mechanisms are completely independent of IFN signaling.
For instance, we have found that STAT1 can regulate the constitutive
expression of MHC class I by an IL-7-dependent but IFN-independent
pathway (39) and that STAT1 can regulate the proliferation
and differentiation of chondrocytes in response to fibroblast growth
factor (FGF) (40).
An example of overlap between basal and IFN-induced regulation of gene
expression is caspase 1. Caspase 1 has been shown to be involved in
IFN--induced apoptosis in a STAT1-dependent manner (41, 42). We found that caspase 1, as well as its upstream activator
caspase 11, was also affected under basal, non-IFN-induced conditions
by the absence of either IFN receptors or STAT1, suggesting that the
levels of these proteins are maintained by constitutive signaling
through the IFN pathway. Caspase 1 and caspase 11 belong to the same
subfamily of caspase genes and share a similar substrate specificity.
Gene targeting experiments have revealed that both caspase 1 and
caspase 11 are primarily important for the regulation of inflammatory
immune responses, and caspase 1-/- and caspase
11-/- mice are highly resistant to endotoxic
shock (4, 5, 43). Because caspase 1 is activated through a
direct physical interaction with caspase 11, caspase 11-deficient cells
are resistant to apoptosis induced by caspase 1 overexpression, and the
production of IL-1 and IL-1ß is blocked after LPS stimulation,
which is a critical event mediated by caspase 1 during toxic shock
(5). The decreased level of caspase 1 and caspase 11 in
STAT1-/- lymphocytes may contribute to the
reduced apoptosis observed. However, similar to caspase
1-/- or caspase 11-/-
cells, mutation of STAT1 did not protect thymocytes from
gamma-irradiation- or dexamethasone-induced cell death (data not
shown), confirming that the major avenues leading to lymphocyte
apoptosis are independent of STAT1. Recently, a family of inhibitor of
apoptosis (IAP) proteins were found to suppress cell death perhaps by
binding directly to caspases and inhibit their function
(44). Although the role of STAT1 in the regulation of IAP
proteins is not clear, an involvement of IAP in enhanced survival of
STAT1-/- lymphocytes cannot be excluded.
A dependence on STAT1 for production of caspase 1, 2, and 3 has been
observed in mutagenized human fibrosarcoma U3A cells (45).
Interestingly, it was reported that STAT1 function did not require
tyrosine phosphorylation or dimerization for maintenance of caspase
gene expression, suggesting a non-cytokine-dependent role for STAT1 in
these cells. In contrast to this report, we found reduced levels of
caspase 1 and 11, but not of 2 and 3, in
STAT1-/- murine lymphocytes. Moreover, we found
that loss of IFN responsiveness, either type I or type II, also led to
reduced levels of caspase 1 and 11, suggesting a standard,
cytokine-dependent role for STAT1 in this process. The differences
between mouse lymphocytes and human fibrosarcomas for regulation of
caspase gene expression by STAT1 remain to be determined.
Interestingly, the levels of bcl-2-related genes were not altered in
the lymphocytes lacking STAT1, although it has been shown that IFN-
up-regulates bak in an adenocarcinoma cell line (42).
The in vitro proliferation of lymphocytes from wild-type, GKO, and
STAT1-/- mice in the presence or absence of
exogenous IFN- or of anti-IFN- Abs confirmed the known role
for IFN- in regulation of cell growth. However, the differences
between disruption of STAT1 and selective modulation of IFN-
responsiveness revealed that additional mechanisms beyond loss of
IFN--mediated antiproliferation contribute to the enhanced
proliferation of STAT1-/- cells. GKO
lymphocytes unable to secrete IFN- grew more rapidly than wild-type
cells but still slower than STAT1-/- cells.
Moreover, wild-type cells treated with neutralizing Ab against IFN-
proliferated similarly to GKO cells but still more slower than
STAT1-/- cells. Hyperproliferation of
STAT1-/- lymphocytes was not due to an abnormal
regulation of IL-2R-chain expression required for high affinity
receptor expression as seen in STAT5-deficient mice (21, 22) since normal levels of IL-2R were expressed in the
absence of STAT1 (data not shown). The fact that the hyperproliferation
was also observed in other cell types, e.g., fibroblasts and
transformed pre-B cells, which do not express IL-2R or IFN-,
suggests that multiple STAT1-dependent processes are involved.
The faster down-regulation of the p27 CDK inhibitor and higher
up-regulation of CDK2 in STAT1-/- cells after
stimulation pinpointed altered levels of cell cycle modulators as an
underlying mechanism of enhanced proliferation. p27 is highly expressed
in T cells and is rapidly eliminated upon mitogenic stimulation,
leading to progression of the cell cycle from quiescence
(G0) to S phase (17, 18). An
enhanced mitogenic activation has also been documented in thymocytes
deficient in p27 (46). Thus, the faster kinetics of
degradation of p27 protein in STAT1-/- cells
may contribute to the promotion of T cell proliferation at early stages
of stimulation. Indeed, the CDK2 kinase activity was found to be higher
in STAT1-/- lymphocytes than that of wild-type
at 24 h of stimulation whereas protein levels of CDK2 were
comparable between these two mice (Fig. 4, A and
B), suggesting that an earlier release of inhibition of p27
may explain an enhanced proliferation rate at this time (Fig. 3A). CDK2, one of the targets of p27 CDK inhibitor, is
necessary for entry into S phase (47). The increased
levels of nuclear CDK2 in STAT1-/- cells after
48 h stimulation would greatly facilitate cell cycle progression
(Figs. 3 and 4), providing an important driving force for
proliferation. Although a direct role of STAT1 in the regulation
of CDK inhibitors and CDKs remains to be determined, IFN- has
been shown to suppress IL-2-mediated decline of P27 and up-regulation
of the protein levels of several CDKs, including CDK2 in T
lymphocytes (48).
Although decreased apoptosis and enhanced proliferation were observed in STAT1-/- lymphocytes in vitro, mutant animals were nonetheless capable of maintaining normal lymphoid homeostasis in vivo. Moreover, no significant increase in spontaneous lymphoproliferative disease occurred in these animals. One likely reason for this difference is that in vitro growth conditions exaggerate the loss of normal growth regulation. For instance, rapid lymphocyte apoptosis occurs in vitro for cells that would survive if left in a lymphoid organ. Presumably, survival signals provided by the organ microenvironment override the induction of apoptosis observed in vitro.
A potential in vivo correlate of altered lymphocyte growth control was
observed in chemical carcinogen-treated mice.
STAT1-/- mice displayed a higher and more rapid
incidence of thymic tumor induction than their wild-type counterparts.
A similar observation of increased incidence of fibrosarcomas induced
by the carcinogen methylcholanthrene has been reported by Schreiber and
colleagues (49). They provided evidence that at least part
of the increased susceptibility to methylcholanthrene was due to
impaired IFN- responsiveness of the induced tumors, suggesting a
defect in immune surveillance. However, given the altered growth and
survival parameters of STAT1-/- lymphocytes in
culture, defects in intrinsic growth control may also have contributed
to the observed increased tumor incidence. For instance, it has
recently been reported that STAT1-dependent genes are expressed in
cultured epithelial cells at confluence and during senescence, again
suggesting a general role for this pathway in cellular growth control
independent of classical inducers of IFN (50). These
results suggest that STAT1 contributes to cellular growth control in
both direct and indirect pathways. Impairment of these controls may
contribute to immune dysfunction and malignancy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David E. Levy, Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016. E-mail address:
3 Abbreviations used in this paper: bak, bcl-2 antagonist/killer; GKO, IFN- knockout; BH, bcl-2 homology; mAPO, mouse apoptosis; IAP, inhibitor of apoptosis.
Received for publication September 10, 1999. Accepted for publication November 22, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 I. Najjar, P. O. Schischmanoff, F. Baran-Marszak, P.-A. Deglesne, I. Youlyouz-Marfak, M. Pampin, J. Feuillard, G. W. Bornkamm, M. K. Chelbi-Alix, and R. Fagard Novel function of STAT1{beta} in B cells: induction of cell death by a mechanism different from that of STAT1{alpha} J. Leukoc. Biol., December 1, 2008; 84(6): 1604 - 1612. [Abstract] [Full Text] [PDF]
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 A. Hosui and L. Hennighausen Genomic dissection of the cytokine-controlled STAT5 signaling network in liver Physiol Genomics, July 1, 2008; 34(2): 135 - 143. [Abstract] [Full Text] [PDF]
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 D. de Totero, R. Meazza, M. Capaia, M. Fabbi, B. Azzarone, E. Balleari, M. Gobbi, G. Cutrona, M. Ferrarini, and S. Ferrini The opposite effects of IL-15 and IL-21 on CLL B cells correlate with differential activation of the JAK/STAT and ERK1/2 pathways Blood, January 15, 2008; 111(2): 517 - 524. [Abstract] [Full Text] [PDF]
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 N. N. Khodarev, A. J. Minn, E. V. Efimova, T. E. Darga, E. Labay, M. Beckett, H. J. Mauceri, B. Roizman, and R. R. Weichselbaum Signal Transducer and Activator of Transcription 1 Regulates Both Cytotoxic and Prosurvival Functions in Tumor Cells Cancer Res., October 1, 2007; 67(19): 9214 - 9220. [Abstract] [Full Text] [PDF]
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B. A. Stout, K. Melendez, J. Seagrave, M. J. Holtzman, B. Wilson, J. Xiang, and Y. Tesfaigzi STAT1 Activation Causes Translocation of Bax to the Endoplasmic Reticulum during the Resolution of Airway Mucous Cell Hyperplasia by IFN-{gamma} J. Immunol., June 15, 2007; 178(12): 8107 - 8116. [Abstract] [Full Text] [PDF]
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N. Cuesta, Q. M. Nhu, E. Zudaire, S. Polumuri, F. Cuttitta, and S. N. Vogel IFN Regulatory Factor-2 Regulates Macrophage Apoptosis through a STAT1/3- and Caspase-1-Dependent Mechanism J. Immunol., March 15, 2007; 178(6): 3602 - 3611. [Abstract] [Full Text] [PDF]
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D. Konforte and C. J. Paige Identification of Cellular Intermediates and Molecular Pathways Induced by IL-21 in Human B Cells J. Immunol., December 15, 2006; 177(12): 8381 - 8392. [Abstract] [Full Text] [PDF]
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W.-C. Chou, D. E. Levy, and C.-K. Lee STAT3 positively regulates an early step in B-cell development Blood, November 1, 2006; 108(9): 3005 - 3011. [Abstract] [Full Text] [PDF]
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 A. Garcia-Sastre and C. A. Biron Type 1 interferons and the virus-host relationship: a lesson in detente . Science, May 12, 2006; 312(5775): 879 - 882. [Abstract] [Full Text] [PDF]
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D. de Totero, R. Meazza, S. Zupo, G. Cutrona, S. Matis, M. Colombo, E. Balleari, I. Pierri, M. Fabbi, M. Capaia, et al. Interleukin-21 receptor (IL-21R) is up-regulated by CD40 triggering and mediates proapoptotic signals in chronic lymphocytic leukemia B cells Blood, May 1, 2006; 107(9): 3708 - 3715. [Abstract] [Full Text] [PDF]
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 C. A. Gysemans, L. Ladriere, H. Callewaert, J. Rasschaert, D. Flamez, D. E. Levy, P. Matthys, D. L. Eizirik, and C. Mathieu Disruption of the {gamma}-Interferon Signaling Pathway at the Level of Signal Transducer and Activator of Transcription-1 Prevents Immune Destruction of {beta}-cells Diabetes, August 1, 2005; 54(8): 2396 - 2403. [Abstract] [Full Text] [PDF]
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 R. Gimeno, C.-K. Lee, C. Schindler, and D. E. Levy Stat1 and Stat2 but Not Stat3 Arbitrate Contradictory Growth Signals Elicited by Alpha/Beta Interferon in T Lymphocytes Mol. Cell. Biol., July 1, 2005; 25(13): 5456 - 5465. [Abstract] [Full Text] [PDF]
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 A. Takeda, S. Hamano, H. Shiraishi, T. Yoshimura, H. Ogata, K. Ishii, T. Ishibashi, A. Yoshimura, and H. Yoshida WSX-1 over-expression in CD4+ T cells leads to hyperproliferation and cytokine hyperproduction in response to TCR stimulation Int. Immunol., July 1, 2005; 17(7): 889 - 897. [Abstract] [Full Text] [PDF]
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 A. Huang, Y.-Y. Zhang, K. Chen, K. Hatakeyama, and J. F. Keaney Jr Cytokine-Stimulated GTP Cyclohydrolase I Expression in Endothelial Cells Requires Coordinated Activation of Nuclear Factor-{kappa}B and Stat1/Stat3 Circ. Res., February 4, 2005; 96(2): 164 - 171. [Abstract] [Full Text] [PDF]
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Y. Tanabe, T. Nishibori, L. Su, R. M. Arduini, D. P. Baker, and M. David Cutting Edge: Role of STAT1, STAT3, and STAT5 in IFN-{alpha}{beta} Responses in T Lymphocytes J. Immunol., January 15, 2005; 174(2): 609 - 613. [Abstract] [Full Text] [PDF]
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A. Halupa, M. L. Bailey, K. Huang, N. N. Iscove, D. E. Levy, and D. L. Barber A novel role for STAT1 in regulating murine erythropoiesis: deletion of STAT1 results in overall reduction of erythroid progenitors and alters their distribution Blood, January 15, 2005; 105(2): 552 - 561. [Abstract] [Full Text] [PDF]
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A. V. Villarino, E. Huang, and C. A. Hunter Understanding the Pro- and Anti-Inflammatory Properties of IL-27 J. Immunol., July 15, 2004; 173(2): 715 - 720. [Abstract] [Full Text] [PDF]
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 P. A. Townsend, T. M. Scarabelli, S. M. Davidson, R. A. Knight, D. S. Latchman, and A. Stephanou STAT-1 Interacts with p53 to Enhance DNA Damage-induced Apoptosis J. Biol. Chem., February 13, 2004; 279(7): 5811 - 5820. [Abstract] [Full Text] [PDF]
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J. J. Sironi and T. Ouchi STAT1-induced Apoptosis Is Mediated by Caspases 2, 3, and 7 J. Biol. Chem., February 6, 2004; 279(6): 4066 - 4074. [Abstract] [Full Text] [PDF]
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K. Schroder, P. J. Hertzog, T. Ravasi, and D. A. Hume Interferon-{gamma}: an overview of signals, mechanisms and functions J. Leukoc. Biol., February 1, 2004; 75(2): 163 - 189. [Abstract] [Full Text] [PDF]
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 D. W. Sternberg and D. G. Gilliland The Role of Signal Transducer and Activator of Transcription Factors in Leukemogenesis J. Clin. Oncol., January 15, 2004; 22(2): 361 - 371. [Abstract] [Full Text] [PDF]
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 T. Nishibori, Y. Tanabe, L. Su, and M. David Impaired Development of CD4+ CD25+ Regulatory T Cells in the Absence of STAT1: Increased Susceptibility to Autoimmune Disease J. Exp. Med., January 5, 2004; 199(1): 25 - 34. [Abstract] [Full Text] [PDF]
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L. A. Lieberman, M. Banica, S. L. Reiner, and C. A. Hunter STAT1 Plays a Critical Role in the Regulation of Antimicrobial Effector Mechanisms, but Not in the Development of Th1-Type Responses during Toxoplasmosis J. Immunol., January 1, 2004; 172(1): 457 - 463. [Abstract] [Full Text] [PDF]
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 T. E. Battle, W. G. Wierda, L. Z. Rassenti, D. Zahrieh, D. Neuberg, T. J. Kipps, and D. A. Frank In Vivo Activation of Signal Transducer and Activator of Transcription 1 after CD154 Gene Therapy for Chronic Lymphocytic Leukemia Is Associated with Clinical and Immunologic Response Clin. Cancer Res., June 1, 2003; 9(6): 2166 - 2172. [Abstract] [Full Text] [PDF]
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E. Dondi, L. Rogge, G. Lutfalla, G. Uze, and S. Pellegrini Down-Modulation of Responses to Type I IFN Upon T Cell Activation J. Immunol., January 15, 2003; 170(2): 749 - 756. [Abstract] [Full Text] [PDF]
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R. Schauvliege, J. Vanrobaeys, P. Schotte, and R. Beyaert Caspase-11 Gene Expression in Response to Lipopolysaccharide and Interferon-gamma Requires Nuclear Factor-kappa B and Signal Transducer and Activator of Transcription (STAT) 1 J. Biol. Chem., October 25, 2002; 277(44): 41624 - 41630. [Abstract] [Full Text] [PDF]
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A. Widschwendter, S. Tonko-Geymayer, T. Welte, G. Daxenbichler, C. Marth, and W. Doppler Prognostic Significance of Signal Transducer and Activator of Transcription 1 Activation in Breast Cancer Clin. Cancer Res., October 1, 2002; 8(10): 3065 - 3074. [Abstract] [Full Text] [PDF]
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S. Agrawal, M. L. Agarwal, M. Chatterjee-Kishore, G. R. Stark, and G. M. Chisolm Stat1-Dependent, p53-Independent Expression of p21waf1 Modulates Oxysterol-Induced Apoptosis Mol. Cell. Biol., April 1, 2002; 22(7): 1981 - 1992. [Abstract] [Full Text] [PDF]
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D. E. Levy Whence Interferon? Variety in the Production of Interferon in Response to Viral Infection J. Exp. Med., February 19, 2002; 195(4): F15 - F18. [Full Text] [PDF]
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 J. Maier, C. Kincaid, A. Pagenstecher, and I. L. Campbell Regulation of Signal Transducer and Activator of Transcription and Suppressor of Cytokine-Signaling Gene Expression in the Brain of Mice with Astrocyte-Targeted Production of Interleukin-12 or Experimental Autoimmune Encephalomyelitis Am. J. Pathol., January 1, 2002; 160(1): 271 - 288. [Abstract] [Full Text] [PDF]
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P. Bernabei, E. M. Coccia, L. Rigamonti, M. Bosticardo, G. Forni, S. Pestka, C. D. Krause, A. Battistini, and F. Novelli Interferon-{gamma} receptor 2 expression as the deciding factor in human T, B, and myeloid cell proliferation or death J. Leukoc. Biol., December 1, 2001; 70(6): 950 - 960. [Abstract] [Full Text] [PDF]
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H. Yoshikawa, Y. Nakajima, and K. Tasaka IFN-{gamma} Induces the Apoptosis of WEHI 279 and Normal Pre-B Cell Lines by Expressing Direct Inhibitor of Apoptosis Protein Binding Protein with Low pI J. Immunol., September 1, 2001; 167(5): 2487 - 2495. [Abstract] [Full Text] [PDF]
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 C. V. Ramana, M. P. Gil, Y. Han, R. M. Ransohoff, R. D. Schreiber, and G. R. Stark Stat1-independent regulation of gene expression in response to IFN-gamma PNAS, June 5, 2001; 98(12): 6674 - 6679. [Abstract] [Full Text] [PDF]
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 M. Sahni, R. Raz, J. D. Coffin, D. Levy, and C. Basilico STAT1 mediates the increased apoptosis and reduced chondrocyte proliferation in mice overexpressing FGF2 Development, June 1, 2001; 128(11): 2119 - 2129. [Abstract] [Full Text] [PDF]
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C.-K. Lee, D. T. Rao, R. Gertner, R. Gimeno, A. B. Frey, and D. E. Levy Distinct Requirements for IFNs and STAT1 in NK Cell Function J. Immunol., October 1, 2000; 165(7): 3571 - 3577. [Abstract] [Full Text] [PDF]
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) and STAT1-/- (
) mice was measured
by counting viable cells at various time points following seeding of
5 x 104 cells per dish. The percentage of cells in S
phase, as determined by flow cytometry, was averaged for two
STAT1-/- cell lines and three wild-type cell lines
measured three times (inset).